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Advances in Stem Cell Biology Series Editor Alexander Birbrair

Advances in Stem Cell Biology

iPSCs for Studying Infectious Diseases, Volume 8

Edited by Alexander Birbrair

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices

Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-823808-0 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Elizabeth Brown Editorial Project Manager: Billie Jean Fernandez Production Project Manager: Omer Mukthar Cover Designer: Mark Rogers Typeset by TNQ Technologies

This book is dedicated to my mother, Marina Sobolevsky, of blessed memory, who passed away during the creation of this volume. Professor of Mathematics at the State University of Ceara´ (UECE), she was loved by her colleagues and students, whom she inspired by her unique manner of teaching. All success in my career and personal life I owe to her.

My father Lev Birbrair and my beloved mom Marina Sobolevsky of blessed memory (July 28, 1959eJune 3, 2020)

Contributors Serkan Belkaya Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, C¸ankaya, Ankara, Turkey David C. Bloom Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, United States Guglielmo Bove Schaller Research Group, Center of Infectious Diseases, Department of Virology, Heidelberg University Hospital, Heidelberg, Germany Adriana Bozzi Instituto Rene´ Rachou, FIOCRUZ, Belo Horizonte, Brazil; Departamento de Cieˆncias Biolo´gicas, Universidade Estadual de Santa Cruz, UESC, Ilhe´us, Brazil Kevin M. Coombs University of Manitoba, Department of Medical Microbiology and Infectious Diseases, Winnipeg, MB, Canada; Manitoba Centre for Proteomics & Systems Biology, Winnipeg, MB, Canada; Children’s Hospital Research Institute of Manitoba, Winnipeg, MB, Canada Viet Loan Dao Thi Schaller Research Group, Center of Infectious Diseases, Department of Virology, Heidelberg University Hospital, Heidelberg, Germany Matthew J. Demers Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States Leonardo D’Aiuto Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States Jessica L. Forbester Division of Infection and Immunity/Systems Immunity University Research Institute, Cardiff University, Cardiff, United Kingdom; MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom Eric C. Freundt Department of Biology, The University of Tampa, Tampa, FL, United States Sandra K. Halonen Department of Microbiology and Immunology, Montana State University, Bozeman, MT, United States




Brandon J. Kim University of Alabama, Department of Biological Sciences, Tuscaloosa, AL, United States Paul R. Kinchington Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; Department of Molecular Microbiology and Genetics, University of Pittsburgh, Pittsburgh, PA, United States Thomas E. Lane Department of Neurobiology & Behavior, University of California, Irvine, Irvine, CA, United States Jeanne F. Loring Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, United States Laura L. McIntyre Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, CA, United States James McNulty Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON, Canada Ann-Kathrin Mehnert Schaller Research Group, Center of Infectious Diseases, Department of Virology, Heidelberg University Hospital, Heidelberg, Germany Vishwajit L. Nimgaonkar Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States Warren C. Plaisted Genomics Institute of the Novartis Research Foundation, San Diego, CA, United States Pavan Rajanahalli Department of Biology, The University of Tampa, Tampa, FL, United States Duncan R. Smith Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhon Pathom, Thailand David A. Stevens California Institute for Medical Research, San Jose, CA, United States; Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA, United States


Craig M. Walsh Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, CA, United States Maribeth A. Wesesky Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States Ali Zahedi-Amiri University of Manitoba, Department of Medical Microbiology and Infectious Diseases, Winnipeg, MB, Canada; Manitoba Centre for Proteomics & Systems Biology, Winnipeg, MB, Canada Wenxiao Zheng Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; Department of Psychiatry, The Second Xiangya Hospital, Xiangya School of Medicine, Central South University, Changsha, China


About the editor Alexander Birbrair Dr. Alexander Birbrair received his bachelor’s biomedical degree from Santa Cruz State University in Brazil. He completed his PhD in Neuroscience, in the field of stem cell biology, at the Wake Forest School of Medicine under the mentorship of Osvaldo Delbono. Then, he joined as a postdoc in stem cell biology at Paul Frenette’s laboratory at Albert Einstein School of Medicine in New York. In 2016, he was appointed faculty at Federal University of Minas Gerais in Brazil, where he started his own lab. His laboratory is interested in understanding how the cellular components of different tissues function and control disease progression. His group explores the roles of specific cell populations in the tissue microenvironment by using state-of-the-art techniques. His research is funded by the Serrapilheira Institute, CNPq, CAPES, and FAPEMIG. In 2018, Alexander was elected affiliate member of the Brazilian Academy of Sciences (ABC), and in 2019, he was elected member of the Global Young Academy (GYA). He is the Founding Editor and Editor-in-Chief of Current Tissue Microenvironment Reports and Associate Editor of Molecular Biotechnology. Alexander also serves in the editorial board of several other international journals: Stem Cell Reviews and Reports, Stem Cell Research, Stem Cells and Development, and Histology and Histopathology.


Preface This book’s initial title was “iPSCs: Recent Advances.” Nevertheless, because of the ongoing strong interest in this theme, we were capable of collecting more chapters than would fit in one single volume, covering induced pluripotent stem cells (iPSCs) biology from different perspectives. Therefore, the book was subdivided into several volumes. This volume “iPSCs for Studying Infectious Diseases” offers contributions by known scientists and clinicians in the multidisciplinary areas of biological and medical research. The chapters bring up-to-date comprehensive overviews of current advances in the field. This book describes the use of iPSCs to model several infectious diseases in vitro, enabling us to study the cellular and molecular mechanisms involved in different infectious pathologies. Further insights into these mechanisms will have important implications for our understanding of infectious disease appearance, development, and progression. The authors focus on the modern state-of-the-art methodologies and the leading-edge concepts in the field of stem cell biology. In recent years, remarkable progress has been made in the obtention of iPSCs and their differentiation into several cell types, tissues, and organs using state-of-the-art techniques. These advantages facilitated identification of key targets and definition of the molecular basis of several disorders. Thus, the present book is an attempt to describe the most recent developments in the area of iPSCs biology, which is one of the rising hot topics in the field of molecular and cellular biology today. Here, we present a selected collection of detailed chapters on what we know so far about the use of iPSCs for modeling multiple infectious diseases. Eleven chapters written by experts in the field summarize the present knowledge about iPSCs for studying infectious diseases. Duncan R. Smith from Mahidol University gives a historical perspective on the application of iPSCs in virology. Thomas E. Lane and colleagues from University of California discuss the use of iPSCs in coronavirus-induced neurologic disease. Ali Zahedi-Amiri and Kevin M. Coombs from University of Manitoba describe iPSCs for modeling influenza infection. Leonardo D’Aiuto and colleagues from University of Pittsburgh compile our understanding of iPSCs for modeling of herpes simplex virus 1 infections. Serkan Belkaya from Bilkent University updates us with what we know about iPSCs for modeling coxsackievirus infection. Eric C. Freundt and Pavan Rajanahalli from The University of Tampa summarize current knowledge on iPSCs to model Theiler’s murine encephalomyelitis virus infection. Viet Loan Dao Thi and colleagues from Heidelberg University address the importance of iPSCs for modeling of hepatotropic pathogen infection. Sandra K. Halonen from Montana State University talks about the use of human iPSCs to study the neuropathogenesis of Toxoplasma gondii. Adriana Bozzi and David A Stevens from Stanford University focus on iPSCs for modeling Chagas disease. Brandon J Kim from the University of Alabama presents the use of iPSCs to study hostepathogen interactions with




Streptococcus agalactiae and Neisseria meningitidis. Finally, Jessica L Forbester from the University of Oxford updates us on the use of iPSCs for modeling of Salmonella infection. It is hoped that the articles published in this book will become a source of reference and inspiration for future research ideas. I would like to express my deep gratitude to my wife Veranika Ushakova, and Ms. Billie Jean Fernandez and Ms. Elisabeth Brown from Elsevier, who helped at every step of the execution of this project. Alexander Birbrair Editor


The application of iPSCs to questions in virology: a historical perspective

1 Duncan R. Smith

Institute of Molecular Biosciences, Mahidol University, Salaya, Nakhon Pathom, Thailand

Chapter outline A brief history of virology ............................................................................................ 2 Viruses as obligate parasites....................................................................................... 2 The advent of cell biology ........................................................................................... 3 Stem cells, embryonic stem cells, and induced pluripotent stem cells ........................... 5 Current applications of iPSCs to virology...................................................................... 6 The family Caliciviridae .............................................................................................. 9 The family Coronaviridae........................................................................................... 10 The family Flaviviridae .............................................................................................. 11 The family Hepadnaviridae ........................................................................................ 13 The family Hepeviridae ............................................................................................. 13 The family Herpesviridae........................................................................................... 13 The family Orthomyxoviridae...................................................................................... 15 The family Paramyxoviridae....................................................................................... 15 The family Picornaviridae.......................................................................................... 16 The family Polyomaviridae......................................................................................... 16 The family Retroviridae ............................................................................................. 17 The family Togaviridae .............................................................................................. 17 Future directions....................................................................................................... 18 Acknowledgment....................................................................................................... 18 References ............................................................................................................... 18 Abstract Viruses are obligate parasites in that they can only replicate within a living host cell. Thus the science of virology is largely dependent upon the requirement to be able to grow and propagate such host cells. While it is relatively simple to be able to grow and maintain suitable host cells for viruses that infect prokaryotic cells, the situation is more complicated when eukaryotic host cells are required for viral propagation. Studies on eukaryotic viruses are thus often a compromise between the ease of propagation of the host cell and the fidelity of the propagated cells to the bona fide host cell. Until recently the choice was largely between primary cells iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00008-0 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 1 The application of iPSCs to questions

(high fidelity, low ease of propagation) or immortalized and transformed cells (low fidelity, high ease of propagation). More recently, the discovery of induced pluripotent stem cells (iPSCs), which have high fidelity and relatively high ease of propagation, has introduced a third option. This chapter will present the historical context of the application of iPSCs to questions in virology and describe how these cells are currently being used. Keywords: Caliciviridae; Cell culture; Coronaviridae; Flaviviridae; Hepadnaviridae; Hepeviridae; Herpesviridae; Induced pluripotent stem cells; Orthomyxoviridae; Paramyxoviridae; Picornaviridae; Polyomaviridae; Retroviridae; Togaviridae; Virology.

A brief history of virology The field of microbiology has existed for nearly 350 years and is considered to have formally started in 1676 when the Dutch scientist Antionie van Leeuwenhoek first observed microbial life using handmade microscopes. The field of virology as a distinct subfield of microbiology has had a much shorter history. The roots of virology lie in the work of Dimitri Ivanovsky (1846e1920), who, in 1892, demonstrated the presence of a causal agent of tobacco mosaic disease that was smaller than any previously described infectious particle. From this point on, viruses were largely defined as an infectious agent that would pass through a filter that retained bacteria and required living cells rather than culture medium for propagation. The physical nature of the infectious agent remained largely unknown until Kausche, Pfankuck, and Ruska observed discrete particles of tobacco mosaic virus using an electron microscope in 1939 (Kausche et al., 1939). Even in the absence of an understanding of the nature of viruses, a number of viruses had been identified as disease agents before 1931 including foot and mouth disease virus by Leoffler and Frosch in 1898, yellow fever virus by Walter Reed in 1900, and rabies virus by Remlinger and colleagues in 1903. Even more strikingly, vaccines had been developed for a number of diseases that we now know are viral in origin including the use of cowpox virus for vaccination against smallpox by Jenner in 1796 and a vaccination against rabies developed by Pasteur in 1885 (see Fig. 1.1).

Viruses as obligate parasites One of the defining moments in virology was when Milton Rivers proposed that viruses are obligate parasites (Rivers, 1927). Although initially controversial, the proposal accounted for the fact that successful virus amplification had previously only been achieved in embryonated eggs or laboratory animals. Maitland and Maitland demonstrated propagation of vaccinia virus in minced chicken kidney in a mixture of chicken serum and Tyrode’s solution (Maitland and Maitland, 1928), although they believed that this did not constitute a cell culture system. However, Rivers, Haagen, and Muckenfuss showed the requirement for live cells using a

The advent of cell biology

FIGURE 1.1 A brief history of virology. The figure shows some of the key points on the path to defining virology as a distinct area of study.

similar system (Rivers et al., 1929). Li and Rivers subsequently established that the virus could grow in minced chicken embryo tissue in Tyrode’s solution (a chemically defined medium), removing the need for a plasma component (Li and Rivers, 1930).

The advent of cell biology The use of minced animal tissues in defined media dominated much of virology in the 1940s and 1950. Importantly, Enders, Weller, and Robbins showed that poliovirus could be grown in cultured cells that were not nerve cells (Weller et al., 1949), and this was instrumental in developing the first polio vaccines, with the original injectable Salk inactivated vaccine (Salk et al., 1955) and the oral live attenuated Sabin vaccine (Sabin et al., 1960) to protect against poliomyelitis being produced in minced rhesus macaque monkey kidney cells. However, as eloquently stated by Tom Curtis, “By 1960, scientists and vaccine manufacturers knew that monkey kidneys were sewers of simian viruses” (Curtis, 2004). In particular, it is estimated that millions around the world were exposed to polio vaccines contaminated with Simian virus 40 (SV40). Questions over the safety of polio vaccines led to a shift of production to African green money kidneys cells and finally to a vaccine produced in the well-characterized Vero cell line (Montagnon, 1989). The polio vaccine story highlighted the problem of using primary cellsdthe possible presence of endogenous viruses. A second major drawback of using primary cells is their relatively limited useful life span. Primary cells are not able to replicate indefinitely, and after a period in culture, the cells become senescent and eventually die and thus must be continually replaced with newly sourced tissues. The concept of a defined life span for cells was first promoted by Leonard Hayflick based on his work with normal



CHAPTER 1 The application of iPSCs to questions

human diploid cells. Hayflick proposed that normal somatic cells had an inherent replication capacity of 40 þ 10 cells divisions, after which the cells become senescent and die (Hayflick, 1965). This intrinsic replication capacity is now termed the “Hayflick limit,” and in 2009, Blackburn, Greider, and Szostak shared the Nobel Prize in Physiology or Medicine for their work on telomeres and telomerase, an enzyme linked with the biological counting mechanism of cellular replication (Varela and Blasco, 2010). The Hayflick limit was proposed to explain the behavior of normal diploid cells, as there were already cell lines that did not conform to this limit. The first bona fide cell line capable of continuous culture was the mouse strain L, generated by W.R. Earle in 1940 from mouse subcutaneous areolar and adipose tissue (Earle et al., 1943). A clone from this strain (L929) generated in 1948 from the 95th subculture was subsequently the first cloned cell line developed (Sanford et al., 1948). In the following years, a number of immortalized or transformed cell lines capable of continuous growth were produced, including HeLa (Scherer et al., 1953), CHO (Tjio and Puck, 1958), MDCK (the isolation of this line was not published by Madin and Darby, but it was subsequently used (Green, 1962) and characterized (Gaush et al., 1966) by others), and WI-38 (Hayflick, 1965), the last of which was developed by Hayflick himself. Currently there are a large number of cell lines capable of continuous growth. A main central repository for cell lines, the American Type Culture collection (ATCC), maintains over 4000 cell lines. These cells are easy to propagate and expand and have thus driven virus research for the last 60 or more years. Cell lines are either immortalized or immortalized and transformed. Immortalized cells generally have achieved stable telomeres through the expression of telomerase activity (Bodnar et al., 1998), while transformed cells additionally have undergone neoplastic transformation. In this regard, as these cells have acquired properties not normally possessed by the corresponding primary cell, immortalized and transformed cells cannot be considered as “normal” cells. In particular, transformed cells often express proteins not normally found in the original cell type and conversely can fail to express proteins that are normally expressed (Pan et al., 2009). The ability of a virus to productively infect a particular cell depends upon the susceptibility of the cell, as well as the permissiveness of the cell. Susceptibility indicates that a particular virus can enter into a cell, while permissiveness indicates that viral replication, packaging, and cellular egress can occur. In this regard, the deranged protein expression found in immortalized and transformed cells can lead to the derivation of susceptible and permissive cell lines from tissues that are not normally target tissues of infection. Conversely, cell lines derived from a known viral target tissue might be refractory to infection. Much of virology is therefore dependent upon less than satisfactory model systems in which virus/cell line pairings are based on utility, rather than being a reflection of true tropism. That said, it should be noted that a similar criticism applies to studies on human pathogenic viruses conducted in animals, in which the pathology may only poorly reflect the pathogenesis seen in humans (Ruiz et al., 2017).

Stem cells, embryonic stem cells, and induced pluripotent stem cells

Stem cells, embryonic stem cells, and induced pluripotent stem cells A stem cell has the capacity to self-renew and to give rise to all of the differentiated cell types of the organism. This concept is almost as old as the field of virology. In his book Anthropogenie, published in 1874, Ernst Haeckel (1834e1919) proposed that a fertilized egg be called a “stammzelle” (or stem cell) (Haeckel, 1874). Around the same time, the field of hematopoiesis (the generation of the cells of the blood) was revolutionized after Paul Erlich (1845e1915) developed the methods to specifically stain different blood cell types (for a review of Erlich’s contributions to histochemistry, see (Buchwalow et al., 2015)). In particular, this work triggered a debate as to whether red and white blood cells had a common precursor. On the side of those who believed in a single precursor, Pappenheim (Pappenheim, 1896) used the term “stem cell” to describe the postulated precursor. In the following years, a number of studies pointed toward the existence of a blood stem cell. For example, Florence Sabin working with irradiated animals provided strong evidence of blood stem cells, but did not identify the cells specifically (Sabin et al., 1932). In 1963, Till and McCulloch published a study (Becker et al., 1963) that showed that one type of cell in the blood was capable of differentiating into three distinct lineages (erythrocytic, granulocytic, and megakaryocytic). While not directly using the term “stem cell,” the first identification of stem cells is commonly credited to them. However, hematopoietic stem cells are not totipotent (capable of differentiating into all cell types including extraembryonic tissues) or pluripotent (capable of differentiating into cells of the three germ layers), but they are multipotent (capable of differentiating into a number of related cell types). The first pluripotent stem cells were isolated and cultured by Evans and Kaufmann from mouse blastocysts (Evans and Kaufman, 1981), and the first human pluripotent stem cells were produced from human blastocysts in 1998 by James Thompson (Thomson et al., 1998). Human embryonic stem cells are produced from potentially viable human embryos, and as such their production and use remain controversial (Lo and Parham, 2009). In 2006, Takahashi and Yamanaka provided a solution to the problems associated with the use of embryonic stem cells. Working with 24 genes identified as being important to embryonic cell function, they showed that the presence of four of these genes was sufficient to reprogram a mouse somatic cell to an embryonic stem cell-like phenotype (Takahashi and Yamanaka, 2006). These four factors, called the Yamanaka factors, consisted of Oct3/4, Sox2, Klf4, and c-Myc. These firstgeneration cells, however, were not fully pluripotent in that they could neither produce functional chimeras nor contribute to the germ line (Takahashi and Yamanaka, 2006). Improved methodologies published the following year by three groups (Maherali et al., 2007; Okita et al., 2007; Wernig et al., 2007) were able to generate fully pluripotent cells, termed induced pluripotent stem cells (iPSC), which were functionally identical to embryonic stem cells. In the same year, human iPSCs generated from somatic cells (fibroblasts) were reported from Yamanaka’s group



CHAPTER 1 The application of iPSCs to questions

using the same factors (Takahashi et al., 2007), as well as by the group of James Thompson using Oct4, Sox2, Nanog, and Lin28 (Yu et al., 2007). There is considerable research ongoing in developing iPSCs using different factors, cell types, and protocols, as well as the development of protocols to differentiate iPSCs into different cell types (Liu et al., 2020). However, crucially, the development and widespread use of iPSCs put these cells into the hands of virologists who, for the first time, were able to look at the cellular events ongoing during virus infection in a cell line that could be differentiated into a bona fide cell type (See Fig. 1.2).

Current applications of iPSCs to virology A search of the relevant literature undertaken in late March 2020 identified more than 100 studies that used iPSCs to address questions in virology (Table 1.1). Not included in the analysis were studies that used viruses such as a lentivirus (Takenaka et al., 2010) or Sendai virus (Simara et al., 2014) to generate iPSCs, or those that use a virus as a tool to investigate non-infection-related questions (Naaman et al., 2018). Collectively, the studies investigated 25 different viruses in 18 genera belonging to 12 virus families and utilized a number of different cell types (Fig. 1.3). More than half of the studies investigated only three viruses, namely Zika virus (ZIKV),

FIGURE 1.2 The application of iPSCs to questions in virology. The figure shows the overall route through which iPSCs are reprogrammed from somatic cells and can be used in virology.

Current applications of iPSCs to virology

Table 1.1 Studies in virology utilizing iPSCs, ordered alphabetically by virus family. Family




Caliciviridae Coronaviridae

Norovirus Betacoronavirus

Sato et al. (2019) Mangale et al. (2017)



Norwalk virus Mouse hepatitis virus (murine coronavirus) Dengue virus

Murray Valley encephalitis virus Usutu virus West Nile virus

Zika virus

Desole et al. (2019); Lang et al. (2016); Manh et al. (2018); Muffat et al. (2018) Fortuna et al. (2018) Salinas et al. (2017) Desole et al. (2019); Fortuna et al. (2018); Huang et al. (2019) Desole et al. (2019); Muffat et al. (2018); Abreu et al. (2018); Alimonti et al. (2018); Caires-Junior et al. (2018); Fong et al. (2017); Gabriel et al. (2017); Garcez et al. (2017); Goodfellow et al. (2018); Lanko et al. (2017); Ledur et al. (2020); Liu et al. (2019); Mesci et al. (2018); Qian et al. (2016); Rolfe et al. (2016); RosaFernandes et al. (2019); Salinas et al. (2017a,b); Simonin et al. (2019); Souza et al. (2016); Tan et al. (2019); Tang et al. (2016); Tricot et al. (2018); Wells et al. (2016); Xu et al. (2016); Zhang et al. (2016); Zhou et al. (2017); Anfasa et al. (2017) Continued



CHAPTER 1 The application of iPSCs to questions

Table 1.1 Studies in virology utilizing iPSCs, ordered alphabetically by virus family.dcont’d Family




Related, unclassified

Fitzroy River virus

Fortuna et al. (2018)


Bamaga virus Hepatitis C virus



Hepatitis B virus



Hepatitis E virus





Herpes simplex virus


Varicella zoster virus

Fortuna et al. (2018) Carpentier et al. (2014); Irudayam et al. (2015); Ito et al. (2017); Kishta et al. (2016); Moriguchi (2015); Moriguchi et al. (2010); Sakurai et al. (2017a,b); Sa-Ngiamsuntorn et al. (2016, 2017); Schobel et al. (2018); Schwartz et al. (2012); Si-Tayeb et al. (2012); Sourisseau et al. (2013); Wu and Dao Thi (2019); Wu et al. (2012, 2014); Yoshida et al. (2011) Chang et al. (2016); Kaneko et al. (2016); Miyakawa et al. (2015); Nie et al. (2018); Sakurai et al. (2017a,b); Shlomai et al. (2014); Xia et al. (2017); Xiong et al. (2019); Yuan et al. (2018) Helsen et al. (2016); Todt et al. (2018); Wu et al. (2018); Zhou et al. (2017) Brown et al. (2019); Poole et al. (2019) D’Aiuto et al. (2019); D’Aiuto et al. (2018); D’Aiuto et al. (2015); D’Aiuto et al. (2017); Lafaille et al. (2015); McClain et al. (2015); McNulty et al. (2016); Zimmer et al. (2018) McClain et al. (2015); McNulty et al. (2016); Baird et al. (2013); Lee et al. (2012)

The family Caliciviridae

Table 1.1 Studies in virology utilizing iPSCs, ordered alphabetically by virus family.dcont’d Family





Influenza virus A

Influenza A virus



Measles morbillivirus Newcastle disease virus Theiler’s murine encephalomyelitis virus Coxsackievirus B3

Ciancanelli et al. (2015); Lim et al. (2019); Zahedi-Amiri et al. (2019) Hubner et al. (2017)

Orthoavulavirus Picornaviridae








Alphavirus Rubivirus

Enterovirus D68 Merkel cell polyomavirus Human immunodeficiency virus

Chikungunya virus Rubella virus

Shittu et al. (2016); Susta et al. (2016) Benner et al. (2016)

Hubner et al. (2017); Lin et al. (2016); Sharma et al. (2014) Hixon et al. (2019) Cheng et al. (2017) Alvarez-Carbonell et al. (2019); Kang et al. (2015); Liao et al. (2015); Ni et al. (2011, 2014); Ye et al. (2014) Ferreira et al. (2019) Hubner et al. (2017)

Hepatitis C virus (HCV), and Hepatitis B virus (HBV). The family Flaviviridae accounted for over half of all studies, and ZIKV alone was the subject of a quarter of all studies.

The family Caliciviridae The family Caliciviridae consists of 11 genera, Bavovirus, Lagovirus, Minovirus, Nacovirus, Nebovirus, Norovirus, Recovirus, Salovirus, Sapovirus, Valovirus, and Vesivirus (Vinje et al., 2019). The viruses in this family are nonenveloped with a single-stranded, positive sense RNA genome. In terms of human health, the genus Norovirus is the most important. This genus contains a single virus species, Norwalk virus, but noroviruses are genetically very diverse with multiple genogroups and genotypes (Atmar, 2010). Noroviruses are transmitted primarily by the fecaleoral route and can cause both endemic and epidemic gastroenteritis. Noroviruses have traditionally been very difficult to culture, and it was only recently that a



CHAPTER 1 The application of iPSCs to questions

FIGURE 1.3 The utilization of iPSCs. iPSCs and cells differentiated from them have been used in studies on a number of different viruses.

methodology was established to culture noroviruses using stem-cell-derived epithelial cell cultures, with the stem cells being obtained from intestinal crypts from tissues obtained at biopsy or surgery (Ettayebi et al., 2016). To overcome the limitations of a culture system requiring adult stem cells, Sato and colleagues successfully derived intestinal epithelial cells from iPSCs (Sato et al., 2019). It is likely that iPSCs will result in rapid advances in our understanding of noroviruses given this significant advance.

The family Coronaviridae The family Coronaviridae has two subfamilies, the Letovirinae and the Orthocoronavirinae. The subfamily Orthocoronavirinae contains four genera, Alphacoronavirus, Betacoronavirus, Deltacoronavirus, and Gammacoronavirus (ICTV Master Species

The family Flaviviridae

list 2018b.v2, available at talk.ictvonline.org/files/master-species-list/m/msl/8266). The viruses in this family of 39 species consist of enveloped viruses with a positive sense, single-stranded RNA genome. Members of genus Betacoronavirus include the Severe acute respiratory syndrome-related coronavirus, and both SARS and SARS-CoV2 belong to this species of virus (Coronaviridae Study Group, 2020). The genus Betacoronavirus also contains the species Murine coronavirus, to which mouse hepatitis virus, a common virus infecting laboratory mice, belongs. Mangale and colleagues undertook a comparative analysis of the susceptibility to mouse hepatitis virus of ex vivo derived neural precursor cells (NPC) and NPCs derived through differentiation of iPSCs (Mangale et al., 2017). The authors found that although the iPSC-NPCs were functionally equivalent, there was reduced susceptibility to the neurotropic mouse hepatitis virus. This potentially has implications in using NPCs to treat neurological disorders.

The family Flaviviridae The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Hepacivirus, and Pegivirus (Simmonds et al., 2017), and collectively the family has more than 60 virus species assigned to it (Simmonds et al., 2017). The members of this family all have a single-stranded positive sense RNA as their genomic material, and the viruses are enveloped. The family includes a number of viral species that are significant human pathogens with broad distribution including yellow fever virus, dengue virus, West Nile virus, and Zika virus in the genus Flavivirus, and Hepatitis C virus in the genus Hepacivirus. Studies have been undertaken on five members of the genus Flavivirus, including dengue virus (DENV), Murray Valley encephalitis virus (MVEV), Usutu virus (USUV), West Nile virus (WNV), and Zika virus (ZIKV), as well as on two related viruses not formally assigned to the genus, namely Fitzroy River virus (FRV) and Bamaga virus (BgV). As noted earlier, the majority of studies on members of this genus were undertaken on ZIKV (Desole et al., 2019; Muffat et al., 2018; Abreu et al., 2018; Alimonti et al., 2018; Caires-Junior et al., 2018; Fong et al., 2017; Gabriel et al., 2017; Garcez et al., 2017; Goodfellow et al., 2018; Lanko et al., 2017; Ledur et al., 2020; Liu et al., 2019; Mesci et al., 2018; Qian et al., 2016; Rolfe et al., 2016; Rosa-Fernandes et al., 2019; Salinas et al., 2017a,b; Simonin et al., 2019; Souza et al., 2016; Tan et al., 2019; Tang et al., 2016; Tricot et al., 2018; Wells et al., 2016; Xu et al., 2016; Zhang et al., 2016; Zhou et al., 2017; Anfasa et al., 2017). ZIKV is a recently emerged arthropod borne virus (Wikan and Smith, 2016) that is transmitted by Aedes species mosquitoes. Although ZIKV was first identified in 1947 (Dick et al., 1952), it remained an obscure virus until approximately 2013 when the virus emerged from Southeast Asia initially to French Polynesia (Cao-Lormeau et al., 2014) and then to many countries and territories around the world (Baud et al., 2017). Although human infection with ZIKV is either asymptomatic or results in relatively mild and selflimiting symptoms, when a pregnant woman is infected, particularly in the first or



CHAPTER 1 The application of iPSCs to questions

second trimester, infection of the fetus can result in a number of abnormalities termed congenital Zika syndrome (CZS), of which the most marked presentation is microcephaly (Martines et al., 2016). Perhaps unsurprisingly, the main focus on studies undertaken with ZIKV has centered on understanding the fetal pathobiology of the disease. Such studies have focused on neural development and neurogenesis and the impact of ZIKV on neural progenitor cells (Caires-Junior et al., 2018; Gabriel et al., 2017; Garcez et al., 2017; Goodfellow et al., 2018; Lanko et al., 2017; Liu et al., 2019; Qian et al., 2016; Rolfe et al., 2016; Rosa-Fernandes et al., 2019; Souza et al., 2016; Tang et al., 2016; Zhou et al., 2017) (Anfasa et al., 2017) (Desole et al., 2019; Mesci et al., 2018), other brain resident cell types such as astrocytes (Ledur et al., 2020) and microglial cells (Abreu et al., 2018) as well as non-brain-resident cell types including human retinal pigment epithelium (Salinas et al., 2017a,b; Simonin et al., 2019) and hepatocytes (Tricot et al., 2018). Other studies have investigated how ZIKV crosses the bloodebrain barrier (Alimonti et al., 2018), how ZIKV gets into cells (Tan et al., 2019; Wells et al., 2016), and the identification of potential therapeutic agents (Xu et al., 2016). Other studies on members of the genus Flavivirus have investigated DENV in target cell types including dendritic cells (Manh et al., 2018) and hepatocyte-like cells (Lang et al., 2016). Significantly, Desole and colleagues showed that, in addition to ZIKV, both DENV and WNV were able to infect pluripotent stem cells (Desole et al., 2019), suggesting that other factors in addition to tropism are responsible for the pathobiology of ZIKV in mediating microcephaly in CZS. This is supported by a study in iPSC-derived human neuronal stem cells, which showed that these cells are able to be infected by USUV (Salinas et al., 2017a,b). Although USUV is not commonly associated with human infection, two cases with neurological involvement in immunocompromised patients have been reported (reviewed in (Smith, 2017)). The issue of neurotropism of viruses was also addressed by Fortuna and colleagues, who used cortical neurons differentiated from equine iPSCs and neurotropic (WNV and MVEV) and nonneurotropic (FRV and BgV) flaviviruses (Fortuna et al., 2018). The remaining study investigated inflammatory responses to WNV using a virulent and less pathogenic isolates of WNV (Huang et al., 2019). The genus Hepacivirus contains 14 viral species (Hepacivirus AeN), of which the most studied is hepatitis C virus (HCV), which belongs to the species Hepacivirus C (Smith et al., 2016). Hepatitis C is primarily spread by direct blood-to-blood contact, and it is estimated that some 100 million people around the world show serologic evidence of past or present HCV infection, and around 71 million people worldwide have chronic HCV infection (Center for Disease Analysis, 2015). Since its discovery in 1989 (Choo et al., 1989), HCV has been difficult to culture (Duverlie and Wychowski, 2007). One culture system was developed using HCV cloned strain JFH1 and Huh7 cells (Wakita et al., 2005; Zhong et al., 2005), but other systems have relied on the introduction of adaptive mutations. It is little surprise therefore that the HCV community rapidly employed iPSCs to produce hepatocyte-like cells that were used to study HCV replication and pathogenesis (Carpentier et al., 2014; Irudayam et al., 2015; Ito et al., 2017;

The family Herpesviridae

Sakurai et al., 2017a,b; Sa-Ngiamsuntorn et al., 2016, 2017; Schobel et al., 2018; Schwartz et al., 2012; Si-Tayeb et al., 2012; Sourisseau et al., 2013; Wu and Dao Thi, 2019; Wu et al., 2012, 2014; Yoshida et al., 2011) and to explore treatment modalities (Kishta et al., 2016; Moriguchi, 2015; Moriguchi et al., 2010).

The family Hepadnaviridae The family Hepadnaviridae consists of two genera, Avihepadnavirus and Orthohepadnavirus. The genus Orthohepadnavirus contains nine virus species, of which the best characterized is Hepatitis B virus. Hepatitis B virus (HBV) is an enveloped virus with a genome of partially double-stranded circular DNA (Liang, 2009). It has been estimated that around one-third of the world’s population has been infected with HBV, and approximately three-quarters of a million people die each year from HBV (Jefferies et al., 2018). As with HCV virus, a suitable host cell for propagation of the virus has been problematic (reviewed in (Verrier et al., 2016)), and as with HCV, the majority of papers utilizing iPSC have focused on developing infection models (Kaneko et al., 2016; Nie et al., 2018; Sakurai et al., 2017a,b; Shlomai et al., 2014; Xia et al., 2017; Yuan et al., 2018) and understanding the mechanisms of HBV pathogenesis (Chang et al., 2016; Miyakawa et al., 2015; Xiong et al., 2019).

The family Hepeviridae The family Hepevridae contains two genera, Orthohepevirus and Piscihepevirus. The genus Orthohepevirus contains four viral species, Orthohepevirus AeD. Human hepatitis E virus, which causes acute viral hepatitis, belongs to Orthohepevirus A (Purdy et al., 2017). The virus, which was first identified in 1983 (Balayan et al., 1983), is transmitted predominantly via the fecaleoral route, often through contaminated water, although infection can also occur through the consumption of infected animals (Purdy et al., 2017). The virus contains a positive sense, single-stranded RNA genome, and the transmissible virus is nonenveloped, although there is a quasienveloped form in the blood (reviewed in (Yin and Feng, 2019)). As with other hepatitis viruses, culture of hepatitis E virus is difficult (Meister et al., 2019), and iPSCs have been used to establish models of viral replication (Helsen et al., 2016; Wu et al., 2018), evaluate drugs (Todt et al., 2018; Dao Thi et al., 2016), and further define viral tropism (Zhou et al., 2017).

The family Herpesviridae The family Herpesviridae has three subfamilies, Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae. The subfamily Alphaherpesvirinae contains five



CHAPTER 1 The application of iPSCs to questions

genera (Iltovirus, Mardivirus, Scutavirus, Simplexvirus, and Varicellovirus), while the subfamily Betaherpesvirinae contains four genera (Cytomegalovirus, Muromegalovirus, Proboscivirus, and Roseolovirus), and the subfamily Gammaherpesvirinae contains four genera (Lymphocryptovirus, Macavirus, Percavirus, and Rhadinovirus) as detailed in the ICTV Master Species list 2018b.v2 (available at talk.ictvonline.org/files/master-species-list/m/msl/8266). The members of this family are enveloped viruses with a double-stranded DNA genome. The family contains a number of well-known human pathogens including those that cause glandular fever (species Human gammaherpesvirus 4, commonly referred to as EpsteineBarr virus), chickenpox (species Human alphaherpesvirus 3, but commonly referred to as varicella zoster virus), and herpes (species Human alphaherpesvirus 1 and 2, but commonly referred to as herpes simplex virus 1 and 2). Human betaherpesvirus 5 (commonly referred to as human cytomegalovirus) is a leading cause of congenital infection. Infection in early pregnancy can lead to sensorineural hearing loss, visual impairment, cognitive defects, and sometime death (Manicklal et al., 2013). Infection in childhood is common, and seroprevalence rates among women of reproductive range vary from 40%e50% to >90% around the world (Manicklal et al., 2013). Once a person is infected, they remain infected for life, and the latent infection can be reactivated (Dupont and Reeves, 2016). iPSCs have been applied both to investigate the effect of the virus on neural cell development (Brown et al., 2019) and to understand the mechanisms underlying reactivation of the virus (Poole et al., 2019). Human alphaherpesvirus 1 and 2 (herpes simplex virus 1 and 2) are associated with oral and genital herpes. While the majority of oral herpes is caused by herpes simplex 1 and the majority of genital herpes by herpes simplex 2, herpes simplex 1 can be transmitted to the genital area (Herpes simplex virus - Fact sheet). Studies have suggested that more than 60% of the world population have been infected with herpes simplex 1, and the infection is lifelong (Herpes simplex virus - Fact sheet). In addition to the typical herpes lesions, infection can occasionally result in encephalitis in adults (Modi et al., 2017), and neonatal infection can also can have highly deleterious effects, including neurodevelopmental impairment or death. To date, all studies investigating herpes simplex virus have used Human alphaherpesvirus 1 (herpes simplex virus 1) (D’Aiuto et al., 2019; D’Aiuto et al., 2018; D’Aiuto et al., 2015; D’Aiuto et al., 2017; Lafaille et al., 2015; McClain et al., 2015; McNulty et al., 2016; Zimmer et al., 2018). The studies have primarily modeled central nervous system infection (D’Aiuto et al., 2019; D’Aiuto et al., 2015; Zimmer et al., 2018) or used iPSCs as a drug screening platform (D’Aiuto et al., 2017; McClain et al., 2015; McNulty et al., 2016). Two studies additionally investigated the effects of drugs against Human alphaherpesvirus 3 (varicella zoster virus) in addition to herpes simplex 1 (McClain et al., 2015; McNulty et al., 2016). Two further studies have investigated varicella zoster virus, and both investigated interactions with neurons (Baird et al., 2013; Lee et al., 2012), which are believed to be where varicella zoster virus establishes latent infection (Kennedy et al., 1998).

The family Paramyxoviridae

The family Orthomyxoviridae The family Orthomyxoviridae contains seven genera, Alphainfluenzavirus, Betainfluenzavirus, Deltainfluenzavirus, Gammainfluenzavirus, Isavirus, Quaranjavirus, and Thogotovirus, as detailed in the ICTV Master Species list 2018b.v2 (available at talk.ictvonline.org/files/master-species-list/m/msl/8266). The members of this family are enveloped viruses with genomes consisting of six to eight segments of linear, negative sense RNA. The genus Alphainfluenzavirus contains a single viral species Influenza A virus, which causes both seasonal and pandemic influenza (Peteranderl et al., 2016). Two studies have used patient-specific iPSCs to understand the impact of genetic deficiency on disease presentation upon influenza A infection (Ciancanelli et al., 2015; Lim et al., 2019), while a third study used iPSCs to investigate possible mechanisms by which influenza A virus could induce developmental abnormalities in a developing fetus (Zahedi-Amiri et al., 2019).

The family Paramyxoviridae The family Paramyxoviridae is a family of enveloped viruses with a singlestranded, negative sense RNA genome. Viruses of this family infect mammals, birds, reptiles, and fish (Rima et al., 2019). The family contains four subfamilies (Avulavirinae, Metaparamyxovirinae, Orthoparamyxovirinae, Rubulavirinae), composed of 14 genera. Measles virus (subfamily Orthoparamyxovirinae, genus Morobillivirus, species Measles morbillivirus) causes measles, a vaccinepreventable highly contagious disease spread by coughs, sneezes, or contact with infected secretions (Keller et al., 2019). Measles virus was one of three viruses (measles, rubella, and coxsackievirus B3) used by Hubner and colleagues (Hubner et al., 2017) to investigate the impact of virus infection on early embryonic development. Interestingly, while congenital measles is comparatively rare (Betta Ragazzi et al., 2005), measles virus was shown to be cytopathogenic to iPSCs (Hubner et al., 2017), a result that contrasts with the observation of Naaman and colleagues, who found measles virus to be a suitable potential vector for investigating factors regulating lineage differentiation (Naaman et al., 2018). Newcastle disease virus (subfamily Avulavirinae, genus Orthoavulavirus, species Avian orthoavulavirus 1) is a highly contagious disease of birds that has considerable impact on the domestic poultry industry despite the availability of a number of vaccines (Mayers et al., 2017). The group of Claudio Afonso has used repeated rounds of infection of chicken iPSC (cIPSC) with Newcastle disease virus to select ciPSC with increased tolerance to the virus (Susta et al., 2016), as well as to develop an improved platform for vaccine production (Shittu et al., 2016).



CHAPTER 1 The application of iPSCs to questions

The family Picornaviridae The family Picornaviridae contains more than 75 viral species distributed in more than 30 genera (Zell et al., 2017). The viruses in this family are nonenveloped with a single-stranded positive sense RNA genome. Genera include the genus Cardiovirus, which has three virus species (Cardiovirus AeC) and the genus Enterovirus, which has 15 virus species (Enterovirus AeL and Rhinovirus AeC). Theiler’s murine encephalomyelitis virus (species Cardiovirus B) is a mouse virus that induces a demyelinating disease and is used in a model for multiple sclerosis. Benner and colleagues (Benner et al., 2016) used iPSCs to produce oligodendrocyte progenitor cells to study the effects of Theiler’s murine encephalomyelitis virus infection. Using global transcriptome profiling, they identified a transcription factor (Olig2) that is required for oligodendrocyte progenitor cells that was downregulated upon infection. Coxsackievirus B3 of the genus Enterovirus (species Enterovirus B) is the main etiologic agent of viral myocarditis (Esfandiarei and McManus, 2008) and has been associated with neonatal infections that affect multiple organs including the CNS and the heart (Muehlenbachs et al., 2015). To date, two studies have used iPSCs to understand viral myocarditis (Lin et al., 2016; Sharma et al., 2014), and one has used them to understand impairment of human embryogenesis, together with two other viruses, measles virus and rubella virus (Hubner et al., 2017). Enterovirus D68 (a member of the species Enterovirus D) is an emerging virus that has been linked to cases of acute flaccid myelitis, primarily in children (Holm-Hansen et al., 2016). Although enterovirus D68 is primarily a respiratory virus, the virus has tropism toward respiratory, intestinal, and neural tissues (Royston et al., 2018). To understand the neurotropism of the virus, Hixon and colleagues (Hixon et al., 2019) used iPSCs to investigate central nervous system invasion through neuronal pathways.

The family Polyomaviridae The family Polyomaviridae contains four genera, Alphapolyomavirus, Betapolyomavirus, Gammapolyomavirus, and Deltapolyomavirus (Polyomaviridae Study Group et al., 2016). Viruses in the family Polyomaviridae are nonenveloped viruses with a small circular double-stranded DNA genome. The genus Alphapolyomavirus contains human polyomavirus 1, commonly referred to as Merkel cell polyomavirus. This virus was discovered only in 2008 (Feng et al., 2008) and although high rates of seropositivity are found in the general population (Pastrana et al., 2009), Merkel cell polyomavirus is associated with the development of a rare neuroendocrine skin cancer, Merkel cell carcinoma. In investigating how Merkel cell polyomavirus can lead to cancer, Cheng and colleagues (Cheng et al., 2017) showed that a viral protein (small T antigen) can act as a reprograming factor generating iPSCs. This study additionally provides a possible explanation as to why the oncogenic Merkel cell polyoma virus is limited to induction of Merkel cell carcinoma.

The family Togaviridae

The family Retroviridae The family Retroviridae contains two subfamilies (Orthoretrovirinae and Spumaretrovirinae) that contain 11 genera. Human immunodeficiency viruses (HIV; subfamily Orthoretrovirinae, genus Lentivirus, species Human immunodeficiency virus 1e2) are causative agents of acquired immunodeficiency syndrome (AIDS). The viruses are enveloped with two identical copies of a single-stranded, positive sense RNA as the genetic material (Lu et al., 2011). HIV is predominantly transmitted by direct exchange of bodily fluids, sexual contact across mucosal membranes, and through vertical transmission from mother to child (Shaw and Hunter, 2012). Studies on HIV that have utilized iPSC have variously looked at generating cells resistant to HIV infection through modulation of the HIV CCR5 coreceptor (Kang et al., 2015; Ye et al., 2014), or through integration of a cellular defense system that maintains cells as refractory to infection (Liao et al., 2015) or to improve suppression of HIV (Ni et al., 2011, 2014), and to investigate the mechanism of latency (Alvarez-Carbonell et al., 2019).

The family Togaviridae The family Togaviridae contains two genera, Alphavirus and Rubivirus (Chen et al., 2018). The viruses of this family are enveloped and possess a single-stranded, positive sense RNA genome. While the genus Alphavirus contains more than 30 virus species, many of which are mosquito-transmitted, the genus Rubivirus contains only a single species, rubella virus (Chen et al., 2018), which is transmitted by a respiratory route. The Alphavirus chikungunya virus (species Chikungunya virus) is a mosquito-transmitted human pathogen that has been responsible for both endemic and pandemic outbreaks of chikungunya fever (Weaver and Lecuit, 2015). Infection with chikungunya virus can result in long-term debilitating arthralgia (Runowska et al., 2018), and, as with many mosquito-transmitted viruses, there is no effective drug to treat infections. Ferreira and colleagues (Ferreira et al., 2019) investigated the potential of the anti-hepatitis C drug sofosbuvir (Heo and Deeks, 2018) as an anti-chikungunya virus gent in multiple systems including iPSC-derived astrocytes and showed that sofosbuvir had the potential for retargeting toward chikungunya virus. Rubella virus causes a vaccine-preventable disease (commonly referred to as German measles). The disease in children and adults can be asymptomatic or associated with symptoms such as a rash, fever, sore throat, and fatigue (Bakshi and Cooper, 1989). Where congenital infection occurs during early pregnancy, congenital rubella syndrome can occur, leading to significant developmental deficits. As part of a comparative study on three viruses capable of congenital infection (miscarriage-associated coxsackievirus, measles virus, and rubella virus), Hubner and colleagues (Hubner et al., 2017) showed that rubella virus was able to replicate in iPSCs over several passages and did not impair subsequent differentiation into the ectoderm, endoderm, and mesoderm, in contrast with the two other two viruses.



CHAPTER 1 The application of iPSCs to questions

Future directions Although the application of iPSCs to questions in virology has been heavily weighted to relatively few viruses, studies have been conducted on a surprisingly large number of different viruses in a number of different genera (Table 1.1), and it is likely that the number of different viruses being investigated in iPSC-derived culture systems will increase over time. A number of studies have used iPSCs to develop improved virus culture systems, and others have been able to interrogate systems that more faithfully replicate bona fide cell types. As established iPSC lines become more common, and the protocols to differentiate them become more widely available, it is probable that culture systems will be developed for many viruses that are currently difficult to culture. Given the fidelity of iPSC-generated cells, it is likely that many studies previously undertaken in transformed cells (such as proteomic analysis, transcriptomics analysis) may need to be reinvestigated in iPSC-generated cells. Indeed, in the near future it might be that studies are not considered reliable unless validated in cells generated from iPCS. Although not highlighted in this chapter, many studies have used organoids developed from iPSCs. Organoids are multicellular in vitro tissues with three-dimensionality (Clevers, 2016) containing multiple cell types representative of a particular organ or tissue. These systems allow interrogation of virus pathology in a highly representative 3D structure with heterologous cellecell contacts. In particular, virus transmission between cell types remains relatively underexplored. iPSCs are highly manipulatable genetically, and as such it might be possible to generate systems whereby cells from markedly different lineages could be cocultured, allowing even more realistic model systems to be generated. The introduction of continuous cell lines had a huge impact on virology studies, and it is almost certain that the introduction of iPSC will have a similar impact on the field of virology in time to come.

Acknowledgment The author would like to thank Wannapa Sornjai, Suwipa Ramphan, and Janejira Jaratsittisin (Mahidol University, Thailand) for help with figures and Professor David Murphy (University of Bristol, UK) for a critical reading of the manuscript. The author is supported by the National Research Council of Thailand and Mahidol University (NRCT5-RSA63015-03), the Thailand Research Fund (IRN58W0002), the National Science and Technology Development Agency (FDA-CO-2561-6820-TH), and Mahidol University.

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Transplantation of iPSCderived neural progenitor cells promotes clinical recovery and repair in response to murine coronavirus-induced neurologic disease


Craig M. Walsh1, Warren C. Plaisted2, Laura L. McIntyre1, Jeanne F. Loring3, Thomas E. Lane4 Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, CA, United States; 2Genomics Institute of the Novartis Research Foundation, San Diego, CA, United States; 3Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA, United States; 4Department of Neurobiology & Behavior, University of California, Irvine, Irvine, CA, United States 1

Chapter outline Introduction .............................................................................................................. 32 Infection with a neurotropic coronavirus as a model of neuroinflammation and demyelination ........................................................................................34 The impact of human neural precursor cell transplantation in demyelinating disease models.............................................................................................37 The influence of adaptive immunity on stem/progenitor cell remyelination .........39 Conclusions.............................................................................................................. 41 References ............................................................................................................... 42 Abstract Intracranial inoculation of susceptible strains of mice with the neuroadapted JHM strain of mouse hepatitis virus (JHMV, a member of the Coronaviridae family of viruses) results in an acute encephalomyelitis characterized by widespread growth of virus in astrocytes, microglia, and oligodendrocytes with relative sparing of neurons. Virus-specific CD4þ and CD8þ T cells infiltrate into the central nervous system in response to infection and control viral replication through secretion of interferon gamma as well as cytolytic activity. Nonetheless, virus persists in white iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00005-5 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 2 Transplantation of iPSC-derived neural progenitor cells

matter tracts, and animals develop an immune-mediated demyelinating disease in which both T cells and macrophages amplify white matter damage. For the past decade, we have explored the therapeutic potential of human neural progenitor cells derived from pluripotent stem cells in promoting clinical recovery associated with remyelination of demyelinated axons following intraspinal transplantation. This chapter highlights recent studies from our laboratories demonstrating that tissue repair is associated with the emergence of regulatory T cells in response to transplantation of NPCs. Keywords: Coronavirus; Demyelination; Immunomodulation; Inducible pluripotent stem cells; Intraspinal transplantation; Neural progenitor cells; Regulatory T cells; Remyelination.

Introduction Characterized by progressive destruction of myelin, multiple sclerosis is a chronic autoimmune disease of the central nervous system (CNS) associated with inflammatory destruction (Steinman, 2014). Genetic and environmental factors are associated with disease susceptibility, although the causes of MS remain poorly understood (Oksenberg et al., 1993; Poser, 1994). Clinical symptoms, including impaired motor skills, loss of vision, behavior changes, paralysis, and cognitive deterioration have been associated with the accumulation of myelin lesions (Lassmann et al., 2007; Neumann et al., 2002; Prineas and Graham, 1981). Activated CD4þ and CD8þ T cells and macrophages accumulate within these lesions and along with activated microglia release proinflammatory cytokines that impair oligodendrocyte function (Lassmann et al., 2007; Traugott et al., 1983). MS disease-modifying therapies (DMTs) target such immune populations within the CNS. Such DMTs limit the activation or infiltration of autoreactive T lymphocytes into the CNS that occurs in relapsing-remitting MS (Weinshenker et al., 1989). Such therapies have been largely ineffective for treatment of chronic forms of MS, but Ocrelizumab (anti-CD20) (Frampton, 2017), a drug that targets B lymphocytes, has recently been approved for treatment of progressive MS. The impairment of remyelination that occurs in MS patients is primarily due to a failure in the maturation of oligodendrocyte precursor cells (OPCs). While OPCs are found at high density within subacute lesions early during MS progression (Chang et al., 2000), these OPCs are incapable of fully repairing these lesions. Instead, remyelination induced by OPCs that mature in demyelinated lesions promotes the generation of thin myelin sheaths, resulting in what are termed “shadow plaques” (Chang et al., 2000; Halfpenny et al., 2002; Lassmann, 1983; Lucchinetti et al., 1999; Prineas et al., 1989; Roy et al., 1999; Schlesinger, 1909). Given this impaired OPC differentiation and remyelination capacity within MS lesions, understanding the influence of the microenvironment within such lesions is of profound clinical significance. Effective OPC-mediated remyelination depends on removal of myelin debris by phagocytic cells, including inflammatory macrophages (Healy et al., 2017; Karamita et al., 2017), neutrophils (Lindborg et al., 2017) and resident microglia


(Karamita et al., 2017; Kucharova and Stallcup, 2017; Zhu et al., 2016). Efficient clearance of myelin by macrophages has been found to be age-dependent in mice, as macrophages from older mice show impaired myelin phagocytosis relative to younger mice. Using parabiosis studies, it was shown that older mice demonstrated enhanced remyelination when fused with younger mice (Ruckh et al., 2012), and this was attributed to increased phagocytosis of myelin debris by the donor macrophages provided by the younger mice. Deficiencies in lipid processing have been proposed to explain this impaired myelin debris clearance in older macrophages (Cantuti-Castelvetri et al., 2018). While younger macrophages were able to efficiently engulf and process the rapid release of lipids associated with demyelinating injury, older macrophages had muted lipid processing, leading to the formation of cholesterol crystals that caused phagolysosomal rupture and activation of inflammasomes. The activation of these inflammasomes was found to exacerbate the inflammatory process within myelin lesions and prevented OPC maturation into functional oligodendrocytes capable of forming new myelin. As a potential means to promote remyelination, OPCs have been targeted by pharmacologic agents that may enhance their maturation to overcome limited natural remyelination in MS patients. High-throughput screening of small-molecule libraries has led to the identification of several compounds that enhance OPC maturation in vivo (Deshmukh et al., 2013; Mei et al., 2014, 2016). In autoimmune encephalomyelitis (EAE) preclinical mouse studies, the antimuscarinic receptor compound benztropine was found to promote an increase in OPC maturation and remyelination (Deshmukh et al., 2013), thus lending credence to the potential of this approach. In addition to this small-molecule approach, there is increasing interest in applying stem cell technology to cell-based therapies for treatment of MS. Neural stem cells (NSCs) and neural progenitor cells (NPCs) derived from fetal brain or from pluripotent stem cells are defined by their ability to differentiate into astrocytes, oligodendrocytes, and multiple types of neurons (Gage, 2000). NPCs are generally considered to be in a slightly more mature state than NSCs, but they are diverse, there is no definitive nomenclature, and they have a wide variety of gene expression phenotypes (Muller et al., 2008). These precursor cells have been found to proliferate, migrate, and differentiate in models of acute spinal cord injury, inflammatory demyelination, and stroke, responding to tissue damage in these models (Picard-Riera et al., 2002; Yagita et al., 2001; Zhang et al., 2004). Transplants of these precursors have also been shown to induce locomotor recovery in chronic spinal cord injury mouse models (Salazar et al., 2010), and they improve cognition in Alzheimer’s disease (AD) mouse models, attributable in part to their production of brain-derived neurotrophic factor (BDNF) (Ager et al., 2015; Blurton-Jones et al., 2009). NPC/NSC transplantation into murine and primate models of Huntington’s disease (HD) rescues motor skill impairment via mature striatal neuron differentiation (Dunnett et al., 2000; Kendall et al., 1998; Palfi et al., 1998; Reidling et al., 2018). Intrathecal and intravenous administration of human NPCs has been shown to promote immunomodulatory effects that limit disease severity in nonhuman primate EAE (Pluchino et al., 2009). Moreover, a



CHAPTER 2 Transplantation of iPSC-derived neural progenitor cells

small-scale clinical study of children with PelizaeuseMerzbacher disease (PMD), a rare hypomyelination disorder in children, demonstrated that frontal-lobe transplantation of human fetal-derived NSCs led to measurable gains in motor function and/or cognition, and this was found to be associated with remyelination (Gupta et al., 2012). While still quite preliminary, these results are encouraging and underscore the potential efficacy of cell therapies to promote myelin regeneration in a variety of demyelinating disease contexts.

Infection with a neurotropic coronavirus as a model of neuroinflammation and demyelination As an additional mouse model of demyelinating disease, intracranial inoculation of C56BL/6 mice with JHMV, a neurotropic strain of mouse hepatitis virus of the Coronaviridae family causes widespread dissemination of virus throughout the brain and spinal cord (Bergmann et al., 2006; Glass et al., 2004; Hosking and Lane, 2009). While neurons are spared, oligodendrocytes, astrocytes, and microglia are susceptible to infection resulting in widespread distribution of the virus throughout the brain (Fleming et al., 1986) (Fig. 2.1A), and this provokes an orchestrated inflammatory

FIGURE 2.1 JHMV infection of the CNS results in rapid spread of virus. (A) In situ hybridization showing distribution of viral RNA in brains of JHMV-infected mice at days 3 and 7 postinfection. Paraffin-embedded brain sections from infected mice at indicated time points were probed with 35S-labeled antisense riboprobes specific for either JHMV. Signal was detected by autoradiography following a 5-day exposure to film. (B) Cartoon depiction of immune response following intracranial (i.c.) infection of the CNS of susceptible C57BL6 with JHMV. Cellular components of the innate immune response e.g., neutrophils, macrophages, and natural killer (NK) cells are rapidly mobilized and migrate to the CNS and contribute to opening the bloodebrain barrier and controlling viral replication. Infiltrating CD4þ and CD8þ T cells reduce viral titers below level of detection through IFN-g secretion and cytolytic activity. Neutralizing virus-specific antibody is required to suppress viral recrudescence during chronic disease.


response within the CNS characterized by entry of neutrophils, NK cells, macrophages, and T cells (Fig. 2.1B). T cells are the drivers of this inflammation, and virus-specific CD4þ T cells help to promote CD8þ T cell expansion in the periphery as well as bloodebrain barrier breakdown and subsequent trafficking of cytotoxic T cells (CTLs) into the CNS (Phares et al., 2012; Zhou et al., 2005). CD4þ Th1 cells also control viral spread through the release of IFN-g. This Th1-produced IFN-g promotes major histocompatibility complex (MHC) Class II expression on microglia and mutes viral replication (Bergmann et al., 2003; Gonzalez et al., 2006; Parra et al., 1999; Phares et al., 2012; Ramakrishna et al., 2004). CD4þ T cell depletion impairs CD8þ T cell viability, the ability of these CTLs to control viral replication (Phares et al., 2012). As the major cytolytic effector within the CNS during JHMV infection, a peak in virus-specific CD8þ T cells CNS residency coincides with viral clearance from glia (Lin et al., 1997; Parra et al., 1999; Ramakrishna et al., 2004). While CD8þ effector T cells are required for resolution of JHMV infections, it is has been found that a colony-stimulating factor 1 receptor (CSF1R) antagonist that depletes microglia resulted in a loss of early infection control, supporting the hypothesis that microglia play a key role in limiting early infection within the CNS to prevent clinical disease and death (Wheeler et al., 2018). Moreover, microglia ablation impaired T cell responses, resulting in elevated CNS viral titers. Thus, microglia play dual roles in serving as a first-line innate immune defense and subsequently enhance T cell responses to limit JHMV infection. In mice that survive acute intracranial JHMV infection, chronic infection leads to an immune-mediated chronic demyelinating disease, with clinical symptoms of ataxia and partial-to-complete hind limb paralysis that peaks at 14e21 days after the initial infection. Histologically, spinal cords from mice chronically infected with JHMV undergo demyelination (Fig. 2.2AeD), though this demyelination shows that oligodendrocyte dysfunction and loss of myelin integrity within white matter tracts are not caused by widespread death mature oligodendrocytes, but rather, are closely associated with viral antigen presentation via MHC-I and MHC-II and the influx of inflammatory leukocytes(Redwine et al., 2001; Stohlman and Hinton, 2001; Wu and Perlman, 1999). Notably, during this chronic stage of infection, a lack of infectious virions suggests that ongoing glial infection is not likely the cause of impaired remyelination. Rather, it is likely that viral RNA quasi-species that persists in chronically infected mice leads to sustained inflammation and demyelination (Adami et al., 1995; Fleming et al., 1995; Rowe et al., 1997). Lesion formation, primarily localized to the lateral funiculus and posterior funiculus, was observed using Luxol Fast Blue staining (Wang et al., 1992). Axonal degeneration within the white matter tracts has been observed in the spinal cords of JHMV-infected mice, as assessed by SMI-32 or Bielschowsky’s silver impregnation stain, and this was found to occur at the same time as demyelination, whereas axon damage has been suggested to precede oligodendrocyte dysregulation in MS (Dandekar et al., 2001; Das Sarma et al., 2009).



CHAPTER 2 Transplantation of iPSC-derived neural progenitor cells

FIGURE 2.2 JHMV-induced demyelination and axonal damage. 5wToluidine blue stained spinal cord sections from (A) control (day 0, DO) and (B) day 28 (D28) postinfection. Demyelination is spread throughout ventral funiculus and lateral white matter columns with notable loss of toluidine blue staining. Electron microscopy reveals extensive loss of myelin sheath at (D) day 28 p.i. compared to (C) noninfected control mice in which thick myelin sheaths are present. Boxed areas in panels A and B indicate regions analyzed for electron microscopic analysis.

Several reports have proposed that, rather than direct killing of oligodendrocytes by virus-induced cytolysis, T cells and macrophages are responsible for demyelination during chronic JHMV infection. Supporting this, JHMV infection of RAG1/ immunodeficient mice that lack functional T and B lymphocytes leads to only limited demyelination despite extensive viral replication within oligodendrocytes (Pewe and Perlman, 2002; Wu and Perlman, 1999). Additionally, extensive demyelination occurs when such mice are administered JHMV-sensitized splenocytes by adoptive transfer from wild-type mice. Subsequent reports have demonstrated that both CD4þ and CD8þ T cells are responsible for demyelination in following intracranial inoculation of JHMV (Pewe and Perlman, 2002) (Lane et al., 2000). Unlike the case for neurotropic virus strains such as Theiler’s virus, other factors, including epitope spreading and the recruitment and activation of autoreactive T cells specific for CNS antigens, do not appear to cause demyelination following CNS infection by JHMV. Thus, JHMV intracranial infection provides a unique mouse model to characterize the complex interplay of inflammatory and regenerative cell types that distinctly impact pathology in demyelinating disease.


The impact of human neural precursor cell transplantation in demyelinating disease models With an overarching goal of developing treatment strategies for human MS patients, we have sought to evaluate the impact of human neural precursor cells (hNPCs) and other stem and progenitor cells in both the JHMV and EAE mouse demyelinating disease models. In early studies, limited clinical recovery was observed following the engraftment of predifferentiated human OPCs into mice following establishment of chronic JHMV-mediated demyelinating disease (Hatch et al., 2009). Despite the administration of immunosuppressive drugs, engrafted cells were rejected within 14 days of transplantation, and this was accompanied by only weak remyelination near the transplant site (Hatch et al., 2009). This was in stark contrast to earlier studies in which human embryonic stem cell (hESC)-derived OPCs were found to promote remyelination and significant clinical recovery in a rat spinal cord injury model (Keirstead et al., 2005). It is noteworthy that human neural stem cells at a more immature stage have been found to exert neuroprotective effects in both mouse and nonhuman primate models of EAE, implying that less mature human neural lineage cells are more functional than differentiated progenitor cells in the context of the damaged CNS (Aharonowiz et al., 2008; Pluchino et al., 2009). A major barrier to the development of cell replacement therapies for treatment of demyelinating diseases is the source of neural lineage cells for transplantation. As described earlier, transplantation of xenogeneic neural stem/progenitor cells leads to their rapid immune-mediated rejection. Instead, the differentiation of human induced pluripotent stem cell (iPSC)-derived NPCs could overcome this issue, particularly if such cells are developed from autologous cells from the patient, as this would most likely block immune-mediated rejection. To address this, these “embryoid body” (EB) NPCs derived from human iPSCs were transplanted stereotactically into the spinal cords of JHMV-infected mice, and while the cells were rapidly rejected in this xenogeneic transplant model, focal remyelination near the transplant site was observed (Fig. 2.3A and B) (Plaisted et al., 2016). Accompanying this focal remyelination was a reduction in the population of CD4þ T cells recruited near the transplant site and a concomitant increase in Foxp3þ CD4þ CD25þ regulatory T cells (Fig. 2.3C). Demonstrating that these regulatory T cells (Tregs) were not simply bystanders but were involved in the protective response, ablation of Tregs using the anti-CD25 antibody PC61.5 muted recovery as assessed using Luxol Fast Blue staining (Fig. 2.4AeC). Likely due to the highly focal nature of tissue regeneration observed, we did not observe statistically significant clinical recovery. Nevertheless, these findings support the hypothesis that Tregs induced by human iPSC-derived NPCs enhanced remyelination, either by muting inflammation or by directly activating the maturation of regenerative populations within the CNS such as OPCs (Dombrowski et al., 2017). These observations suggest that methods to enhance the recruitment of Tregs into sites of ongoing demyelination and other forms of CNS damage may be protective, not simply through blockade of ongoing inflammation, but also through induction of endogenous tissue regenerative mechanisms.


FIGURE 2.3 Focal remyelination in animals transplanted with EB-NPCs. (A) Representative electron micrographs of coronal spinal cord sections from HBSS, fibroblast, and EB-NPC-injected mice. (B) Analysis of the ratio of the axon diameter versus total fiber diameter (g-ratio) confirmed enhanced remyelination at the transplant site of EB-NPC-injected mice compared to controls (P < .001; 300 axons were measured per experimental group). (C) Quantification of the number of CD4þFOXP3þ Tregs demonstrated a significant (P < .05) increase in the CLNs of EB-NPC transplanted mice compared to controls at day 5 p.t. Data is presented as average  SEM and was analyzed using one-way ANOVA followed by Tukey’s multiple comparison test.

FIGURE 2.4 Regulatory T cells are necessary for EB-NPC-induced myelin sparing. (A) Representative spinal cord sections stained with LFB and H&E. Outlined areas highlight demyelination. (B) Quantification of white matter damage revealed PC61.5treated EB-NPC-transplanted mice did not have reduced demyelination when compared to nontreated EB-NPC-transplanted mice. All data is presented as the average  SEM and was analyzed using one-way ANOVA followed by Tukey’s multiple comparison test; *P < .05, **P < .01, ***P < .001.


In the aforementioned studies, the cells used were generated using an embryoidbody-based technique that resulted in the expression of PAX6, a marker of NPC cells. However, when hESC-derived “neural precursor-like cells” (NPLCs) that lacked expression of PAX6 were stereotactically transplanted into the spinal cords of mice bearing JHMV-induced demyelinating disease, clinical and histological improvement was observed out to 6 months posttransplant (Chen et al., 2014) even though these NPLCs were rejected within 8 days of transplant. In contrast to the studies that employed human iPSC-derived NPCs, the remyelinated axons were distributed both rostrally and caudally, rather than localized to the region of cell delivery. As with human-iPSC-derived NPCs, hNPLC engraftment led to a reduction in overall numbers of T cells, but with an increase in the numbers of CD4þ Foxp3þ CD25þ Tregs (Chen et al., 2014). Treg depletion mediated by PC61.5 administration abrogated the therapeutic benefits of hNPLC transplantation, demonstrating here as well that Tregs were necessary for histological and clinical recovery. These PAX6-negative NPLCs were not neural precursor cells, at least as they have been traditionally defined. Instead, they were developed using a protocol that enhances the differentiation of peripheral neural lineage cells rather than CNS neural lineage derivatives, as confirmed by gene expression studies demonstrating distinct gene expression signatures in human iPSC-derived NPCs versus NPLCs (Plaisted et al., 2016). Further analyses of the gene expression signatures of these cells yielded clues to the differences in disease-modifying activity of NPLCs versus NPCs, with the observation that NPLCs with the strongest ability to promote clinical recovery had high levels of TGF-b2 (Chen et al., 2014). An antiinflammatory cytokine, TGF-b2 has previously been shown to promote the differentiation of peripheral Tregs (Marie et al., 2005). Tregs have also been found to play vital roles during both acute and chronic JHMV infection (Anghelina et al., 2009). IL-10-expressing virus-specific Tregs limit the responsiveness of virus-specific CD4þ effector T cells, and Treg ablation increases mortality; these findings support the hypothesis that, during acute JHMV infection, Tregs limit immune pathology without interfering with viral clearance. Adoptive transfer of Tregs into mice undergoing chronic CNS infection by JHMV led to an attenuation of clinical and histological disease by muting neuroinflammation and subsequent demyelination (Trandem et al., 2010).

The influence of adaptive immunity on stem/progenitor cell remyelination As described earlier, the therapeutic potential of stem and progenitor transplantation has been tested in a variety of neurological disorders. The current lack of therapeutic options for those suffering from progressive MS makes this approach particularly attractive. MS, however, is a complex disease requiring examination of not only the potential to repair damage to the CNS but also the interactions that occur



CHAPTER 2 Transplantation of iPSC-derived neural progenitor cells

between the immune system and transplanted cells. Further complicating these endeavors is the wide variety of available cell sources, derivation and differentiation protocols available to generate cells with therapeutic potential. This makes MS a disease of interest as it highlights the therapeutic potential of NPCs to repair damage to the CNS as well as the interactions associated with transplanting foreign cells into the CNS. There has been extensive use of human fetal NPCs in both animal and human studies examining the potential of cell-based therapies to repair damage to the CNS. Human derived NPCs have shown efficacy in primate models of demyelination, improving disease pathology and clinical outcome (Pluchino et al., 2009). Studies in mice utilizing transplantation of syngeneic fetal NPCs show long-term engraftment and differentiation of transplanted NPCs throughout the spinal cord. However, in order to prevent immune rejection, these studies often must intentionally suppress the immune system or prevent immune rejection through the use of syngeneic transplants, an issue that may be difficult to achieve in MS patients (see aforementioned). While such studies effectively examine the potential of transplanted cells to directly repair damage through integration and differentiation, they fail to address immune interactions that occur such as the role of the immune system in both tissue repair and the potential for chronic transplant rejection. Rejection of transplanted cells may not, in fact, be as detrimental as previously thought. In studies performed employing both JMHV and EAE to model demyelinating disease, human NPC transplantation has shown therapeutic potential in the absence of long-term engraftment. Indeed, although hNPCs are found to be quickly rejected following engraftment, improved clinical scores and diminished pathology have been observed (Plaisted et al., 2016, McIntyre et al., 2020), including an increase in focal remyelination (Fig. 2.3). Unlike the case for previous studies employing immune suppression, immune deficiency, or syngeneic stem cells, improvements observed in pathology following immune-mediated rejection were not based on cell replacement directly and instead, required immune function. Mice transplanted with human NPCs showed decreased T cell infiltration into the CNS and increased regulatory T cell (Tregs) recruitment in both the JHMV and EAE demyelination models. Histological improvement was dependent on Tregs since depletion of Tregs abrogated the impact of human NPC transplantation. In mice, this response appears to be due, in part, to presentation of human NPC-derived antigens, resulting in greater Treg recruitment and activation within demyelinating lesions (McIntyre et al., manuscript in preparation). A major question remains: how might human stem/progenitor cells that are transplanted but rejected prior to engraftment and differentiation into cells that mediate repair function? One possibility is that such transplanted cells promote the recruitment and/or activation of Tregs that suppress pathological inflammation, thereby allowing for endogenous repair to occur without further immunopathology. This is unlikely given that immune suppression alone does not result in meaningful remyelination and


CNS regeneration. An alternative explanation is that Tregs directly influence the regenerative process within the CNS. Notably, Tregs have been found to be capable of inducing OPC maturation into mature functional oligodendrocytes capable of remyelination. These studies reveal the importance of studying NPC transplantation in clinically relevant models that accurately model potential interactions with systems outside the CNS such as the immune system. While it might be important to examine the therapeutic potential of stem/progenitor cells to promote remyelination and regeneration when isolated from the influence of the immune system, such models do not provide a complete picture of the potential outcomes and, in our opinion, do not adequately recapitulate the environment confronted by these cells in a translational context. For effective therapeutic implementation, it is evident that neural lineage stem and progenitor cells of different origins must be examined, not only for their ability to directly promote tissue regeneration, but also for potential impacts of the immune system in this process (Plaisted et al., 2016, McIntyre et al., 2020).

Conclusions Research with transplantation of human neural stem/progenitor cells such as NPCs and NPLCs highlights significant promise for the treatment of neurologic disorders such as MS. Given rather limited therapeutic approaches for treating chronic forms of MS such as secondary progressive MS, stem-cell-based therapies may offer significant advantages over those that are solely focused on immune suppression. As described in this chapter, a major barrier to clinical translation of this approach is the identification of effective stem/progenitor cell types capable of promoting remyelination and neurologic repair. In designing cell therapies for human disease, it is important to standardize criteria for defining cell types to be used for transplantation. Our analysis of gene expression profiles of a variety of human precursors and stem cells revealed that they are very diverse; for example, while pluripotent stem cells were very similar to each other, cells that had been designated as neural stem cells were clustered into multiple subgroups (Muller et al., 2008). Thus, the provenance and protocols used to generate stem cells for transplantation appear to be significant issues. A second caveat that must be overcome for effective cellreplacement therapy is the difficulty in developing abundant supplies of such cells necessary for patient engraftment. As highlighted here, these issues may potentially be overcome through the regenerative influence of Tregs that are recruited into the damaged CNS following stem cell transplantation. Further research employing animal models such as JHMV and EAE, as well as preclinical experiments with human tissues (e.g., peripheral blood lymphocytes), will help to clarify the impact of immune:stem cell interactions in remyelination and may offer new therapeutic strategies for the treatment of nervous system diseases such as MS.



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CHAPTER 2 Transplantation of iPSC-derived neural progenitor cells

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CHAPTER 2 Transplantation of iPSC-derived neural progenitor cells

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iPSCs for modeling influenza infection


Ali Zahedi-Amiri1, 2, Kevin M. Coombs1, 2, 3 1

University of Manitoba, Department of Medical Microbiology and Infectious Diseases, Winnipeg, MB, Canada; 2Manitoba Centre for Proteomics & Systems Biology, Winnipeg, MB, Canada; 3 Children’s Hospital Research Institute of Manitoba, Winnipeg, MB, Canada

Chapter outline Introduction .............................................................................................................. 48 IAV-induced cell death in iPSCs................................................................................. 49 Differentiation potentials of IAV-infected iPSCs ........................................................... 55 iPSC-derived tissues and organoids for modeling influenza infection ........................... 60 Concluding remarks .................................................................................................. 62 Acknowledgments ..................................................................................................... 62 References ............................................................................................................... 62 Abstract The cell reprogramming technology has been revolutionized by recent advances in generating induced pluripotent stem cells (iPSCs), which not only hold great promise in regenerative medicine and cell therapy but also in modeling different disease conditions, including viral infections. Influenza A virus (IAV) remains a global threat to the human population, and how IAV affects humans during the embryonic stage is poorly understood, despite the ambiguities on transplacental passage and links to congenital defects. Moreover, the virus’ tropism for many extrapulmonary tissues has largely remained elusive. The capacity of iPSCs for in vitro simulation of embryogenesis, and the potential of these cells to differentiate into many other cell types, provides a valuable resource to study IAV-mediated alterations in various cell states and types. In this review, we discuss the potential applications of iPSCs in the molecular characterization of IAV-induced cell death through modeling both virus-impaired embryonic development and cell-specific death signals. In addition, recent IAV-infected iPSC proteomic data were reanalyzed here to predict the effects that this virus might have on molecules controlling differentiation pathways toward germ layer formation and embryogenesis. We also address how iPSC-derived cells and organoids could overcome some challenges associated with using cell lines or primary cells for studying IAV pathogenesis. We highlight the significance of iPSC-based models in identifying common and cellspecific mechanisms facilitating IAV infection, which might contribute to the development of efficient antiviral treatments against this pathogen. iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00011-0 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 3 iPSCs for modeling influenza infection

Keywords: Apoptosis; Aptamers; Autophagy; Bioinformatics; Blastocyst; Chemokines; Cytokines; Embryology; Epithelialemesenchymal transition; Immune modulation; In vitro modeling; Infertility; Influenza; Pluripotent stem cells; Proteomics; Virus.

Introduction Influenza A virus (IAV) is a highly contagious pathogen known to cause seasonal epidemics and sporadic pandemics among the human population. Over the past century, about 100 million people have died from influenza infection (Taubenberger et al., 2007; Murray et al., 1996). During this period, mutations of several specific human-adapted strains, as well as highly pathogenic avian strains of IAV, have contributed to clinical complications, ranging from mild to acute respiratory infections (Taubenberger and Morens, 2009). As an enveloped virus and a member of the family Orthomyxoviridae, IAV is comprised of an eight-segment genome containing negative-sense single-stranded RNA [()ssRNA)] that codes for several viral proteins. Depending on antigenic specifications of Neuraminidase (NA) and Hemagglutinin (HA) genes encoding surface proteins, IAVs currently can be serologically sorted into 11 NA and 18 HA subtypes (Tong et al., 2012; Schild et al., 1980). Most influenza pandemics have been linked to two subtypes of NA (N1, N2) and three subtypes of HA (H1, H2, H3), even though at least six other subtypes of each of these two surface glycoproteins may cause human infection (Tong et al., 2013). Nevertheless, considering the evidence on human infections with some highly pathogenic variations of avian viruses such as H5N1 and H7N9, different subtypes of IAV constantly remain as a global concern and might result in potential pandemics and epidemics (Richard et al., 2014). Recent epidemiological studies from influenza pandemics and seasonal epidemics have reported high fatality rates in pregnant women and in the fetus, indicating that both mothers and neonates are among high-risk groups that might be more severely influenced by IAV complications (Engels et al., 2017). Apart from different cases of influenza-triggered miscarriage and stillbirth, influenza infection during pregnancy was assumed multiple times to directly cause certain developmental abnormalities in offspring, including cleft lip, neural tube defects, cardiac malformations, hydrocephaly, digestive system defects, and limb reduction anomalies (Rasmussen et al., 2012; Luteijn et al., 2014; Leck et al., 1969; Coffey and Jessop, 1963; Czeizel et al., 2008). Despite the unknown molecular mechanisms mediating IAV-induced teratogenic effects, some postmortem histopathological reports from pregnant women have revealed that a few IAV subtypes such as H3N2, H5N1, and H1N1 are capable of extrapulmonary spread into various maternal and fetal tissues, such as histiocytes, cytotrophoblasts, fetal lung cells, fetal cardiomyocytes, and fetal liver macrophages (Gu et al., 2007; Yawn et al., 1971; Lieberman et al., 2011; Jewett, 1974). Secondary viremia is noted to happen sporadically during pregnancy. Thus, transplacental passage of influenza is debated as one imminent clinical manifestation of IAV infection in pregnant women. However,

IAV-induced cell death in iPSCs

transplacental passage might be trimester-specific or could occur asymptomatically. Furthermore, natural cellular functions and embryonic cell differentiation capacity could be influenced by fetomaternal viral interference in molecular pathways regulating embryogenesis and fetal development. IAV replicates productively in some multipotent and unipotent stem cells that play roles in embryonic development and tissue regeneration (Khatri and Chattha, 2015; Khatri et al., 2012; Pringproa et al., 2015). Although mouse embryonic stem cells (mESCs) are not fully permissive to IAV replication, these cells not only lose their viability even at low multiplicity of infection (MOI), but also allow limited viral protein production, suggesting the potential susceptibility of the blastocyst’s inner cell mass (ICM) to IAV (Wash et al., 2012). ESCs are pluripotent cells that exist temporarily undifferentiated inside the ICM of a blastocyst before commencing embryogenesis through giving rise to germ layers. Because some of the influenza-triggered congenital malformations are presumed to originate from preimplantation stages, modeling early pregnancy using pluripotent stem cells (PSCs) might provide insight into the possible effects of IAV infection on embryonic development. An intrablastocyst infection could also be modeled by induced pluripotent stem cells (iPSCs), which are derived via reprogramming somatic cells and look promising as moral and practical alternatives compared to ESCs. Moreover, the differentiation capacities of these cells make them useful to generate various stem cells and terminally differentiated cell populations for studying cell-specific responses to IAV infection. The present chapter is orientated toward the potential applications of iPSCs and iPSC-derived cells in modeling influenza infection.

IAV-induced cell death in iPSCs The termination of blastocyst growth causes early pregnancy failure. Possible consequences of maternal IAV infection at the time of conception or during preimplantation stages have not been well studied so far. One of the main questions regards whether direct intrablastocyst infection with IAV could result in destruction of ESCs or impaired embryogenesis. Since iPSCs resemble ESCs residing in the ICM of a blastocyst, these cells have the capacity to characterize intrablastocyst vulnerability to viral infection experimentally. IAV is a highly cytolytic pathogen and capable of inducing cell death in a variety of mammalian cells (Roulston et al., 1999; Takizawa et al., 1996; Mori et al., 1995). However, cell death and survival signals under the pluripotency state, especially in the course of viral infections, remain to be elucidated (Hossini et al., 2016). IAV cannot productively replicate in human induced pluripotent stem cells (hiPSCs), despite hijacking the cellular transcription and translation machinery (Zahedi-Amiri et al., 2019). The entry of IAV ribonucleoproteins into the nuclei of these cells is associated with cytopathological effects as well as reduced levels of cell viability within 24 h of infection, showing the early induction of cell death



CHAPTER 3 iPSCs for modeling influenza infection

in hiPSCs after exposure to influenza. Apoptosis, which is considered the most common sort of programmed cell death, can be activated via intrinsic and extrinsic pathways in a variety of IAV-infected host cells. Although the form and pathway of IAV-triggered cell death might be cell-type-specific, influenza NS1 protein is known to temporarily suppress apoptosis through the PI3k-Akt signaling pathway in the vast majority of somatic and differentiated cells at early stages of infection to increase viral replication (Chen et al., 2001; Zhirnov et al., 2002; Ehrhardt et al., 2007). During the next stages of infection, IAV induces apoptotic or necrotic cell death to facilitate the release of progeny virions for infecting other cells (Yeganeh et al., 2018). Such a difference indicates that IAV-induced cytological deformities and cell death in PSCs might occur faster than in somatic cells, regardless of limited viral replication under the pluripotency state. The stress-induced mitochondrial damage can stimulate the activation of the intrinsic apoptotic pathway, which is regulated by the Bcl-2 family of proteins involving both proapoptotic and antiapoptotic proteins (Levine et al., 2008; Ouyang et al., 2012). In IAV-infected hiPSCs, downregulation of antiapoptotic regulator Bcl-2, the stable expression of proapoptotic regulator Bax, and transient activation of apoptosis regulator p53 might show the potential initiation of apoptosis; however, the restricted cleavage of some apoptotic executioners such as caspases-3, -7, and -9 suggests that this virus does not activate intrinsic apoptosis in hiPSCs by 24 h (Zahedi-Amiri et al., 2019). The specific protein expression profiles of apoptotic executioners and regulators after IAV infection of PSCs highlight the possible involvement of another caspase-independent pathway that induces cell death within the first day of infection. IAV has been reported to promote virion uncoating through altering and utilizing autophagy, a mechanism that regulates the recycling of dysfunctional cellular components, and its activation might be associated with cell death (Gannage´ et al., 2009; Zhou et al., 2009; Comber et al., 2011; Kroemer and Levine, 2008). IAV has been shown to overactivate autophagy in hiPSCs, but there is insufficient proof to directly link this mechanism to the demonstrated cell death in these cells (Zahedi-Amiri et al., 2019). Therefore, it is not clear whether IAV-modulated cell death in PSCs could be triggered via apoptotic or nonapoptotic pathways, and further investigations are required to define the exact mechanism contributing to the cellular demise of hiPSCs after IAV infection. Based on our proteomic screening of IAV-infected hiPSCs (Zahedi-Amiri et al., 2019), we designed a graphic overview representing all significantly altered proteins that are predicted to activate or suppress the most common types of cell death (Fig. 3.1). IAV-mediated dysregulations in the hiPSC proteome by 1 day postinfection might involve activation of autophagy and apoptosis along with inhibition of necrosis. The aptamer-based technology that was used for proteomic screening of IAV-infected hiPSCs was selective and could not report most of the viral-induced changes in the proteome, suggesting that expression profiles for many other proteins controlling different types of cell death remain unknown. Nevertheless, analyzing proteineprotein interaction networks of molecules influencing any type of cell death helps identify signaling pathways linked to these proteins. For example, among those interacting proteins that were found

Reanalyses of proteomic data sets reveal that IAV affects the expression of specific proteins in a way that activates autophagy and caspaseindependent apoptosis while inhibiting necrosis. Predictions of activation or inhibition relationships between molecules and cell death variants are established by both functional analysis of each molecule in literature and Ingenuity Pathway Analysis (IPA) bioinformatics software (QIAGEN, Germany). Interacting proteins corresponding to each type of cell death are connected to indicate their link to various signaling pathways.

IAV-induced cell death in iPSCs

FIGURE 3.1 Overview of IAV-affected proteins that are predicted to differentially influence different types of cell death in hiPSCs.



CHAPTER 3 iPSCs for modeling influenza infection

to exclusively (e.g., not involving necrosis or autophagy) regulate apoptosis in IAVinfected hiPSCs, CSK, ERBB3, FER, INSR, and ROR1 are connected to the IL-15 signaling pathway, which usually facilitates the demise of virally infected cells by natural killer cells (Di Sabatino et al., 2011). Such a pathway-based analysis reflects only a small part of virus-induced dysregulations in various pathways and suggests the need for examining other member molecules of potentially affected pathways that have not been studied in PSCs under the influence of IAV. This approach ultimately not only determines whether these pathways could be substantially targeted by IAV but also highlights the potential direct or indirect roles of other member molecules in such pathways for mediating virus-induced cell death. Different RNA viruses do not evenly affect cell viability and might induce different types of cell death in PSCs. For example, Measles, which is also a () ssRNA virus like IAV, causes enlargement of the nucleus and forms balloon-like structures within the hiPSC colonies, ultimately leading to floating syncytial debris and low levels of cell viability(Hu€bner et al., 2017). Similarly, in vitro infection with another ()ssRNA virus such as La Crosse virus severely reduces the viability of mESCs by causing cell rounding and detachment from culture vessels (Wang et al., 2014). These murine pluripotent cells were not noticed to be cytopathogenically influenced by Sendai virus, another ()ssRNA virus (Wang et al., 2013). Among positive-sense genome (þ)ssRNA viruses that have been tested in mESCs, Chikungunya virus was reported to induce cell death, while West Nile virus does not appear to result in lytic cell death (Wang et al., 2013, 2014). Different studies showed that Rubella virus, which also has a (þ)ssRNA genome, contributes to apoptosis in a variety of cell types through a p53-dependent mechanism (Megyeri et al., 1999). However, Rubella virus was demonstrated to establish a noncytopathogenic replication in iPSC lines (Hu€bner et al., 2017; Bilz et al., 2019). Despite evidence on cytolytic effects of some RNA viruses on PSCs, types of cell death associated with each of these viruses are not yet characterized under the pluripotency state. In the case of IAV-infected hiPSCs, proteomic findings accentuating the involvement of a caspase-independent pathway or autophagy in causing IAVinduced cell death have low specificity because of being analyzed by bioinformatic predictions for any cell type (Zahedi-Amiri et al., 2019). Overcoming such challenges requires not only further cell-specific in vitro validations but also identifying potential IAV-provoked alterations of other regulators in targeted pathways by using large-scale omics approaches. The restricted replication of IAV in mESCs and hiPSCs theoretically suggests that a small number of ESCs within the ICM might be damaged by this virus in the case of low-input multiplicities of infection. Depending on IAV subtype, trophoblast cells that protectively surround a blastocyst can undergo significant apoptoticinduced cytopathic effects after IAV infection or allow only IAV protein production without significant viral release and cytopathology (Yawn et al., 1971; Trinh et al., 2009; Komine Aizawa et al., 2012). Moreover, although IAV proteins and mRNA were isolated several times from both maternal and fetal tissues, the extrapulmonary replication of this virus does not appear to be as considerable as its replication in

IAV-induced cell death in iPSCs

respiratory cells. Consequently, irrespective of fewer barriers within the blastocyst at the preimplantation stage, enough infectious IAV particles might not reach the ICM to infect all ESCs. This raises the question of whether the possible destruction of a small percentage of these cells by IAV-induced cell death could influence the implantation process of a blastocyst. Marikawa et al. described an embryoid bodies (EBs)-based model similar to the gastrulation stage, which are known as gastruloids (Marikawa et al., 2009). Such postimplantation models were further studied by others and additional information was obtained regarding symmetry breaking, axial elongation, and germ layer specification in aggregates of murine ESCs and iPSCs (Van den Brink et al., 2014; Beccari et al., 2018). The gastruloid model was later supported by the assembly and addition of extraembryonic trophoblast tissue (Harrison et al., 2017). Nevertheless, these models cannot mimic preimplantation phases and are not capable of generating all embryonic tissues, such as trophectoderm for implantation and other tissues for later steps in embryogenesis, suggesting that other models need to be developed for determining the effect of IAV on implantation and early stages of embryonic development. Rivron et al. have recently reported the formation of the first synthetic blastocysts by combining only mouse stem cells, including trophoblasts stem cells (TSCs) and ESCs (Rivron et al., 2018). These artificial structures are named blastoids and resemble real blastocysts at E3.5 according to single-cell RNA sequencing. As shown in Fig. 3.2, this model is shaped using microwell platforms to assemble independently grown TSCs and ESCs in a mixture containing the inducers of self-organization, such as WNT activator and cAMP analogue (Rivron et al., 2018; Vrij et al., 2019). Interestingly, blastoids can be successfully implanted into the uterus of pseudopregnant mice. Nesting of these blastocyst-like structures is followed by further growth and differentiation, leading to the formation of deciduae with vascular permeability and the fusion of trophoblasts with the maternal vascular system (Rivron et al., 2018). Despite differentiation into postimplantation trophoblast populations and expression of related extraembryonic markers, successful implantation of blastoids does not support the later stages of embryogenesis. However, due to the similarity of the preimplantation stage between mice and humans, the blastoid can be considered an appropriate model to study the impact of IAV-induced cell death on the blastocyst as these synthetic structures simulate the pre- and early postimplantation phases of development. Blastoids have mass production capacity through bioengineering technologies, and their initial separated cell culture only involves a few in vitro blastocyst destructions for first-time isolation of cells. To solve ethical dilemmas raised before on the direct extraction of animal blastocyst compartments such as ESCs, iPSCs originating from murine somatic cells can be used as an alternative source for generating blastoids through an ICM-free approach. In Fig. 3.2, this approach is technically compared to the original idea of blastoid that utilizes the components of an actual murine blastocyst. Several studies have shown the generation of trophoblasts from both mouse and human iPSCs. This idea was further followed up by using 3D microstructured scaffolds to morphologically resemble the trophectoderm at the late-stage blastocyst (Okeyo et al., 2018). Considering the initial separated culture of cells in the blastoid


54 CHAPTER 3 iPSCs for modeling influenza infection

FIGURE 3.2 Application of iPSCs in developing ICM-free blastoid model. Murine iPSCs can be reprogrammed from somatic cells to generate a blastoid model without involving the components of a real blastocyst. The aggregation of iPSC-derived TSCs with iPSCs in microwell platforms results in the formation of blastoids through using WNT activator and cAMP analogue as inducers of self-organization. In addition to several uses in drug testing, embryology, studying infertility, contraception, and disease modeling, the blastoid model has the capacity for intrablastocyst modeling of IAV infection to determine the type of and effects of IAVinduced cell death during pre- and postimplantation stages.

Differentiation potentials of IAV-infected iPSCs

system, it is likely that the combination of iPSCs and iPSC-derived trophoblasts in microwell platforms results in the formation of blastoids that are functionally and physically equivalent to the first-generation ones engineered by using ESCs and TSCs. Such an iPSC-based model mimics in utero implantation and could pave the way for characterizing the type of cell death that IAV induces in PSCs residing in the blastocyst ICM. Determining the mechanisms and attributes of virus-mediated cell death under the pluripotency state not only helps in understanding whether IAV can terminate blastocyst implantation or growth, but also sheds new light on cellular targets that might inhibit intrablastocyst infection with this virus. Although a developing fetus is progressively protected against pathogens by the placenta, some systematic reviews and epidemiolocal assessments have suggested some links between spontaneous miscarriages and maternal IAV infection within the second and third trimesters (Stanwell-Smith et al., 1994). During these postembryonic stages of development, various multipotent and unipotent stem/progenitor cells differentiate into several tissues after germ layer specifications, which ultimately contribute to organogenesis. Potential disruption of stem cell self-renewal and differentiation by virus-induced cell death is a possibility that could lead to defects in fetal growth and miscarriage. It is not completely clear whether IAV can cause cytolytic effects and cell death in different types of progenitor stem cells while the fetus is developing. Infection of ESC-derived neural progenitor cells (NPCs) with an IAV H5N1 subtype resulted in cytoplasmic vacuolation, damaged nuclear membrane, and shrinkage with chromatin condensation (Pringproa et al., 2015). Even though these observations confirm the cytopathic effects of IAV, the type of cell death that could be caused by this virus in NPCs remains unknown, which continues to add to the ambiguities surrounding the molecular mechanisms of IAV-induced cell death in different types of stem cells. In addition to PSCderived organoids, different types of progenitor stem cells can be separately derived in vitro through differentiation of iPSCs in order to discover cell-specific pathways of IAV-triggered cell death. Regardless of developmental aspects, the differentiation capacity of iPSCs also allows the 3D generation of terminally differentiated adult tissues that cannot be easily cultured as primary cells after isolation but are known as common targets for different IAV subtypes, such as lung epithelial cells (Ciancanelli et al., 2015; Ghaedi et al., 2015). Taken together, similarities and differences of virus-associated death signals under various cell potency conditions can be compared in iPSC-based models, which provide valuable information concerning the molecular mechanisms that IAV exploits for eliminating embryonic, fetal, and adult cells.

Differentiation potentials of IAV-infected iPSCs Many questions remain unanswered regarding the effect different viruses have on ESC pluripotency and differentiation capacitates. Apart from virus-induced cell death, some viruses are capable of subverting the host cell proteome, which raises



CHAPTER 3 iPSCs for modeling influenza infection

questions about the possibility of virus-mediated dysregulations in different signaling pathways governing embryogenesis. ESCs give rise to epiblast and hypoblast layers, eventually differentiating into the primary embryonic germ layers endoderm, mesoderm, and ectoderm during gastrulation. iPSCs have great promise for in vitro modeling of differentiation into three germ layers (Takahashi et al., 2007). Nevertheless, similar to many other viruses, the role that IAV may play in terms of affecting stem cell differentiation remains largely unknown. Determining the level of pluripotency in infected PSCs could shed some light on the possibility of spontaneous or abnormal differentiation. The infection of hiPSCs with IAV has been recently shown to result in the activation of autophagy and decreased expressions of pluripotency regulators such as NANOG, SOX2, and Oct-4A (Zahedi-Amiri et al., 2019). In order to generate a complete picture of affected cell potency by IAV, such demonstrations at the protein level require the assessment of other pluripotency markers such as SSEA4, TRA-1-81, and TRA-160, which affect the carbohydrates making up glycolipids or glycoproteins expressed on undifferentiated PSC surfaces. SSEA4 is formed of a glycosphingolipid that contains a terminal sialic acid residue, while TRA-1-60 and TRA-1-81 are known as keratin sulfate proteoglycan epitopes on PODXL, which carries sialic acid as its terminal sugar residue (Henderson et al., 2002; Draper et al., 2002; Fong et al., 2009). Unlike the TRA-1-81 epitope that is neuraminidase-resistant, one of the two forms of TRA-1-60 is sensitive to neuraminidase, despite the indistinguishable cellular reactivity of both forms (Badcock et al., 1999). Interestingly, IAV entry into host cells initiates with the binding of the viral HA protein to the sialic acid receptor on the surface of the targeted cell (Stegmann, 2000). Moreover, during the stage of viral release, IAV NA protein, possessing sialidase function, cleaves sialic acid residues on the cell surface to block the attachment of HA proteins of the newly synthesized enveloped virions to these receptors, ultimately facilitating the budding of progeny particles (Gamblin and Skehel, 2010). It is not clear whether these two viral surface glycoproteins can interact with pluripotency epitope sialic acid residues on the cell membrane either at entry or release steps of the IAV life cycle. For example, due to the presence of sialic acid on terminal sugar residue, the neuraminidasesensitive variant of the TRA-1-60 epitope could not only be targeted by HA for viral entry but could also be digested by NA sialidase activity during viral release, suggesting that IAV proteins might influence various types of pluripotency regulators. The restricted replication of IAV and several other viruses in PSCs is assumed to be caused by the underdeveloped glycosylation system in these cells that might hinder the maturation of HA and NA (Wash et al., 2012; Smith-Arica et al., 2003; Wagner et al., 2000; Satomaa et al., 2009; Atwood Iii et al., 2008). Likewise, the neuraminidase-resistant type of TRA-1-60, as well as TRA-1-81 epitopes, could also potentially act as intrinsic barriers to NA protein, which hypothetically must result in decreased viral shedding. Pluripotency is a complex network that will not be simple to evaluate just based on a few markers. Different viruses might use diverse mechanisms to alter other regulators of such a network. Measles virus, which is the only other tested ()ssRNA virus against pluripotency, has not been proven to

Differentiation potentials of IAV-infected iPSCs

affect the expression of OCT4 and SOX2 in infected hiPSCs (Hu€bner et al., 2017). Similarly, (þ)ssRNA viruses such as Rubella and Coxsackievirus also do not alter this intrinsic PSC feature (Hu€bner et al., 2017; Bilz et al., 2019). However, most viruses have not yet been studied at sufficient detail for determining their potential impacts on various pluripotency mediators. Although the recent selective proteomic screening of IAV-infected hiPSCs provided some clues, the expression profiles of several other components that indirectly regulate the pluripotency network have not been characterized after viral infection. In addition, specifying the interactions between IAV proteins and any pluripotency regulator proteins (e.g., immunoprecipitation) might reveal whether this virus has a direct or indirect effect on this feature of PSCs, eventually revealing the mechanism by which IAV possibly influences the differentiation of these cells. The wide-scale evaluation of pluripotency in IAV-infected PSCs could provide an image on potential effects this virus has on the differentiation capacity, but cannot practically contribute to understanding how IAV might abnormally force or interrupt the differentiation as well as stem cell fate commitment. Despite the potential of iPSCs to differentiate into three germ layers and their derivatives, IAV has never been specifically tested for its ability to affect this feature of PSCs. A few other RNA viruses have been reported to influence the formation of certain germ layers in vitro. For example, iPSCs infected with Measles virus and Coxsackievirus were incapable of differentiation into ectoderm, endoderm, and mesoderm layers, probably because of lytic viral replication cycles (Hu€bner et al., 2017). Rubella virus was also noted to impair the differentiation of these cells to the endoderm layer, even though it did not cause apparent disruption in the formation of mesoderm and ectoderm layers (Bilz et al., 2019). None of these viruses are known to reduce pluripotency according to the assessment of a few markers, which might highlight the probability of virus-induced impairments in germ layer formation without altering the pluripotency. Because IAV can diminish the levels of pluripotency, the differentiation range of PSCs could be either normally or defectively restricted to certain derivatives by this virus. Irrespective of the fact that this hypothesis has not been tested so far, our aptamer-based selective proteomic analyses of IAV-infected hiPSCs recently showed some useful information concerning virus-triggered alterations in the expression of some proteins that act as mediators or inducer signals for differentiation into the three germ layers and subsequent derivatives (ZahediAmiri et al., 2019). This approach can help predict differentiation to which germ layer would be likely more forced or affected by IAV. As illustrated in the left panel of Fig. 3.3, a few molecules that are known to be more expressed in a certain germ layer can be dysregulated by IAV in hiPSCs in ways to involve either activation or inhibition functions toward the formation of a related germ layer and its derivatives. According to abnormal phenotypes observed by several studies in knockout models, downregulation of some germ-layer-specific proteins such as TK1, GFRA1, and ADSL could negatively influence the formation or differentiation pathways of at least one other germ layer that is not known to express an adequate amount of the altered protein. For example, lack of TK1 causes a wide range of anomalies that



CHAPTER 3 iPSCs for modeling influenza infection

FIGURE 3.3 Possible connections between IAV-induced dysregulations in the proteome of hiPSCs and differentiation into three germ layers and their derivatives. IAV was found to cause both upregulation and downregulation of specific proteins in hiPSCs that play crucial roles in dominating pluripotency and differentiation. Based on previously reported knockout models, activation and inhibition networks are developed here for each molecule to predict the impact of virus-mediated protein expression patterns on pluripotency and germ layer formation. The germ layer- and pluripotencyspecific proteins are shown in the left panel, some of which can influence other layers without being dominantly expressed in them. The right panel represents proteins that are initially expressed in more than one target tissue but might mediate both inhibition and activation effects toward various germ layers.

Differentiation potentials of IAV-infected iPSCs

can originate from mesoderm derivatives, but Dobrovolsky et al. demonstrated that Tk(/) knockout mice develop defects in digestive and respiratory systems, which rise from endoderm, a layer that does not express this protein (Dobrovolsky et al., 2003). Such multifunctionalities could also be observed among other proteins that are simultaneously detectable in two germ layers or under the pluripotency state and in one germ layer (Fig. 3.3, right panel). For example, endodermal cells do not initially express HAT1, STAT3, ERBB3, INSR, METAP1, CSK, and SERPINF1, but their absence or insufficiencies during embryogenesis lead to abnormalities that involve endoderm derivatives, most notably including but not limited to abnormal lung development, abnormal pancreas morphology, abnormal intestinal morphology and polyps, respiratory failure, and enlarged liver (Okamoto et al., 2004; Lee et al., 2009; Riethmacher et al., 1997; Erickson et al., 1997; Doll et al., 2003). This tissue expression-independent pattern of protein function not only might highlight these molecules as potential primary inducers or indirect regulators of differentiation across various germ layers but also reflect the range of negative effects that IAV might cause on proteins controlling the formation of these layers and their cellular subpopulations. Although the selective proteomic screening of IAV-infected hiPSC was limited to measuring the expression of w1300 proteins, complementary functional analyses predict that proteins functioning in the ectoderm and mesoderm are the most affected by virus-induced changes, whereas proteins expressed in the endoderm are less influenced by IAV for either differentiation activation or inhibition. Considering in vivo evidence on defects that emerge from lack of these ectodermal and mesodermal genes, their protein downregulation by IAV under the pluripotency state might suggest the capability of this virus for targeting differentiation into mesoderm and ectoderm layers. Such a possibility could strengthen the potential link between maternal IAV infection and developmental malformations such as cardiac and neural tube defects, which might originate from embryonic abnormalities in mesoderm and ectoderm derivatives. How persistent and stable are IAV-induced alterations in the proteome of PSCs along their differentiation process, and could this virus significantly maintain any dysregulated protein activation or inhibition functions in germ layers? Another important question is whether such an affected pluripotency proteome could cause abnormal or premature differentiation in PSCs. SOMAScan proteomic screening indicates that this virus could not only decrease the expression of proteins governing the pluripotency network, but also could activate a few key proteins with developmental roles that are not normally expressed in undifferentiated PSCs, including FGF1, NOTCH1, TF, and MRC2 (Fig. 3.3). This supports the hypothesis that IAV, if not directly responsible for abnormal differentiation or cell fate commitment in PSCs, could reduce the extent of differentiation capacity in these cells via targeting pluripotency. Furthermore, elevated expression of CXCL11, IL19, IL-29, and CCL5 in hiPSCs after IAV infection might also suggest the loss of pluripotency and altered cell state, as PSCs do not secrete such cytokines following various viral or bacterial infections, probably because of mutual inhibition between the interferon system and the pluripotent state, which involves RNA interference mechanism for



CHAPTER 3 iPSCs for modeling influenza infection

reinforcing underdeveloped innate immunity in these cells (Zahedi-Amiri et al., 2019; Guo, 2017). Lack of sufficient expression profiles hinders determining the effects IAV has on the function of many other specific markers as well as molecules that play roles in the formation and differentiation of germ layers and their derivatives. iPSCs, which have been shown to be as promising as ESCs in differentiation potentials, could be considered a practical resource for testing the impact of IAV on differentiation and formation of these three germ layers via embryoid bodies (EBs). Nevertheless, such a strategy requires implementation together with large-scale proteomic analysis (e.g., by mass spectrometry) to measure all IAV-mediated changes in the proteome during both the pluripotency state and the differentiation process toward germ layers. This approach not only provides valuable information concerning the persistence and efficiency of dysregulations that IAV triggers, but also might contribute to discovering the underlying molecular mechanisms that allow IAV to express its negative impacts on embryogenesis.

iPSC-derived tissues and organoids for modeling influenza infection While historically appropriate for original model development, it is increasingly recognized that studying various aspects of any viral infection in immortalized transformed cell lines raises doubt about whether such cells are sufficiently physiologically relevant to accurately mimic all original features in the tissue normally targeted by the virus. Such interference with normal cellular senescence involves mutations that might result in different gene expression patterns, especially in cases of mutagenized cancerous cells (Marx, 2014; Kleensang et al., 2016; Lorsch et al., 2014; Masters and ATCCSDOWASN, 2010). For example, A549 cells, which were isolated from adenocarcinoma human alveolar basal epithelial cells, are considered a well-known cancerous cell line that has been used widely for understanding IAV replication in lung tissues. A549 cells have contributed markedly to the in vitro characterization of IAV growth kinetics as well as to evaluation of infection-mediated proteomic changes. However, the relatively unique genetic profile and different phenotypical aspects of this cancer-derived cell line probably hinder the precise modeling of all molecular alterations that IAV induces in the normal host-targeted tissues (Coombs et al., 2019; Kondo et al., 2015; Kroeker et al., 2013; Kroeker et al., 2012; Pan et al., 2009). Moreover, even though primary cells overcome ambiguities about physiological relevance, challenges such as limited passage potential and difficulties in isolation might limit the study of IAV pathogenesis in these potentially more relevant cells. Alternatively, because of extensive differentiation and proliferation capacities, iPSCs could open a window of opportunity to more accurate in vitro modeling of IAV infection through generating a wide range of cell types and organoids. A few other viruses have been tested with various iPSC-derived cells. For instance,

iPSC-derived tissues and organoids for modeling influenza infection

replication levels of human cytomegalovirus were explored in neural cells differentiated from iPSCs, such as neural stem cells, neural progenitor cells, and neurons (Belzile et al., 2014; D’Aiuto et al., 2012). Likewise, iPSCs have been used for generating neural progenitor cells and sensory neurons to determine cell permissiveness and viral latency for neurotropic viruses such as herpes simplex virus and varicella zoster virus (Lee et al., 2012). Differentiation of iPSCs to these neural lineages allows feasible modeling of diseases such as viral encephalitis, in which the disease phenotype involves difficult-to-access tissues in the human brain. Viral hepatitis also affects poorly accessible cells within the liver, but has been practically modeled by using hepatitis B and C viruses against iPSC derivatives, including hepatocytes and hepatic progenitor cells (Shlomai et al., 2014; Wu et al., 2012; Schwartz et al., 2012; Carpentier et al., 2014; Liu et al., 2011). Another example was the infection of iPSC-derived cardiomyocytes with coxsackievirus that assists in studying viral myocarditis (Sharma et al., 2014). Several studies have generated patient-specific iPSC-derived T cells, monocytes, macrophages, and NK cells, to develop cellbased immunotherapy and antiviral strategies against human immunodeficiency virus (Liao et al., 2015; Ni et al., 2011, 2014; Kamata et al., 2010; Kambal et al., 2011; Ye et al., 2014; Jerebtsova et al., 2012). The patient’s iPSCs were also used for deriving pulmonary epithelial cells to assess the effect of interferon regulatory factor 7 (IRF7) mutations on the respiratory tract of individuals infected with pandemic H1N1 2009 influenza virus (Ciancanelli et al., 2015). Similarly, a few research groups have differentiated knockout and wild-type iPSCs into macrophages and myeloid cells to study the roles of other interferon response boosters such as interferon-induced transmembrane protein (IFITM3) and interferon regulatory factor 5 (IRF5) in restricting influenza infection (Wellington, 2019; Forbester et al., 2020). Nevertheless, depending on subtypes and strains, IAV tropism might not be limited to respiratory and immune cells. For example, in addition to the neuroinvasivity of IAV H5N1 and H7N1 viruses in different animal models, subtype H1N1, H3N2, and H5N1 RNA also have been detected in the human central nervous system (Morishima et al., 2002; Fujimoto et al., 1998; Pabbaraju et al., 2014; de Jong et al., 2005; Shinya et al., 2011; Jang et al., 2009; Chaves et al., 2014). Clinical studies suggest that IAV-associated complications could either directly or indirectly affect different tissues and organs (Sellers et al., 2017). The cellular mechanisms and molecular pathways aiding the extrapulmonary spread of IAV are not yet fully understood in human infections. Apart from the capability of differentiation into respiratory cell lineages as the main target for IAV, iPSCs have the potential to create almost all cell types that this virus might infect, ranging from progenitor stem cells to terminally differentiated adult tissues. Additionally, iPSC-derived organoids are another technological breakthrough in stem cell biology that allow the 3D formation of miniaturized organs for modeling infectious diseases (Fatehullah et al., 2016). The infectivity of avian influenza H7N9 and pandemic 2009 H1N1 viruses has been examined in human airway organoids that were derived from tissueresident adult stem cells and contained components of respiratory epithelium such as beating ciliated, basal, goblet, and club cells (Zhou et al., 2018). Despite the



CHAPTER 3 iPSCs for modeling influenza infection

development of iPSC-derived lung organoids that include upper airway epithelium together with surrounding smooth muscles and myofibroblasts (Dye et al., 2015), IAV still remains one of the poorly studied pathogens in respiratory system organoids. Although some aspects of IAV pathogenesis such as inflammatory responses could not be easily investigated in these systems due to lack of immune cells, recent advances in generating different types of iPSC-derived organoids could help to reveal IAV-mediated effects on both organogenesis and various adult tissues.

Concluding remarks In the present review, we discuss potential applications of iPSCs for in vitro modeling of IAV infection. iPSC-based cultures could be used not only to compare IAV-triggered cell death in various cell types with different potency but also to determine the effects of this virus on stem cell differentiation and embryogenesis, which might contribute to discovering the possible link between developmental defects and maternal IAV infection during early pregnancy. Enormous numbers and types of iPSC-derived cells, as well as of some iPSC-based organoids, provide an extensive resource to generate alternatives to many inaccessible or difficult-to-isolate human tissues. Further studies need to be carried out in combination with novel omics approaches to characterize the possible subtype- and strain-specific effects of IAV on unique features of iPSCs and derivatives. Hopefully, modeling virus-induced alterations using iPSCs will help in development of antiviral strategies against cell-specific molecular mechanisms that IAV exploits to expand its pathogenesis.

Acknowledgments This work was supported by grants MT-11630 and PAN-83159 from the Canadian Institutes of Health Research to K.M.C. The authors thank Protiti Khan for expert technical assistance. Contributions A.Z.-A. wrote the first draft, and both the authors edited the final manuscript.

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CHAPTER 3 iPSCs for modeling influenza infection

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Human induced pluripotent stem cells for modeling of herpes simplex virus 1 infections


Leonardo D’Aiuto1, Paul R. Kinchington2, 3, James McNulty4, Wenxiao Zheng1, 5, Matthew J. Demers1, Maribeth A. Wesesky1, David C. Bloom6, Vishwajit L. Nimgaonkar1 1

Department of Psychiatry, Western Psychiatric Institute and Clinic, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; 2Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; 3Department of Molecular Microbiology and Genetics, University of Pittsburgh, Pittsburgh, PA, United States; 4Department of Chemistry and Chemical Biology, McMaster University, Hamilton, ON, Canada; 5Department of Psychiatry, The Second Xiangya Hospital, Xiangya School of Medicine, Central South University, Changsha, China; 6Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, United States

Chapter outline Introduction .............................................................................................................. 70 HiPSC-based models of HSV-1 latency and reactivation in human CNS-like neurons .......................................................................................................74 HiPSCs to model the interaction of HSV-1 with neural progenitor cells (NPCs)...80 Use of hiPSCs for antiherpetic drug screening.................................................80 Concluding remarks .................................................................................................. 84 Future directions....................................................................................................... 86 References ............................................................................................................... 87 Abstract Herpes simplex virus, type 1 (HSV-1) infection is highly prevalent and globally affects approximately 67% of people under age 50. It causes substantial morbidity, including recurrent ocular disease, cold sores, and rare cases of devastating encephalitis. Its pathogenicity stems primarily from the ability of the virus to cause recurrent lytic lesions, a consequence of its ability to persist for life as a latent infection in neurons. Latency is an elusive, poorly understood, and to date, untreatable process that involves a dynamic interplay between the virus and host cells. While considerable knowledge of the HSV-1 latent state has been gleaned from small animal modeling, we are now at the cusp of needing to examine HSV-1 in more human relevant models, as HSV-1 is a human-specific virus, and the iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00012-2 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 4 Human induced pluripotent stem cells

underlying mechanisms of latency have presumably evolved over millennia through human hostevirus interactions. Neuron-virus studies have been substantially hampered by the absence of satisfactory human models. Human induced pluripotent stem cell (hiPSC) and embryonic stem cell (hESC) technologies are profoundly changing this picture, as they can provide human neuron models to examine the interaction of pathogens with their hosts. They are also aiding in the development of novel therapeutic approaches. Herein, we describe the use of hiPSCs to derive human models of HSV-1 acute and latent infections and to generate human neuronal platforms for antiviral drug screening. Keywords: Acyclovir resistance; Antiviral drug screening; Encephalitis; Herpes simplex virus 1 (HSV-1); HSV-1 latency; HSV-1 reactivation; Human embryonic stem cells (hESCs); Human induced pluripotent stem cells (hiPSCs); Neural progenitor cells; Neurons; Three-dimensional (3D) neuronal cultures; Trans-dihydrolycoricidine; Two-dimensional (2D) neuronal cultures; Varicella zoster virus (VZV).

Introduction Members of the Herpesviridae family are enveloped, double-stranded DNA viruses that can have a range of clinical manifestations in humans, ranging from entirely asymptomatic to causing clinical diseases with severe to fatal complications. The first report of herpes infections goes back to classical Greece, where the term “herpein” was coined by Hippocrates as a description of skin lesions that seemed to “creep” on the skin in the genital region. The presence of painful lesions around the mouth was initially described by the Roman physician Aulus Cornelius Celsus. This medical condition was later called “herpes labialis” by the physician Herodotus (Newton, 2018). Hundreds of Herpesviruses have been identified in multiple mammalian, avian, reptilian, and fish species, but there are at least nine herpesviruses that are known to infect humans. By electron microscopy, all herpesviruses have a unique and characteristic structural morphology that is comprised of four components. The central core contains a large double-stranded linear DNA genome (ranging from 120 to 250 kb) encapsulated by an icosahedral-shaped nucleocapsid composed of 162 capsomeres. This is surrounded by the distinguishing feature known as the tegument, a proteinaceous and rather amorphous structure that contains numerous proteins that function to facilitate entry, intracellular capsid transport, the start of viral gene expression, the counteracting of innate host responses, and the adaption of the host cell to infection. The outer host-cell-derived lipid membrane envelope contains numerous virus-encoded glycoproteins that mediate virus egress from the producer cells and attachment/entry to new host cells (Kelly et al., 2009). Herpesviruses are divided into one of three subfamilies, mainly based on the gene arrangements on the genome, but also on the host range, duration of the productive cycle, and the site of latency (IARC Working Group, 1997). Members of Alphaherpesvirinae (a) generally exhibit a wide host range and a short replication cycle, with many establishing latency primarily, but not exclusively, in sensory ganglia. Human


alphaherpesviruses include the closely related herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and the more distant varicella zoster virus (VZV). All use neuronal cells to establish persistence. Betaherpesvirinae (b) members are characterized by a longer replication cycle, a more restricted host range, and the ability to establish latency in hematopoietic and progenitor cells committed to the myeloid lineage (Collins-McMillen and Goodrum, 2017). This subfamily includes the human Cytomegalovirus and the human herpesviruses 6A, 6B and 7 (HHV-6A, HHV-6B, HHV-7). Gammaherpesvirinae (g) include the Rhadinovirus human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus; and the Lymphocryptovirus human herpesvirus 4 (HHV-4), otherwise known as Epsteine Barr Virus. These have a highly restricted host range, a long productive cycle, and the ability to establish latency in lymphoid organs and cells of B lineage. While alphaherpesviruses establish latency in nondividing cells, beta and gammaherpesviruses establish latency in proliferating cells and thus have means to replicate the genome during latency (Lagunoff, 2016). Recent work has suggested the potential of human alphaherpesviruses to persist in cells of nonneuronal origin though this remains controversial (Cohen et al., 2020). The majority of individuals worldwide become infected with several human herpesviruses (Sehrawat et al., 2018; Virgin et al., 2009) in the course of their lifetimes and may be considered as part of the human “virome.” The most extensively studied herpesvirus is HSV-1, which most often causes epithelial skin or mucosal infections in humans, with an estimated 3.6 billion people infected worldwide (Looker et al., 2015; Shrivastava et al., 2019). HSV-1 also infects the cornea and is the main cause of infectious blindness in westernized societies (Kaye et al., 2000). HSV-1 can also infect the central nervous system (CNS), which may result in sporadic encephalitis (Venkatesan and Murphy, 2018), a potentially devastating disease that can often result in cognitive impairment and/or death (Young et al., 2010). There are growing concerns about perinatal HSV-1 CNS infections (Young et al., 2010). All herpesviruses are characterized by the ability to cause two modes of infections, lytic and latent (Evans et al., 2013). Species-specific latent infections seem to represent the strategy by which more than 100 herpesviruses diversified from a common ancestor (Umene and Sakaoka, 1999). In lytic infections, most virus genes are expressed into proteins to result in production of progeny virus. In contrast, latent infections involve little viral gene expression, but allow the virus to persist in the host indefinitely. This can then reactivate to renew virus production, leading to recurrent disease or asymptomatic shedding. For HSV-1, the lytic infection in mucosal and epithelial tissues results in infection of proximal neuronal axons infiltrating the site of replication (Gilden et al., 2007), using specific axon receptors that include the herpesvirus entry mediator (HVEM). The capsid becomes partially detegumented and then retrogradely transported to the neuronal nuclei in sensory ganglia, where it undergoes either further lytic spread or entry into the latent state. The lytic/latent decision process is not entirely clear, but they are influenced by the delivery of the incoming virus tegument proteins to the neuronal nucleus, since a lack of efficient delivery correlates with a preferred onset of latency (Hafezi et al., 2012).



CHAPTER 4 Human induced pluripotent stem cells

Unlike the replicating HSV genome, the latent HSV-1 genome is retained in the nuclei as a heterochromatically silenced episome with no free DNA ends. Viral chromatin is predominantly repressive to drive the downregulation of most viral lytic gene expression, with the exception of latency-associated transcripts (LATs) and their splicing products (Stevens et al., 1987; Spivack and Fraser, 1987). LATs run opposite and partially antisense to the RNA of a gene encoding a critical viral regulatory protein known as ICP0 that is predominantly prolytic in its activity (Stevens et al., 1987). Similar transcripts are found for several of the alphaherpesviruses, including VZV (Depledge et al., 2018). It is now recognized that the HSV-1 latent state is not entirely antigenically silent, since there is rare sporadic expression of some HSV-1 lytic genes at low frequency that drives the activation of virus-specific CD8þ T cells that reside in the ganglia after latency has been established (Ma et al., 2014). During latency, HSV-1 affects the host neuronal transcriptional circuits by upregulating several host genes with antiviral activity (Ma et al., 2014). These observations suggest that the suppression of productive infection during latency may be the consequence of an inhibitory host response opposing HSV-1 lytic infection and reactivation, rather than a totally inactive viral state. Studies in animal models have shown that during latency, the HSV-1 genome is highly enriched in heterochromatin repressive marks H3K9me3 and H3K27me3 (Cliffe et al., 2009; Kwiatkowski et al., 2009). In contrast, the viral region encoding the LAT is enriched in epigenetic marks typical of transcriptionally active regions, such as H3K4me2, H3K9ac, and H3K14ac (Bloom et al., 2010). There is substantial evidence that this region is insulated by CTCF marks in order to prevent transcriptional silencing (Bloom et al., 2010), but heterochromatin silencing is more dynamic than the preconceived total level of gene repression that was once thought to occur. HSV-1 latent infections cannot, as yet, be effectively targeted for efficient removal to prevent reactivation and recurrent disease. Latency has played a pivotal role in the evolution of herpesviruses. In some individuals, the latent genome can reactivate in response to various different stimuli, including stress, illness, UV exposure, hormonal changes, or trauma (Wilson and Mohr, 2012). A unified pathway for the different stimuli is not well resolved, but it is likely that all would result in reorganizational changes of the host and viral chromatin machinery that then may allow expression of virus proteins. Given that some IE-expressed proteins counteract antiviral host responses, it is thought that their expression levels are critical to lytic/latent decisions (Wilson and Mohr, 2012). One particularly attractive model (Kim et al., 2012) has proposed that dynamic chromatin organizations can trigger the unregulated expression of many genes outside of the typical regulatory cascade seen in lytic infections, in a process known as “animation.” This may re-enter the latent stage (possibly as a result of triggered innate and adaptive immune responses acting to target the gene repression release) or may progress to a regulated expression of the full genome resulting in virus production and assembly. Intriguingly, viral progeny generated upon reactivation may not be fully assembled until it is anterogradely transported to the axons near the periphery. There, final assembly takes place to release virus at the periphery and cause recurrent lesions and disease (Enquist et al., 1998).


Virus can also be transported to the CNS (Doll et al., 2019) via tentorial nerves that innervate the meninges of the anterior and middle cranial fossa (Gilden et al., 2007). Alternative routes, such as ocular, nasal, and hematogenous spread, can be utilized by HSV-1 to reach the CNS (Margolis et al., 1989; Duarte et al., 2019). A possible complication of HSV-1 migration to CNS is the development of encephalitis (HSE), a brain parenchyma inflammation, which causes abnormalities in the temporal and frontal lobe structures and is associated with severe neurologic dysfunction (Skelly et al., 2012; Singh et al., 2016). What is the fate of HSV-1 when it gains access to human CNS, and what determines if it does or does not cause encephalitis? Postmortem studies provided evidence to suggest the presence of HSV-1 genomes in brain tissues of seropositive individuals without any history of encephalitis or neurologic disease (Olsson et al., 2016), suggesting that HSV-1 may even establish genomes in the neuron of the CNS. This hypothesis, in turn, poses the question as to whether HSV-1 has the ability to reactivate only from neurons of peripheral nervous system (PNS), or if this ability is extended likewise to CNS neurons. In support of this hypothesis, there are data suggesting that HSV-1 can be experimentally reactivated from latently infected murine and shrew brainstems (Li et al., 2016, Sekizawa and Openshaw, 1984, Yao et al., 2014), though there are also data suggesting that in vivo this may more frequently involve reactivation from trigeminal ganglia (Doll et al., 2019). These possibilities are discussed in more detail later in this chapter. While the medical community considers viral pathogens as vectors of opportunistic parasitic disease, the ability of HSV-1 in its latent stage, to initiate an immune response, may contribute to host defense. In this sense, HSV-1 may cause acute viral infection in the lytic phase, while the chronic latent infection may have evolved as a commensalism in the human microbiome. Nonetheless, the presence of such an unpredictable guest in the CNS is unappealing, given the capricious nature of viral reactivation and the host pathologies associated with active lytic infection. While animal models have been instrumental to understanding the viral life cycles and to elucidate the mechanisms underlying their pathogenesis in the CNS, it is now recognized that animal models do not mimic all the events occurring in the natural host. This is driving the need to complement studies from animal models with human-specific systems. Indeed, directed differentiation technologies of human induced pluripotent stem cells (hiPSCs) and embryonic stem cell (hESCs) are revolutionizing the abilities to study human-specific hostepathogen interactions in disease-relevant terminally differentiated cell types. Furthermore, recent advances in stem cell differentiation protocols are allowing the study of pathogenic mechanisms in physiologically relevant three-dimensional (3D) culture systems that more closely mimic architectural features of differentiating organs. Here we review the use of hiPSCs to (i) generate human in vitro models of HSV-1 acute and latent infections of CNS cells, and (ii) to identify novel antiherpetic drugs that might be effective to treat HSV infections, more specifically to HSV CNS diseases such as encephalitis.



CHAPTER 4 Human induced pluripotent stem cells

HiPSC-based models of HSV-1 latency and reactivation in human CNS-like neurons The target of CNS neuron studies is herpes simplex encephalitis (HSE), which represents one of the gravest complications caused by HSV-1. HSE symptomatology includes fever, confusion, coma, and altered mental status. HSE patients often manifest with bizarre behavior, personality changes, anosmia, and gustatory hallucinations due to typical involvement of the frontal and temporal lobes. Accompanying inflammation in the orbitofrontal and temporal lobes is characteristic of HSE, and progressive temporal lobe edema can lead to hemorrhagic necrosis of the temporal lobe and possible death. Korsakoff’s amnesia has also been described in HSE survivors (Hunkin and Parkin, 1993). HiPSC-based in vitro models have provided evidence that TLR3 pathway plays an important intrinsic protective anti-HSV-1 immunity in the CNS (Lafaille et al., 2012; Zimmer et al., 2018). An important role in the controlling immunity of CNS neurons to HSV-1 is also played by SNORA31, which encodes an H/ACA small nucleolar RNA with largely unknown function (Lafaille et al., 2019). HSE can be caused by primary infection but is most commonly associated with viral reactivation. HSV-1 can gain the access to the CNS by retrograde transport through the trigeminal or olfactory nerves after viral reactivation in the trigeminal ganglia after an initial ocular infection or may reach the CNS by hematogenous dissemination route (Margolis et al., 1989; Shukla et al., 2012; Mori et al., 2005; Burgos et al., 2005). The question of whether HSE may result from viral reactivation from latency established in the CNS remains an area of some debate. For long time, a widely accepted notion was that the CNS cannot be a source of HSV-1 latency, but the difficulty in detecting viral reactivation from the CNS complicates this issue. Evidence from animal models indicates that the CNS can be a reservoir of HSV-1 latency (Chen et al., 2006; Drummond et al., 1994). In a mouse model of HSE, latency-associated transcript (LAT) could be detected in the brain stem, olfactory bulbs, cerebrum, and cerebellum on postinoculation day 42, indicating that HSV-1 can establish latency in the CNS (Drummond et al., 1994). Furthermore, Chen et al. (2006) used a new methodological approach (“dissociation method” instead of the classical “mincing method”) to show that HSV-1 could be efficiently recovered from the brainstem, olfactory bulb, and frontal cortex, providing evidence that HSV-1 can establish latency in the CNS (Chen et al., 2006). To further investigate whether HSV-1 latency can be established in human CNS neurons, a human model of HSV-1 latency and reactivation was generated using hiPSC-derived functional neurons exhibiting features of dorsolateral prefrontal cortex pyramidal neurons (D’Aiuto et al., 2014). These neurons were generated from neural progenitor cells (NPCs) differentiated from hiPSCs that express the NPC markers nestin, SOX1, PAX6, and musashi. Subsequent neuronal differentiation from NPCs was derived by culture in neurobasal medium supplemented with 2% B27, brain-derived neurotrophic factor (BDNF) at 10 ng/mL, CHIR99021 3 mM, forskolin 10 mM, dorsomorphin 1 mM, GibcoTM Antibiotic/Antimycotic 1x. After


4 days, CHIR99021, forskolin, and dorsomorphin were withdrawn. After 6 weeks of differentiation in these culture conditions (culture medium was changed every other day), the majority of cells showed morphological features of neurons and generated membrane currents, which were detected in response to glutamate, a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA) (D’Aiuto et al., 2014; Dimitrion et al., 2017). The neuronal cells stained for TUJ1 (neuron-specific class III beta-tubulin), MAP2 (microtubule-associated protein 2; a marker for dendrites), VGLUT1 (vesicular glutamate transporter 1), and NR1 (subunit of the NMDA receptor) (D’Aiuto et al., 2014). The expression of GAD1 (glutamic acid decarboxylase 1) was detected in a small fraction of neurons. These hiPSC-derived neurons possess features of cortical neurons from layer 3 of the dorsolateral prefrontal cortex (DLPFC) (Hayes and Lewis, 1992, Arion et al., 2007) in that subpopulations of neurons stained for Calbindin and CART (Cocaineand amphetamine-regulated transcript) (whose expression is higher in layer 3 of pyramidal cells in the DLPFC compared with layer 5 pyramidal cells). A low staining level staining for FEZ2 and HS3ST2 (both exhibiting higher expression level in layer 5 pyramidal cells vs. layer 3 cells) was detected. Using mass spectroscopy, we also observed robust expression of pre- and postsynaptic proteins such as SYN1, SYN2, SYNJ1, and VAMP2 and ligand-gated channels. Approximately 30% of the cells stained for glial fibrillary acidic protein (GFAP) and show features of radial glial cells (Dimitrion et al., 2017); cells with features of astrocytes were infrequent. HSV infections were monitored using a genetically engineered HSV-1 based on the KOS strain that expressed enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP) as reporter genes for two different lytic promoters. These are the immediate early promoters driving the ICP0 E3 ubiquitin ligase regulatory protein and the true late glycoprotein C (gC), respectively (Ramachandran et al., 2008). EGFP expression indicates the initiation of lytic cycles, while mRFP expression indicates commitment to viral DNA replication, since true late regulated genes such as that for gC require the onset of DNA replication for the initiation of transcription. Importantly the absence of these fluorescent protein genes in cells containing viral DNA is taken to indicate latent state. We first developed an HSV-1 latency model in hiPSC-derived 2D CNS-like neurons (D’Aiuto et al., 2015), by adapting a protocol developed previously to establish latent infections of human sensory neurons isolated from aborted fetuses (Wigdahl et al., 1984) (Fig. 4.1). HiPSC-derived neurons were infected with purified HSV-1 at low multiplicities of infection (MOI of 0.3) in the presence of the antivirals (E)-5-(2bromovinyl)-20 -deoxyuridine (5BVdU, 30 mM) and interferon-a (IFN-a, 125 U/mL) for 7 days to block any sporadic lytic infections that might arise (Fig. 4.2), which is a typical result in most HSV infections of neurons. The two antivirals thus result in latent events being favored. Cells infected at the same MOI in the absence of 5BVdU þ IFN-a (acute infection) routinely result in a lytic infection that then takes over and causes cytopathic effect in the entire culture. In antiviral-induced latency, features of HSV-1 latency were routinely observed, including the downregulation of HSV-1 lytic genes, the suppression of virus production, and a drastic reduction of



CHAPTER 4 Human induced pluripotent stem cells

FIGURE 4.1 Establishment of HSV-1 acute and latent infections in hiPSC-derived cortical and reactivation from latency.


viral DNA copy number when compared directly to unblocked lytic infections (D’Aiuto et al., 2015, 2019). A chromatin immunoprecipitation assay to evaluate histone methylation status of H3K4 and H3K27 at the ICP0, ICP4, gC, and LAT promoters revealed a 50- to 100-fold increase in the repressive marker H3K27me3 and a corresponding loss of the H3K4me3 permissive marker at these loci. A significant enrichment of the polycomb group protein Bmi1 at the same HSV-1 promoter regions was found in this latency model. The enrichment of H3K27me3 and Bmi1 and the reduction of H3K4me3 indicated that HSV-1 genome acquired heterochromatic features that were similar to those described in animal model systems, particularly in the murine model (Kwiatkowski et al., 2009; Bloom et al., 2010). Removal of 5BVdU þ IFN-a for 5 days did not routinely lead to spontaneous HSV-1 reactivation, indicating that most of the neurons in these cultures harbored stable latent infections as seen in murine and rat ex vivo primary neuron culture systems (D’Aiuto et al., 2015, 2019) (Fig. 4.1). HSV-1 reactivation after initial 7-day treatment with 5BVdU þ IFN-a was, however, consistently observed when neurons where subsequently cultured 5 days with the chromatin modifier and type 1 histone deacetylase inhibitor, sodium butyrate (NaB, 5 mM) (Fig. 4.1). Vero plaque assay showed the renewed production of infectious virus, indicating that the model HSV-1 latent state in hiPSC neurons is reversible. HSV-1 chromatin in latently infected neuronal cultures was significantly less accessible to micrococcal nuclease when compared to acutely infected cultures, providing additional evidence that HSV-1 genome is organized into heterochromatin in this model. Taken together, these results indicated that HSV-1 can establish latency in human CNS neurons in vitro. This supported similar evidence obtained from human neuronal latency platforms-based hESC (Pourchet et al., 2017) and human mesencephalic (LUHMES) cell line (Edwards and Bloom, 2019). An important issue of this hiPSC-based 2D monolayer latency model is the contrast of efficient viral reactivation in latently infected cultures exposed to NaB, in comparison with the collective results seen from in vivo mouse models. The latter generally show a lack of efficacious reactivation of HSV-1 from the CNS and indeed from peripheral ganglia as well (Li et al., 2016; Rock and Fraser, 1983). Importantly, it has been suggested that the inefficient reactivation of HSV from murine and rodent systems is the consequence of genetic differences in the human host factors interacting with viral transactivators of the IE genes (Kim et al., 2012). On the basis of these


(A) Schematic representation of culture treatment paradigm (Newton, 2018). acute infection: neurons are infected with HSV-1 (KOS) for 24 h (Kelly et al., 2009); latent infection: neuronal cultures are infected with HSV-1 in the presence of 5BVdU and IFN-a for 7 days (IARC Working Group, 1997); reactivation from latency: antivirals 5BVdU and IFN-a are removed from latently infected cultures and viral reactivation is triggered by treatment with sodium butyrate (NaB) for 5 days. Infected cells are indicated in green. (B) Flow cytometry analysis of the percentage of EGFP-positive cells in uninfected, HSV-1 acutely and latently infected neuronal cultures and after viral reactivation.



CHAPTER 4 Human induced pluripotent stem cells

FIGURE 4.2 Flow chart illustrating acute infection of brain organoids. Insets depict micrographs of formalin-fixed, paraffin-embedded section coimmunostained with Tuj1 and nestin (top) and ICP4 and MAP2 (bottom). Nuclei were counterstained with Hoechst 33342.

results, we hypothesized that a more physiologically relevant culture system was needed to model viral reactivation. We therefore developed processes to model human brain organoids, which represent 3D culture systems that recapitulate architectural features of a developing brain. These were generated from hiPSC-derived NPCs seeded at a density of 3  105 cells/well in Millicell 96-well cell culture insert plates (Millipore) and cultured in neurobasal medium supplemented with 2% B27, BDNF at 10 ng/mL, CHIR99021 at 3 mM, forskolin at 10 mM, dorsomorphin at 1 mM, GibcoÔ Antibiotic/Antimycotic 1X. After 4 days, CHIR99021, forskolin, and dorsomorphin were withdrawn and the cells were cultured for additional 4 weeks. During the process of differentiation under these conditions, NPCs selfassembled into round, quasispheroidal structures. These structures were manually detached and transferred singularly into low-attachment 24-well plates (where they transformed into spheroidal structures in a few hours) and cultured for up to 20 months. The diameter of these structures was similar in all the culture wells


(w1.3 mm). Immunohistochemistry (IHC) analysis revealed an elaborate, 3D organized cytoarchitectural arrangement of NPCs and neurons. Importantly, the distribution of the different cell types in the outer layer and central region of the organoids showed similarity to the laminar organization of the CNS. Cells in the outer region (50e150 mm from the edge range) of the organoid expressed markers typical of brain ventricular and subventricular zones, including nestin, vimentin, and the homeodomain transcription factor Cux2. The neuronal markers Tuj1, MAP2, or Tau were localized to cells located in more central regions of the organoid and expressed markers found in neocortical and hippocampal neurons, including Calbindin, Ctip2 (a transcription factor expressed in the cortical layer 5 and in granule cells in the dentate gyrus). A robust differentiation of NPCs in neurons was evidenced by expressing the glutamatergic marker VGlut1, and a fraction of neuronal cells expressed the dopaminergic marker tyrosine hydroxylase (TH). We first asked if the organoids could be efficiently infected by HSV-1. IHC analysis of HSV-1 KOS-infected organoids (1500 pfu/organoid) showed cells expressing the HSV-1 immediate early gene ICP4 throughout the organoids at 48 h postinfection, indicating that they could be infected by HSV-1 and likely produced progeny virus (Fig. 4.2). Brain organoids infected with the aforementioned recombinant HSV-1 strain KOS carrying EGFP and RFP reporter genes (1500 pfu/organoid) expressed the fluorescent reporter genes by 48 h postinfection (hpi), with fluorescence expanded to the entire organoid. Conversely, in the presence of the antivirals 5BVdU þ IFN-a, no fluorescent cells were observed in most infected organoids (86%) after 11 days. However, genomes were present and detectable using PCR. When these organoids were subsequently cultured in the absence of 5BVdU þ IFN-a to evaluate spontaneous reactivation or were exposed to sodium butyrate (NaB, 5 mM), trichostatin A (TSA, 1 mM) or the phosphatidylinositol 3-kinase inhibitor LY294002 (PI3Ki, 20 mM) to induce viral reactivation, no fluorescent cells were detected after 22 days of treatment. In agreement with an indicated lack of reactivation, no such treated organoids were positive for expression of the mRNA of the HSV-1 IE gene ICP4. The continued presence of viral DNA in subsequently prepared paraffin sections was confirmed by quantitative PCR (D’Aiuto et al., 2019). Collectively, these results suggest that organoids can host latent genomes, but that they appear to be more difficult to stimulate in reactivating HSV-1 to the same stimuli that reactivate HSV-1 from peripheral neurons. This is in marked contrast to the high-frequency reactivation observed in latently infected 2D cultures exposed to NaB. We speculate that the inefficiency of HSV-1 reactivation in the CNS model may be the consequence of a need for a different set of stimuli to drive reactivation. We did observe spontaneous HSV-1 reactivation in a small fraction (17%) of the organoids infected with the antivirals 5BVdU þ IFN-a, starting from day 8 postinfection. IHC analysis of the neuronal marker MAP2 and the viral protein ICP4 on formalin-fixed paraffin sections of these organoids showed that viral reactivation was accompanied by (i) abnormal subcellular distribution of MAP2; (ii) loss of neuronal processes; (iii) cellecell fusion causing the formation of neuronal syncytia. These data suggest that HSV-1 induces CNS cellecell fusion (D’Aiuto et al., 2019).



CHAPTER 4 Human induced pluripotent stem cells

HiPSCs to model the interaction of HSV-1 with neural progenitor cells (NPCs) A key question remaining was if neurons are the only CNS cell types where HSV-1 can establish latency? We were particularly aware that HSV-1 induces damage in CNS regions associated with memory formation, which include the hippocampus and associated limbic structures (Beers et al., 1995; Ando et al., 2008; Jonker et al., 2014). Adult neurogenesis occurs in the subgranular zone (SGZ) of the hippocampus and the subventricular zone (SVZ) lining the walls of the lateral ventricles (Taupin and Gage, 2002). The new neurons generated from the neural stem/progenitor cells residing in these regions contribute to learning and memory (Gonc¸alves et al., 2016). The hippocampus is known to be highly vulnerable to HSV-1 infection (D’Aiuto et al., 2017). Our study, along with those of others, has shown that NPCs are susceptible to HSV-1 in vitro (D’Aiuto et al., 2015; D’Aiuto et al., 2017; Menendez et al., 2016). To investigate whether HSV-1 latency can be established in cells that represent early stages of neuronal differentiation, 2D and 3D models of HSV1 infection of hiPSC-derived NPCs were developed and generated (Zheng et al., 2020). These models revealed that HSV-1 establishes a persistent infection in NPCs, that is, like that seen in the organoids, relatively refractory to reactivation. On the basis of these results, it can be hypothesized that the ability of HSV-1 to reactivate from latency under the given stimuli normally used to induce reactivation from peripheral neuron models is acquired during differentiation of NPCs into the respective classes of neuron subtypes.

Use of hiPSCs for antiherpetic drug screening A huge goal of the herpesvirus field is to obtain a vaccine that can prevent the initial infection or reactivation from latency. Despite vigorous efforts, an effective vaccine for HSV-1 has yet to be FDA licensed. This is, on reflection, not entirely surprising given the fact that many experience reactivated HSV-1 diseases, often quite severe, even though they have a fully formed adaptive immune response that developed upon the primary infection. It is well recognized that HSV-1 has a host of immune evasion strategies that allow it to initiate productive infections repeatedly in the presence of adaptive immunity (Chew et al., 2009). A second goal of the herpesvirus field is to obtain an antiviral that may target the latent state. This too has been unobtainable, and the best we have done to date is to develop safe antivirals that can reduce lytic replication. In particular, nucleoside analogs have been well developed to target processes involved in lytic infections. The most effective are based on purine nucleoside analogs Acyclovir (ACV) and Penciclovir or their respective esterified and more orally bioavailable prodrugs Valacyclovir (VAL) and Famciclovir. These agents are selective and only active in HSV-infected cells. They are activated only in virus-infected cells when the viral thymidine kinase (TK) is expressed, which initiates the first phosphorylation of the drugs to the monophosphate form. The downstream triphosphorylated nucleotide then acts by selectively blocking the activity of the viral polymerase directly or by acting as a chain terminator


upon its incorporation into replicating DNA. Advent of these drugs constituted a turning point in HSV-1 antiviral therapy and significantly reduced the mortality rate of HSV encephalitis patients (from 70% to 19%). However, a large portion of antiviral-treated encephalitis survivors still suffer from permanent neurological sequelae. The antivirals are not effective against the quiescent latent state, so there remains a huge, as yet incurable reservoir of infection. They have proven to be relatively safe and have been used in prophylactic therapies to reduce frequency of reactivation and subsequent disease (Bentley et al., 2008) although this can lead to the development of antiviral resistance (van Velzen et al., 2013). Furthermore, the rising prevalence of genital HSV-1 infections is heightening concerns for fetal infections, particularly those that can lead to CNS infections that affect neurodevelopment as a consequence of the high susceptibility of NPCs to HSV-1 (D’Aiuto et al., 2015; D’Aiuto et al., 2017; Menendez et al., 2016). Current antiviral treatment improves the chances of survival from neonatal encephalitis (decreasing the mortality to 15%) but only 50% of survivors develop normally, and total mortality rate from disseminated infection remains high (30%) (Kimberlin et al., 2001a, 2001b). Our in vitro studies indicate that there are relatively low potencies of ACV for inhibiting lytic HSV-1 infection in NPCs (Zheng et al., 2020). Hence, there is a need to identify new antiviral drugs that can efficiently abort HSV-1 infections in neuronal precursor cells before they undergo neuronal differentiation. New antiviral searches are also warranted because of the rising incidence of HSV-1 resistance to ACV derivatives, particularly in immune-compromised patients. In immunocompetent patients, prevalence of resistance is approximately 0.5%, but in the immunocompromised, ACV resistance increases to 15.7% (Frobert et al., 2014). Resistance to ACV can develop from mutations in UL23 and/or UL30 genes encoding the thymidine kinase (TK) and the catalytic subunit of viral DNA polymerase, respectively. Virus resistance in UL23 arises when TK is deleted, reduced, or changed in substrate specificity (Piret and Boivin, 2014). Mutations in UL30 have also been identified in clinical isolates, arising by single amino acid substitutions in regions responsible for nucleotide recognition, binding, and catalysis (Piret and Boivin, 2011). Viral resistance has also been documented with the nonnucleoside antivirals that act through the helicase primase (Piret and Boivin, 2014; Burrel et al., 2013; Hussin et al., 2013; Leonard et al., 2010). Foscarnet, the only approved second-line antiviral drug, is a mimic of pyrophosphate and can only be administered intravenously. It is considerably more toxic than ACV or VAL (Leonard et al., 2010; AIDS Research Group, 1992; Markham and Faulds, 1994) but is used in serious infections. Neurotoxicity is a possible side effect of ACV in patients with renal failure (Chowdhury et al., 2016; Berry and Venkatesan, 2014). As such, new anti-HSV-1 drugs remains a public health need, particularly agents that are potent, well tolerated, and have a mechanism of action different from that used by ACV. In screening for novel effective compounds, the choice of a cellular platform is critical. The use of disease-relevant cell types should represent a fundamental



CHAPTER 4 Human induced pluripotent stem cells

requirement for a drug screening campaign. Most anti-HSV drug-screening platforms employ the monkey line Vero or easy-to-grow epithelial or fibroblast cell lines (Leary et al., 2002; Matza-Porges et al., 2014; Piret et al., 2016; Ma et al., 2019), which is reasonable given that HSV-1 lytic infections primarily occur in epithelial or mucosal cells. However, considering the fact that HSV-1 infections cause profound damage to peripheral and CNS neuronal structures, we argue that CNS cell models be considered for antiviral screening. As pointed out earlier, hiPSCs have provided the unprecedented opportunity to generate 2D and 3D disease-relevant cell types in quantities compatible with high-throughput drug screening platforms. We promoted the employment of hiPSCs to identify novel antiherpetic drugs in 2015 to investigate the antiviral activity of nonnucleoside agents exhibiting lysosomotropic properties (McClain et al., 2015). This study was motivated by earlier reports indicating the efficacy of lysosomotropic drugs such as chloroquine (CQ) and bafilomycin A1 (BFLA) (Harley et al., 2001) in inhibiting HSV-1 lytic infections. These agents are thought to interfere with viral packaging and maturation, by altering the pH in the trans-Golgi network and endocytic network (Harley et al., 2001; Nieland et al., 2004). Antiviral activity and cytotoxicity were initially assessed for three compounds, 30N12, 16F19, and 4F17, using neuronal cultures generated from hiPSCs (McClain et al., 2015). We used the HSV-1 dual fluorescent reporter virus just detailed so that the percentage of cells expressing the reporter genes in the infected neuronal cultures in the presence or absence of the tested compounds can be quantified and analyzed by flow cytometry. All compounds showed antiviral potency against HSV-1 in hiPSC-derived neurons, with effective 50% inhibition concentration of 30N12, 16F19, and 4F17 at 9.27 mM, 0.42 mM, and 41.35 mM, respectively. The EC50 of ACV in the same infected hiPSC-derived neurons is 0.27 mM. As with ACV, these compounds also were able to prevent viral reactivation frequency from in vitro latently infected hiPSC-derived neurons. Intriguingly, all also exhibited high efficacy against the related human alpha herpesvirus VZV in ARPE-19 cells, and compound 4F17 could also inhibit HCMV infections in human foreskin fibroblast cells. This data suggested that these agents had potential for further development and use against multiple herpesviruses. We also addressed antiviral activity of several synthetic derivatives of alkaloid compounds originally isolated from Amaryllidaceae. Plants of the Amaryllidaceae family produce many alkaloids with known biological activities (Ding et al., 2017; Jin, 2013, 2016; Evidente et al., 2009). A subclass of these alkaloids, lycorane, had been reported to have inhibitory effects against several different viral pathogens, including HSV-1 (Ieven et al., 1982; Li et al., 2005; Hwang et al., 2008; Zou et al., 2009; McNulty and Zepeda-Vela´zquez, 2014; Gabrielsen et al., 1992; He et al., 2013; Vrijsen et al., 1986; Renard-Nozaki et al., 1989). Based on these earlier reports, we investigated the natural alkaloids pancratistatin, narcislasine, trans-dihydrolycoricidine, as well as several synthetic versions of natural alkaloids and analogs (trans-dihydrolycoricidine, 3-epi-trans-dihydrolycoricidine, 3-deoxytrans-dihydrolycoricidine, and trans-dihydronarciclasine) in the HSV-1-infected hiPSC-derived neuron platform. Three alkaloid compounds exhibited robust


antiviral activity and demonstrated superior activity over acyclovir (ACV) in neurons. Fluorescent in situ hybridization (FISH) revealed that they inhibited HSV-1 DNA replication and the accumulation of replicated genomes to suppress lytic infection. Conversely, none of the trans-dihydrolycoricidine-truncated derivatives exhibited anti-HSV-1 activity, suggesting that the C3-b-hydroxyl group, which is found only in natural products, is required for HSV-1 antiviral activity. Due to its simple structure when compared to pancratistatin or narciclasine, the synthetic version of trans-dihydrolycoricidine was extensively investigated to determine if it would exhibit antiviral effects. A deeper characterization indicated that this compound either (1) influences one or more stages of HSV-1 lytic cycles at or before DNA replication or (2) inhibits multiple stages of the viral replication process. Importantly, this compound could efficiently reduce viral reactivation frequency from latency in an hiPSC-based in vitro platform. The EC50 of trans-dihydrolycoricidine was estimated to be 0.10 mM, similar to the reported values for ACV for HSV-1 (EC50 ¼ 0.07 mM). At concentrations from 100 nM to 50 mM, trans-dihydrolycoricidine or ACV did not significantly affect the viability of the hiPSCderived neurons. Indeed, these showed a similar selective efficacy to ACV. Select derivatives also inhibited other herpesviruses; for example, trans-dihydrolycoricidine caused an approximate 220-fold reduction at 10 mM in luciferase counts per second in ARPE-19 pigment epithelial cells, which were infected with a VZVexpressed luciferase from the late ORF9 promoter. This is considerably improved over the less effective concentrations of ACV against VZV (50 mM) which only resulted in a 1.8-fold reduction. VZV is considerably more resistant to ACV compared to HSV. The EC50 of this compound for VZV was estimated to be 0.15 mM. No significant changes in cell viability were observed at tested concentrations for trans-dihydrolycoricidine and ACV (McNulty et al., 2016). A later study showed that trans-dihydrolycoricidine, also referred to as R430, effectively prevented HSV-1 reactivation in a murine model of HSV-1 latency (D’Aiuto et al., 2018a). This finding complements the results from the hiPSCbased model of latent HSV-1 infection that has the potential for development. Importantly, R430 was shown to inhibit ACV-resistant HSV-1 strains lacking thymidine kinase and also reduces growth of PAA-resistant HSV in hiPSC-derived NPCs. R430 also displayed antiviral effects against other DNA viruses (HSV-2, HCMV, MCMV, HBV) and RNA viruses (Brazilian and Cambodian Zika strains of ZIKV, HCV) (D’Aiuto et al., 2018a). To build on our drug-screening process, we wanted to assess whether concerns regarding drug activity in different cell types would be reflected in the reality of our findings. To that end we expanded the number of compounds tested at once and directly compared results in Vero cells, hiPSC-derived NPCs and hiPSCderived neurons (D’Aiuto et al., 2017). Seventy-three compounds were tested, including lysosomotropic agents, amaryllis alkaloids, quinazolinones, nostadiones, and epigenetic inhibitors. Each compound was tested at two concentrations (10 and 50 mM) and used a 50% or greater inhibition as a success criterion for each concentration. At 10 mM, only seven drugs reduced HSV-1 infection by



CHAPTER 4 Human induced pluripotent stem cells

75%e95% in neurons but were less effective in NPCs and Vero cells. At 50 mM 19 out of 73 drugs inhibited HSV-1 in neurons, and 14 of these were also effective in NPCs. Surprisingly, only 5 of these 19 were effective in Vero cells, suggesting that there is a cellular specific component to some of these antivirals. This comparative drug screening identified new compounds exhibiting anti-HSV-1 activity in neurons, such as the quinazolinones, using an hiPSC-based cellular platform that would not have been discovered using Vero cells (D’Aiuto et al., 2017). Thus, while screening in a continuous epithelial line such as Vero cells may detect some efficacious compounds, novel important lead compounds may require specific and perhaps more relevant cell types, particularly if the drugs act by targeting a host cell process that is utilized by the virus for its own replication. These results highlight the pivotal importance of a careful selection of the cell types to employ in an antiviral drug screening campaign. A main drawback of the 2D culture systems is their inability to adequately mimic a natural tissue environment. This important limitation has repercussion on the predictability of in vivo response of drugs discovered using 2D cellular platforms. In fact, there is increasing evidence about the superior predictive power of 3D cultures when compared to 2D monolayers. This, in turn, has fueled current research to develop 3D cellular platforms for drug screening. Starting from hiPSCs, we developed in 96-well plates an 3D cell platform, named A-3D, exhibiting features of a developing cortex (D’Aiuto et al., 2018b). A schematic representation of the differentiation strategy to generated the A-3D cultures is depicted in Fig. 4.3. The z-stacks of this multilayered culture system can be rapidly acquired using a laser-powered confocal instrumentation amendable to high-throughput assays (Cellinsight CX7 LZR, Thermo Fisher). The suitability of this 3D cell platform for high-throughput antiviral drug screening has been evaluated by determining the EC50 of acyclovir on cells infected with recombinant HSV-1 expressing EGFP and RFP reporters. Flow cytometry (FC) and CX7 LZR were employed to analyze the percentage of infected cells in culture wells exposed to increasing concentration of acyclovir (from 0.1 to 50 mM). The acyclovir EC50 estimated using FC and CX7 LZR was similar (3.144 and 3.121 mM, respectively), but the latter allowed to reduce the timing for culture plates analysis by fivefold (D’Aiuto et al., 2018b). These results showed the feasibility of performing a rapid and robust antiherpetic drug screening using an hiPSC-derived 3D cellular platform.

Concluding remarks HSV-1 has been one of the most heavily studied herpesviruses and has become apparent that behaviors seen in nonhuman culture often misrepresent the actions of the virus in humans. It has also become clear that, while HSV replicates quite well in many animal model systems, animal models do lack certain human specificities that are seen in humans and, in some cases, show behaviors and activities that do not represent human disease. A classic example is the human versus murine

Concluding remarks

FIGURE 4.3 Scheme of antiviral drug screening in hiPSC-derived 2D and 3D neuronal platforms. (A) HiPSC-derived NPCs (hiPSC-NPCs) are seeded in Matrigel-coated 96-well plates at the density of 2  104 cells/well and differentiated into neurons (hiPSC-neurons). 2D neuronal cultures are then infected with a modified KOS HSV-1 strain that expresses EGFP from the viral immediate early ICP0 promoter and monomeric red fluorescent protein (mRFP) from the promoter of the true late regulated Glycoprotein C (gC) at an MOI of 0.3. After 2 h, the inocula are removed, the cells are washed and exposed to drugs. The percentage of cells expressing the fluorescent reporter genes, cell viability is analyzed at 48 h postinfection using flow cytometry. (B) Adherent 3D neuronal cultures are generated



CHAPTER 4 Human induced pluripotent stem cells

cellular responses regarding necroptosis (Guo et al., 2015; Huang et al., 2015). Recent years have witnessed an expansion in the use of hiPSC-derived 2D and 3D culture systems generated from patient-specific cells for disease modeling and development of novel therapeutic approaches. However, by and large, these models are at their infancy, and there is plenty of room for development of more complex ex vivo and in vitro models for 3D modeling of virus-induced disease. Although their goal is to complement animal models of the disease, the use of hiPSC technologies in virology is still not widely accepted enough to be part of the routine methods. Part of this is a result of the difficulties setting up the stem cell differentiations platforms. Several factors contribute to the limited use of hiPSCs in virology, which include costs related to hiPSCs maintenance and their differentiation protocols in specific cell types that are relevant to human infection. However, despite these practical and economical demands, it is likely accurate to predict that hiPSC-based technologies and direct cell-conversion strategies (transdifferentiation) (Horisawa and Suzuki, 2020) will play a prominent role in the investigation of aspects of pathogenehost interaction in a human context.

Future directions There is a good deal of evidence that links neurotropic viruses to neurodegenerative diseases. For example, HSV-1 is an important risk factor for Alzheimer’s disease (AD), an age-related neurodegenerative disease affecting more than 56 million people worldwide (Alzheimer’s Disease Facts and Figures, 2020). This link was suggested by the discovery of HSV-1 remnants, including virus-specific molecules and DNA, in amyloid plaques in the brain tissue of AD cases. Furthermore, experimental studies report that HSV-1 latency/reactivation cycles are associated with increased neuronal b-amyloid and phosphorylated tau in vitro and in vivo and that these changes appear to be attenuated by antiviral treatments. These data support a role in the pathogenic virus hypothesis, which states that continuous but “restricted” episodes of HSV-1 reactivation contribute to the onset of AD pathology. Specifically, HSV-1 infection initiates and/or accelerates production of the Ab peptide form ending in amino acid 42 (Ab42), a highly fibrillogenic Ab form driving amyloid plaque formation in AD. Recent evidence that the Ab42 form has antiviral and antibacterial properties (Bourgade et al., 2016) suggests that its increased


by seeding hiPSC-derived NPCs into Matrigel-coated optically clear 96-well plates at the density of 3  105 cells/well. NPCs are then cultured in two neuronal differentiation media for 4 weeks. During this period differentiating cells form a multilayered 3D structure (whose thickness goes up to 60 mm) composed of neurons sandwiched between NPCs. To perform drug screening, 3D cultures are infected at MOI 0.3 for 2 h. After inocula removal and cell washings, cultures are exposed to compounds. After 48 h the percentage of infected cells, cell viability and other parameters are analyzed using high content screening.


production could be an initial protective response to prevent the cell-to-cell spread of the viral infection. Recent studies are showing that hiPSC-derived 3D cultures may be instrumental in investigating the role of HSV-1 in the development of AD pathology (Cairns et al., 2020; Eimer et al., 2018). The veracity of the hypothesis that herpesviruses have a mechanistic involvement in the development of AD pathology will motivate the development and use of antiherpetic drugs as prevention therapy or slow the progression of the disease, which are effective in the target tissuedthe CNS. The generation of complex hiPSC-derived 3D culture platforms representative of the different CNS cell types for drug screening will improve the predictability of antiviral drug activity in vivo. The applicability of the currently developed HSV-1/hiPSC model system to investigate the pathology of other CNS active viruses is timely and highly relevant. This could include investigations into the effect viruses such as Zika and could even extend to human corona virus in addition to herpesviruses on the development of 3D cellular architectures. Indeed a neuronal involvement of the new human coronavirus responsible for the 2019e20 pandemic is emerging (Yang et al., 2020, Aghagoli et al., 2020). Furthermore, the role of emerging viral pathogens such as SARSCoV-2 in gaining entry to neurons through ACE2 receptors (Baig et al., 2020) is ripe for investigation and is expected to permit the discovery of novel antiviral compounds in a relevant host cell. The role of coronaviruses in causing neuronal cell death requires immediate investigation, and this effect is believed to be a separate issue to that contributing to viral encephalitis (Netland et al., 2008).

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CHAPTER 4 Human induced pluripotent stem cells

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CHAPTER 4 Human induced pluripotent stem cells

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iPSCs for modeling coxsackievirus infection


Serkan Belkaya Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, C¸ankaya, Ankara, Turkey

Chapter outline Biology of coxsackieviruses ...................................................................................... 96 Coxsackievirus-associated disease in humans............................................................ 98 Experimental models for coxsackievirus infection ....................................................... 99 iPSC modeling of coxsackievirus infection ...............................................................101 Concluding remarks and future perspectives ............................................................106 References .............................................................................................................106 Abstract Coxsackieviruses are common human pathogens and can lead to life-threatening diseases, particularly in children. Coxsackievirus has been studied for over 70 years, yet there is currently no specific medication or vaccine available against its infection. Our understanding of coxsackievirus pathogenesis is extremely limited by the unavailability of experimental models that faithfully recapitulate coxsackievirus-related disease in humans. In this context, induced pluripotent stem cell (iPSC) technology provides an unparalleled tool to model natural viral infections in physiologically relevant cells and facilitate the development of novel therapies for infectious diseases. The present chapter reviews advances in the iPSC modeling of coxsackievirus infection. First, an overview of the general aspects of coxsackievirus biology including a brief history, classification, life cycle, and tissue tropism of coxsackieviruses is provided. Various coxsackievirus-associated diseases in humans are also summarized. Finally, in vitro and in vivo experimental models utilized to study coxsackievirus infection are discussed, with a particular focus on iPSCs and the utility of iPSC-derived differentiated cells as human disease models. Keywords: CAR; Cardiomyocyte,; Coxsackievirus; CVB3; CXADR; DAF; Disease modeling; Enterovirus; Hand-foot-and-mouth disease; Infectious diseases; Interferon; iPSC; Meningitis; Myocarditis; Pancreatitis; Picornavirus; Viral immunity; Viral infection.

iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00004-3 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 5 iPSCs for modeling coxsackievirus infection

Biology of coxsackieviruses The coxsackieviruses were first identified from fecal specimens of children suffering from paralysis following a poliomyelitis outbreak in the town of Coxsackie, New York in 1947 and from cases of nonparalytic poliomyelitis in 1948. These virus isolates were found to induce paralysis in newborn mice but not in adult mice or primates (Dalldorf and Sickles, 1948; Melnick et al., 1949). Based on their tissue tropism and pathogenicity in newborn mice, coxsackieviruses were empirically classified into two groups: group A (Coxsackievirus A; CVA) with 23 known different serotypes and group B (Coxsackievirus B; CVB) with six known different serotypes (Dalldorf, 1950; Gifford and Dalldorf, 1951). CVA isolates infected skeletal muscle causing acute myositis with flaccid paralysis in newborn mice, whereas CVB induced a spastic paralysis with broader pathological lesions of tissues including the brain, heart, pancreas, and skeletal muscle (Dalldorf, 1950; Gifford and Dalldorf, 1951). Coxsackieviruses represent small, nonenveloped RNA viruses that belong to the Enteroviridae genus within the Picornaviridae family. They are composed of w7.4 kb linear, single-stranded positive-sense RNA genome, which contains a large (w6.5 kb) open reading frame (ORF) flanked by untranslated regions (UTRs) at both 30 and 50 termini. The ORF is translated as a monocistronic messenger RNA into a single polyprotein composed of four structural proteins (VP1, VP2, VP3, and VP4) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) by the host cell ribosome (Rueckert, 1996; Melnick, 1996). The two viral proteases, 2A and 3C, cotranslationally cleave this viral polyprotein into several processing intermediates and mature functional proteins (Rueckert, 1996). The structural proteins, VP1eVP4, assemble to form the icosahedral virus capsid, whereas nonstructural proteins are involved in viral replication and manipulation of the host cell biology (Badorff et al., 1999; Belov et al., 2007; Chau et al., 2007; Clark et al., 1993; Doedens and Kirkegaard, 1995; Etchison et al., 1982; Hsu et al., 2010; Ilnytska et al., 2013; Joachims et al., 1999; Kerekatte et al., 1999; Krausslich et al., 1987; Lanke et al., 2009; van Kuppeveld et al., 1997; Ventoso et al., 1998; Yalamanchili et al., 1997a,b). The virally encoded RNA-dependent RNA polymerase, 3D, converts the single-stranded positive-sense viral RNA genome to negative-sense RNA intermediates and uses these intermediates as templates to synthesize more copies of single-stranded positive-sense RNA genome for translation of more viral proteins and generation of progeny viruses (Rueckert, 1996). Newly generated mature coxsackieviruses are released, usually by lysis of the infected host cell, to initiate new cycles of infection in neighboring target cells. Coxsackievirus invasion begins with viral entry into the target cell by attachment of virus to cell surface receptors and its subsequent receptor-mediated internalization. Various host-cell receptors have been reported for different serotypes of coxsackieviruses to be involved in the virus attachment and internalization processes. Some CVA serotypes including CVA2, CVA3, CVA4, CVA5, CVA6, CVA10, and

Biology of coxsackieviruses

CVA12 require kringle containing transmembrane protein 1 (KREMEN1) as host cell entry receptor (Staring et al., 2018). Human KREMEN1 is broadly detected in many tissues with the highest expression in the esophagus and skin (Fagerberg et al., 2014). CVA7, CVA14, and CVA16 depend on binding to scavenger receptor B2 (SCARB2 or CD36L2) (Yamayoshi et al., 2009). CVA16 also uses selectin P ligand (SELPLG, or CD162) and heparan sulfate chains for attachment to target cell surface (Nishimura et al., 2009; Zhang et al., 2017b). SCARB2 is ubiquitously expressed in all human tissues with higher expression in the brain, gastrointestinal tract, and lung, whereas SELPLG is mainly found in lymphoid cells, lung macrophages, neurons, and microglial cells (He et al., 2014; Jiao et al., 2014; Yu et al., 2014). CVA13, CVA15, CVA18, CVA20, CVA21, and CVA24 bind to intercellular adhesion molecule 1 (ICAM-1 or CD54) as the entry receptor (Baggen et al., 2018; Newcombe et al., 2003; Shafren et al., 1997). Human ICAM-1 is extensively expressed in the lung, particularly in alveolar and bronchial cells (Cunningham and Kirby, 1995; Milne et al., 1994). CVA24 also utilizes sialic-acid-linked surface proteins, such as SELPLG to enter the cell (Baggen et al., 2018; Mistry et al., 2011; Nilsson et al., 2008). Overall, receptor preferences for virus attachment and cellular internalization greatly vary among different CVA serotypes, and entry receptors for some CVA serotypes still remain undiscovered. All CVB serotypes can utilize the coxsackie adenovirus receptor (CAR), encoded by the CXADR gene, as the primary receptor for viral entry into the target cells (Bergelson et al., 1997a,b; Carson et al., 1997; Tomko et al., 1997). Human CXADR is broadly expressed in many tissues including the brain, heart, gastrointestinal tract, pancreas, and skin (Fagerberg et al., 2014; Tomko et al., 1997). CAR is a transmembrane protein found at tight junctions between epithelial cells and the intercalated discs between cardiomyocytes (Cohen et al., 2001; Shaw et al., 2004). CVB1, CVB3, and CVB5 also bind to a coreceptor, decay accelerating factor (DAF or CD55), for initial attachment to the cell surface. DAF is highly expressed in cardiac muscle and various epithelial tissues including corneal, conjunctival, intestinal, oral mucosal, placental, and respiratory epithelium (Fagerberg et al., 2014; Holmes et al., 1990; Medof et al., 1987; Shieh and Bergelson, 2002; Zimmermann et al., 1990), where CAR is located at the sites of cell-to-cell contacts and is therefore not readily accessible for CVB (Bergelson et al., 1994, 1995). Coupling of CVB to DAF induces the clustering of DAF and movement of CVB-DAF complex from the apical surface of polarized cells toward the tight junctions, where CVB can bind to CAR and enter the cell (Shieh and Bergelson, 2002; Bergelson et al., 1995; Coyne and Bergelson, 2006). Similarly, CVA21 can utilize DAF as a coreceptor for attachment to the host-cell surface (Shafren et al., 1997). However, a strain variant of CVB3 (CVB3-PD) infects CAR- and DAF-negative cells by using heparin sulfates for binding and internalization (Zautner et al., 2003, 2006). Overall, a wide range of surface receptors are utilized by coxsackieviruses for host cell entry, suggesting a heterogeneous cell and tissue tropism among their serotypes based on the expression patterns of entry receptors in the host organism. Nevertheless, specific receptor expression alone does not suffice to account for the susceptibility and



CHAPTER 5 iPSCs for modeling coxsackievirus infection

permissiveness to coxsackievirus infection in a tissue, as both intracellular factors and extracellular tissue milieu are involved in regulating viral entry and replication (Cheung et al., 2005; Harvala et al., 2005; Schneider-Schaulies, 2000; Wessely et al., 2001; Crowell et al., 1988).

Coxsackievirus-associated disease in humans Coxsackieviruses are transmitted through the fecaleoral route and can cause a wide spectrum of diseases in humans of all ages (Fig. 5.1) (Grist and Reid, 1988). However, neonates and children are mostly and most severely affected (Grist and Reid, 1988; Hammond, 1988; Pallansch, 1988). Coxsackievirus infection in children typically presents as either asymptomatic or a nonspecific febrile illness. Exanthems, such as hand-foot-and-mouth disease (HFMD), herpangina, and undifferentiated rash, are more common coxsackievirus-associated clinical manifestations during childhood, particularly related to CVA serotypes (Rocchi and Volpi, 1988; Modlin and Rotbart, 1997; Romero, 2008; Romero and Modlin, 2015). In rare cases, severe inflammatory diseases of the brain, heart, liver, lung, or pancreas can occur. Infection of the central nervous system (CNS) by coxsackieviruses, mostly CVB serotypes, can cause aseptic meningitis and less frequently acute flaccid myelitis and encephalitis (Modlin and Rotbart, 1997; Romero, 2008; Romero and Modlin, 2015; Bell et al., 1988). Other rare, life-threatening pediatric illnesses associated

FIGURE 5.1 Coxsackievirus-associated clinical syndromes in humans.

Experimental models for coxsackievirus infection

with coxsackieviruses are inflammatory heart diseases, such as myocarditis and pericarditis, resulting from the infection of the heart muscle, particularly with CVB3 (Knowlton, 2008; Levine et al., 2010; Reyes and Lerner, 1988; Baboonian et al., 1997). Affected children may suddenly die from heart failure or have to undergo heart transplantation. Some may develop sequelae such as chronic constrictive pericarditis following acute pericarditis and dilated cardiomyopathy upon acute myocarditis, both requiring long-term therapy for heart failure (Knowlton, 2008; Levine et al., 2010; Reyes and Lerner, 1988; Baboonian et al., 1997). Coxsackieviruses are also associated with acute hepatitis, respiratory tract syndromes, and severe pancreatitis, which may progress into chronic pancreatitis and type I diabetes mellitus, in humans (Modlin and Rotbart, 1997; Romero, 2008; Romero and Modlin, 2015; Drescher and Tracy, 2008; Toniolo et al., 1988). Moreover, coxsackieviruses, like polioviruses, can cause various muscle disorders including arthritis, myositis, pleurodynia, and rhabdomyolysis (Modlin and Rotbart, 1997; Romero, 2008; Romero and Modlin, 2015; Messacar et al., 2018; Cohen, 2015). Acute hemorrhagic conjunctivitis is another coxsackievirus-associated clinical manifestation, following infection with a pathogenic variant of CVA24 (Rocchi and Volpi, 1988; Wright et al., 1992; Ghafoor and Burney, 1987). Finally, coxsackievirus infection during pregnancy can lead to increased risk of miscarriage and stillbirth or neonatal morbidity (Grist and Reid, 1988; Axelsson et al., 1993; Frisk and Diderholm, 1992; Hwang et al., 2014; Ogilvie and Tearne, 1980; Yu et al., 2015; Goldenberg et al., 2010; Bendig et al., 2003; Cheng et al., 2006; Euscher et al., 2001). Pathogenesis of coxsackievirus-associated disease in humans still remains elusive, with not one clinically approved vaccine or antiviral medications available for the prevention or treatment of coxsackievirus infections in humans.

Experimental models for coxsackievirus infection Current knowledge of the mechanisms of human coxsackievirus-associated diseases mostly comes from in vitro cellular systems and in vivo animal models. In vitro modeling of coxsackievirus infection, which generally comprises transformed cell lines, has enabled the identification of virus entry receptors and the elucidation of the intracellular viral life cycle. Infection studies with cell lines have also provided invaluable information on how coxsackieviruses can lead to biological, biochemical, morphological, and physiological changes in infected cells, including virus-induced immune responses, modulation of host transcriptional and translational machinery, cytopathic effects, and changes in membrane permeability and cell metabolism. Moreover, nontransformed human embryonic stem cell lines were found to be susceptible and permissive to coxsackieviruses and proposed as a noncancerous cellbased in vitro model alternative to tumor-derived cell lines (Scassa et al., 2011; Romorini et al., 2012). Coxsackievirus infection was shown ex vivo utilizing human primary cells such as conjunctival epithelial cells, endothelial cells, fetal heart cells, hepatocytes, pancreatic cells, and renal cells obtained from healthy donors,



CHAPTER 5 iPSCs for modeling coxsackievirus infection

individuals underwent resection, or brain-dead individuals(Chehadeh et al., 2000; Lee et al., 2011; Lind et al., 2013, 2014; Sane et al., 2013; Roivainen et al., 2000; Kandolf et al., 1985; Conaldi et al., 1997a,b). Persistent infection of coxsackieviruses has also been described in a number of cell lines and primary cells (Roivainen et al., 2000; Conaldi et al., 1997a,b; Alidjinou et al., 2015, 2017; Desailloud et al., 2009; Harrath et al., 2004; Jaidane et al., 2012; Lietzen et al., 2019; Pinkert et al., 2011; Zhang et al., 2013; Heim et al., 1992). Yet, in vitro infection systems lack the complexity and dynamics of an organism at cellular, histological, and systemic levels. In this context, in vivo animal models have been used for better understanding of coxsackievirus-associated pathologies. Several neonatal murine models of coxsackievirus A infection, such as CVA4, CVA6, CVA10, and CVA16, have been described, mostly damaging the CNS and skeletal muscles although high viral titers were detected in many organs without inducing pathogenic effects (Yang et al., 2016; Li et al., 2017; Zhang et al., 2017a, 2019; Mao et al., 2012; Huang et al., 2015). CVA16 also induced neurological manifestations in young gerbils (Sun et al., 2016), suggesting these rodents as an alternative in vivo model for coxsackievirus infection. Interestingly, CVA16 infection in mice did not lead to any lesions in the oral mucosa or limbs, which are typical characteristics of HFMD in humans. On the other hand, rhesus macaques, which are phylogenetically much closer to humans than rodents, displayed vesicles over the hands, feet, and oral mucosa upon infection with CVA16, but, unlike rodents, did not develop any CNS lesions (Wang et al., 2017). Thus, these contradictory findings among different animal models of CVA infection raise questions about their relevance for coxsackievirus-associated diseases in humans. Coxsackievirus B infection has also been extensively studied in vivo. In newborn mice, and compared to CVA, CVB causes much broader and simultaneous pathological lesions of different tissues including the brain, heart, liver, lung, and pancreas (Dalldorf, 1950; Gifford and Dalldorf, 1951; Grodums and Dempster, 1962; Liu et al., 2013; Wang et al., 2014). A neonatal mouse model of CVB3 infection revealed that neural stem cells and myeloid cells are the primary targets at initial stages of infection (Feuer et al., 2003, 2005; Tabor-Godwin et al., 2010). Porcine models of CVB infection have also been reported, but pigs did not develop any symptoms of CNS infection unlike mice and humans (Garland and Mann, 1974; Lai et al., 1979). CVB3 and CVB4 are frequently associated with pancreatitis in humans and thought to trigger the development of type I diabetes (Drescher and Tracy, 2008; Toniolo et al., 1988; Huber and Ramsingh, 2004). Most CVB infections in mice can cause acute pancreatitis (Huber and Ramsingh, 2004; Hong et al., 2017; Moon et al., 2005); however, human diabetes-like symptoms were only observed in diabetes-induced or -susceptible mouse lines and patas monkeys (Drescher et al., 2004; Yoon et al., 1978, 1986; Benkahla et al., 2019; Wegner et al., 1985; Stone et al., 2018; Jaidane et al., 2009). CVB serotypes, in particular CVB3, which is one of the most commonly implicated viral culprits of acute myocarditis in humans, can also infect cardiomyocytes and induce myocarditis in mice and nonhuman primates (Reyes and Lerner, 1988; Cammock et al., 2013; Hoshino et al., 1983; Paque et al., 1981).

iPSC modeling of coxsackievirus infection

Several mechanisms have been proposed based on experimental murine models to explain the pathogenesis of CVB3-induced myocarditis, including (i) direct destruction of cardiomyocytes by viruses, (ii) cardiac damage by activated immune cells upon viral infection, and (iii) virus-induced autoimmunity against cardiac cells (Badorff et al., 1999; Chow et al., 1992; McManus et al., 1993; Hashimoto et al., 1983; Henke et al., 1995; Wolfgram et al., 1985; Gauntt et al., 1991, 1993; Horwitz et al., 2000; Esfandiarei and McManus, 2008). However, whether these animal model-based mechanisms underlie coxsackievirus-induced myocarditis in humans is unclear. The progression and outcome of coxsackievirus infection in experimental animal models can be dramatically affected by animal species, laboratory strains, clinical coxsackievirus isolates, and the infection route and virus dose utilized. This hampers the clinical translation of findings to humans (Loria, 1988; Minnich and Ray, 1980; Chow et al., 1991; Wolfgram et al., 1986). Nevertheless, animal studies have provided useful insights into the mechanisms of coxsackievirusinduced pathologies and nonclinical efficacy evaluation and safety assessment of vaccines and medications tested against coxsackieviruses.

iPSC modeling of coxsackievirus infection The discovery of induced pluripotent stem cells (iPSCs) reprogrammed from somatic cells, such as dermal fibroblasts, keratinocytes, circulating T cells, and renal tubular epithelial cells, and their differentiation into almost any specific cell lineage in the human body have been revolutionary for in vitro disease modeling (Aasen et al., 2008; Dambrot et al., 2013; Raab et al., 2014; Staerk et al., 2010; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Yu et al., 2007; Gaignerie et al., 2018; Loh et al., 2009). iPSC technology has enabled a simple, robust, and plentiful production of disease-relevant cells from somatic cells, generating “disease in a dish” models. This is particularly important for those diseases in which disease-related primary cells and tissues from patients are not routinely accessible or easy to obtain and/or maintain in sufficient quantities for ex vivo study. Of note, iPSC-derived differentiated cells are generally immature, exhibiting fetal-like characteristics, which makes them more suitable for modeling early-age onset diseases than lateonset diseases in humans. Various cardiovascular, hematologic, hepatic, infectious, metabolic, muscular, neurologic, pulmonary, renal, retinal, rheumatic, and vascular diseases have been modeled using disease-relevant cells and tissues derived from iPSCs of healthy individuals and patients (Rowe and Daley, 2019; Peng et al., 2019; Cho et al., 2019; Paik et al., 2020; Corbett and Duncan, 2019; Kakinuma and Watanabe, 2019; Calvert and Ryan Firth, 2020; Papapetrou, 2019; Wiegand and Banerjee, 2019; Onder and Daley, 2012; van Mil et al., 2018; Trevisan et al., 2015; Liu et al., 2018). These iPSC-based disease models are being utilized for drug screening, biomarker discovery, and gene editing for the future development of personalized cell replacement therapies.



CHAPTER 5 iPSCs for modeling coxsackievirus infection

There is a significant lack of information available about the active infection stages of viral diseases in humans. In this regard, iPSCs create an unprecedented in vitro model to better understand various aspects of a natural viral infection in humans, including the virus entry, viral replication, virus-induced cellular injury, host cell-intrinsic antiviral immunity, and the genetic basis of individual susceptibility (or resistance) to viruses. For instance, patient-specific iPSC-derived CNS and lung epithelial cells were utilized for infection with herpes simplex and influenza viruses as proof-of-principle cellular models to establish the causality between inborn defects in interferon (IFN) immunity and clinically relevant cellular phenotypes in children suffering from herpes simplex encephalitis or isolated severe influenza, respectively (Ciancanelli et al., 2015; Lafaille et al., 2012). Acute viral myocarditis is another life-threatening illness of childhood, which often follows infection with coxsackieviruses, in particular CVB3 (Knowlton, 2008; Levine et al., 2010; Reyes and Lerner, 1988; Baboonian et al., 1997). Many affected children recover spontaneously, but some develop dilated cardiomyopathy and may need long-term therapy for heart failure or undergo heart transplantation. In fulminant cases, sudden cardiac death occurs (Knowlton, 2008; Levine et al., 2010; Reyes and Lerner, 1988; Baboonian et al., 1997). Yet, the mechanisms of coxsackievirusinduced myocarditis in humans have remained obscure due to limitations of available experimental models. Human primary cardiomyocyte-based modeling has not been possible, as human cardiac tissue samples are difficult to obtain routinely, and isolated cardiomyocytes, which do not proliferate, cannot be maintained in vitro for long periods of time (Mitcheson et al., 1998). On the other hand, cancer cell lines such as noncardiac HEK293 and HeLa cells or cardiac AC16, HL-1, and H9c2 cells are proliferative, thus do not recapitulate human primary cardiomyocytes (Fechner et al., 2007; Li et al., 2014; Shah et al., 2017; Marchant et al., 2008). Lastly, animal modeling of coxsackievirus-induced myocarditis is substantially hindered by interspecies differences in cardiovascular phenotypes, such as electrophysiology, heart rate and metabolism, and immune systems between mice and humans, and by intraspecies differences in susceptibility to CVB3 among various mouse strains (Chow et al., 1991; Wolfgram et al., 1986; Casanova and Abel, 2004; Doevendans et al., 1998; Seok et al., 2013; Shay et al., 2013). The derivation of cardiomyocytes from human iPSCs has facilitated the study of natural coxsackievirus infection in a physiologically relevant cellular model of acute viral myocarditis in humans. Coxsackievirus infection in iPSC-derived cardiomyocytes was first reported in 2014 (Sharma et al., 2014). This report utilized iPSCs reprogrammed from primary dermal fibroblasts or peripheral mononuclear blood cells of different healthy individuals to generate cardiomyocytes using a two-dimensional (2D) monolayer differentiation protocol as previously established (Churko et al., 2013; Lian et al., 2012; Sun et al., 2012). Differentiated cardiomyocytes were infected with a modified CVB3 strain ectopically expressing Renilla luciferase, which enables the monitoring of viral replication by luminescence (Lanke et al., 2009). Both human iPSCs and iPSC-derived cardiomyocytes from healthy individuals or patients with dilated cardiomyopathy had detectable CAR expression on their cell surfaces. Of note, CXADR

iPSC modeling of coxsackievirus infection

mRNA levels in iPSC-derived cardiomyocytes were found to be dramatically lower (w30 times) than in left ventricular myocardial tissues obtained from adult organ donors, which can be attributed to immaturity of differentiated cardiomyocytes (Lundy et al., 2013). Evident with surface CAR expression, human iPSC-derived cardiomyocytes were susceptible and permissive to CVB3 infection, displaying virus-induced cytopathic effects, impaired beating, irregular intracellular calcium transients, and eventual cell death following virus proliferation. Pretreatment of cardiomyocytes with exogenous IFN-b1 reduced CVB3 replication in a dose-dependent manner, consistent with the protective effect of IFNs against CVB3 infection observed in human primary cardiomyocytes and myocardial fibroblasts, and contractile embryoid bodies derived from human embryonic stem cells (Scassa et al., 2011; Kandolf et al., 1985; Heim et al., 1992, 1995, 1996, 1997). Gene expression analysis in CVB3-infected iPSC-derived cardiomyocytes revealed that IFN-b1 priming of cardiomyocytes confers protection against CVB3 by activating various intracellular mechanisms involved in viral clearance. Moreover, ribavirin (an inhibitor of viral DNA synthesis (Heim et al., 1997; Crotty et al., 2000)), fluoxetine (a selective serotonin reuptake inhibitor (Zuo et al., 2012)), and pyrrolidine dithiocarbamate (an inhibitor of the ubiquitineproteasome proteolytic pathway and coxsackievirus RNA-dependent RNA polymerase, 3D (Si et al., 2005; Lanke et al., 2007)) alleviated CVB3 proliferation in iPSC-derived cardiomyocytes, indicating the promise of these cells for antiviral drug screening and toxicity testing against coxsackieviruses. This study also revealed that human iPSC-derived cardiomyocytes are more susceptible and permissive to CVB3 than murine cardiac HL-1 cells, evident with low levels of Cxadr expression and viral replication in HL-1 cells, suggesting human iPSC-derived cardiomyocytes as a more natural and physiological model for coxsackievirus infection compared to tumor-derived cell lines. Modeling CVB3 infection in iPSC-derived cardiomyocytes was further extended by another study deciphering the impact of altered antiviral immunity in cardiomyocytes against CVB3 (Belkaya et al., 2017). This study utilized control iPSCs generated from healthy individuals and mutant iPSC lines bearing naturally occurring deleterious mutations in Toll like-receptor 3 (TLR3) or signal transducer and activator of transcription 1 (STAT1), genes involved in IFN-mediated antiviral immunity, derived from young patients with severe viral diseases other than coxsackievirus infection (Chapgier et al., 2006; Guo et al., 2011). Cardiomyocyte differentiation was performed using a modified version of the original differentiation protocol described elsewhere (Yang et al., 2008). Consistent with the previous report, CVB3 (Nancy strain) replicated and induced cytotoxicity in control iPSCderived cardiomyocytes generated from healthy individuals. Control cardiomyocytes were also infected with a mutant strain of Vesicular stomatitis virus (VSV; Indiana strain), which is not able to shutdown host antiviral immunity (Stojdl et al., 2003), in order to assess virus-induced transcriptomic changes compared to mock- and CVB3-infected control cardiomyocytes. Mutant VSV infection resulted in elevated expression of genes involved in IFN-mediated virus clearance pathways, indicating an intact antiviral immunity in human iPSC-derived cardiomyocytes.



CHAPTER 5 iPSCs for modeling coxsackievirus infection

On the other hand, CVB3 did not lead to any alterations in the expression of IFNs or IFN-stimulated genes but instead induced genes associated with the unfolded protein response and cellular senescence in cardiomyocytes. Several studies in mice suggested the involvement of the innate immune system in controlling coxsackievirus infection in the myocardium (Chow et al., 1992; Mena et al., 1999). In particular, deficiency of proteins involved in antiviral IFN immunity, such as Ifn-b, type I IFN receptor (Ifnr), Tlr3, and Unc-93 homolog B1 (Unc93b1), caused increased vulnerability to CVB3 infection and virus-induced myocardial injury in mice (Wessely et al., 2001; Deonarain et al., 2004; Negishi et al., 2008; Lafferty et al., 2015). In contrast, human cardiomyocytes derived from iPSC lines deficient in TLR3 or STAT1 were not more or less vulnerable to CVB3 infection, with no obvious differences in viral replication and virus-induced cytotoxicity compared to control cardiomyocytes. Priming with exogenous IFN-a2b reduced CVB3 proliferation and its cytopathic effects in control and TLR3-deficient cardiomyocytes, but not in STAT1-deficient cardiomyocytes as might be expected. Collectively, human iPSC-derived cardiomyocytes were responsive to exogenous IFNs; however, there was no impact of altered cardiomyocyte-intrinsic TLR3/IFN-mediated antiviral immunity on predisposition to CVB3 infection. Overall these studies demonstrated the suitability of human iPSC-derived cardiomyocytes for the investigation of cellintrinsic innate immune responses and exogenous antiviral mechanisms against coxsackieviruses. Coxsackieviruses are frequently associated with spontaneous abortion in women, with unclear pathogenesis (Axelsson et al., 1993; Frisk and Diderholm, 1992; Hwang et al., 2014). CVB3 infection during early gestation in mice was also shown to induce pregnancy losses, attributed to high levels of CAR expression in the uterus and embryos (Hwang et al., 2014). In order to model how viruses can affect early human embryogenesis, infection with various miscarriage-associated viruses including CVB3 was studied in human iPSCs, as they can represent pluripotent cells at the early stages of human development (Hubner et al., 2017). This study utilized two commercially available human iPSC lines, a viral-integration-free episomal iPSC line and a lentiviral iPSC line, both displaying similar metabolic activities and comparable levels of pluripotency markers as well as surface CAR expression. iPSCs were infected with a recombinant CVB3 strain, ectopically expressing enhanced green fluorescent protein (EGFP) for direct visualization of infected cells (Slifka et al., 2001; Feuer et al., 2002). CVB3 replicated and induced cytopathic effects in both iPSC lines. CVB3 infection also led to altered metabolic activities of iPSCs, evident with increased basal oxygen consumption rates, respiratory capacities, and ATP production. This study provided evidence that human iPSC modeling of coxsackievirus infection can further advance our understanding of virus-induced pathologies in the uterus and embryo leading to miscarriage, stillbirth, or congenital morbidities.

Utility of human iPSC-derived specific cell types as a powerful in vitro system to model coxsackievirus infection and discover novel or better antiviral therapies.

iPSC modeling of coxsackievirus infection




CHAPTER 5 iPSCs for modeling coxsackievirus infection

Concluding remarks and future perspectives Coxsackieviruses can cause a wide range of devastating illnesses in humans, particularly in children, with almost no information available concerning the early stages of coxsackievirus infection and virus-induced histopathologies. Modeling human natural coxsackievirus infection using iPSC-derived relevant cells paves the way for studying many aspects of the virus biology and virus-induced cellular phenotypes clinically related to coxsackievirus-associated diseases. These studies will provide novel insights into coxsackievirus pathogenesis and permit patient-specific modeling of coxsackievirus-related disease. The use of human iPSC-based models will also facilitate the screening for novel antiviral drugs against coxsackieviruses and the testing for preclinical efficacy and toxicity of such drugs (Fig. 5.2). Nevertheless, iPSC-derived cells, which are 2D monolayers of cells like all other conventional cell culture lines, lack the complex microenvironment of an organ in vivo. Animal models suggest the involvement of multiple cell types in disease progression in response to coxsackievirus infection. Accordingly, the utility of threedimensional iPSC modeling systems, such as engineered tissues, organoids, and organs-on-chips, will allow for the study of coxsackievirus infection within a pathophysiologically relevant tissue environment and ultimately expand our knowledge on coxsackievirusehost interaction and coxsackievirus-associated diseases.

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Pluripotent stem-cellderived oligodendrocyte progenitors to model demyelination caused by Theiler’s murine encephalomyelitis virus and other viruses


Eric C. Freundt, Pavan Rajanahalli Department of Biology, The University of Tampa, Tampa, FL, United States

Chapter outline Importance of myelin in the CNS..............................................................................122 Virus-induced demyelination....................................................................................123 Steps to myelination: OPC proliferation, migration, and maturation ............................124 Disruption of myelination by viruses.........................................................................126 Theiler’s murine encephalomyelitis virus (TMEV)...........................................126 John Cunningham virus (JCV) ......................................................................129 Mouse hepatitis virus (MHV)........................................................................130 Murine leukemia virus (MLV) .......................................................................131 Canine distemper virus (CDV) ......................................................................132 Zika virus...................................................................................................132 HHV-6.......................................................................................................133 Semliki Forest virus (SFV) ...........................................................................134 Induced pluripotent stem cells (iPSCs) as a model system to study demyelinating viruses ...................................................................................................................134 Challenges with isolation of primary OPCs ....................................................134 Advantages of iPSCs ...................................................................................135 Brain organoids with iPSCs to model viral CNS infection................................136 Conclusions and future perspectives ........................................................................137 References .............................................................................................................138

iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00007-9 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 6 Pluripotent stem-cell-derived oligodendrocyte progenitors

Abstract There is considerable interest in understanding the molecular mechanisms that govern differentiation of neural progenitor cells (NPCs) into oligodendrocyte progenitor cells (OPCs), and finally mature, myelinating oligodendrocytes as new discoveries in these pathways could allow for targeted therapies to increase remyelination in diseases such as multiple sclerosis (MS). Numerous studies demonstrate that a variety of distinct oligodendrotropic viruses, or viruses that infect oligodendrocytes, can interfere with the development of OPCs, OPC migration in the central nervous system (CNS), differentiation and ultimately myelination, and pluripotent stem cells have emerged as powerful tools to study virus infections in these cell types. This chapter reviews cellular proteins and pathways involved in oligodendrocyte differentiation and how viruses disrupt maturation of oligodendrocytes and myelination, with a focus on Theiler’s murine encephalomyelitis virus (TMEV), a virus that causes a demyelinating disease with pathological similarities to MS. Pure populations of myelinogenic OPCs derived from pluripotent stem cells have been useful to investigate disruption of OPC maturation and myelination by TMEV. Pluripotent stem cells may also be used to study viruseoligodendrocyte interactions for other viruses and further elucidate OPC maturation, myelin formation, and virus-induced demyelination. Keywords: Brain organoid; Canine distemper virus; Demyelination; Human herpesvirus 6; iPSC; John Cunningham virus; Mouse hepatitis virus; Murine leukemia virus; Myelin; Oligodendrocyte progenitor; Semliki Forest virus; Theiler’s murine encephalomyelitis virus; Virus; Zika virus.

Importance of myelin in the CNS Myelin is an extension of the plasma membrane of the oligodendrocyte that wraps numerous times around surrounding axons. Evolution of myelination in vertebrates allowed for rapid propagation of nerve impulses by enabling salutatory conduction. Without myelination, an action potential travels along unmyelinated axons in invertebrates at a rate of 1 m/s, whereas that speed is 50e100x faster in myelinated axons of equivalent diameter (Zalc, 2006). Myelin forms a dense multilayered structure, which is discontinuous because the membranes derived from different oligodendrocytes do not fuse. The small interruptions in myelin sheaths, known as nodes of Ranvier, are the location of axonal voltage-gated sodium channels and enable salutatory conduction. Myelin is primarily composed of lipids (70%e85%) with relatively low protein content. Proteins found within myelin include myelin basic protein (MBP) and proteolipid protein (PLP), which are predominant, and additional proteins such as 20 :30 Cyclic nucleotide-30 -phosphodiesterase (CNPase), myelin-associated glycoprotein (MAG), and myelin-oligodendrocyte glycoprotein (MOG), which are present in lesser quantities. In addition to its role as an electrical insulator, myelin also supports the maintenance of axons. For example, neurological defects in mice deficient in

Virus-induced demyelination

CNPase, a relatively minor component of myelin specifically expressed by oligodendrocytes, are not due to demyelination but result instead from axonal degeneration (Lappe-Siefke et al., 2003). CNPase-deficient mice exhibit axonal degeneration due to a subtle change in myelin expressed by the oligodendrocyte that likely disrupts cytoplasmic channels in myelin and transport of metabolites to the axon (Stassart et al., 2018). Oligodendrocytes also support axonal energy metabolism through NDMA receptors that regulate GLUT1 trafficking and glucose import. When NDMA is lacking on oligodendrocytes, mice develop axonopathy and neurological dysfunction despite myelination (Saab et al., 2016).

Virus-induced demyelination Relatively few viruses invade the central nervous system (CNS) and even fewer directly infect oligodendrocytes (Fazakerley and Walker, 2003). Most of the viruses that infect oligodendrocytes do so rarely and only after successfully crossing the bloodebrain barrier into the CNS. For example, Theiler’s murine encephalomyelitis virus (TMEV) infects oligodendrocytes and causes a demyelinating disease in susceptible strains of mice, but without intracranial inoculation, rates of CNS infection are extremely low (1/1000 to 1/10,000) (Brahic, 2010) However, those viruses that are known to infect oligodendrocytes lead to demyelination frequently. It is also important to note that additional oligodendrotropic viruses likely exist but have not been identified because they do not result in overt disease. Demyelinating diseases are characterized by a loss of myelination of axons in the CNS. Viruses that infect oligodendrocytes often lead to demyelination, which is likely due to both direct effects of the virus and initiation of an antiviral immune response that may turn autoreactive and begin to target myelin epitopes. How much of demyelination, and the amount of subsequent remyelination, is due to direct effects of viral infection, including induction of innate immune responses within oligodendrocytes, and how much demyelination is due to the subsequent cellmediated immune response, including activated macrophages, cytotoxic T lymphocytes, and cytokines? The answer to this question certainly depends on the particular virus, but studies addressing this question may lead to greater understanding of normal myelination. In some virus infections, ultimate destruction of oligodendrocytes depends on an inflammatory response, such as in TMEV and mouse hepatitis virus (MHV) infections, as discussed further, whereas others do not. Virus-induced demyelinating diseases vary with respect to the amount of remyelination that can occur following the initial insult. Remyelination is the process whereby oligodendrocyte progenitor cells (OPCs) are recruited to the site where oligodendrocytes have been destroyed and undergo maturation and production of new myelin. Remyelination is limited in some human demyelinating diseases, such as MS, but stimulation of this process may ameliorate neurological symptoms. Pharmacological stimulation of OPC proliferation to promote remyelination in vivo has promising therapeutic potential in diseases such as MS (Najm et al., 2015).



CHAPTER 6 Pluripotent stem-cell-derived oligodendrocyte progenitors

Steps to myelination: OPC proliferation, migration, and maturation Oligodendrocytes originate from neuroepithelial precursors, which also give rise to neurons and astrocytes, and develop based on a complex program of transcriptional and epigenetic regulation (reviewed in (Emery and Lu, 2015). Prior to differentiation into postmitotic oligodendrocytes, OPCs migrate from the ventral ventricular zone of the telencephalon, where they originate, to sites of myelination (Kessaris et al., 2006). Migration of OPCs occurs in a Wnt-dependent manner along blood vessels and depends on expression of the chemokine (C-X-C motif) receptor 4 (Cxcr4) on OPCs, which likely interacts with stromal derived factor 1 (Sdf-1), also known as CXCL12, on endothelial cells (Tsai et al., 2016; Banisadr et al., 2011; Dziembowska et al., 2005). Single-cell RNA sequencing showed that these migrating OPCs display a unique transcriptional profile (Marques et al., 2016). Many transcription factors and other molecules involved in OPC growth and maturation have been identified (Fig. 6.1). The coexpression of Olig2 and Nkx2.2 induces oligodendrocyte maturation, whereas depletion of Olig2 prevents oligodendrocytes from forming (Fancy et al., 2004). Similarly, depletion of Sox8 and Sox9 prevents oligodendrocyte formation, and loss of Sox9 alone resulted in delayed appearance of OPCs (Stolt et al., 2005). Sox10 interacts with Olig1 and Olig2 to induce transcription of MBP (Li et al., 2007). Oligodendrocyte maturation occurs due to simultaneous upregulation of transcription factors for differentiation with downregulation of inhibitors, including Hes5, Id2, Id4, Sox5, and Sox6 (Liu et al., 2006; Wang et al., 2001; Samanta and Kessler, 2004; Stolt et al., 2006). Additionally, maturation depends on the Wnt/b-catenin pathway. Adenomatous polyposis coli (APC) negatively regulates b-catenin and loss of APC prevented OPC maturation while reducing OPC proliferation (Lang et al., 2013). b-catenin binds Tcf4, which is highly expressed in pro-oligos (Cahoy et al., 2008). Tcf4 is a transcription factor activated by Wnt signaling in OPCs and is later downregulated in mature oligodendrocytes (Fancy et al., 2009; Fu et al., 2009). Zfp488 is also highly expressed in prooligos and influences differentiation (Soundarapandian et al., 2011). Additional transcription factors, such as Nkx2-2, Olig1, Ascl1, Yin Yang 1 (YY1), Zfhx1b, PRMT5, and Sox10, are also required for maturation (Qi et al., 2001; Stolt et al., 2002; Xin et al., 2005; He et al., 2007; Sugimori et al., 2008; Weng et al., 2012). For example, myelin regulatory factor, Myrf, is strongly induced in early stages of oligodendrocyte maturation and controls expression of many genes involved in myelination, including myelin proteins MBP and PLP (Bujalka et al., 2013; Emery and Lu, 2015). Many genes activated by Myrf1 are also activated by Olig2 and Sox10, suggesting that there is at least some functional overlap between them (Emery and Lu, 2015). Olig1/2 also activates Smad7 through targeting Smadinteracting protein-1 (Sip1), and Smad7 is then required for oligodendrocyte differentiation (Weng et al., 2012). Several key G-protein-coupled receptors also play a role in oligodendrocyte differentiation, including GPR17, GPR37, and GPR56

Steps to myelination: OPC proliferation, migration, and maturation

FIGURE 6.1 TMEV disrupts multiple genes that contribute to OPC proliferation, maturation, and differentiation. Representative genes were up- or downregulated in OPCs infected by TMEV. Gene expression levels as determined by RNA-seq (Benner et al., 2016). Genes involved in OPC proliferation are highlighted in green, those involved in OPC to immature oligodendrocyte differentiation are highlighted in red, genes highlighted in blue are involved in oligodendrocyte maturation. This figure was generated with images from Servier Medical Art (https://smart.servier.com) under a Creative Commons attribution 3.0 unported license.

(Chen et al., 2009; Yang et al., 2016a; Ackerman et al., 2015; Giera et al., 2015). Finally, micro-RNAs have also been described that enhance oligodendrocyte development and myelination, such as mIR-219 (Dugas et al., 2010). mIR-146 also promotes OPC differentiation, likely through its role as a negative regulator of TLR2 and interleukin-1 receptor-associated kinase 1 signaling (Zhang et al., 2019). Thus, many cellular transcription factors and cellular proteins have been identified as important in the steps leading to myelination, and it is important to consider how viruses that infect OPCs and oligodendrocytes affect the expression or function of these factors.



CHAPTER 6 Pluripotent stem-cell-derived oligodendrocyte progenitors

Disruption of myelination by viruses Theiler’s murine encephalomyelitis virus (TMEV) TMEV is a member of the Cardiovirus genus in the family Picornaviridae and was discovered by Max Theiler in 1937 (Theiler, 1937). The genome of TMEV is singlestranded positive-polarity RNA and approximately 8100 nucleotides. The genome of TMEV is organized similarly to other picornaviruses and includes both a 50 and 30 untranslated region. Translation is initiated at an internal ribosomal entry site and produces a polyprotein that is later cleaved to generate 12 proteins that provide nonstructural and structural functions. In addition, TMEV produces an out-of-frame protein, called L*, from the 50 end of its genome that is important for promoting macrophage infection and enabling the virus to persist, which may be due in part to its activity as an inhibitor of RNAse L (Sorgeloos et al., 2013). A second out of frame small protein, called 2B*, is also produced due to a frameshift but is only 14 amino acids in length, and no function has yet been ascribed to 2B* (Loughran et al., 2011). TMEV is found throughout the world as a common pathogen of wild mice and rats (Lipton, 1975). The virus is spread by fecal oral means and when transmitted in this fashion rarely enters the CNS or causes CNS disease. In contrast to its natural route of infection, however, TMEV causes reliable neurological disease when inoculated intracerebrally, the outcome of which depends on the virus strain and the genetic background of the host. Strains of TMEV are divided into two groups according the degree of neurovirulence. A highly neurovirulent group, called George Davis 7, includes the GDVII and FA strains and causes a severe encephalitis that is often fatal for the mouse. Strains showing lower neurovirulence are called the Theiler’s original strains and include strains Daniels (DA), BeAn, WW, TO4, Yale, and 4727. These strains of TMEV cause an initial encephalitis characterized by infection of neurons and a small proportion of glial cells that persists for approximately 2 weeks and is then resolved. However, in certain strains of mice, the virus may persist and establish a chronic infection that manifests in late disease (about 30e40 days post infection) that is characterized by focal, demyelinating lesions in the white matter and is accompanied by inflammatory infiltrates composed of CD4þ and CD8þ T cells, B cells, and activated macrophages. This chronic disease manifests in weakness of the hind limbs and ultimately spastic paralysis. The chronic phase of the disease shares clinical and histopathological features in common with multiple sclerosis (MS) in humans and has been used extensively as a model for progressive MS. In addition to TMEV-induced demyelinating disease (TMEV-IDD), TMEV has also been utilized as a disease model for both epilepsy and myocarditis (reviewed in (Gerhauser et al., 2019). TMEV shows a marked shift in the types of cells that it infects during early and late disease in the CNS. During the encephalitic phase of disease, the virus is found mainly in neurons in the hippocampus, cortex, and spinal cord, while few oligodendrocytes astrocytes and macrophages can be observed to have been infected.

Disruption of myelination by viruses

During late disease, however, TMEV is found primarily in the glial cells of white matter, including macrophages, astrocytes, and oligodendrocytes, with very few neurons still showing evidence of virus infection (Aubert et al., 1987; Brahic et al., 1981; Lipton et al., 1995; Rossi et al., 1997; Rodriguez et al., 1983). The main reservoir for TMEV during persistent infection is macrophages, although oligodendrocytes may also harbor virus during persistence (Rossi et al., 1997). The reason for this change in cell type specificity during early and late infection is likely due to the immune response being able to efficiently clear the virus from neurons during early infection but not being able to eliminate the reservoir in glial cells during the chronic stages of infection. Support for this hypothesis comes from studies using Scid mice, which failed to clear TMEV infection from neurons, and reconstitution of immune cells demonstrated that cell-mediated responses as opposed to antibody-mediated responses were essential for clearing the virus from neurons (Njenga et al., 1997). The genetic determinants of whether a mouse will clear TMEV or whether the virus will persist and establish an MS-like disease have been extensively studied, and the topic is well reviewed elsewhere (Brahic et al., 2005). Major determinants of resistance are involved in regulating the immune response and include the H2 class I genes, which present antigen to cytotoxic T lymphocyte and therefore regulate destruction of infected cells. Another gene that contributes to susceptibility to chronic disease in mice is NeST, a long intergenic noncoding RNA, which recruits histone H3 lysine 4 methyltransferases and enhances expression of IFN-g. Although this gene was discovered by careful mapping of loci that regulate susceptibility to TMEV, NeST contributes to immune regulation more broadly and affects the immune response to both bacterial and viral pathogens (Gomez et al., 2013). In addition to these genes, which are involved in the host response to the virus, genes that contribute to myelin formation are also determinants of resistance. For example, shiverer C3H mice, which have a deletion in MBP that dramatically reduces myelin formation, are resistant to persistent infection but wild-type C3H mice are susceptible. Similarly, rumpshaker mice, which have a point mutation in PLP, are also able to clear TMEV and are not chronically infected (Bihl et al., 1997; Roussarie et al., 2007). The mechanism by which these mutations in myelin proteins are able to prevent viral persistence can be explained by studies that examined how the virus spreads from axon to oligodendrocyte in the optic nerve of mice. In these experiments, TMEV was introduced into the eye and transited down axons of retinal ganglion cells and into oligodendrocytes and astrocytes within the optic nerve. Infection of oligodendrocytes in the optic nerve was greatly diminished in shiverer mice, indicating that infection of oligodendrocytes through myelin was a necessary step to establish persistent infection (Roussarie et al., 2007; Brahic and Roussarie, 2009). The cause of demyelination during chronic infection by TMEV has also been extensively studied. While the majority of studies have focused on the contribution of the immune response to destruction of oligodendrocytes, direct infection of OPCs and oligodendrocytes is also thought to play a role. Regarding the immune-mediated



CHAPTER 6 Pluripotent stem-cell-derived oligodendrocyte progenitors

mechanisms, bystander cell killing of oligodendrocytes by cytokine secretion, including tumor necrosis factor alpha, from activated monocytes and microglial cells has been documented. These activated macrophages can be seen as having phagocytosed a large amount of myelin (Oleszak et al., 1997). Epitope spreading, in which virus-reactive T cells lead to recognition of self-epitopes, has also been documented to occur in TMEV-IDD. For example, CD4þ T cells in TMEV-IDD have been shown to recognize epitopes from PLP, a major protein component of myelin and later epitopes from MBP (Neville et al., 2002). Remyelination does occur in the corpus callosum during early phases of TMEV disease and is mediated by neural stem cells in the subventricular zone and local OPCs. NSCs in the SVZ are stimulated to proliferate and differentiate into OPCs that then migrate to sites of myelin damage. Purified myelin was shown to stimulate generation of oligodendrocytes from NSCs, and secretion of Wnt7a by M2c microglia also contributed to differentiation of NSCs into OPCs. Despite the occurrence of remyelination in the early phases of disease, remyelination is rare and incomplete during the chronic phase of TMEV infection and is likely caused by a reduction in OPC differentiation and maturation (Murray et al., 2001). A study of chronically infected SJL/J mice revealed that areas of OPCs showed high levels of phosphorylated STAT3, which shifts OPC differentiation from oligodendrocytes to astrocytes (Sun et al., 2015). This change in JAK-STAT signaling was not shown to occur in infected cells, although other studies of TMEV-infected OPCs and cell lines have demonstrated similar downmodulation of gene pathways important for OPC proliferation and maturation (Benner et al., 2016; Pringproa et al., 2010; Qi et al., 2001). Remyelination can be induced in TMEV-infected mice using an oligodendrocytemyelin targeting monoclonal antibody (Warrington et al., 2007). Remyelination in this model was also shown to result in preservation of functional spinal cord axons (Wootla et al., 2016). In addition, remyelination was recently shown to occur when chronically infected mice were given extracellular vesicles (EVs) from mesenchymal stem cells and was associated with increased myelin expression and a concurrent reduction in motor impairment (Laso-Garcia et al., 2018). Together, these studies demonstrate that a pool of OPCs exists that is capable of remyelination despite the chronic inflammatory state in the CNS and viral persistence in glial cells. Evidence for direct infection and depletion of oligodendrocytes as a contributor to TMEV-IDD comes from multiple lines of investigation. Nude mice, which lack a robust T cell compartment, also develop demyelinating disease when infected with the DA strain of TMEV (Rosenthal et al., 1986; Roos and Wollmann, 1984). In vitro, TMEV preferentially infected an immature OPC cell line compared to cells that had been differentiated and expressed MBP and was also found to inhibit OPC differentiation (Pringproa et al., 2010). TMEV can infect primary oligodendrocytes in vitro, although the virus does not replicate well in these cells. In contrast, the virus replicates well in an immature oligodendroglial cell line, but caused decreased expression of MBP and MOG (Qi and Dal Canto, 1996). Infection of OPCs with TMEV

Disruption of myelination by viruses

FIGURE 6.2 Development of OPCs into myelinating oligodendrocytes. Genes that contribute to each step are shown in blue. Viruses that disrupt these steps are shown in red. *Viruses for which there is strong evidence for the immune response in demyelination. This figure was generated with images from Servier Medical Art (https://smart.servier.com) under a Creative Commons attribution 3.0 unported license.

showed that Olig2 is specifically downregulated by the virus in addition to other genes that influence OPC differentiation, such as Nkx2.2, Sox8, and Sox10 (Benner et al., 2016). A separate report also found that differentiation was inhibited by TMEV (Pringproa et al., 2010) (Fig. 6.2). Thus, TMEV may limit remyelination by infecting OPCs and preventing their maturation. Pluripotent stem cells have been useful to create highly pure populations of expandable OPCs that retain characteristics of primary OPCs and are able to differentiate into mature oligodendrocytes (Najm et al., 2011). These cells have been used as a model to understand how TMEV infection impacts gene expression (Benner et al., 2016). Numerous genes important for OPC proliferation, such as Olig2, Nkx2-2, and Id2, were suppressed by infection. Additionally, genes important for maturation and differentiation of OPCs, including Olig1, Sox11, and Egr1, were also dysregulated. While Olig1 was suppressed by TMEV, Egr1, which is normally downregulated in OPCs in early stages of differentiation to oligodendrocytes, was found to increase in expression (Yao et al., 1995; Zeger et al., 2007). Similarly, TMEV caused upregulation of Sox11, which acts as a repressor of oligodendrocyte differentiation (Swiss et al., 2011). These data suggest that infection by TMEV may prevent remyelination by modifying the genes essential for OPCs to both proliferate and mature into oligodendrocytes.

John Cunningham virus (JCV) JCV is a ubiquitous member of the Polyomaviridae; more than half of the adult population is infected and vast majority remains asymptomatic (Swanson and McGavern, 2015). However, immunosuppression can allow the virus to cause a disease that is characterized by demyelination called progressive multifocal leukoencephalopathy (PML), which is often fatal (Pietropaolo et al., 2018).



CHAPTER 6 Pluripotent stem-cell-derived oligodendrocyte progenitors

Many aspects of the pathogenesis of this infection remain to be elucidated, due in part to the lack of an animal model, although the virus appears to infect peripheral blood mononuclear cells and can spread hematogenously across the BBB to infect oligodendrocytes in the CNS (Wollebo et al., 2015). Intriguingly, rearrangement of a noncoding regulatory region in JCV DNA is associated with its ability to cause disease. Destruction of oligodendrocytes is progressive and, unlike TMEV-IDD, remyelination does not appear to occur (Tan and Koralnik, 2010). JC virus replicates well in a human OPC cell line in vitro and depends on Akt signaling (Peterson et al., 2017). Expression of the agnoprotein of JCV in the CG-4 progenitor cells impaired their differentiation into mature oligodendrocytes by inducing apoptosis (Merabova et al., 2008). In a separate report, JCV infected primary OPCs and prevented differentiation as measured by expression of MBP, PLP, and PDGFRa. Thus, in addition to infecting and disrupting mature oligodendrocytes, JCV may also prevent remyelination by disrupting OPC differentiation (Darbinyan et al., 2013). Coinfection of patients with JVC and human immunodeficiency virus type 1 (HIV-1) may further contribute to destruction of oligodendrocytes through the activity of HIV-1 proteins (reviewed in (Liu et al., 2016). Although HIV-1 was reported to infect culture human oligodendrocytes in one report, other studies failed to detect infection (Albright et al., 1996; Sharpless et al., 1992; Takahashi et al., 1996). However, infection of oligodendrocytes by HIV-1 may not be necessary to cause their destruction. In particular, HIV-1 Tat can be secreted and taken up by oligodendrocytes, where it then acts to stimulate transcription from JCV early and late gene promoters (Daniel et al., 2004; Wright et al., 2013). When added in vitro, the envelope glycoprotein 120 (gp120) decreased arborization and length of oligodendrocyte processes, although MBP expression was unaffected (Kimura-Kuroda et al., 1994). However, at least in culture, oligodendrocyte development did not appear to be perturbed by a lytic infection of microglial cells as oligodendrocytes continued to express GalC and extended membrane processes for up to 28 days in culture (Sharpless et al., 1992). Thus, the role of HIV-1 in disruption of oligodendrocytes remains an open question.

Mouse hepatitis virus (MHV) MHV is single-stranded positive-polarity RNA enveloped virus in the family Coronaviridae. The virus causes various diseases depending on the strain and route of inoculation, with some strains causing encephalitis with a subsequent demyelinating disease that has similarities to the pathology of MS. There is a large body of literature that supports a role for the immune response in MHV-induced demyelination, although the exact mechanism is not fully understood (reviewed in (Bender and Weiss, 2010). A recent study compared a demyelinating strain of MHV, RSA59, with a nondemyelinating strain, RSMHV2, for their abilities to infect OPCs and oligodendrocytes in vitro and in vivo. The strains infected OPCs equally well in vitro, although the demyelinating strain infected mature oligodendrocytes much more efficiently.

Disruption of myelination by viruses

Both strains led to decrease in GalC expression in mature oligodendrocytes. In the spinal cord, the demyelinating strain also infected oligodendrocytes in greater numbers (Kenyon et al., 2015). These data indicate that direct infection of oligodendrocytes correlates with demyelination. However, although MHV directly infects oligodendrocytes, demyelination requires functional lymphocytes. In RAG-deficient mice, demyelination was absent in infected mice but occurred following adoptive transfer of splenocytes from MHV-immunized mice (Wu and Perlman, 1999). Infection of oligodendrocytes for MHV-induced demyelination therefore appears necessary but not sufficient for disease. A few reports demonstrate that infection of oligodendrocytes can trigger apoptosis during entry by activating the Fas pathway (Liu and Zhang, 2007). Mitochondria are also involved in MHV-induced apoptosis, as overexpression of Bcl-xL partially rescued cells from apoptosis and led to persistence of viral RNA (Liu et al., 2006). Remyelination occurs after the single major episode of demyelination and often leads to recovery of the animal. Additionally, remyelination can be induced in the context of chronic MHV infection by injecting neural stem cells (Totoiu et al., 2004) (Hardison et al., 2006). Neural stem cells were injected into mice with demyelination migrated to lesions and differentiated into mature oligodendrocytes (Carbajal et al., 2010). These data suggest that the development and maturation of OPCs into myelinating oligodendrocytes are not perturbed during the chronic phase of MHV infection and indicate that the destruction of mature oligodendrocytes, likely by the immune response to viral antigen in these cells, may be the underlying cause of demyelination. Intriguingly, SARS-CoV-2, also a member of Coronaviridae and the cause of the COVID-19 pandemic, has been recently reported to cause demyelinating lesions in a single case report (Zanin et al., 2020). However, other reports of neurological symptoms in SARS-CoV-2 infection have not been successful in detecting viral RNA in cerebrospinal fluid (Helms et al., 2020). The frequency of demyelination in COVID19 patients remains to be determined.

Murine leukemia virus (MLV) Certain strains of MLV, which is classified as a retrovirus, cause a neurodegenerative disease that is noninflammatory and similar in many respects to vacuolar disease caused by prions. The env gene is a major determinant of virulence and is necessary and sufficient for neurodegeneration (Kay et al., 1993; Li et al., 2011). A recent study demonstrated that murine leukemia virus infects oligodendrocyte precursor cells and inhibits their maturation. In this intriguing study, the authors demonstrate that MLV efficiently infected NG2þ and Olig2þ OPCs, although few mature oligodendrocytes were infected. To determine why fewer oligodendrocytes were infected, NPCs expressing PLP-EGFP were infected ex vivo and transferred into a PO mouse brainstem. Fewer oligodendrocytes were detected in the mouse



CHAPTER 6 Pluripotent stem-cell-derived oligodendrocyte progenitors

receiving infected NPCs, which suggests that the OPCs were unable to differentiate. Importantly, differentiation into astrocytes appeared to be unaffected (Li et al., 2016b). These results are consistent with a separate study that identified Olig2þ cells but not PLPþ or MBPþ cells as primary target of the virus in the CNS (Clase et al., 2006). Although the mechanism by which MLV prevents OPC maturation has yet to be determined, other studies have also demonstrated a similar effect of retrovirus proteins on OPC maturation. For example, expression of the Env protein by human endogenous retrovirus type W (HERV-W) family, also known as multiplesclerosis-associated retrovirus (MSRV), inhibits OPC differentiation and remyelination through activation of TLR4 (Kremer et al., 2013). Intriguingly, a neutralizing monoclonal antibody against the Env protein was able to restore OPC differentiation and myelin production (Kremer et al., 2015). Moreover, exposure of NPCs to Tat from HIV-1 reduced expression of Olig2 (Hahn et al., 2012).

Canine distemper virus (CDV) CDV is an enveloped, nonsegmented negative-polarity RNA virus and a member of the genus Morbillivirus in the Paramyxoviridae family. Primary demyelination is noninflammatory and a common pathological feature in affected canines (Vandevelde and Zurbriggen, 2005). The virus initially replicates in the lymphoid tissue, resulting in immunosuppression, and can later spread to the epithelium and the CNS through infection of enervating mononuclear cells. Demyelination appears approximately 3 weeks post infection and may progress due to autoimmunity in the chronic stage of disease (Vandevelde et al., 1982a; Higgins et al., 1989; Vandevelde et al., 1982b). Infection of oligodendrocytes is much less common than infection of astrocytes (Mutinelli et al., 1989), and a restricted infection of oligodendrocytes was observed in vitro (Zurbriggen et al., 1993). Despite inefficient replication, oligodendrocytes infected with CDV in vitro undergo metabolic changes and decrease expression of PLP, MBP, and MOG (Glaus et al., 1990; Graber et al., 1995). Coculture with macrophages and CDV-immune complexes resulted in decreased myelination, and this deficiency was rescued when macrophages were depleted from the culture, indicating that bystander destruction of oligodendrocytes by macrophages may also play a role (Botteron et al., 1992). The mechanisms by which virus replication is restricted and how the virus disrupts oligodendrocytes remain unresolved. Early axonal damage that precedes demyelination can be detected during CDV infection, suggesting that the CDV-induced demyelination may be caused by an inside-out model, in which disruption of the axon leads to degeneration of the myelinating oligodendrocyte, and has also been proposed as a model in TMEV-IDD (Lempp et al., 2014; Tsunoda and Fujinami, 2002).

Zika virus Zika virus (ZIKV) is a member of Flaviviridae and is transmitted by Aedes mosquitos. The virus was recently responsible for an epidemic in the Americas.

Disruption of myelination by viruses

Although ZIKV rarely causes CNS infection in adults, trans-placental transmission and infection of the fetus result in a high percentage of abnormalities, including microcephaly, congenital malformations, and fetal demise (Brasil et al., 2016). The virus preferentially infects NPCs, and this tropism is likely to explain its ability to impair brain development (Tang et al., 2016). White matter is also severely disrupted in congenital ZIKV infection within the first trimester, including absence of myelination and Olig2-positive cells (Chimelli et al., 2017). Some oligodendrocytes could be detected, however, in one case that had been infected in the last trimester. Depletion of oligodendrocytes could be explained by viral destruction of NPCs. However, studies also demonstrate that the ZIKV can infect other cells of the oligodendrocyte lineage. One report documented that ZIKV preferentially infected mature oligodendrocytes and OPCs compared to microglial cells, astrocytes, and neurons in in vitro myelinating cultures prepared from spinal cord of mice lacking the receptor for type I interferon (Ifnar1). In cultures from both wild-type and Ifnar1-deficient mice, the number of infected mature oligodendrocytes was higher than the number of OPCs, suggesting that the virus may preferentially infect oligodendrocytes. Moreover, myelination was particularly disrupted. The amount of PLP and MOG staining was decreased despite normal density of oligodendrocytes (Cumberworth et al., 2017). Disruption of myelination in these cultures was likely due to both an arrest of myelination and loss of myelin from virus-induced cell death. In a separate report examining virus infection of primary human tissue, ZIKV preferentially infected OPCs and other cell types, including neural stem cells, astrocytes, and microglial cells, but did not infect neurons efficiently (Retallack et al., 2016). Thus, it is plausible that ZIKV may have direct effects on myelination independent of its effects on other neural cells. Although the clinical relevance of this is uncertain, instances of acute disseminated encephalomyelitis, which is characterized by inflammatory demyelination, following ZIKV infection have been reported (Roth et al., 2017; Brito Ferreira et al., 2017).

HHV-6 Human herpesvirus 6 is a betaherpesvirus that is associated with pediatric encephalitis. HHV-6, like several other viruses, has been evaluated as a potential environmental contributor to MS (Leibovitch and Jacobson, 2014). A neurovirulent strain of HHV-6, designated HHV-6A, was able to productively infect the oligodendrocyte cell line (MO3.13) in vitro and seemed to cause a latent infection; DNA persisted for over 60 days in culture but no detectible HHV-6A RNA or protein could be detected after day 30 (Ahlqvist et al., 2005). Moreover, infection of human OPCs with HHV-6A has been reported. Infected OPCs were not killed and the proliferation of the cells was decreased. These cells also displayed an increase in expression of GalC, a marker of differentiation (Dietrich et al., 2004). Expression of the Human Herpesvirus 6A latency associate transcript U94A prevented migration of OPCs in vitro but did not affect proliferation or the ability of these cells to differentiate.



CHAPTER 6 Pluripotent stem-cell-derived oligodendrocyte progenitors

Furthermore, expression of U94A inhibited migration of OPCs to demyelinated lesions (Campbell et al., 2017). The authors of this study suggest a mechanism whereby HHV-6 may inhibit remyelination in patients with MS without being present in demyelinated lesions. HHV-6B, which typically causes exanthema subitum, can also cause encephalitis, particularly in transplant patients, and infects oligodendrocytes (Albright et al., 1998). An additional virus in herpesviridae, HSV-1, has recently been shown to infect cells of the OL lineage. The oligodendroglial cell line KG-1C supports productive infection of HSV-1 (Bello-Morales et al., 2005). OPCs were able to be infected although efficiency of infection increased as cells were differentiated. Infection of OPCs promoted rather than prevented their differentiation (Bello-Morales et al., 2014). It is unclear whether these results relate to encephalitis caused by HSV-1 or potential association with demyelinating disease.

Semliki Forest virus (SFV) SFV is an alphavirus in the family Togaviridae and as such has a positive-polarity RNA genome and an enveloped capsid. The virus is spread by mosquitos and can infect humans, resulting in a mild febrile disease. In mice, however, some strains of SFV are neuroinvasive and cause a demyelinating disease once in the CNS (Fazakerley et al., 2006). SFV also directly infects oligodendrocytes, although virus infection of these cells may not be directly cytopathic. Demyelination was observed but reduced in infected nude mice, whereas infected beige mice (lacking natural killer cells) displayed enhanced demyelination. Remyelination occurred rapidly in nude mice (Gates et al., 1984). Infected oligodendrocytes appeared to dedifferentiate in the optic nerve (Butt et al., 1996). Other studies, however, did not observe demyelination in the absence of an inflammatory immune response (Fazakerley and Webb, 1987). Intracerebral inoculation of SFV strain A7(74) led to widespread infection of CNPase-positive oligodendrocytes in the white matter of the corpus callosum. In this system, myelin loss was not dependent on an inflammatory response (Fazakerley et al., 2006). SFV A7 (74) has also been modified to express fluorescent proteins and used as a tool to study oligodendrocytes in culture. In one such study, SFV vectors were shown to infect NG2-positive, CNPase-negative pro-oligos in addition to mature oligodendrocytes in a hippocampal slice culture in vitro (Haber et al., 2009). It is unclear whether OPCs were also infected by SFV, as the infected cells shown all appear to have developed multiple membrane extensions.

Induced pluripotent stem cells (iPSCs) as a model system to study demyelinating viruses Challenges with isolation of primary OPCs Deriving induced pluripotent stem cells (iPSCs) from mice or human somatic cells and differentiating to oligodendrocyte progenitor cells (OPCs) seem to be

iPSCs as a model system

advantageous over primary OPCs (p-OPCs) to model TMEV infection (Cefalo et al., 2016). Obtaining sufficient quantities of p-OPCs from rats, mice, and humans is challenging, thereby limiting the experimental design and replicates. Multiple methods for isolating p-OPCs are used including fluorescence-activated cell sorting (FACS) by specific markers, isolation based on adherent properties, differential centrifugation, and immunopanning (Chen et al., 2007). Isolating OPCs from mice is more challenging compared to rats since their cell surface antigens are different. Differentiation of mouse OPCs gives rise to multiple cell types of the glia. Increasing pure populations of p-OPC cell numbers with static cultures postisolation need to be improved. In mice, OPCs can be isolated using specific oligodendrocyte (OL) markers such as neuro-glial antigen-2 (NG2), which is a proteoglycan, A2B5, which is a cell surface ganglioside, platelet-derived growth factor receptoralpha (PDGF-Ra), fatty acid binding protein-7 (FABP-7), and neural cell adhesion molecule (NCAM). In addition, pre OLs, immature and mature OLs are removed using O4, GalC, RAN2, and MOG markers (Kuhn et al., 2019). Immunopanning methods rely on specific monoclonal antibodies that need high purity, which can be very expensive, and multiple antibodies are required to eliminate microglia, astrocytes, immature and mature oligodendrocytes to isolate majority of the cells containing OPCs (Ogawa et al., 2011). Rat p-OPC isolation uses multiple steps that include exposing the rat brain cell suspension to a series of FBS concentrations followed by rocking/shaking and adding conditioned medium from neuroblastoma cells to get adherent OPCs that can further be expanded minimally (Zhu et al., 2014). It is important to know the source of the isolated OPCs from the rat brain as expression of Foxg1 and Hoxc8 identifies OPCs from the forebrain and the spinal cord, respectively (Horiuchi et al., 2017). Cell numbers of p-OPCs can be increased and maintained by immortalizing cell lines in animal models. Some cell lines such as Oli-neu, CG-4, and OLN-93 may be suited, but constitutive expression of viral proteins during immortalizing cells can introduce confounding effects that make it difficult to study cellular responses to virus infection. Another alternative is to generate reporter rodent models using fluorescent tagged OPC markers (MBP, OLIG2, SOX10), these models also have similar issues with cost and animal handling (Li et al., 2016a).

Advantages of iPSCs To date, iPSCs have been a robust in vitro model system used for drug discovery and development, disease modeling (disease in a dish), regenerative and personalized medicine (Rowe and Daley, 2019). But they also serve as valuable tools for investigating the molecular mechanisms for many viral infections (Kim et al., 2019). Yamanaka’s seminal paper in 2006 for inventing iPSCs changed the perspectives of using animals as model organisms to pluripotent stem cells in a dish (Takahashi and Yamanaka, 2006). iPSCs have typical characteristics that include unlimited expansion, self-renewing capacity, maintaining their pluripotent state, and differentiating into most tissues of the body (ectoderm, endoderm, and mesoderm). iPSCs



CHAPTER 6 Pluripotent stem-cell-derived oligodendrocyte progenitors

also have a more robust DNA repair mechanism that is not activated in other somatic cells, thereby conferring advanced mechanisms for repairing stress-related DNA mutations (Yoshimura, 2019). Most of the early iPSCs developed use an engineered lentivirus that is made nonreplicative containing the Yamanaka factors responsible for reprogramming gene expression in somatic cells to a more pluripotent state that shows close similarities in gene expression with embryonic stem cells (Teshigawara et al., 2017). This holds true for human, mouse, and rat. However, recent advances in the discovery of small molecules, mRNA, and protein-based iPSC generation has replaced lentiviral-induced reprogramming with virus-free generation of iPSCs (Steinle et al., 2017). Even though the efficiency of colony formation and iPSC generation is decreased compared to virus-induced reprogramming, these iPSCs have a more stable genome (Martin, 2017). Nonintegrating viruses such as the Sendai virus used to make iPSCs have become more popular in modeling diseases in vitro (Tai et al., 2018). It is also important not to have any integrated viral DNA in the iPSCs to study and model viral infections. A cumulative approach on cost-effectiveness, reduced labor time and space needs, sensitivity to stressors, and an abundant supply of cells (iPSCs) make them a more suitable system than mammalian model organisms for basic and applied research (D’Antonio et al., 2017).

Brain organoids with iPSCs to model viral CNS infection Human iPSC-derived brain organoids generated in vitro can depict and mimic similar developmental stages, cellecell interactions/organization in vitro (Pasca, 2019). After embryoid body formation through aggregation, spinner flasks are used to amplify spheroids (>30 days) to grow cerebral and region-specific organoids (Yang et al., 2016b). OPCs can be developed by exposing these spheroids to growth factor cocktail (PDGF, EGF, bFGF, N2/B27, T3, and arachidonic acid). Guided differentiation of entire brain organoids, which have restricted cell types such as OPCs or OLs, can further be fused with spheroids (loaded with TMEV or other viruses) to study viral entry, persistence, and their life cycle in vitro (Pasca, 2019). Quantification studies can be achieved by 3D live cell imaging, RNA-seq, GWAS, and exomeseq. As stated, shiverer mutations (in myelin) prevent transit of TMEV from axon to myelin and thereby prevent viral persistence (Roussarie et al., 2007). These interactions can be better understood in vitro using cerebral organoids and organoids containing OLs, microglia, and astrocytes (Antonucci and Gehrke, 2019). Due to the flexibility of starter iPSCs, which can be genetically engineered using CRISPRCas9, single protein changes can be developed and stage-specific infection of TMEV and other oligodendrotropic viruses can be achieved. CXCR2 (and its ligand CXCL1) is shown to play a protective role to OLs during TMEV and the John Howard Mueller (JHM) strain of mouse hepatitis viral infection (JHMV) (Marro et al., 2019; Hosking et al., 2010). We can identify clues of myelin protection, induction of apoptosis, chemokine -induced host defense, or disease exacerbation by altering CXCR2 gene expression levels in cerebral and OPC organoids (Bergmann et al., 2006).

Conclusions and future perspectives

Conclusions and future perspectives The mechanisms behind how the viruses discussed in this chapter disrupt myelination are ready to be explored using iPSC-derived OPCs, pro-OLs, oligodendrocytes, and other cells in brain organoids. These cells overcome many experimental limitations posed by primary culture, such as maintenance of mouse breeding colonies, timed matings, microdissection, enzymatic disruption, and immunopanning. iPSCderived OPCs can be produced in large numbers and enable transcriptome profiling to uncover which genes are dysregulated as a result of virus infection (Lager et al., 2018). Moreover, profiling gene expression in these cells may uncover new gene candidates that regulate OPC migration, proliferation, and differentiation. Studies using RNA-sequencing in stem-cell-derived OPCs infected with TMEV were done in mouse epiblast-derived stem cells (Benner et al., 2016). These cells, although capable of showing how TMEV disrupts OPC gene expression, were somewhat challenging to grow, less accessible, and limited to a few genetic backgrounds (Najm et al., 2011). An additional advantage of iPSC-generated OPCs and oligodendrocytes is that they can be produced from any wild-type or mutant genetic backgrounds. For example, OPCs were produced from shiverer mice, which were deficient to ensheath axons in coculture (Lager et al., 2018). This advancement will enable virologists to discern whether there are cell-intrinsic factors in OPCs from different strains that affect the ability of viruses to infect these cells and disrupt myelination. The viruses included in this chapter are diverse in terms of host range, type of genome, replication strategies, and pathogenesis. Yet, despite these differences, these viruses all have the ability to infect cells of the oligodendrocyte lineage and disrupt myelination. Although these viruses do not appear to share a common mechanism for disrupting myelination, there are some similarities that are notable. For example, in both TMEV and MHV infections, demyelination occurs through immune-mediated mechanisms but direct infection of oligodendrocytes is a requirement for disease. In contrast, viruses such as CDV and MLV cause demyelination in the absence of a detectable inflammatory response. What are the reasons for these differences? Studies that utilize iPSC-derived OPC and oligodendrocytes to examine gene expression in these different models may be useful to determine why inflammation is required in some infections and not others. While it is challenging to determine the precise contribution of viral gene expression versus the host’s response to infection in disruption of oligodendrocytes, more work is necessary to understand these interactions. A clear role exists for the immune system in demyelinating diseases, although the cases presented here demonstrate that direct infection of oligodendrocytes also plays a role. Drugs that stimulate OPC proliferation to promote remyelination in vivo have promise for demyelinating diseases, and these drugs can also be useful tools to explore the relative contributions of the immune response to remyelination (Najm et al., 2015). For example, clobetasol, a drug that induces OPC proliferation, possesses immunomodulatory effects. However, compounds that promote OPC proliferation without



CHAPTER 6 Pluripotent stem-cell-derived oligodendrocyte progenitors

modulating the immune response, such as miconazole, could be useful to discern whether destruction of oligodendrocytes in virus infections and lack of remyelination are immune-mediated or due to direct infection of OPCs. The mechanism by which TMEV causes such a profound shift in gene expression in OPCs remains unknown. While a specific virus protein could modulate gene expression, such as the Leader protein, which disrupts nuclear-cytoplasmic trafficking (Reviewed in (Freundt et al., 2018), a more generalizable mechanism may be that the signaling cascade necessary for OPC maturation is superseded by innate immune signaling, such as induction of type I interferon and other stress-related kinases that can be triggered by virus infection. Interferon has been shown to modulate myelination in TMEV-IDD, but the effect is somewhat complicated; while shortterm treatment caused a reduction in demyelination, long-term treatment led to greater demyelination. Remyelination was also observed and increased in longterm treatment, which suggests that OPC development can occur normally in the presence of type I interferon (Njenga et al., 2000). Furthermore, induction of innate immune signaling seems to have different outcomes depending on the receptors involved. For example, while TLR2 and TLR4 activation inhibits oligodendrocyte maturation, activation of TLR3 by poly I:C resulted in increased MBP expression in OPCs (Yamashita et al., 2017). Thus, how these viruses have similar effects on OPC differentiation despite interacting with distinct innate-signaling molecules remains an open question. As many fundamental discoveries in biology have come from studying the pathways that are perturbed by viruses, it is likely that continued investigation into viral infection of OPCs will lead to novel insights about how oligodendrocytes develop and function in the CNS.

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iPSCs for modeling hepatotropic pathogen infections


Guglielmo Bovea, Ann-Kathrin Mehnerta, Viet Loan Dao Thi Schaller Research Group, Center of Infectious Diseases, Department of Virology, Heidelberg University Hospital, Heidelberg, Germany

Chapter outline The liver is a target organ for many pathogens .........................................................150 Hepatitis viruses.....................................................................................................154 Hepatitis A virus (HAV) ...............................................................................154 Current knowledge of the HAV life cycle.................................................... 156 Established and novel in vitro and in vivo models to study HAV infection ....... 157 Open questions in HAV research.............................................................. 158 Hepatitis B virus (HBV)...............................................................................158 Current knowledge of the HBV life cycle.................................................... 158 Established and novel in vitro and in vivo models to study HBV infection....... 159 Open questions in HBV research ............................................................. 161 Hepatitis C virus (HCV) ...............................................................................161 Current knowledge of the HCV life cycle.................................................... 162 Established and novel in vitro and in vivo models to study HCV infection ....... 163 Open questions in HCV research.............................................................. 164 Hepatitis delta virus (HDV) ..........................................................................164 Current knowledge of the HDV life cycle.................................................... 165 Established and novel in vitro and in vivo models to study HDV infection....... 166 Open questions in HDV research ............................................................. 166 Hepatitis E virus (HEV) ...............................................................................167 Current knowledge of the HEV life cycle .................................................... 168 Established and novel in vitro and in vivo models to study HEV infection ....... 169 Open questions in HEV research .............................................................. 170 Plasmodium............................................................................................................170 Current state of knowledge on Plasmodium liver stage ...................................171


Equal contribution.

iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00013-4 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

Established and novel in vitro and in vivo model systems for the Plasmodium LS..........................................................................................172 Open questions Plasmodium liver stage research...........................................173 Addressing open questions in hepatotropic infection research with HLCs ...................173 Systems integrating diverse hepatic cell types to improve liver pathogenesis studies ...................................................................................................................175 3D systems to study hepatotropic infections .............................................................177 Personalized modeling and treatment of hepatotropic infections................................180 Limitations of iPSCs and future directions for the study of hepatotropic infections ......181 Outlook ..................................................................................................................182 Acknowledgments ...................................................................................................183 References .............................................................................................................183 Abstract The liver acts as an important filter for the body and is thus exposed to a wide range of pathogens. In order to study a hepatotropic pathogen’s intricate life cycle as well as associated disease and pathogenesis, authentic culture systems are vital. As such, human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells have recently been proposed as an attractive alternative to conventionally used primary human hepatocytes and hepatoma cell lines. Here, we summarize current knowledge on the biology of major liver pathogens, including hepatotropic viruses AeE as well as the malaria parasite Plasmodium. We discuss the available systems used to study them, remaining questions in the field, and how iPSC technology may help to solve them. We also summarize limitations of available iPSC-derived hepatic systems and recent efforts to overcome them. With their ability to provide a universally susceptible system for personalized, complex, and at the same time isogenic infection models, we believe that iPSCs have the potential to transform the future of hepatotropic infectious disease medicine. Keywords: Antiviral treatment; Cell polarization; Cell culture adaptation; Cocultures; Disease models; Hepatitis virus; Hepatocyte-like cells; Innate immunity; iPSC/ESC; Liver architecture; Liver organoids; Pathogenehost interactions; Patient isolates; Personalized models; Plasmodium.

The liver is a target organ for many pathogens The liver is the body’s largest gland and plays an active role in maintaining physiological homeostasis. Many viral, bacterial, and parasitic pathogens infect the liver cells, including hepatitis AeE viruses (HAVeHEV) and the parasite Plasmodium (Fig. 7.1). Infection with these pathogens often leads to inflammation of liver tissue, referred to as hepatitis. Depending on the pathogen and its course of infection, illness can range from asymptomatic and self-limiting to fulminant and chronic disease. Chronic virus infection, often entailing persistent liver injury, can lead to fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). In addition, hepatic metabolic functions are altered, as reflected by the deposition of free fatty acids in the liver cells. The ensuing oxidative stress response can result in fatty liver

The liver is a target organ for many pathogens

FIGURE 7.1 Pathogen transmission and schematic structure of the liver. Nonenveloped HAV and HEV are transmitted fecal-orally and have to cross the intestinal barrier. They likely infect intestinal epithelial cells and are released as quasi-enveloped particles into the bloodstream where they can reach the liver. In the liver, HAV and HEV infect the hepatocytes and are released as quasi-enveloped particles back into the blood or as highly infectious, nonenveloped particles into the bile. Enveloped HBV, HCV, and HDV particles are transmitted directly through the blood. When they reach the liver, they also infect the hepatocytes, from which they are released as enveloped particles back into the blood and potentially as noninfectious, nonenveloped particles into the bile. Plasmodium sporozoites are injected by mosquitos into the skin where they can enter the blood vessels. The transformation of sporozoites into merozoites occurs in the liver inside the hepatocytes. Merozoites can then invade the erythrocytes and initiate the disease. HSCs, hepatic stellate cells; KCs, Kupffer cells; LSECs, liver sinusoidal endothelial cells; pDCs, plasmacytoid dendritic cells.



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

disease called hepatic steatosis. According to the World Health Organization (WHO), chronic hepatitis B and C, for example, affect about 325 million people worldwide and are responsible for w1.4 million deaths per year (World Health Organization, 2017). In order to treat infections and their associated diseases effectively, it is mandatory to study and understand a pathogen’s biology as well as its interaction with the host cell. The major target cell types for hepatotropic infections are the liver epithelial cells (hepatocytes). While most epithelial cells are columnar polarized, with a single apical opposite of a single basal membrane (Fig. 7.3), hepatocytes have a special polarization with multiple apical and basal membranes (Figs. 7.1e7.3, further details in part 6.) (reviewed in Treyer and Mu¨sch (2013)). This polarity plays a major role in the entry and release mechanisms of all hepatotropic viruses. Another important aspect of host-pathogen interaction is the innate immune response, the first-line host defense (reviewed in Hoffmann et al. (2015)). It is essentially based on recognition of pathogen-associated molecular patterns (PAMPs) by specific pattern recognition receptors (PRRs), which initiates a complex signal transduction cascade. This cascade leads to the expression and secretion of interferons (IFNs), e.g., type I (IFNa and -b) and type III (IFN-l) IFNs. IFN signaling leads to the expression of IFNstimulated genes (ISGs) via Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. ISGs can then interfere with the early and late stages of the virus life cycle, which is critical for the elimination of virus infections (reviewed in Schneider et al. (2014)).

FIGURE 7.2 Applications of differentiated cells from iPSCs for infectious disease modeling.

The liver is a target organ for many pathogens

FIGURE 7.3 iPSC-derived coculture systems of increasing complexity. Patient-specific iPSCs obtained by cell reprogramming or available iPSC lines genetically modified by CRISPR-Cas9 to harbor a single nucleotide polymorphism (SNP) of interest can be differentiated to a variety of liver cell types. These cell types can be cultured in different 2D and 3D coculture setups, including direct 2D coculture of nonparenchymal cells (NPCs) with nonpolarized HLCs (left), 2D contact-free coculture of NPCs with columnar polarized HLCs on Transwells (middle), and 3D coculture of NPCs with iPSCderived liver organoids (right).

A major issue of hepatotropic infectious disease research has historically been the lack of appropriate cell culture models (reviewed in Wang et al. (2019)). Hepatoma cell lines are the most widely used culture model in the field. While they are easy and relatively cheap to culture, they pose limitations. For example, the hepatoma cell line Huh-7.5, a subclone of the Huh-7 cell line, carries a mutation in the innate immune sensor retinoic acid-inducible gene I (RIG-I) (Sumpter et al., 2005). The HepG2 cell line expresses only to low levels the metabolizing enzymes of the cytochrome P450 (CYP450) family and is therefore unsuited for drug evaluations (Westerink and Schoonen, 2007). This aberrant nature of cancerous hepatoma cells often renders them nonpermissive to infection and thus limits investigations of the pathogen’s life cycle as well as the effect of the infection on the cellular proliferation, metabolism, and apoptosis pathways.



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

The use of primary human hepatocytes (PHHs), the most physiologically relevant cell system, is restricted by limited availability and high donor-to-donor variability (Rogue et al., 2012). As PHHs do not proliferate in vitro, their genetic manipulation and thus, studies of host factors of interest are restricted (reviewed in Bhatia et al., 2014). Furthermore, PHHs quickly dedifferentiate upon plating in cell culture and therefore limit studies of chronic infection. Isogenic complex systems that better mimic the cellular diversity of the liver (Fig. 7.1) are difficult to generate with either system, as cancer cell lines are derived from different donors, and obtaining different primary cell types from the same donor is extremely difficult. Recent advances have been made in differentiating human embryonic or induced pluripotent stem cells (ESCs/iPSCs) to a variety of cell types, including hepatocyte-like cells (HLCs) (Si-Tayeb et al., 2010) (Fig. 7.2). With their ability of self-renewal, the stem cell origin provides superior reproducibility and less donor-to-donor variability than PHHs, while HLCs provide better physiological responses than hepatoma cells (reviewed in Schwartz et al. (2014)). In this chapter, we summarize the latest research on hepatotropic pathogens, discuss the challenges and open questions, and how iPSC systems may help in addressing some of them.

Hepatitis viruses Hepatitis viruses (HAVeHEV) can infect the liver and often cause severe diseases. While they all mainly infect hepatocytes (Fig. 7.1), they differ in their biological properties and pathogenesis. Infection with hepatitis viruses can lead to both acute and chronic disease (Table 7.1), with the latter posing an increased risk of developing malignant HCC. As millions of people are infected each year (World Health Organization, 2017), hepatitis viruses represent a major threat to the global population.

Hepatitis A virus (HAV) HAV was discovered in 1973 (Feinstone et al., 1973) and is a nonenveloped, positive-sense, single-stranded RNA virus, classified in the Hepatovirus genus within the Picornaviridae family (Zell et al., 2017). Six major genotypes (GTs) exist (Smith and Simmonds, 2018) of which three infect humans and cause an estimated 1.4 million cases of hepatitis A every year (Havelaar et al., 2015). Transmission principally occurs fecal-orally via the ingestion of contaminated food and water (Fig. 7.1), but virus spread through sexual contact and drug use is also possible (reviewed in Jacobsen (2018)). HAV infections are usually acute and self-limiting. While frequently asymptomatic in children, most adults develop symptomatic illness with severe or fulminant hepatic manifestations (reviewed in Shin and Jeong (2018)). Therapy is generally supportive, without specific treatment (Shin and Jeong, 2018). Despite improvements in hygiene and the availability of an efficient

Table 7.1 Hepatitis viruses overview. Structure Genome size Virus particle Transmission Course Annual infections Chronic infections Incubation timea Vaccine






(þ) ssRNA 7.5 kb Non-/quasienveloped Fecal-oral

rcDNA 3.2 kb Enveloped

(þ) ssRNA 9.6 kb Enveloped

() ssRNA 1.7 kb Enveloped

Sexual/parenteral/ vertical Acute/chronic e

Sexual/parenteral/ vertical Chronic 1.75 mln (World Health Organization, 2017)


(þ) ssRNA 7.2 kb Non-/quasienveloped Fecal-oral


257 mln (World Health Organization, 2017) 6e24



Acute 1.4 mln (Havelaar et al., 2015) e

Acute/chronic e

Acute/chronic 20 mln (World Health Organization, 2017)

71 mln (World Health Organization, 2017) 2e24

12e70 mln (Stockdale et al., 2020; Chen et al., 2019) 2e10



Yes (HBV)



Number of weeks before symptoms onset; kb, kilobases; mln, million; (), negative-sense; (þ), positive-sense; e, no estimates available; rcDNA, relaxed circular DNA; ssRNA, single-stranded RNA.


Hepatitis viruses 155


CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

vaccine, recent outbreaks in developed countries have been reported (Snyder et al., 2019). As global tourism, human migration, anti-vaccine campaigns, and food consumption are increasing, HAV infection remains a potential menace to public health.

Current knowledge of the HAV life cycle It was only recently discovered that HAVexists in two different forms: nonenveloped (nHAV) in stool and, despite the absence of viral glycoproteins, in a so-called quasienveloped (eHAV) form in the bloodstream (Feng et al., 2013) (Fig. 7.1). After ingestion, nHAV first crosses the intestinal barrier, by either translocation (Dotzauer et al., 2005) or direct infection of enterocytes, which are columnar polarized (Fig. 7.3). The intestinal Caco-2 cell line, for example, is permissive for HAV infection (Hirai-Yuki et al., 2016; Blank et al., 2000). In case of infection, it is hypothesized that nHAV enters enterocytes apically. After entry, the genome replicates, progeny virus is assembled and then secreted from the enterocyte’s basal membrane. The into the bloodstream released virus is likely quasi-enveloped (Feng and Lemon, 2014). n/eHAV particles can then reach the liver and infect the hepatocytes from their basolateral membrane (Fig. 7.1). Yet, secretion from polarized infected Caco-2 cells occurred almost exclusively from the apical side (Hirai-Yuki et al., 2016; Blank et al., 2000). This suggests that only a small fraction reaches the liver; however, validation with primary systems would be desirable. Initial attachment to hepatocytes is mediated by the interaction of phosphatidylserine (PtdSer) on eHAV particles with the T-cell immunoglobulin and mucin domain 1 (TIM1) protein, also known as HAV cellular receptor 1 (HAVCR1) (Kaplan et al., 1996; Feigelstock et al., 1998; Moller-Tank et al., 2013). Yet, TIM1 expression was not essential for productive infection of Huh-7.5 and Vero cells (Das et al., 2017). An elegant study by Rivera-Serrano et al. showed that early steps of entry into hepatocytes of both nHAV and eHAV are mediated by integrin b1 and clathrin-dependent endocytosis (Rivera-Serrano et al., 2019). While nHAV subsequently enters the cell from late endosomes, eHAV proceeds to lysosomes where the quasi-envelope is enzymatically degraded (Rivera-Serrano et al., 2019). For both virus forms, the binding of the capsid to the GD1a ganglioside receptor (Das et al., 2020) and the disruption of endolysosomal membranes by the membrane-penetrating activity of the capsid protein VP4 (Shukla et al., 2014) may lead to uncoating. The replication cycle starts with translation of the HAV polyprotein, which is processed by the HAV cysteine protease 3Cpro (Schultheiss et al., 1994; Gosert et al., 1996) to yield four structural (VP4, VP2, VP3, and VP1pX) and six nonstructural proteins (2A, 2B, 3A, 3B, 3Cpro, and 3Dpol) (Lemon et al., 2017). HAV genome replication by the RNA-dependent RNA polymerase (RdRp) 3Dpol likely takes place within a virus-induced tubular-vesicular network at the endoplasmic reticulum (ER) (Teterina et al., 1997; Gosert et al., 2000). New capsids are assembled from 12 pentamers, composed of protomers containing the VP4-2 precursor VP0, VP3, and VP1pX (Stapleton et al., 1993). The latter is cleaved by a cellular protease to separate pX from VP1 (Graff et al., 1999). Interestingly, pX can only be found as an

Hepatitis viruses

extension of VP1 on the capsid of eHAV but not on nHAV particles (Feng et al., 2013; Anderson and Ross, 1990). Packaging of the RNA genome is preceded by cleavage of VP0 (Bishop and Anderson, 1993), but the mechanism of encapsidation is poorly understood. HAV hijacks the endosomal sorting complexes required for transport (ESCRT) pathway for its progeny release: The lipid quasi-envelope is acquired by budding into endosomes in multivesicular bodies (MVBs), which is mediated by the interaction between pX and the apoptosis-linked gene 2-interacting protein X (ALIX) (Feng et al., 2013; Jiang et al., 2020). Fusion of MVBs with the plasma membrane releases eHAV from hepatocytes basolaterally into the bloodstream and apically into the bile (Fig. 7.1) where the quasi-envelope is likely removed by bile acids (Hirai-Yuki et al., 2016). Using a subclone of HepG2 cells, which was selected for its ability to columnar polarize in Transwells (Snooks et al., 2008), Hirai-Yuki et al. showed that similar amounts of eHAV progeny were released from both apical and basal membranes (Hirai-Yuki et al., 2016). Only treatment with exogenous human bile acids converted eHAV to nHAV, which is in agreement with a study showing that HepG2 cells do not produce matured bile acids (Everson and Polokoff, 1986).

Established and novel in vitro and in vivo models to study HAV infection Many HAV studies are based on HepG2 and Huh-7.5 cell lines as well as PHHs; nonhepatic cells are also permissive for HAV (reviewed in Kanda et al. (2020)). Since primary HAV isolates replicate inefficiently in cell culture, HAV strains have been adapted to a variety of primate and nonprimate cell lines by serial passaging (Provost and Hilleman, 1979; Daemer et al., 1981; Binn et al., 1984). During this process, the viral genome acquires mutations, which can result in altered biology. This encludes enhanced replication efficiency and cytopathicity (Emerson et al., 1993; Lemon et al., 1991; Zhang et al., 1995). Based on these adapted strains, subgenomic replicons have been generated by replacing the capsid-encoding sequence with reporter genes (Gauss-Mu¨ller and Kusov, 2002; Esser-Nobis et al., 2015; Yang et al., 2007). Of note, Huh-7 cells stably replicating a selectable HAV replicon were cured by IFN and became thereafter more permissive to HAV infection than the parental line (Konduru and Kaplan, 2006). The use of iPSC-derived HLCs for HAV research has been limited so far. We recently developed a differentiation protocol to generate ESC/iPSC-derived columnar polarized HLCs with defined apical and basal membranes on Transwell filters (Dao Thi et al., 2020) (Fig. 7.3). Polarized HLCs were permissive for infection with the cell-culture-adapted HAV HM175/18f strain but showed cytolytic effects (Dao Thi et al., 2020). Less cytopathic, nonadapted HAV isolates are likely better suited for these studies, but this remains to be validated. In vivo, chimpanzees were fundamental for immunological studies (Lanford et al., 2011) and vaccine development (Purcell et al., 1992). Small animal models permissive to HAV infection, such as pigs and shrews (Song et al., 2016; Zhan et al., 1981), or to HAV-related viruses, such as ducks and seals (Ou et al., 2017; Anthony et al., 2015), were also explored. A recent breakthrough study demonstrated



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

that mice lacking expression of the IFN-a/b receptor 1 or the mitochondrial antiviral signaling protein (MAVS) can support HAV replication to high viral titers (Hirai-Yuki et al., 2016).

Open questions in HAV research Numerous steps of the HAV life cycle remain unexplored. The HAV cell entry receptor needs to be identified (Das et al., 2019). Detailed mechanistic insights into virus uncoating, genome encapsidation, and virus secretion are lacking. Directional infection and release from both intestinal cells and hepatocytes remain to be characterized in greater detail, ideally with primary polarized cell systems. The precise roles of innate immune recognition and its suppression in liver injury as well as the acute nature of HAV infection remain elusive. Cytotoxic CD8-positive T-cells and secretion of interleukin-15 (IL-15) have recently been implicated in mediating liver damage during HAV infection (Kim et al., 2018), offering some potential for clinical exploitation of the latter as a therapeutic target. Whole-exome sequencing revealed the association of an interleukin-18-binding protein (IL18BP) gene deletion with fulminant liver disease in a young individual (Belkaya et al., 2019). However, the precise mechanisms and the range of cell types involved are unknown.

Hepatitis B virus (HBV) First evidence of HBV was discovered in 1965 with the Australia antigen (Blumberg et al., 1965), and only a few years later, it was linked to viral hepatitis (Blumberg et al., 1969). HBV is a hepatotropic virus with a DNA genome. There are ten different HBV genotypes (AeJ) in the Orthohepadnavirus genus within the Hepadnaviridae family(Magnius et al., 2020). Transmission mainly occurs perinatally and through contaminated body fluids (reviewed in Yuen et al. (2018)) (Fig. 7.1). In immune-competent adults, HBV infection is mostly self-limiting with rare cases of fulminant acute hepatitis (Yuen et al., 2018). If not cleared, which is the case in more than 90% of infants under one year of age, the infection becomes chronic (Yuen et al., 2018). Fluctuations in virus replication and associated immune responses cause liver damage, which can progress to cirrhosis and HCC (reviewed in Xie (2017)). Despite the introduction of an efficient vaccine in 1981 (reviewed in Meireles et al. (2015)), an estimated 257 million people are still chronic carriers of HBV (World Health Organization, 2017). Current treatment includes administration of IFN-a and antiviral nucleos(t)ide analogues (reviewed in Lok (2018)). Even though the latter suppress the viral load in serum and slow the disease progression, complete elimination is usually not achieved (Lok, 2018).

Current knowledge of the HBV life cycle In serum, HBV can be found as DNA-containing infectious virions (Dane particles) (Dane et al., 1970) (Fig. 7.1), noninfectious RNA-containing particles (reviewed in Hu and Liu (2017)), as well as nucleic-acid-free filamentous and spherical subviral particles (SVPs) (Hu and Liu, 2017). The partially double-stranded, relaxed circular

Hepatitis viruses

DNA (rcDNA) contains four partially overlapping open reading frames (ORFs) (reviewed in Yuen et al. (2018)) that encode seven proteins: polymerase (Pol), capsid protein (C), hepatitis B e antigen (HBeAg), X protein (HBx), and three forms of the hepatitis B surface antigen (HBsAg), including large (LHBs), medium (MHBs), and small HBsAg (SHBs). Heparan sulfate proteoglycans (HSPGs) mediate HBV attachment to hepatocytes (Schulze et al., 2007), followed by binding of the LHBs PreS1 region to the sodium taurocholate cotransporting polypeptide (NTCP) receptor (Ni et al., 2014; Yan et al., 2012). Entry probably proceeds by clathrin-mediated endocytosis and membrane fusion in late endosomes (reviewed in Herrscher et al. (2020)). Through microtubular transport (Rabe et al., 2006), the capsid reaches the nuclear pore complex where it dissociates, releasing the rcDNA and associated Pol into the nucleus with the help of importin a/b (reviewed in Gallucci and Kann (2017)). The genome is converted into covalently closed circular DNA (cccDNA) by the cellular DNA repair machinery (reviewed in Schreiner and Nassal (2017)). In the nucleus, the cccDNA is shielded from the cellular innate immune response and can persist for a long time, serving as a reservoir in chronic carriers (reviewed in Nassal (2015)). Integration into recurrent sites of the host genome can lead to chromosomal instability (reviewed in Tu et al. (2017)). In addition, it provides replicationindependent expression of mutated or truncated forms of HBsAg (Wang et al., 2003; Liu et al., 2009) and HBx (Kumar et al., 1996), which have been shown to stimulate hepatocyte expansion (Fan et al., 2001) and induce stem-cell like properties (Ng et al., 2016), respectively. The cccDNA hijacks the cellular RNA polymerase II (RNA pol II) (Rall et al., 1983) to produce a pregenomic RNA (pgRNA), which encodes Pol, C, and HBeAg, as well as three subgenomic RNAs, which encode the remaining viral proteins (reviewed in Seeger and Mason (2015)). Interaction of Pol with the 50 -end of the pgRNA (Pollack and Ganem, 1993) triggers encapsidation after which the pgRNA is reverse transcribed into rcDNA (Seeger and Mason, 2015). Mature nucleocapsids can interact with HBsAgs on the ER (Bruss and Thomssen, 1994) and bud into MVBs (Watanabe et al., 2007), releasing enveloped virions through the ESCRT pathway (Lambert et al., 2007) into the bloodstream (Fig. 7.1). Additionally secreted HBeAg and SVPs decorated with HBsAg can act as immunological decoys in the blood (Rydell et al., 2017; Walsh and Locarnini, 2012). The presence of noninfectious HBV DNA (Zheng et al., 2015) and HBsAgs (Hoefs et al., 1980) in the feces of chronic carriers suggests that some HBV particles are also released apically from infected hepatocytes into the bile (Fig. 7.1).

Established and novel in vitro and in vivo models to study HBV infection For years, HBV studies were limited to PHHs and differentiated HepaRG cell culture models (reviewed in Li et al. (2020)). As HBV did not readily infect hepatoma cells, plasmid transfection to overcome entry (Sells et al., 1987; Tsurimoto et al., 1987) or cell lines with integrated HBV genomic DNA that produced infectious particles (Tsurimoto et al., 1987; Ladner et al., 1997; Weiss et al., 1996) were the only tools to study HBV biology.



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

In a major breakthrough and with the help of peptides mimicking the N-terminus of the LHBs (Myrcludex B) (Ni et al., 2014), the bile acid transporter NTCP was discovered as the HBV entry receptor (Ni et al., 2014; Yan et al., 2012). Ectopic expression of NTCP rendered human hepatoma cells (Ni et al., 2014; Yan et al., 2012) and even a mouse hepatic cell line (Lempp et al., 2016) susceptible to HBV infection, enabling an unprecedented number of molecular HBV studies. Bipotent HepaRG cells become susceptible to HBV infection upon differentiation to hepatocyte-like cells (Gripon et al., 2002) or ectopic expression of NTCP (Sakurai et al., 2017). Primary tupaia hepatocytes (PTHs) also support HBV infection and helped identify the HBV entry receptor (Yan et al., 2012). PHHs naturally express NTCP (Yan et al., 2012) and support primary isolate infection (Gripon et al., 1988; Winer et al., 2017). Several systems such as micropatterned (MPCC) (Shlomai et al., 2014) and self-assembled (SACC) (Winer et al., 2017) PHH cocultures with mouse fibroblasts, three-dimensional (3D) microfluidics devices (Ortega-Prieto et al., 2018), or the application of a combination of five chemicals (Xiang et al., 2019) improved the maintenance of PHHs and enabled chronic HBV studies. Comparable to PHHs, ESC/iPSC-derived HLCs also express NTCP (Xia et al., 2017). In 2014, Shlomai et al. demonstrated that iPSC-derived HLCs were permissive to HBV isolate infection, followed by other studies confirming the utility of HLCs in HBV research (Sakurai et al., 2017; Kaneko et al., 2016). Only recently, Xia et al. reported enhanced infection efficiency of ESC/iPSC-derived HLCs owing to an improved differentiation protocol (Xia et al., 2017). Their HLCs supported HBV spread and longterm culturing while maintaining a stable differentiation state, thus enabling chronic HBV studies. In vivo, HBV research is restricted by the narrow host tropism (Guo et al., 2018). Chimpanzees taught us about innate immunity (Wieland et al., 2004) and chronicity (Ruiz-Opazo et al., 1982) in HBV infection and facilitated the development of the HBV vaccine (Buynak et al., 1976). Natural hosts of closely related hepdnaviruses including woodchuck (Tennant and Gerin, 2001) or duck HBV (Schultz et al., 2004) can serve as surrogate animal models. However, these viruses use different receptors (Tong et al., 1999) and differ in the levels of cccDNA established after infection (Zhang et al., 2003). Moreover, duck hepatitis B virus lacks the HBx protein (Chang et al., 2001). Tree shrews are permissive to human HBV (Wang et al., 2012; Walter et al., 1996). Transgenic mice expressing single HBV proteins such as HBsAg and HBx or the complete HBV genome were created to study pathogenesis and test antiviral agents (reviewed in Hwang and Park (2018)). Additionally, HBV mouse models can be generated by hydrodynamic injection with HBV-encoding plasmids or infection with adeno-associated viruses (AAVs) carrying a replicationcompetent HBV genome (Hwang and Park, 2018). They proved useful for investigating the impact of innate and adaptive immunity on HBV clearance (Chou et al., 2015; Yang et al., 2014) and mimicking chronicity upon adenoviral delivery of recombinant cccDNA (Li et al., 2018). To study liver injury and validate new potential drugs (Volz et al., 2013), human liver chimeric mice are frequently used (reviewed in Sun and Li (2017)).

Hepatitis viruses

Open questions in HBV research Despite intensive studies, the determinants of HBV chronicity are not fully understood. It is assumed that age-related progression to chronicity is based on immature immune responses and tolerance in infants (Chou et al., 2015; Publicover et al., 2013; Publicover et al., 2011); however, more studies are needed to support this hypothesis. Myrcludex B blocks HBV dissemination and suppresses cccDNA amplification in the liver of humanized mice (Volz et al., 2013). This indicates that HBV could spread by de novo infection rather than cell-to-cell spread within the liver, but it remains to be proven. The persistence of cccDNA certainly represents the greatest hurdle in HBV eradication, and the poor knowledge of cccDNA biology is a major obstacle. The depletion of cccDNA depletion in vitro (Tu et al., 2020) and in vivo (Allweiss et al., 2018), occurring during hepatocyte proliferation could have therapeutic potential but remains to be shown. Novel treatment strategies aim at eliminating this viral reservoir (reviewed in Bloom et al. (2018)), for instance, by CRISPR-Cas9-based cccDNA degradation (Kostyushev et al., 2019), inactivation (Yang et al., 2020), or drugs targeting cccDNA transcription (Hayakawa et al., 2020; Sekiba et al., 2019; Ren et al., 2019). Although evidence of a type III IFN response in HBV-infected hepatocytes exists (Sato et al., 2015), HBV is generally considered a stealth virus, since innate immune recognition is weak (reviewed in Megahed et al. (2020)). It further remains controversial whether IFN-a treatment can induce cccDNA degradation in patients, as suggested in vitro (Lucifora et al., 2014). Unlike HCV, it is also not understood why chronic HBV rarely leads to fibrosis (Wong et al., 2013). Single nucleotide polymorphisms (SNPs) in the Toll-like receptor 3 (TLR3 (Geng et al., 2016; Fischer et al., 2018) and microRNA-122 (Liu et al., 2014; Zhou et al., 2015) genes were related to increased risk of HBV infection and chronic disease progression, but their effect on HBV replication is not clear.

Hepatitis C virus (HCV) Discovered in 1989 (Choo et al., 1989), HCV is an enveloped, positive-sense, singlestranded virus from the Hepacivirus genus, classified in the Flaviviridae family (Simmonds et al., 2017). There are eight HCV genotypes with 86 subtypes (Borgia et al., 2018), and a great diversity of genetic variants exists in patients (so-called quasispecies) (reviewed in Farci (2011)). HCV is mainly transmitted parenterally, but also from mother to child, and through sexual contact (reviewed in Spearman et al. (2019)) (Fig. 7.1). HCV infections can be acute or asymptomatic, but can become chronic in 60%e80% of cases (Modi and Liang Hepatitis, 2008). When not cleared, subclinical HCV infection persists, potentially leading to hepatic cirrhosis and HCC (Spearman et al., 2019). Globally, w71 million people are chronic HCV carriers (World Health Organization, 2017). Direct-acting antiviral agents (DAAs) can cure the disease with clearance rates close to 100% (reviewed in Holmes et al. (2019)). However, they are less efficient against GT3 (McPhee, 2019), can cause the emergence of resistance-associated substitutions in HCV



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

strains, and reinfection is still possible (reviewed in Wyles and Luetkemeyer (2017)). A vaccine is currently not available, and its development is still hampered by the genetic heterogeneity of HCV (reviewed in Bailey et al. (2019)).

Current knowledge of the HCV life cycle The tissue and host tropism of HCV is exceptionally narrow. This is determined by the complex cell entry process during HCV infection (reviewed in Miao et al. (2017)) that involves four essential cell receptors: cluster of differentiation 81 (CD81) (Pileri et al., 1998), lipoprotein receptor scavenger receptor class B type I (SR-BI) (Scarselli et al., 2002), and the tight junction proteins claudin-1 (CLDN1) (Evans et al., 2007) and occludin (OCLN) (Ploss et al., 2009). Through the endocytic pathway (Blanchard et al., 2006), HCV reaches late endosomes where the low pH likely triggers membrane fusion activity in glycoprotein E1, causing virus uncoating (Banda et al., 2019). The HCV genome (w9.6 kilobases, kb) encodes a polyprotein that is processed by viral and host proteases to yield three structural (core, E1, E2) and seven nonstructural proteins (p7, NS2, NS3-4A, NS4B, NS5A, NS5B) (reviewed in Manns et al. (2017)). HCV replication then occurs in close proximity to the ER (Manns et al., 2017). NS4B alters the ER surface (Egger et al., 2002), leading to the formation of double-membrane vesicles (DMVs) within a so-called membranous web (Romero-Brey et al., 2012). HCV isolates naturally need low levels of phosphatidylinositol 4-kinase alpha (PI4KA) (Berger et al., 2011) to replicate, but HCV cell culture strains can adapt to the high levels of PI4KA in hepatoma cells (Harak et al., 2016). In DMVs, hidden from cellular sensors, the error-prone NS5B polymerase produces negative-sense and positivesense HCV RNA strands (Manns et al., 2017). Double-stranded RNA intermediates of replication can be secreted via exosomes, thereby reducing the innate immune response (Gru¨nvogel et al., 2018). Progeny virus assembly, orchestrated by p7, NS2, and NS5A (Popescu et al., 2011; Gentzsch et al., 2013; Hughes et al., 2009), occurs on ER-wrapped lipid droplets (LDs) (Ogawa et al., 2009). HCV virions bud into the ER to acquire their envelope and are then released though the very-low-density lipoprotein (VLDL) secretory pathway (reviewed in Vieyres and Pietschmann (2019)) as complete HCV particles or minicores (Eng et al., 2009). Mature HCV particles are then composed of capsid, a phospholipid bilayer with E1 and E2 envelope glycoproteins, and VLDL components (thus termed lipovirion) (Vieyres and Pietschmann, 2019). Although the mechanism of lipovirion maturation is not clear, VLDL components such as apolipoprotein E (ApoE) are involved in HCV assembly (Lee et al., 2014), immune evasion (Wrensch et al., 2018), and attachment to SR-BI (Dao Thi et al., 2012). Studies showed preferential secretion of HCV virions from the basolateral membrane of infected polarized hepatocytes (Belouzard et al., 2017). The presence of HCV RNA (Beld et al., 2000; Haruna et al., 2001) and HCV core protein (Heidrich et al., 2016) in feces from chronic patients suggests some level of apical HCV particle release (Fig. 7.1).

Hepatitis viruses

Established and novel in vitro and in vivo models to study HCV infection Similar to other hepatotropic viruses, HCV studies were long hindered by the absence of efficient cell culture systems. Since HCV isolates do not replicate in hepatoma cells without adaptation (reviewed in Kaul and Bartenschlager (2009)), selectable replicon systems with nonstructural proteins replaced by antibiotic markers were established (Lohmann et al., 1999). They enabled HCV replication studies and the development of antiviral drugs, but did not yield infectious particles (reviewed in Lohmann and Bartenschlager (2014)). Importantly, the work of Bukh et al. showed that cell culture-acquired mutations impaired HCV replication in vivo (Bukh et al., 2002). The breakthrough discovery of the JFH-1 (GT2a) strain that replicates in culture without adaptation (Zhong et al., 2005; Uprichard et al., 2006; Kato et al., 2003; Lindenbach et al., 2005) permitted the study of the whole HCV life cycle. Based on this strain, efficient JFH-1-recombinant chimeras were created and used along with Huh-7.5, Huh-7-Lunet, and HepG2 CD81 hepatoma cells (reviewed in Lohmann (2019)) to test DAAs (Gottwein et al., 2011) as well as to study HCV entry and assembly (reviewed in Lindenbach (2013); Lindenbach and Rice (2013); Bartenschlager et al. (2011)). Importantly, Huh-7.5 hepatoma cells ectopically expressing SEC14-like lipid binding 2 (SEC14L2) support pan-genotype HCV isolate infection without culture adaption (Saeed et al., 2015), highlighting the drawbacks of using hepatoma cells for studying authentic hepatotropic virus biology. In addition, hepatoma cells do not fully lipidate ApoB100 and mainly produce low-density lipoproteins (LDL) rather than VLDL (Meex et al., 2011), which makes them less ideal for HCV particle maturation and secretion studies. A range of primary hepatocyte systems has been used for HCV infection studies: PTHs (Zhao et al., 2002), human liver slices (Lagaye et al., 2012), and PHHs (Molina et al., 2008), with the latter supporting primary isolate infection. MPCC of PHHs can recapitulate the entire HCV life cycle (Ploss et al., 2010). Notably, ESC/iPSC-derived HLCs are also permissive to HCV infection (Wu et al., 2012; Sa-Ngiamsuntorn et al., 2016; Carpentier et al., 2014; Carpentier et al., 2020). We and others showed that ESC/iPSC-derived HLCs produce fully lipidated ApoB (Dao Thi et al., 2020; Carpentier et al., 2014, Scho¨bel et al., 2018), suggesting that HLCs, unlike hepatoma cells, have a functional VLDL biosynthesis pathway. The IFN response blunts HCV replication in both PHHs (Thomas et al., 2012; Yang et al., 2011) and ESC-derived HLCs (Zhou et al., 2014) while it is almost absent in Huh-7.5 cells due to the RIG-I deficiency (Sumpter et al., 2005; Blight et al., 2003). Interestingly, inhibitors of the JAK/STAT pathway increase HCV levels in ESC-derived HLCs (Zhou et al., 2014) and make the immune response finely tunable (Carpentier et al., 2020), allowing studies of chronicity. Another advantage of ESC/iPSC-derived HLCs is the support of infection with HCV patient isolates from different genotypes (Wu et al., 2012) (Fig. 7.2). In vivo, HCV research had long depended on chimpanzees (reviewed in Lanford et al. (2001)). Today, transgenic mice can be created to express HCV antigens,



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

human host cofactors, or the complete HCV genome (reviewed in Burm et al. (2018)). The latter can mimic HCV chronic infection, develop steatosis as well as HCC (Lerat et al., 2002), and demonstrated the role of NS5B in hepatitis Crelated inflammation (Simonin et al., 2013). Human liver chimeric mice supporting HCV infection (reviewed in Yong et al. (2019)) were useful to characterize HCV lipovirions in vivo (Ellis et al., 2013; Andreo et al., 2017) and evaluate DAA efficiency (Kneteman et al., 2006). When these mice were engrafted with autologous immune systems, they showed acute hepatitis symptoms upon HCV infection (Billerbeck et al., 2016) and proved useful for immunity studies (Keng et al., 2016). HCVrelated hepaciviruses and pegiviruses can infect rodents (Billerbeck et al., 2017; Kapoor et al., 2013) and horses (Pfaender et al., 2015), establishing chronic infection (Kapoor et al., 2013; Scheel et al., 2015). Finally, Tupaia (Kayesh et al., 2017) and Zebrafish (Ding et al., 2015) were proposed as alternative animal models.

Open questions in HCV research To date, it is still unclear where HCV virions acquire the lipoprotein components. ApoB and ApoE are thought to bind to HCV particles during virion secretion along the VLDL secretory pathway (Boyer et al., 2014), but studies have shown that secreted ApoB and E can also associate with HCV lipovirions in the extracellular compartment (Denolly et al., 2019; Bankwitz et al., 2017). The viro-lipid nature further challenges the quest for an efficient vaccine, as lipoproteins may alter or shield the particle’s immunogenic entities. The HCV protease NS3/4A efficiently cleaves adaptor proteins MAVS (Meylan et al., 2005) and TRIF (Ferreon et al., 2005; Li et al., 2005), hereby impairing IFN induction. Nevertheless, HCV generates a strong and infection-limiting IFN response in PHHs (Thomas et al., 2012; Yang et al., 2011) and ESC-derived HLCs (Zhou et al., 2014). SNPs in host genes such as ApoE (Wozniak et al., 2002), interferon lambda 3 (IFNL3, formerly known as interleukin 28 B (IL28B)) (Ge et al., 2009), and TLR3 (Zayed et al., 2017; El-Sharawy et al., 2020) have an impact on IFN therapy and infection outcome, but the underlying mechanisms are not fully understood. Infection of PHHs with different HCV isolates is possible (Molina et al., 2008), but remains inefficient. Thus, identifying a highly efficient, pan-genotype, physiological culture system for HCV isolates remains a big challenge. Such a system could contribute to a better understanding of virus-host interactions (Fig. 7.2), e.g., why GT3 but no other GT infection quickly progresses to steatosis (Shrivastava et al., 2016).

Hepatitis delta virus (HDV) The hepatitis delta antigen was first discovered in 1977 (Rizzetto et al., 1977) and subsequently identified to be a virus in 1980 (Rizzetto et al., 1980). HDV is an enveloped virus with a circular, negative-sense, single-stranded RNA genome (reviewed in Mentha et al. (2019)). The eight HDV genotypes are classified in the Deltavirus genus (Magnius et al., 2018). Since HDV depends on HBV as a helper virus, infection occurs by HBV/HDV coinfection or by superinfection of chronic HBV carriers

Hepatitis viruses

(Mentha et al., 2019). The latter almost invariably results in chronic hepatitis D, the most severe form of viral hepatitis with accelerated progression to liver cirrhosis (Mentha et al., 2019). HDV is mainly transmitted through blood products and sexual contact (Mentha et al., 2019) (Fig. 7.1). Current estimates of the global prevalence range from 12 million (Stockdale et al., 2020) to 70 million (Chen et al., 2019) people. Pegylated IFN-a is the recommended therapy (reviewed in Asselah et al. (2020)). The entry inhibitor Hepcludex (Myrcludex B) (Blank et al., 2016) was recently approved by the European Medicines Agency. Its combination with pegylated IFN-a leads to a fast and significant reduction of serum HDV RNA levels and lowers relapse rates, but does not result in complete elimination (Wedemeyer et al., 2019). Even though HBV vaccination can protect uninfected individuals from HDV, chronic HBV carriers remain at high risk for HDV superinfection.

Current knowledge of the HDV life cycle The HDV genome contains a single ORF within the complementary antigenome sequence, encoding the hepatitis delta antigen (HDAg), which is expressed as a small (S-HDAg) and a large isoform (L-HDAg) (Luo et al., 1990). Both associate with the viral genome, which adopts a rod-like secondary structure by internal base pairing (Wang et al., 1986), to form a ribonucleoprotein (RNP) (Ryu et al., 1993; Zuccola et al., 1998). HDV is a defective virus that does not encode its own envelope proteins and depends on HBV or potentially other viruses (Perez-Vargas et al., 2019), whose surface antigens it acquires to yield infectious progeny (Mentha et al., 2019). Similar to HBV, the binding of LHBs to NTCP mediates HDV infection (Ni et al., 2014; Yan et al., 2012). Like HBV, HDV supposedly enters by clathrinmediated endocytosis and fusion in late endosomes (reviewed in Watashi and Wakita (2015)). Both HDAgs contain a nuclear localization sequence (Xia et al., 1992), likely guiding the HDV RNA into the nucleus (Chou et al., 1998; Tavanez et al., 2002). HDV genome replication and mRNA transcription are mediated by the cellular RNA pol II (Chang et al., 2008; Greco-Stewart et al., 2007). The selfcomplementarity of the HDV RNA genome may play a role in adapting the substrate specificity of the cellular enzyme (Greco-Stewart et al., 2007). S-HDAg is essential for replication (Kuo et al., 1989; Yamaguchi et al., 2001) and binds several subunits of RNA pol II (Cao et al., 2009; Yamaguchi et al., 2007). Replication takes place through a rolling-circle mechanism, first generating multimeric linear transcripts of antigenomic polarity (Mentha et al., 2019). Ribozyme-mediated self-cleavage (Kuo et al., 1988) and ligation by a still debated mechanism (Reid and Lazinski, 2000; Sharmeen et al., 1989) create circular antigenome monomers, which then serve as a template for genome production. Editing of an amber stop codon in the antigenome by adenosine deaminase acting on RNA 1 (ADAR1) drives the transition from S- to L-HDAg synthesis (reviewed in Casey (2012)). Since L-HDAg is crucial for virion assembly (Chang et al., 1991) and inhibition of replication (Hwang and Lai, 1994; Modahl and Lai, 2000), antigenome editing represents the switch toward virion release. Farnesylated L-HDAg (Glenn et al., 1992) of newly assembled RNPs interacts with HBsAgs on the ER (Hwang and Lai, 1993), triggering



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

HDV envelopment and release into the bloodstream through an unidentified pathway (Mentha et al., 2019) (Fig. 7.1). Although no evidence supporting the secretion of HDV into the bile is available, the presence of HBV RNA (Zheng et al., 2015; Hoefs et al., 1980) and HCV RNA (Beld et al., 2000) in feces could suggest that some HDV particles are also secreted apically from hepatocytes (Fig. 7.1).

Established and novel in vitro and in vivo models to study HDV infection Similar to HBV, hepatoma cells require ectopic expression of NTCP to support HDV infection (Ni et al., 2014; Yan et al., 2012). Due to the lack of HBsAgs, however, virion assembly and release are not recapitulated in these cells (reviewed in Verrier et al. (2016)). Only recently, Ni et al. established an Huh-7-derived cell line (Huh-7END) for continuous virion production by integrating the HDV antigenome and HBsAgs sequence (Ni et al., 2019). The HepG2-derived HepNB2.7 cell line stably expresses both HBV envelope proteins and NTCP and thus supports the full HDV replication cycle (Lempp et al., 2019). HepaRG cells become susceptible to HDV infection upon differentiation or ectopic NTCP expression (Ni et al., 2014). Both PHHs and PTHs are permissive to HDV infection (Verrier et al., 2016). PHHs in SACC coculture format with stromal cells supported long-term HBV/HDV coand superinfection (Winer et al., 2020). Furthermore, Unzu et al. reported that PHH-derived hepatic progenitors differentiated to hepatocyte-like cells are permissive to HDV infection and replication (Unzu et al., 2019). In addition, we found that iPSC-derived HLCs are permissive to all four HDV genotypes (GT1e4) tested (Qu et al., presented at HBV liver meeting 2019). We also observed a moderate upregulation of several IFNs and ISGs in HDV-infected HLCs, showing the suitability of iPSC-derived HLCs for HDV immune studies (Fig. 7.2). In vivo, chimpanzees can be infected with HBV/HDV (Kos et al., 1991; Negro et al., 1988), and woodchucks support HDV replication if chronically infected with woodchuck hepatitis virus (Ponzetto et al., 1984). Transgenic mice expressing human or humanized NTCP (He et al., 2016, 2015) can be infected with HDV but not HBV. Human liver chimeric mice support HDV monoinfection (Giersch et al., 2014) and HBV/HDV co- and superinfection (Lu¨tgehetmann et al., 2012). AAVdelivered HBV and HDV genome sequences can replicate and produce infectious particles in mice (Sua´rez-Amara´n et al., 2017). Recently, Winer et al. (2018) generated a transgenic mouse model based on coexpression of HBsAgs and human NTCP that allows HDV propagation. However, HDV viral titers were far below those observed in patients (Winer et al., 2018). The use of nonhuman primates is prohibited, human liver chimeric mice are intrinsically unsuited for immune studies, and AAV-injected mice provide only partial reproduction of the physiological infection mechanism. Thus, the ideal small animal model for investigating HBV/HDV co- and superinfection remains to be identified.

Open questions in HDV research Several aspects of the HDV life cycle remain poorly characterized, including entry and uncoating, circularization of the genome and antigenome, localization and

Hepatitis viruses

detailed mechanism of RNP assembly, and virion secretion. It is also not known how the HDV genome persists during cell division (Giersch et al., 2019). In contrast to HBV, HDV appears to stimulate an innate immune response involving IFN-b, IFN-l, and downstream production of ISGs (reviewed in Jung et al. (2020)). Only recently, melanoma differentiation-associated protein 5 (MDA5) has been proposed as the main PRR involved in HDV sensing (Zhang et al., 2018). Yet, the recognized RNA element, which should be cytosolic, remains to be determined. IFN mainly inhibits the early steps of HDV infection (Han et al., 2011), but does not affect HDV genome replication in the nucleus following the establishment of infection (Zhang et al., 2018). Interference of HDV with IFN downstream signaling has been previously suggested (Giersch et al., 2015; Pugnale et al., 2009), potentially providing an explanation for the poor responsiveness to pegylated IFN-a treatment in patients (Rizzetto and Smedile, 2015). Besides, many questions regarding the interaction of HBV and HDV in co- and superinfection remain (reviewed in Shirvani-Dastgerdi and Tacke (2015)). HDV can suppress HBV replication (Shirvani-Dastgerdi and Tacke, 2015), but whether this depends on HBV/HDV competition or on the innate immune response induced by HDV, remains to be clarified. Lastly, the triggers for acceleration of liver cirrhosis in chronic hepatitis D require thorough investigation.

Hepatitis E virus (HEV) HEV was discovered in 1983 (Balayan et al., 1983) and is a nonenveloped, positivesense, single-stranded RNA virus of the Hepeviridae family (Purdy et al., 2017), which infects a wide range of hosts. The human-infecting genotypes are classified in the Orthohepevirus A species (Purdy et al., 2017). GT1 and 2 are transmitted fecal-orally and restricted to human infection, whereas GT3, 4, and 7 can be transmitted zoonotically (reviewed in Kamar et al. (2017)) (Fig. 7.1). GT3 (Boxall et al., 2006) and GT4 (Matsubayashi et al., 2008) human-to-human transmission can occur through blood transfusions. Recently, distantly related Orthohepevirus C GT1 (rat) HEV was also found to infect humans, but the exact transmission routes are unknown (Sridhar et al., 2018; Andonov et al., 2019; Sridhar et al., 2020). Up to 20 million people are infected with HEV annually (World Health Organization, 2017), usually asymptomatic and self-limited (Kamar et al., 2017). Yet, a high mortality has been observed in pregnant women (for GT1 and GT2) (reviewed in Pe´rez-Gracia et al. (2017)) and immunocompromised patients can develop chronic infection with progressive liver injury when infected with GT3 (Kamar et al., 2008) and GT4 (Sridhar et al., 2019). Genotype-dependent extrahepatic manifestations affecting the peripheral nerves, kidney, and pancreas were reported (reviewed in Horvatits and Pischke (2018)). Current therapies are based on off-label pegylated IFN-a and ribavirin (reviewed in Peters van Ton et al. (2015)), which can induce resistance associated with treatment failure (Debing et al., 2014; Lhomme et al., 2015; Todt et al., 2016). The vaccine Hecolin against HEV GT1 has only been licensed in China for a restricted age range (Proffitt, 2012).



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

Current knowledge of the HEV life cycle Similar to HAV, HEV is nonenveloped (nHEV) in feces (Balayan et al., 1983) and quasi-enveloped (eHEV) in the bloodstream (Takahashi et al., 2010; Yin et al., 2016a) (Fig. 7.1). The 50 -capped and 30 -polyadenylated HEV genome contains three major ORFs 1e3, encoding the nonstructural polyprotein, the capsid, and a small phosphoprotein, respectively (Kamar et al., 2017). GT1 encodes an additional protein ORF4 (Nair et al., 2016). HEV primarily infects the liver and intestine (Kamar et al., 2017; Marion et al., 2020). Upon ingestion, HEV likely passes the gastrointestinal barrier by infecting polarized enterocytes from their apical side (Marion et al., 2020; Emerson et al., 2010) (Fig. 7.1). Like HAV, HEV is then released basolaterally into the bloodstream, reaching the liver through the portal vein (reviewed in Yin et al., 2016b) (Fig. 7.1). Release from HEV-infected Caco-2 cells (Emerson et al., 2010) and human primary intestinal cells (PICs) (Marion et al., 2020), however, was predominantly apical. An HEV cell entry receptor has not been described yet, but several host factors such as glucose-regulated protein 78 kDa (GRP78) (Yu et al., 2011), HSPGs (Kalia et al., 2009), ATP synthase subunit b (ATP5B) (Ahmed et al., 2016), asialoglycoprotein receptor (ASGPR) (Zhang et al., 2016), and integrin a3 (Shiota et al., 2019) have been proposed to facilitate HEV entry. Following dynamin-2 and clathrin-mediated endocytosis (Yin et al., 2016a), eHEV possibly enters the cell from the endolysosome, after degradation of the quasi-envelope. In the absence of detectable colocalization with Rab5, nHEV was proposed to uncoat in early endosome vesicles, close to the plasma membrane (Yin et al., 2016a). In the cytosol, the translated ORF1 polyprotein comprises seven domains: methyltransferase, Y domain, papain-like cysteine protease, hypervariable region (HVR), X domain, RNA helicase, and RdRp (Kamar et al., 2017). Whether the polyprotein is processed into individual products remains controversial (reviewed in Parvez (2017)). New viral genomes are produced via a full-length negative-sense RNA intermediate (Kamar et al., 2017). A shorter subgenomic RNA of 2.2 kb is transcribed for expression of ORF2 and ORF3 (Graff et al., 2006). Cytosolic ORF2 can, upon interaction with the HEV genome (Surjit et al., 2004), self-assemble to form the icosahedral capsid (Guu et al., 2009; Xing et al., 2010). The majority of ORF2 is released glycosylated through the secretory pathway, likely acting as a decoy for the immune system (Montpellier et al., 2018; Yin et al., 2018). Apart from a recently identified ion channel activity (Ding et al., 2017), ORF3 may interact with various host factors (Chandra et al., 2010; Wang et al., 2014; Chandra et al., 2011; Chandra et al., 2008). ORF3 undergoes posttranslational palmitoylation, which mediates its association to the ER membrane (Gouttenoire et al., 2018). It is essential for virion release (Emerson et al., 2010; Yamada et al., 2009), likely by interacting with tumor susceptibility gene 101 (Tsg101) and other ESCRT proteins (Nagashima et al., 2011; Nagashima et al., 2011) to facilitate budding into MVBs (Nagashima et al., 2014, 2017). As a result, secreted HEV particles are quasi-enveloped, decorated with ORF3, and protected from neutralizing antibodies (Chapuy-Regaud et al., 2017). When budding from the

Hepatitis viruses

apical side of HEV-infected polarized hepatocytes (Capelli et al., 2019) (Fig. 7.1), the quasi-envelope is removed by the bile acids (Dao Thi et al., 2020). Then, highly infectious nHEV is shed in feces and can spread to the next patient (Yin et al., 2016a).

Established and novel in vitro and in vivo models to study HEV infection HEV research employs hepatic cell lines PLC/PRF/5, Huh-7 (and derivatives), HepaRG, HepG2/C3A, and PHHs, along with nonhepatic lung, intestinal, neuronal, and placental cell lines (reviewed in Fu et al. (2019)). Patient-derived HEV isolates replicate slowly and only to low levels in carcinoma cells (Fu et al., 2019; Meister et al., 2019). In a major breakthrough, Shukla and colleagues adapted the GT3 Kernow-C1 strain by serial passaging to replicate efficiently in HepG2 cells (Shukla et al., 2011). The adapted strain (termed p6) harbors an insertion of a human ribosomal S17 fragment in the HVR region (Shukla et al., 2011, 2012). Even though the insertion altered some aspects of HEV biology (Shukla et al., 2011; Wu et al., 2018), such as widening its tissue and species tropism (Shukla et al., 2011), this strain enabled a wealth of HEV molecular studies. Interestingly, the introduction of a patient-derived ribavirin-resistant mutation in the HEV Kernow-C1/p6 RdRp region further increased viral titers (Debing et al., 2016; Todt et al., 2020). GT1-4 HEV replicons were created by replacing the ORF2/3-coding sequence with reporter genes (Shukla et al., 2012; Emerson et al., 2004; Graff et al., 2005). PHHs (Todt et al., 2020; Oshiro et al., 2014; Yin et al., 2017) and PICs (Marion et al., 2020) are permissive to both cell-culture-adapted strains and primary isolates. In 2016, Helsen et al. showed that ESC/iPSC-derived HLCs support the entire replication cycle of the HEV Kernow-C1/p6 strain. They also showed that nonhepatic progenitor cells support HEV replication, but not infection (Helsen et al., 2016). In addition, we found that iPSC-derived HLCs are permissive for infection with HEV GT1-4 patient-derived isolates (Wu et al., 2018) and that HEV replication induces robust type III IFN signaling and ISG induction in iPSC-derived HLCs (Wu et al., 2018). Ribavirin and sofosbuvir inhibited replication of primary isolates in iPSC-derived HLCs (Wu et al., 2018; Dao Thi et al., 2016), demonstrating the suitability of HLCs for antiviral drug testing (Wu et al., 2018; Dao Thi et al., 2016) (Fig. 7.2). We further demonstrated that ESC/iPSC-derived pol-HLCs are permissive for HEV infection, similar to HAV, and can recapitulate the directionality of in vivo HEV transmission (Fig. 7.2) (Dao Thi et al., 2020). In vivo, nonhuman primates can be infected with the four human HEV genotypes (reviewed in Corneillie et al. (2019)), but do not develop clinical symptoms (Spahr et al., 2018). Other attractive animal models include natural hosts of the zoonotic GTs, including pigs (Cao et al., 2017; Huang et al., 2005), rabbits (Wang et al., 2018), donkeys (Rui et al., 2020), birds (Liu et al., 2020), and as recently reported, rats (Debing et al., 2016). In addition, humanized liver chimeric mice support acute and chronic infection with HEV GT1 and GT3 (Sayed et al., 2017; Allweiss et al., 2016; van de Garde et al., 2016).



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

Open questions in HEV research All basic steps of the HEV life cycle need a better understanding. The receptor for n/eHEV is yet to be identified and may be a key determinant for the wide cell and tissue tropism. Only a few HEV genotypes have the capacity to jump the species barrier, but the underlying mechanisms remain enigmatic (reviewed in Wang and Meng (2020)). Interestingly, HSPGs are essential for the attachment of nHEV to host cells, but dispensable for eHEV infection (Yin et al., 2016a). Moreover, the HEV genome replication site and the putative processing of ORF1 are poorly characterized. Translation of the ORF4 protein under ER stress conditions enhances GT1 replication in Huh-7 cells (Nair et al., 2016); but cancer cells are predisposed to ER stress (Oakes, 2020) and are therefore unsuited for these types of studies. Molecular details of HEV assembly and polarized secretion are unknown. HEV Kernow-C1/p6 infection induces a robust type III IFN response (Wu et al., 2018; Yin et al., 2017), although several HEV proteins have been suggested to interfere with innate immune signaling (reviewed in Lhomme et al. (2020)). TLR4 (Arya et al., 2018) and ApoE (Zhang et al., 2015) polymorphisms were associated with positive and negative outcomes of HEV infection, respectively, but introducing the latter in Huh-7.5 cells did not have any effect on HEV replication (Weller et al., 2016). As hepatoma cells do not have a functional lipoprotein metabolism (Meex et al., 2011), performing such studies in physiological systems may be more beneficial to mimic the natural infection and elucidate the impact of such polymorphisms on HEV infection (Fig. 7.3).

Plasmodium Plasmodium, a genus of unicellular parasitic protozoans of the sporozoan subclass Coccidia, is the causative organism of malaria (Laveran, 1881). Plasmodium parasites are spread by mosquitos (Fig. 7.1). Six species can infect humans, which have caused 228 million malaria cases and 405,000 deaths in 2018 (World Health Organization). More than 90% of both cases and deaths were attributed to subSaharan Africa where P. falciparum is most prevalent and often results in severe clinical manifestations (World Health Organization). P. knowlesi infection can also result in severe disease (World Health Organization,; Cox-Singh et al., 2008), while P. malariae, P. ovale curtisi, and P. ovale wallikeri mostly cause uncomplicated malaria (World Health Organization). P. vivax is the most prevalent species outside of Africa (World Health Organization) and can provoke periodic relapses of disease due to dormant stages (White, 2011). Malaria is primarily treated with artemisinin-based combination therapies (Tse et al., 2019), which can induce resistance (Me´nard et al., 2016). Despite recent advances in identifying compounds targeting various stages of the Plasmodium life cycle (reviewed in Raphemot et al. (2016); Yahiya et al. (2019)), little progress has been made in developing well-tolerated drugs that target the dormant stages to prevent relapses


in patients (reviewed in Campo et al. (2015)). Primaquine and its analogue tafenoquine are the only approved drugs for elimination of P. vivax dormant stages (Tse et al., 2019) but have contraindications (Baird and Hoffman, 2004; Phillips-Howard and Wood, 1996). The leading P. falciparum preerythrocytic vaccine candidate RTS,S is currently under phase IV clinical trial in three African countries (Greenwood and Doumbo, 2016) despite modest results in phase III (RTS S Clinical, 2015).

Current state of knowledge on Plasmodium liver stage Upon the bite of a female Anopheles mosquito, sporozoites are injected into the host skin from where they actively invade the blood vessels (reviewed in Me´nard et al. (2013)) (Fig. 7.1). The sporozoites reach the liver through the bloodstream, where they first enter the hepatocytes. Although it is a crucial amplification step for successful progression of the entire Plasmodium life cycle within the mammalian host, the biology of this so-called liver stage (LS) is poorly understood. After entering the hepatocytes, the parasite resides within an invagination of the host cell membrane, the parasitophorous vacuole (PV) (reviewed in Nyboer et al. (2018)). Productive entry and PV formation are poorly characterized. Initial attachment of the major sporozoite surface protein, the circumsporozoite protein (CSP), to HSPGs (Frevert et al., 1993) appears critical. Further involvement of hepatocyte receptors Ephrin receptor A2 (EphA2) (Kaushansky et al., 2015), CD81 (Silvie et al., 2006; Silvie et al., 2003), and SR-BI (Rodrigues et al., 2008; Yalaoui et al., 2008) has been proposed. P36 is released from specific secretory organelles upon initial attachment (Arredondo et al., 2018) and is the only parasite protein identified so far to potentially interact with EphA2 (Kaushansky et al., 2015); but the importance for hepatocyte infection is controversial (Langlois et al., 2018). Exchange between the metabolically active parasite and the host cell is mediated by the PV membrane (PVM) and a tubulovesicular network (Nyboer et al., 2018). Several parasite proteins are exported to the PVM during LS, likely exerting functions related to nutrient acquisition, host cell remodeling, and protection from immune recognition (Nyboer et al., 2018). However, their precise roles as well as their interaction partners remain to be identified. Once thousands of daughter cells have formed, the PVM breaks down, and progeny-containing vesicles, so-called merosomes, bud into the bloodstream (Fig. 7.1), where they rupture to gain access to erythrocytes (Vaughan and Kappe, 2017; Vaughan et al., 2012). P. vivax and P. ovale can persist in hepatocytes as dormant, nondividing hypnozoites (White, 2011). The mechanisms of hypnozoite formation and reactivation are unclear. The Plasmodium LS is clinically silent, as opposed to the erythrocyte infection stage. It was thus considered to remain undetected by the immune system. Yet, recent studies showed that Plasmodium RNA is recognized by the cytosolic sensor MDA5 (Liehl et al., 2014; Miller et al., 2014). Downstream signaling leads to production of type I IFNs, ISG activation, and recruitment of immune cells (Liehl et al., 2014; Miller et al., 2014; Liehl et al., 2015).



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

Established and novel in vitro and in vivo model systems for the Plasmodium LS In the absence of suitable culture models for the human-pathogenic Plasmodium species, most insight into LS biology has been gained by studying the rodent malaria models P. berghei and P. yoelii (reviewed in De Niz and Heussler (2018)). Among the human species, P. falciparum and P. vivax are the most intensively studied due to their prevalence and clinical relevance. The immortalized cell line HC-04 (Sattabongkot et al., 2006; Tao et al., 2014) and the hepatoma line HepG2-A16 (Hollingdale et al., 1983, 1984, 1985) are often used to explore hepatocyte invasion in vitro, but the infection efficiency with P. falciparum and P. vivax sporozoites remains poor (Chattopadhyay et al., 2010; Tweedell et al., 2019). Recently, a subclone of the HC-04 line was found to be more permissive to infection with P. falciparum sporozoites (Tweedell et al., 2019). Even though HepG2-A16 cells, a subclone of the HepG2 cell line (Schwartz and Rup, 1983), allow invasion of P. falciparum sporozoites (in contrast to the parental line) (Hollingdale et al., 1984; Chattopadhyay et al., 2011), further LS development is only possible in HC-04 cells (Sattabongkot et al., 2006). In contrast, P. vivax has been reported to complete LS development in both HC-04 and HepG2-A16 cells and even to establish nondividing forms (Sattabongkot et al., 2006; Hollingdale et al., 1985; Subramani et al., 2020). Ectopic expression of CD81 was sufficient to render HepG2 cells permissive for LS completion of rodent P. yoelii but not P. falciparum (Silvie et al., 2006). Moreover, several human hepatoma lines such as Huh-7 and HeLa are permissive to rodent P. berghei infection but not to other Plasmodium species (Calvo-Calle et al., 1994; Silvie et al., 2007). Overall, this underlines the major differences in the requirements for productive hepatocyte infection between the different Plasmodium species. Yet, the determinants for the varying susceptibilities remain to be determined, ideally in a universal in vitro culture system. In general, uncontrolled proliferation of immortalized cells together with the low infectivity rates hinder long-term studies, a prerequisite for investigating hypnozoite development and reactivation. PHHs are permissive for the human-pathogenic species P. falciparum and P. vivax (Subramani et al., 2020; Smith et al., 1984; Mazier et al., 1984), as well as for rodent P. berghei and P. yoelii sporozoites (Ng et al., 2014; March et al., 2013). When cultured in MPCC format, PHHs kept a stable hepatic phenotype for up to 6 weeks (Khetani and Bhatia, 2008) and recapitulated full LS development of P. falciparum and P. vivax as well as the formation of hypnozoites (March et al., 2013; Gural et al., 2018). In 2015, Ng et al. showed that iPSC-derived HLCs supported P. falciparum and P. vivax sporozoite infection and maturation of LS parasites (Ng et al., 2015). Yet, they did not investigate whether the LS was completed by the release of daughter cells and whether HLCs could support P. vivax hypnozoite formation (Ng et al., 2015). Of note, Ng et al. reported that infection with P. falciparum could only be cleared by primaquine when iPSC-derived HLCs were pretreated with the smallmolecule FPH1 (March et al., 2013; Ng et al., 2015) which upregulates the drugmetabolizing CYP450 enzymes, likely necessary for the bioactivation of primaquine (Ng et al., 2015; Camarda et al., 2019). In contrast, the immortalized hepatocyte-like

Addressing open questions in hepatotropic infection research with HLCs

cell line imHC was shown to be sensitive to primaquine while supporting LS completion and hypnozoite formation (Pewkliang et al., 2018). More recently, Subramani et al. demonstrated that iPSC-derived HLCs, obtained from peripheral blood mononuclear cells (PBMCs) of P. vivax-infected patients and a healthy donor, as well as ESC-derived HLCs support infection with sporozoites generated from P. vivax clinical isolates, albeit to only low levels (Subramani et al., 2020). In vivo, human liver chimeric mice support completion of the LS for P. falciparum and P. vivax while partially establishing hypnozoites (reviewed in Minkah et al. (2018)). Additional repopulation with human erythrocytes further allows transition from liver to blood stage (Vaughan et al., 2012; Soulard et al., 2015). Polyethylene-glycol-based human ectopic artificial livers based on PHH support infection with P. falciparum sporozoites (Ng et al., 2017), but it remains to be shown if LS development can be completed and whether this system allows P. vivax infection and hypnozoite formation.

Open questions Plasmodium liver stage research All steps of Plasmodium LS remain poorly understood. Future studies need to characterize parasite-host interaction partners involved in productive entry, PV formation, and exchange at the PVM. Further, the determinants for the switch between progeny production and establishment of dormant stages as well as the triggers for reactivation of hypnozoites remain to be identified. In addition, many questions persist regarding innate immune recognition of LS, the role of autophagy-related pathways (reviewed in Agop-Nersesian et al. (2018)), and the impact on the establishment of protective immunity (reviewed in Gowda and Wu (2018)). Numerous polymorphisms in genes encoding endothelial receptors and components of the immune response have been associated with differential outcomes of P. falciparum infection (reviewed in Gowda and Wu (2018); Driss et al. (2011)). Yet, it remains unclear whether and how these differences in host genetics affect the LS. Deepening our understanding of LS biology will certainly advance the search for novel drug targets. Improved hepatic in vitro systems will be also required to validate the effect of potential drug candidates on the Plasmodium LS (Fig. 7.2).

Addressing open questions in hepatotropic infection research with HLCs As outlined in the preceding paragraphs, many life cycle steps remain poorly characterized for the majority of hepatotropic pathogens. For example, cellular determinants of infection such as (co-) entry receptors for HAV, HEV, and Plasmodium sporozoites are yet to be discovered. In order to identify such cellular factors, it is possible to perform genome-wide siRNA/shRNA knockdown or CRISPRCas9-based knockout screens in iPSC-derived HLCs, which can be genetically



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

modified at their pluripotent stage (reviewed in Hockemeyer and Jaenisch (2016)) (Fig. 7.3). Modified differentiated HLCs are then challenged with (reporter) virus or Plasmodium sporozoites to identify host factors that render cells susceptible to infection. These host factors can then be likewise genetically manipulated at the iPSC stage (Fig. 7.3) and subsequently studied in differentiated HLCs to confirm their role during entry of the respective pathogen. In this way, any host determinant of interest can be analyzed. Importantly, HLCs, likely due to their primary-like nature, are permissive for pan-genotype isolates, as shown for HCV (Wu et al., 2012) and HEV (Wu et al., 2018) (Fig. 7.2). As cell culture adaptations can alter virus biology (Lemon et al., 1991; Kaul and Bartenschlager, 2009; Bukh et al., 2002; Shukla et al., 2012; Wu et al., 2018), it would be desirable to corroborate any novel findings with nonadapted virus. Therefore, HLCs also provide an authentic system to study viral determinants. HEV is an ideal example, as the mechanisms for its wide tissue and species tropism are unknown. Genotypic differences might be at play as only some GTs can jump the species barrier, while others cannot (reviewed in Wang and Meng (2020)). Both viral and cellular determinants can be analyzed using the capacity of iPSCs to differentiate into any desired cell type with the same genetic background (Figs. 7.2 and 7.3), followed by challenges with different HEV GTs. Similarly, the differences in host and parasitic determinants for productive infection between the various human-pathogenic and rodent Plasmodium species can be investigated. Another example is HCV. HLCs have a functional lipoprotein metabolism making studies of HCV assembly possible, including where and how it acquires the lipoproteins during particle maturation. This would also help to analyze how the lipovirion composition influences HCV dissemination in the liver (reviewed in Vieyres and Pietschmann (2019)). In addition, the permissiveness of HLCs to HCV isolates of multiple GTs offers the possibility of identifying the underlying mechanisms leading to steatosis in HCV GT3 patients (Shrivastava et al., 2016) and to develop phenotypic resistance assays in cases of DAA treatment failure (reviewed in McPhee (2019); He´zode (2018)). The unprecedented susceptibility and permissiveness of HLCs to the hepatotropic viruses discussed in this chapter provide a uniquely universal system that allows comparisons (Fig. 7.2) and thus, the identification or exclusion of common denominators. For example, an important aspect of virus-host interaction is the innate immune response. With their intact immune signaling, HLCs provide an appropriate system to support studies of innate immune recognition of HDV, HEV, and Plasmodium LS parasites (Fig. 7.2). This will help investigate the stealthy nature of HAV and HBV infections, validate immune evasion strategies of HAV, HCV, and HEV, as well as determine the HDV RNA element recognized by innate immune sensors. This can be achieved by either modulating innate immune sensors and effectors on the genetic level or blunting the innate immune response with specific inhibitors in HLCs and subsequent challenge with the respective virus. Recent success in maintaining HLCs in culture for several weeks opens the door for studying the formation and reactivation of P. vivax hypnozoites as well as chronic

Systems integrating diverse hepatic cell types

HBV, HCV, HDV, and HEV infection. Investigating the cellular alterations in apoptotic and proliferative pathways in infected cells could help in understanding and with that, potentially treating or preventing HCC (Fig. 7.2). Importantly, coinfection of the liver by different hepatotropic viruses such as HBV and HCV (Mavilia and Wu, 2018) or HEV superinfection of chronic HBV carriers (Chen et al., 2016), as well as HBV or HCV coinfection with Plasmodium has been reported (Ouwe-Missi-Oukem-Boyer et al., 2011; Andrade et al., 2011) that can often exacerbate as well as accelerate the hepatic disease (Jamma et al., 2010; Hoan et al., 2015; Tseng et al., 2020). Here, HLCs provide a unique platform to perform virus coinfection studies (Fig. 7.2). Of immediate interest are HBV-HDV coinfections. HLCs constitute a suitable system to determine whether, e.g., an HDV-induced innate immune response may be involved in the interference of HBV replication (Shirvani-Dastgerdi and Tacke, 2015; Alfaiate et al., 2016) and test whether targeting ADAR1 could be useful for suppressing both HBV (Yuan et al., 2020) and HDV (Casey, 2012) replication at the same time. Likewise, HLCs will allow dissection of HBV and HCV interactions during coinfections and elucidate how the elimination of HCV by DAA treatment leads to HBV reactivation, besides the reversal of RIG-I-based HBV suppression (Murai et al., 2020). Similarly, the mechanisms underlying exacerbation of disease progression upon HEV superinfection of chronic hepatitis B carriers (Chen et al., 2016; Hoan et al., 2015; Tseng et al., 2020) can be analyzed.

Systems integrating diverse hepatic cell types to improve liver pathogenesis studies Liver pathogenesis is a central aspect of all hepatotropic infections, which depends on the complex interplay between different cell types in the hepatic environment (reviewed in Irwin (2020)) (Fig. 7.1). Hepatocytes are the major parenchymal cells that carry out anabolic and catabolic activities of nutrients, drugs, and other compounds. Together with the other parenchymal cell type, the biliary epithelial cells (cholangiocytes) (Fig. 7.1), hepatocytes constitute the majority of the liver mass (Irwin, 2020). Nonparenchymal cells (NPCs) include liver sinusoidal endothelial cells (LSECs), liver-resident macrophages (Kupffer cells, KCs), hepatic stellate cells (HSCs), and plasmacytoid dendritic cells (pDCs) (Irwin, 2020) (Fig. 7.1). Upon chronic liver injury, induced by excessive fat, alcohol, or drug consumption, the regenerative wound-healing mechanisms of the liver can lead to fibrosis (reviewed in Cordero-Espinoza and Huch (2018)). Nonparenchymal HSCs play a central role in fibrogenesis. In response to inflammation, HSCs undergo a response known as “activation,” which is the trans-differentiation of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts. Once activated, HSCs excessively produce extracellular matrix (ECM) (reviewed in Tsuchida and Friedman (2017)). In response to liver injury, KCs secrete TGF-b, which amplifies HSC



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

activation by stimulating the expression of profibrotic genes and with that the production of collagen, connective tissue growth factor (CTGF), and NADPH oxidases (NOX) (reviewed in Fabregat and Caballero-Dı´az (2018)). The repeated and excessive accumulation of ECM proteins leads to substitution of the liver parenchyma with scar tissue (reviewed in Hernandez-Gea and Friedman (2011)). Sustained fibrogenic processes can eventually result in cirrhosis, a severe distortion of the liver architecture (Hernandez-Gea and Friedman, 2011), which increases the risk of HCC development (reviewed in Baglieri et al. (2019)). NPCs also play a major role in liver infectious diseases by mediating pathogeninduced inflammation and fibrosis development. For instance, KCs can sense HBV (Cheng et al., 2017), may support HEV replication (Sayed et al., 2020), and are a major source of IL-1b during HCV infection (Shrivastava et al., 2013; Negash et al., 2013). Fibrotic processes in HSCs are directly stimulated by HCV core and NS3 proteins to enhance TGF-b transcription and signaling (Bataller et al., 2004; Sakata et al., 2013; Taniguchi et al., 2004). Besides, NPCs are also exploited by pathogens for traversing the endothelial barrier (Fig. 7.1). HBV can cross the endothelial lining of LSECs by transcytosis to reach the liver parenchyma and infect hepatocytes (Breiner et al., 2001). HCV was shown to bind to the C-type lectin L-SIGN on LSECs, which probably allows enrichment of virus and transmission to neighboring hepatocytes (Ludwig et al., 2004; Gardner et al., 2003; Lozach et al., 2004). Traversal of KCs and several hepatocytes has been suggested to precede productive liver infection by Plasmodium sporozoites (Pradel and Frevert, 2001; Tavares et al., 2013; Frevert et al., 2005). Hepatic cocultures are thus necessary to better model the liver environment, which should lead to a better understanding of the mechanisms resulting in liver injury in general. For example, recent cocultures of the immortalized HSC line LX-2 with hepatoma cells showed that HBsAg stimulation can promote expression of collagen type I in HSCs (Liu et al., 2014) and that Wnt inhibitors can reduce the expression of HBV-induced fibrosis genes (Li et al., 2019). Similarly, cocultures of LX-2 and HCV-infected hepatoma cells showed upregulation of profibrotic pathways in HSCs (Akil et al., 2019; Salloum et al., 2016) and that HSCs can trigger HCV-infected hepatocytes to express proinflammatory cytokines and chemokines, possibly promoting the recruitment of CCR5-positive cells to the site of inflammation (Nishitsuji et al., 2013). Finally, expression of HCV core protein in hepatoma cells increased oxidative stress levels in cocultured LSECs, potentially mediating accelerated progression to fibrosis (Sun et al., 2018). In addition, immune cells are key players in pathogenesis, and a wealth of coculture systems have been developed to study immunological aspects in vitro (Klo¨ss et al., 2017; Liu et al., 2017; Shi et al., 2017; Yoon et al., 2011). These include, for example, how direct contact of primary natural killer cells with HCV-infected hepatoma cells diminishes their functional capacity for degranulation, thus being a potential driver of chronicity (Yoon et al., 2011). They also revealed that HCV RNA-positive exosomes released from infected hepatoma cells can trigger an immune response by pDCs in a cell contact-dependent manner (Dreux et al., 2012).

3D systems to study hepatotropic infections

Finally, coculture of KCs isolated from Plasmodium-infected mouse liver with PHHs showed that KC-secreted hepatocyte growth factor triggered apoptosis of infected hepatocytes and in this way, may be involved in limiting LS infection (Gonc¸alves et al., 2017). The previously listed studies are mainly based on coculturing different cancer cell lines or PHHs with established NPC cell lines or primary NPCs, which, in most cases, all have different genetic backgrounds. In contrast, the same ESC/ iPSC line can be differentiated into a variety of liver-resident cells, thus providing a unique isogenic system with enhanced cellular diversity (Figs. 7.2 and 7.3). ESC/iPSC differentiation protocols to generate all major liver-resident cells have been previously developed: hepatocytes (Si-Tayeb et al., 2010), cholangiocytes (Dianat et al., 2014), endothelial cells (Koui et al., 2017), macrophages (Tasnim et al., 2019), pDCs (Sachamitr et al., 2018), and HSCs (Coll et al., 2018); with various coculture setups of stem cell-derived parenchymal and nonparenchymal cells that have been reported. One example is the coculture of iPSC-derived LSECs and HSCs with liver progenitor cells (LPCs), which promoted LPC self-renewal and hepatic maturation without exogenous cytokines (Koui et al., 2017). In future studies, host factors or SNPs of interests can be genetically modulated in ESCs/ iPSCs (Fig. 7.3). Modified ESCs/iPSCs can then be differentiated to the cell type(s) of choice, including HLCs and/or NPCs, and their role in hepatotropic infections as well as associated pathogenesis investigated in coculture setups. Here, different kinds of setups, such as either direct coculture through cell transfer or insert coculture systems as, e.g., Transwells (Fig. 7.3), will allow dissection of the role of celle cell contacts and secreted soluble factors, respectively. Consequently, cocultures of iPSC-derived cell types could help to better model and understand the underlying mechanisms of disease progression in chronic HBV, HCV, HDV, and HEV patients. Studying differences in the inflammatory responses of NPCs to the different hepatotropic viruses may also help to clarify why HAV and HEV usually lead to acute infection (Shin and Jeong, 2018; Kamar et al., 2017) while HBV, HCV, and HDV infections can become chronic. Finally, they could also help to clarify the role of NPCs in antigen presentation as well as the induction of protective immunity and immune tolerance during malaria (Gowda and Wu, 2018).

3D systems to study hepatotropic infections Apart from the complexity given by the diversity of liver cell types, the unique 3D architecture of the liver (reviewed in Irwin (2020)) should also be considered when modeling hepatotropic infections. This architecture is based on the specialized function of hepatocytes. While forming a crucial cell layer that separates sinusoidal blood from the bile (Fig. 7.1), they manage two countercurrent flow systems: the uptake, processing, and secretion of blood components such as nutrients, bile acids, and amino acids in one direction, with the synthesis and secretion of bile, cholesterol, and phospholipids in the other direction. In order to regulate this exchange,



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

hepatocytes rely, compared to other epithelial cells, on a unique polarization with multiple basal membranes facing the sinusoids and multiple apical membranes between adjacent hepatocytes, forming the bile canaliculi (Figs. 7.1 and 7.3) (reviewed in Treyer and Mu¨sch (2013)). As mentioned before, this polarity plays a major role in entry and release mechanisms of all hepatotropic viruses. For example, HCV entry requires the tight junction proteins CLDN1 (Evans et al., 2007) and OCLN (Ploss et al., 2009). HBV and HDV entry is dependent on basal NTCP expression (Ni et al., 2014; Yan et al., 2012). Enteric viruses HAV and HEV heavily rely on hepatocyte polarity for their transmission (Fig. 7.1), and mechanisms of quasi-envelope removal likely depend on the bile canaliculi environment (Dao Thi et al., 2019). Different attempts have been made to establish polarized hepatocyte systems, and the interaction with ECM plays a major role (reviewed in Gissen and Arias (2015)). While most hepatoma cells cannot be columnar polarized (reviewed in Treyer and Mu¨sch (2013); Decaens et al. (2008)), we found that ESC/iPSC differentiation to HLCs on ECM-coated semipermeable Transwells yielded columnar polarized HLCs (Fig. 7.3), which can recapitulate the in vivo directional release of HEV particles (Dao Thi et al., 2020). While the convenience of accessible basal and apical membranes in this system offers several advantages, iPSC-derived systems in which HLCs acquire their natural hepatic polarization (Figs. 7.1 and 7.3) are also needed. A variety of hepatocyte 3D culture systems have been established. Single cell types such as hepatoma cells or PHHs can spontaneously form spheroids upon cultivation on low-adhesion plates or in Matrigel, collagen, and other supportive matrices (reviewed in Gural et al. (2018)). In this setting, the hepatocytes typically acquire their complex hepatic polarity (Figs. 7.1 and 7.3) and form bile canaliculi. Such spheroids have been shown to be permissive for HBV/HCV infection (Ortega-Prieto et al., 2018; Baktash et al., 2018; Fu et al., 2019; Liu et al., 2014; Molina-Jimenez et al., 2012). Spheroids of PHHs cultured on a 3D biocompatible scaffold also supported P. vivax infection and long-term maintenance (Chua et al., 2019). Yet, spheroids do not integrate the aforementioned complexity of the liver given by the different cell types. Due to the growing knowledge of iPSC differentiation, recent progress has been made in generating so-called liver organoids (LOs) (reviewed in Prior et al. (2019); Cotovio and Fernandes (2020)). Previously, the term organoid was inconsistently used to refer to a variety of 3D culture systems. The most recent definition, however, describes an ideal organoid to contain various stem-cell-derived, organ-specific cell types, which self-assemble into a 3D structure (Fig. 7.3) during the process of differentiation (reviewed in Lancaster and Knoblich (2014)). These organoids can to some extent replicate certain structural and functional characteristics of the in vivo organ (Lancaster and Knoblich, 2014). Since simultaneous differentiation of stem cells into multiple cell types remains challenging, attempts to generate LOs have included, among others, coculture of predifferentiated progenitors as well as combination of iPSC- with non-iPSCderived cell types (Prior et al., 2019; Cotovio and Fernandes, 2020).

3D systems to study hepatotropic infections

The first robust liver bud entirely derived from iPSCs was generated by Takebe et al. (2017). Coculture and further differentiation of iPSC-derived endodermal, endothelial, and mesenchymal progenitor cells gave rise to LOs composed of hepatocytes, endothelial cells, and HSCs. Similar systems generated by coculture of ESCs/iPSCs-derived progenitors with endothelial or mesenchymal cells of nonESC/iPSC origin were described by other groups shortly thereafter (Pettinato et al., 2019; Wang et al., 2019). LOs assembled from endothelial cells, bonemarrow-derived mesenchymal cells, and iPSC-derived endodermal cells were shown to be permissive to HBV infection and recapitulated the induction of hepatic dysfunction (Nie et al., 2018), demonstrating the valuable potential of LOs for the study of hepatotropic pathogens. Various protocols have further achieved codifferentiation of iPSCs into hepatocytes and cholangiocytes (Guan et al., 2017; Wu et al., 2019). More recently, Ouchi et al. (2019) reported codifferentiation of ESCs/iPSCs into hepatocytes, cholangiocytes, KCs, and HSCs via an intermediate step of foregut aggregates. Fully iPSCderived LOs composed of the various liver cell types will help elucidate the roles of cellecell interactions in the pathogenesis of hepatotropic pathogens, replicate pathogen-induced liver injury, and correlate the importance of disease-related SNPs (Fig. 7.3). The addition of other NPCs such as pDCs, for instance, in a 3D coculture setup (Fig. 7.3) will allow more detailed studies on fibrogenic processes and immune response to hepatotropic pathogen infections. The comaintenance and codifferentiation of all different liver cell types within a suitable 3D system (Fig. 7.3), however, remains a challenging task due to the different nutrient and growth factor requirements (Prior et al., 2019; Cotovio and Fernandes, 2020). Another limitation is the lack of vascularization, which limits the LO size due to loss of cell viability and onset of necrosis (reviewed in Grebenyuk and Ranga (2019)). Recent advances in organoid vascularization utilize microfluidic devices on chips or bioreactors harboring 3D aggregates of iPSC-derived HLCs or LOs to mimic the in vivo microvasculature (Freyer et al., 2016; Schepers et al., 2016; Wang et al., 2018; Bhatia and Ingber, 2014). In line with this, Ortega-Prieto and colleagues demonstrated that a 3D microfluidic PHH system was permissive to HBV infection (Ortega-Prieto et al., 2018). Further improvements of LOs include the use of biocompatible scaffolds, which spatially mimic the liver architecture (reviewed in da Silva Morais et al. (2020); Jensen and Teng, (2020)). For example, hepatic iPSC-derived progenitors grown on an inverted colloidal crystal scaffold, which resembled the architecture during liver bud formation, formed LOs that supported infection with HCV (Ng et al., 2018). A recent study further reported the repopulation of decellularized rat liver with iPSC-derived hepatocytes and cholangiocytes as well as vascular endothelial cells, mesenchymal stem cells, and fibroblasts (Takeishi et al., 2020). Eventually, bioprinting of iPSCs (reviewed in Romanazzo et al. (2019)), either in combination with a scaffolding biomaterial or using scaffold-free bioinks, and subsequent differentiation to LOs will be a promising tool for reproducing the complex liver architecture, creating near-physiological mini-liver systems.



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

FaulknereJones and colleagues laid the foundation for this by demonstrating sustained viability and continued differentiation of ESC/iPSC-HLCs following 3D bioprinting (Faulkner-Jones et al., 2015). The advances summarized here should be applied to generate near-physiological, iPSC-derived LOs suitable and permissive for hepatotropic pathogen studies. The combination of iPSC-derived gut organoids with complex iPSC-derived hepatobiliary-pancreatic 3D structures (Koike et al., 2019) through fluidics will finally enable authentic mimicry of the guteliver axis and thus allow, among others, integrative studies of HAV/HEV transmission and other aspects of hepatotropic pathogen dissemination.

Personalized modeling and treatment of hepatotropic infections HLCs have proven useful in testing antiviral drugs against HBV (Xia et al., 2017), HEV (Dao Thi et al., 2016), and HDV (Qu et al., presented at HBV liver meeting 2019). With the possibility of generating iPSCs from donor-derived cells and differentiating them to various cell types, which are then permissive for virus isolate infection from the same donor (Fig. 7.2), iPSCs provide a unique platform for personalized infection modeling and drug testing (reviewed in Davidson et al. (2015); Sayed et al. (2016)) (Figs. 7.2 and 7.3). This was already indicated by the recent combination of patient-derived P. vivax sporozoites and HLCs for the comparison of different platforms to study LS development (Subramani et al., 2020). Comparing iPSCs from healthy donors with donors carrying a SNP of interest, or introducing a SNP of interest in wild-type iPSCs by CRISPR-Cas9 prior to differentiation (Fig. 7.3), will help elucidate the positive and negative impacts of such host genetic differences on pathogenesis and disease progression in either monocultures, cocultures of different cell types, or even 3D systems (Fig. 7.3). Consequently, the underlying processes by which polymorphisms in the TLR3 gene result in differential risk of HBV/HCV infections and HCC development (Geng et al., 2016; El-Sharawy et al., 2020) could be systematically investigated. In addition, the protective effects of the ApoE alleles against HEV infection and HCV-induced liver damage could be clarified (Wozniak et al., 2002; Zhang et al., 2015). Similarly, genetic manipulation of iPSCs may help resolve the role of the IL18BP gene in developing fulminant hepatitis A (Belkaya et al., 2019). The genetic tractability of HLCs may also help assess the impact of various host polymorphisms (Driss et al., 2011) on hepatocyte infection by Plasmodium species and the progression of the LS. If hypnozoites could be identified in HLCs, this could additionally benefit diverse P. vivax relapse models based on differences in host genetics. Overall, genetically amenable iPSCs offer unique opportunities for assessing the effect of host genetic differences in isogenic pairs of wild-type and mutated cells (Fig. 7.3). For example, patient-derived iPSCs harboring a mutation in the LDL receptor

Limitations of iPSCs

(LDLR) were corrected by CRISPR-Cas9-mediated insertion of an LDLR expression cassette and infected with HCV, uncovering a potential role of LDLR in HCV morphogenesis (Caron et al., 2019). Donor-derived HLCs can also be used for personalized drug testing to elucidate the detailed mechanisms of drug responsiveness and assess drug hepatotoxicity, as well as for large-scale screening for specific compounds alleviating hepatic disease phenotypes or targeting virus infection (Fig. 7.2). This becomes particularly important if a polymorphism confers reduced sensitivity to certain antiviral treatments such as the IL28B SNP affecting HCV therapy (Ge et al., 2009). Therapeutic strategies counteracting the effects of certain polymorphisms may have the potential to limit progression to severe disease outcomes. Engraftment of immunocompromised mice with donor-derived or genetically manipulated HLCs (Carpentier et al., 2014; Nagamoto et al., 2016; Yuan et al., 2018) allows the creation of personalized animal models for recapitulating patient-specific disease progression and testing of antiviral therapies (Fig. 7.2). However, the capacity of these systems to faithfully imitate human infection, in the absence of an immune system or other human cell types, is limited. Finally, donor-derived iPSCs carrying a polymorphism associated with fatal or severe outcome of hepatotropic infection could be ex vivo repaired using CRISPR-Cas9, differentiated to HLCs, and then transplanted back into the patient.

Limitations of iPSCs and future directions for the study of hepatotropic infections Current iPSC differentiation protocols yield HLCs that express fetal hepatocyte genes such as alpha-fetoprotein (AFP) as well as CYP3A7, but do not express all liver markers at a level equivalent to adult PHHs (reviewed in Schwartz et al. (2014)). A well-known limitation of iPSC-derived HLCs is also the low expression of CYP450 drug-metabolizing enzymes compared to PHHs (Ulvestad et al., 2013; Medine et al., 2013; Lu et al., 2015), which may limit antiviral drug studies and the use of HLCs for high-throughput drug screenings. This is further hampered by inefficient infection of patient-derived HLCs with P. vivax sporozoites (Subramani et al., 2020), for example. In general, immature HLC phenotypes, challenging reproduction of hepatic polarization, suboptimal metabolic functions, and limited cultivation periods can represent major hurdles of using iPSC technology for hepatotropic pathogen studies. In addition, differentiation and cultivation of HLCs remain expensive, timeconsuming, and difficult compared to conventional cell culture. Recently, small molecules (March et al., 2013; Li et al., 2018; Siller et al., 2015), accurate choice of medium composition (Toba et al., 2020) in combination with optimized protocols (Xia et al., 2017; Sa-Ngiamsuntorn et al., 2016; Ng et al., 2015), differentiation on semipermeable Transwell membranes (Dao Thi et al., 2020), hepatic cocultures (Pettinato et al., 2019; Berger et al., 2015; Freyer et al., 2017) and 3D aggregates (Ardalani et al., 2019; Ogawa et al., 2013)



CHAPTER 7 iPSCs for modeling hepatotropic pathogen infections

have emerged as potential solutions to improve maturity, cellular architecture, and in vitro maintenance of HLCs. For example, HLCs cocultured with murine fibroblasts in the MPCC setup showed enhanced levels of drug-metabolizing enzymes and were better suited for long-term drug toxicity assessment than HLCs grown in monolayers (Berger et al., 2015). Likewise, others have compared iPSC-derived liver 3D spheroids with cells grown in 2D and found increased phenotypic and metabolic responses upon exposure to hepatotoxic drugs (Sirenko et al., 2016).

Outlook Hepatotropic infections remain a major threat to the global population (Asrani et al., 2019). The hepatotropic infectious disease field would highly benefit from an in vitro system that is universally susceptible, genetically tractable, and at the same time physiologically relevant. Ideally, it also replicates the complex liver architecture. Such a system would allow more authentic modeling of infection, pathogenesis, and disease. We believe that ESC/iPSC-derived HLCs harbor several major advantages over conventional hepatocyte systems and thus, have the potential to fill this need (Fig. 7.2). Once differentiated, HLCs are primary-like cells that can better replicate cellular pathways than hepatoma cells. These are critical to improve pathogen-associated molecular studies on the one hand and to increase the understanding of disease progression and pathogenesis on the other hand. At the same time, unlike PHHs, the system is reproducible and genetically tractable (Fig. 7.3). Owing to the aforementioned intrinsic and functional limitations, HLCs may not yet be fit to cover all aspects of hepatotropic pathogen studies. However, better differentiation protocols will hopefully yield HLCs that more closely resemble hepatocytes in the liver. The ability to differentiate ESCs/iPSCs into organoids fuels further hopes for the generation of a vascularized, near-physiological mini-liver system, which accurately mimics the cellular diversity of the liver, as well as its structure and function. Universal permissiveness of such a complex ESC/iPSC-derived liver system to all hepatotropic pathogens will open up unprecedented possibilities for the study of hepatotropic diseases, allowing direct comparisons among the pathogens in terms of disease, liver injury, immune responses, and interaction with the various liver cell types. In addition, ESCs/iPSCs provide an unmatched versatility as they constitute the base unit for the generation of systems with varying complexity, ranging from 2D mono- and cocultures to 3D organoids composed of different cell types (Fig. 7.3) and engraftment in animal models (Fig. 7.2), while simultaneously providing an isogenic background. With the enormous progress made in the past decades in genetic manipulation by CRISPR-Cas9 and next-generation sequencing for rapid analysis of patient genomes (reviewed in Qin (2019)), personalized medicine has become highly feasible. Together with the genetic tractability of iPSCs, the impact of patient-specific SNPs can now be studied in systems of varying complexity (Fig. 7.3). It further holds the potential of repairing SNPs of interest by ex vivo gene therapy.


In summary, ESC/iPSC-derived in vitro systems will provide the field of hepatotropic infectious disease research with universally susceptible, reproducible, physiologically relevant, and genetically tractable models. We believe that iPSCs have a great potential for generating systems of increasing complexity, by which every stage of pathogen infection can be characterized and drug sensitivity could be studied in a personalized fashion, eventually unraveling the remaining mysteries of hepatotropic infectious diseases.

Acknowledgments We thank Xianfang Wu, Ombretta Colasanti, Stephan Urban, Volker Lohmann, Eike Steinmann, Markus Ganter, Zhenfeng Zhang, and Andrew Freistaedter for helpful discussions and critical reading of the manuscript. GB and VLDT were supported by the Schaller foundation. AM was supported by DFGdProject ID 272983813-SFB/TRR179.

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Use of human induced pluripotent stem cells (hiPSC)-derived neuronal models to study the neuropathogenesis of the protozoan parasite, Toxoplasma gondii

Sandra K. Halonen Department of Microbiology and Immunology, Montana State University, Bozeman, MT, United States

Chapter outline Introduction ............................................................................................................216 Overview of principles and methods for generation of neurons from hiPSCs................217 hiPSC reprogramming into neuronal cells via directed differentiation ..............218 2D monolayers hiPSC-derived neuronal models: advantages and limitations ....220 3D neural organoid models..........................................................................220 Limitations of neural organoids ................................................................ 222 Complex neural organoids and future developments in organoid culture........ 222 T. gondii: biology of chronic infection in the brain....................................................223 Bradyzoite and cyst biology .........................................................................224 Pathogenesis of the chronic infection in the brain .........................................226 Potential uses of 2D neuron monolayers to study T. gondii .............................227 Developmental biology of bradyzoites and cysts ......................................... 227 Host/parasite interactions ........................................................................ 227 Neuropathogenesis ................................................................................ 227 Previous studies using hiPSC-derived 2D neuronal culture .............................228 Potential for uses of 3D cerebral organoids to study Toxoplasma gondii...........229 Future trends and direction: use of hiPSC-derived 2D and 3D models to model human parasitic infections ......................................................................................230 References .............................................................................................................231

iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00010-9 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 8 Use of human induced pluripotent stem cells

Abstract The use of in vitro generated neurons derived from human induced pluripotent stem cells (hiPSCs) is an important tool for neurological disease modeling. Two-dimensional (2D) monolayer cultures and three-dimensional (3D) brain organoids, which can more precisely mimic in vivo complexities and cytoarchitecture of the brain, have been used to study neurological disorders such as Parkinson’s disease, schizophrenia, autism spectrum disorder, among others. While significant progress has been made using 2D and 3D hiPSC-derived neuronal models to study neurological disorders, the use of hiPSCs in the study of neuropathogenesis of parasitic diseases has been limited. Toxoplasma gondii is an obligate intracellular parasite that infects neurons in the central nervous system causing a chronic lifelong infection in the brain with approximately one-third of the worlds’ population chronically infected. While chronic Toxoplasmosis has been thought to be asymptomatic, recent evidence indicates that the chronic infection is associated with development of neuropsychiatric disorders such as schizophrenia, neurological disorders such as cryptogenic epilepsy, and cognitive decline in the elderly. Cysts in neurons, containing the bradyzoite form of the parasite, are the dominant forms present in chronic infections and likely the cause of some neurological effects during the chronic infection, yet remain poorly understood aspects of the parasites’ life cycle. The use of 2D and 3D human neuronal models provides opportunities to address questions of host/parasite interactions of the bradyzoites and cysts in neurons and investigate mechanisms of neuropathogenesis of T. gondii. In this review, an overview of the methods of hiPSC-derived 2D and 3D neuronal models is given, followed by a discussion of potentials of these neuronal models to study T. gondii. The use of hiPSC-derived 3D organoids to study pathogenesis of other parasitic infections is also briefly addressed. Keywords: Bradyzoite; Cerebral organoids; Cerebral toxoplasmosis; CNS; Cystogenesis; Disease modeling; Hosteparasite interaction; Human induced pluripotent stem cells; Human neurons; iPSCs; Monolayer hiPSC culture; Neuroparasitic infection; Neuropathogenesis of protozoan parasite; Reprogramming; Toxoplasma gondii.

Introduction The use of in vitro generated neurons derived from induced pluripotent stem cells has emerged as an important tool for neurological disease modeling over the past decade. Human induced pluripotent stem cells (hiPSC) offer distinct advantages over the previous use of cell lines or rodent primary neural cell cultures in that they are self-renewing with long-term culturing capabilities, and they are able to generate functional neuronal and glial cells of human origin. The use of hiPSCs obtained by reprogramming somatic cells also eliminates the ethical concerns associated with the use of embryonic stem cells that have also been used for neuronal disease modeling. Methods for two dimensional (2D) monolayer cultures have been developed using hiPSCs and have been used to study neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease, neuropsychiatric disorders such as schizophrenia and bipolar disorder, and neurodevelopmental

Overview of principles and methods for generation of neurons from hiPSCs

disorders such as autism spectrum disorder, among others (Logan et al., 2019; Liu et al., 2018; D’Souza et al., 2020; Ferrari et al., 2020). More recently, threedimensional (3D) brain organoids have been developed, which can more precisely mimic in vivo complexities and cytoarchitecture of the brain (Dutta et al., 2017; Pacitti et al., 2019; Lancaster and Knoblich, 2014; Worsdorfer et al., 2020). While significant progress has been made using hiPSC-derived systems to model diverse neurological disorders, the use of hiPSCs in the study of neuropathogenesis of infectious parasitic diseases such as cerebral malaria, trypanosomiasis, and toxoplasmosis, all of which cause significant neurological disease worldwide, has been limited. Toxoplasma gondii is an obligate intracellular parasite that infects neurons in the central nervous system (CNS) causing a chronic lifelong infection in the brain. T. gondii is a ubiquitous infection with approximately one-third of the world’s population chronically infected (Hill et al., 2005). While chronic Toxoplasmosis has traditionally been thought to be asymptomatic, recent evidence indicates that the chronic infection is associated with development of neuropsychiatric disorders such as schizophrenia and depression, neurological disorders such as cryptogenic epilepsy and migraines, and cognitive decline in the elderly (Fabiani et al., 2013, 2015; Burgdorf et al., 2019; Sugden et al., 2016; Xiao et al., 2018; Pedersen et al., 2011, 2012; Miman et al., 2010; Palmer, 2007; Yazar et al., 2003; Gajewski et al., 2014, 2016; Brown and Patterson, 2011; Yolken et al., 2009; Hinze-Selch et al., 2010; Flegr, 2010; Beste et al., 2014; Golka et al., 2015; Mendy et al., 2015; Ngoungou et al., 2015). Most previous in vitro neuronal models used to study T. gondii have used neuronal cell lines or rodent primary neural cell cultures, which may not recapitulate the cellular and molecular mechanisms present in human neurons or are derived from in vivo studies in mice chronically infected with T. gondii, which may not be reflective of the complex processes that occur in human brains, especially as relates to behavior (Mammari et al., 2014, 2015; Parlog et al., 2015; Ferguson & Hutchison, 1987a,b). The host/parasite interactions of T. gondii with the neuron host cell and in the CNS are complex and incompletely understood. Many of the advantages afforded by the use of hiPSCs for modeling neurological diseases also pertain to the modeling of neuropathogenesis of T. gondii. In this review, an overview of the principles and methods of hiPSC-derived 2D and 3D neuronal models will be given, followed by a discussion of potentials of use of hiPSC-derived 2D neuronal monolayers and 3D cerebral organoids to study neuropathogenesis of T. gondii. The use and potential of hiPSC-derived 3D organoids to study pathogenesis of other parasitic infections will also be briefly addressed.

Overview of principles and methods for generation of neurons from hiPSCs In 2007, Takahashi and Yamanaka discovered that mature somatic cells such as human fibroblasts could be reprogrammed into induced PSCs (iPSCs) via the



CHAPTER 8 Use of human induced pluripotent stem cells

expression of four transcription factors, SOX2, OCT4, KLF4, and c-Myc, the now so-called Yamanaka factors (Takahashi et al., 2007). Since that time reprogramming of iPSCs derived from many human somatic cell types such as blood and urine that are more easily accessible than fibroblasts has been developed (Cheng et al., 2017; Raab et al., 2014; Kim et al., 2016; Shi et al., 2016). Differentiation protocols into numerous cell types, including neurons, have also been established (Pistollato et al., 2017; Arenas et al., 2015; Chen et al., 2013; Peng and Zeng, 2011; Sances et al., 2016; Jiang et al., 2016; Carpenter et al., 2012; Shi et al., 2012). The discovery of iPSCs and development of protocols to generate neurons from hiPSCs provided a means to generate unlimited human neurons in vitro and facilitated in vitro disease modeling of many neurological diseases. Derivation of human neurons generated from somatic cells that are induced to hiPSCs via introduction of Yamanaka factors is called directed differentiation. An alternative method called direct reprogramming derives human neurons directly from differentiated cells without going through an iPSC (Ladewig et al., 2013). Neurons generated via direct reprogramming are called induced neurons (iNs). Details of direct reprogramming methods can be found in several recent reviews and will not be discussed further in this review as directed differentiation methods have more versatility for disease modeling studies (Caiazzo et al., 2011; Torper et al., 2013; Gascon et al., 2017; Grath and Dai, 2019).

hiPSC reprogramming into neuronal cells via directed differentiation Directed differentiation protocols first derive hiPSCs from somatic cells, such as fibroblasts or hematopoietic cells, via the introduction of Yamanaka factors, followed by neural induction and neuronal differentiation to produce differentiated neurons (Fig. 8.1). The principle of most hiPSC-derived neuron protocols reproduces the events that occur in neurodevelopment (Tao and Zhang, 2016; Reubinoff et al., 2001; Zhang et al., 2001). Namely, hiPSCs are induced to begin differentiation via culture under serum-free medium conditions, which favors neuroderm and limits meso-endoderm cells, resulting in the majority of cells becoming neural stem cells (NSCs). NSCs during development act as self-renewing cells that can differentiate into multiple types of brain cells. Likewise, in vitro NSCs can be differentiated into neurons in neural differentiation medium containing components such as brain-derived neurotrophic factor (BDNF), glial-cell-derived neurotrophic factor (GDNF), dibutyryl-cAMP, and ascorbic acid. Many improvements in directed differentiation of neurons from iPSCs have been developed over the last decade due in part to improved understanding of signaling pathways. For example, initial protocols often used the generation of embryoid bodies (EBs) to derive NSCs and the use of stromal cells or astrocytes as feeders. The use of inhibitors of the SMAD-dependent TGFb and bone morphogenetic protein (BMP) signaling pathways, the so-called “dual SMAD inhibition” method, inhibits the differentiation into cells with a nonneural fate and thus

Overview of principles and methods for generation of neurons from hiPSCs

FIGURE 8.1 Directed differentiation of hiPSC-derived neurons and other CNS cell types. Schematic of derivation of differentiated human neurons and other CNS cell types such as microglia, astrocytes, and oligodendrocytes, from human induced pluripotent stem cells (hiPSC’s) derived from somatic cells. Somatic cells such as fibroblasts or hematopoietic cells are reprogrammed into induced pluripotent stem cells (iPSC) via Yamanaka factors (OCT4, SOX2, KLF4, L-Myc), subjected to neural induction via dual SMAD inhibition to form neural stem cells (NSC), a self-renewing cell population that can be propagated in culture. NSCs when treated with neurotrophic factors produce differentiated neurons (hiPSC-neurons) or alternately can differentiate into Astrocytes or Oligodendrocytes when treated with the appropriate differentiation factors. The iPSCs, prior to neural induction, can also be induced to differentiate microglia, cells of mesodermal origin.

removed the requirements of generating EBs and the use of stromal cells as feeder layers (Chambers et al., 2009a,b). The elimination of the use of EBs, which exhibited batch-to-batch variation, improved the performance of hiPSC protocols. In addition, the elimination of the need for feeder layers created a simpler and more economic solution for generating NSCs. Protocols have also now been established to derive specific neuron subtypes from hiPSC-derived NSCs, based upon the understanding and use of patterning morphogens, which govern morphogenesis during embryogenesis (Tao and Zhang, 2016). For example, during embryogenesis, morphogenesis patterning is coordinated by morphogen gradients along the anterioreposterior (A-P) and dorsale ventral (D-V) axes. Morphogens affecting the A-P patterning include FGFs, WNTs, and retinoic acid (RA) while those that influence D-V patterning include WNTs, BMPs, and sonic hedgehog (SHH). Thus, for example, hiPSC methods to generate midbrain dopaminergic neurons (mDAs), all typically include, in addition to reprogramming toward a stem cell fate through the addition of Yamanaka factors and SMAD signaling inhibitors to suppress mesodermal fate, incorporation of patterning morphogens such as FGF8, SHH, WNT (Ferrari et al., 2020; Arenas et al., 2015). Based upon the knowledge of patterning morphogens, protocols are now established for derivation of diverse neuronal subtypes including glutamatergic, GABAergic, GABAergic/cholinergic, dopaminergic, 5-HT, and motor neurons (Tao and Zhang, 2016). Glial subtypes such as astrocytes and oligodendrocytes can also be differentiated from NSCs generated from hiPSCs (Fig. 8.1). Astrocytes are generated after neurogenesis, relatively late in development, and thus the differentiation process in vitro



CHAPTER 8 Use of human induced pluripotent stem cells

takes a longer period of time. Other complicating factors of astrocyte differentiation are that the molecular signals for astrocyte differentiation and the molecular markers of astrocytes and astrocyte subtypes are less defined (Tao and Zhang, 2016). Thus, hiPSC-derived astrocyte protocols often require functional characterizations to be done as well. Despite these limitations, several protocols for differentiation of astrocytes from hiPSCs have been established with efficiencies ranging up to >90% (Krencik and Zhang, 2011; Serio et al., 2013; Shaltouki et al., 2013). Oligodendrocytes also appear late in development and typically require longer periods of time in culture, but similar to astrocytes several protocols with relatively high efficiency (80%e90%) have been developed (Wang et al., 2013; Gorris et al., 2015; Stacpoole et al., 2013).

2D monolayers hiPSC-derived neuronal models: advantages and limitations The goal of most hiPSC reprogramming methods has been to provide a twodimensional (2D) monolayer of relatively pure (>70%) human neurons for singlecell, population-based assays that are conducive to mechanistic studies, drug and toxicity screenings (Logan et al., 2019; Liu et al., 2018). Advantages of 2D monolayers include good resolution of cell morphology and ease of microscopic analysis, as well as protocols that are relatively easy, inexpensive, fast to produce, and have high reproducibility (Table 8.1). These 2D monolayer neuronal cultures have contributed to disease modeling studies of a wide variety of neurological diseases including neurodegenerative, neurodevelopmental, and neuropsychiatric disorders as well as neurological disorders such as epilepsy (Logan et al., 2019; Liu et al., 2018; Pacitti et al., 2019). In addition to these advantages, hiPSC-derived 2D monolayers are also amenable to CRISPR gene editing enabling elucidation of genetic factors underlying disease risk, cellular processes of disease initiation, and other pathogenesis mechanisms to be addressed (Rehbach et al., 2020). The many advantages of these 2D neuronal cultures conversely create disadvantages including lack of heterogeneous cell types, tissue architecture, and in vivo organ environment (Logan et al., 2019; Liu et al., 2018). Additionally, 2D systems favor stronger interactions with the surface of the culture vessel than cellecell interactions or between the cells and the extracellular matrix (ECM). Cellecell interactions or cellematrix interactions control processes such as cellular differentiation and maturation. Thus, many of the processes that occur in differentiation and development often do not occur in 2D neuron monolayers, limiting their use in modeling of neurological diseases. The limitations of 2D monolayer cultures have driven the development of more complex 3D in vitro models known as neural organoids (Logan et al., 2019; Liu et al., 2018; Pacitti et al., 2019).

3D neural organoid models Organoids are three-dimensional (3D) aggregates derived from iPSCs that contain multiple cell types and have complex tissue organization (Liu et al., 2018; Dutta et al., 2017; Pacitti et al., 2019). In addition to heterogeneous cell types and complex

Overview of principles and methods for generation of neurons from hiPSCs

Table 8.1 Comparison of hiPSC-derived 2D and 3D neuronal models. 2D monolayer

3D monolayer

Production method

Grown on rigid flat substrate as a monolayer

Timing Maturation status ECM

Fast (days to weeks) Immature in metabolic and electrophysiological activity Little-to no cell-ECM interactions Flat morphology Cell functionality partial and variable Monoculture

Embedded in matrigel and undergo self-organization in response to differentiation cues Slow (months) Mature

Cell morphology and functionality Cell types

In vivo-like composition and cell contacts Similar to in vivo

Tissue architecture Variability and reproducibility Genome-editing capabilities Characterization and analysis


Heterogeneous cell types and subtype but limited to cells of ectodermal origin Complex

Low variability and high reproducibility Easy

High variability and low reproducibility Hard

Easy cell retrieval Ease of cellular, molecular, and microscopy analysis



Hard to retrieve cells and analyze phenotypes Microscopy analysis more difficult often requiring confocal microscopy and sectioning Absent

Modified from original source, Liu, C., Oikonomopoulos, A., Sayed, N., Wu, J.C., 2018. Modeling human disease with induced pluripotent stem cells: from 2D to 3D and beyond. Development 145. https://doi.org/10.1242/dev.156166.

tissue architecture, 3D organoids overcome many of the limitations of 2D monolayers including allowing dynamic interactions between cells and ECMs, generating cell maturity, and improved cell functionality (Table 8.1). Brain organoids, also called cerebral organoids, for example, contain distinct cell types such as glial cells as well as neurons and show distinct brain regions (Lancaster and Knoblich, 2014). Cerebral organoids have a wider application for in vitro disease modeling than 2D monolayers and have been used in disease modeling of neurological disorders including Alzheimer’s disease, Parkinson’s disease, and schizophrenia, among others (Choi et al., 2014; Lee et al., 2016; Raja et al., 2016; Tieng et al., 2014; Amin and Pasca, 2018; Stachowiak et al., 2017). Organoids are distinct from 2D neuronal models in that they develop through intrinsic self-organizing processes (Dutta et al., 2017). Cerebral organoids are typically generated from iPSCs grown in suspension culture where they aggregate to form cell clusters. Cells within the aggregates differentiate to develop a



CHAPTER 8 Use of human induced pluripotent stem cells

neuroepithelial phenotype, a type of stem cell capable of differentiating into other cell types. These aggregates are embedded in Matrigel and further cultivated for maturation, resulting in a multilayered cortex-like tissue organization. The neural organoids consist of neuroepithelial stem cell compartments lining fluid-filled cavities that are reminiscent of ventricles and differentiate neural cell types such as radial glial cells, neurons, astrocytes, oligodendrocytes, and ependymal cells. Brain-region-specific organoids can be produced via the targeted manipulation of specific signal pathway pathways (e.g., SHH, Wnt, TGFb) during neural induction (Pacitti et al., 2019). Brain region organoids that have been developed include cortical, midbrain, forebrain, hypothalamus, cerebellar, and hippocampal-choroid plexus organoids with specific brain region organoids used for disease modeling of diverse neurological disorders (Logan et al., 2019; Lancaster and Knoblich, 2014; Jo et al., 2016; Qian et al., 2016; Pasca, 2018; Muguruma et al., 2015; Sakaguchi et al., 2015).

Limitations of neural organoids 3D organoids recapitulate the complex features of the brain and are invaluable in vitro tools to model neurological disease and offer many advantages over 2D monolayers, although they still have limitations (Table 8.1). For one, cerebral organoids are limited in size as necrotic centers occur over time in culture due to insufficient supply of nutrients and oxygen. Bioreactors can be used to oxygenate the culture medium, which allows culture for short periods of time (Qian et al., 2016). As 3D cerebral organoids are generated from iPSCs, they have many of the same limitations of 2D monolayers including differentiation efficiency across cell lines and batch-to-batch variability. Other disadvantages include need for more complex methods for characterization and analysis, and they are less amenable to use of gene editing techniques. Finally, cost, technical difficulties, and the time to generate cerebral organoids, which typically take about 100 days, are significantly greater than for 2D monolayers and important factors to consider with 3D organoid culture.

Complex neural organoids and future developments in organoid culture Cerebral organoids while being more complex and reflective of the cytoarchitecture of brain environment still lack some features of the in vivo environment (Table 8.2). Neural organoids are generated from neuroectodermal progenitor cells, and thus lack a vascular system, which is of endodermal and mesodermal origin (Logan et al., 2019; Pacitti et al., 2019; Worsdorfer et al., 2020). Vascularization of neural organoids has been attempted via the incorporation of iPSC generated endothelial cells into neural organoids but as blood vessels are also comprised of cells of mesenchymal origin, incorporation of endothelial cells is not sufficient (Worsdorfer et al., 2017). An alternative method is the cocultivation of mesenchymal and neural organoids, which has resulted in partially vascularized neural organoids (Worsdorfer et al., 2019). Methods of vascularization of neural organoids are still in development, but would help reduce or eliminate the size limitation of neural organoids as well as accelerating functional maturation of neurons.

T. gondii: biology of chronic infection in the brain

Table 8.2 Some of the outstanding questions on chronic Toxoplasmosis that could be addressed using of 2D and 3D neuronal models. A. Developmental biology of bradyzoites and cyst biology

B. Host/parasite interactions in neurons

C. Neuropathogenesis

2D monolayers

3D organoids

Allow longitudinal studies of bradyzoite/cyst development to gain knowledge of temporal events that occur during bradyzoite differentiation and cyst maturation Allow dynamic and kinetic events occurring within cysts such as bradyzoite motility, intraneuronal trafficking of bradyzoites, patterns of cyst growth, etc., to be discerned Impacts of soma-associated cysts versus dendritic cysts on neuronal functions, etc. Dissect neuronal hosteparasite interaction of bradyzoites; determining direct effects of bradyzoite/cysts on neurons such as apoptosis, etc. Study of bradyzoite/cysts on neurotransmission and specific neurotransmitters using regionspecific neurons Assessment of impacts on neuronal structures such as dendrites and synapses

Allow more mature cyst development and maturation and longer term studies

Screening and evaluation of antibradyzoite and cyst drugs with potential for mechanism of action studies

Enable study of effect on neurotransmission and neuronal networks Investigate interactions between infected neurons and astrocytes Evaluate drug effective against mature cysts

Cerebral organoids, generated from NSCs, also lack immune cells such as microglia, which are of mesodermal origin. Microglia, which exhibit morphological and functional characteristics of in vivo microglia, however, can be generated from hiPSC (Fig. 8.1). hiPSC-generated microglia can then be cocultured with neural organoids (Muffat et al., 2016, 2018). As microglia have important roles in the synaptic plasticity, neurogenesis, homeostatic functions, and immune activity, incorporation of microglia into neurological models may be of importance in many disease-modeling studies (Abud et al., 2017).

T. gondii: biology of chronic infection in the brain T. gondii is an obligate intracellular protozoan parasite that infects cats as the definite host with a wide variety of birds and mammals, including man, able to serve as



CHAPTER 8 Use of human induced pluripotent stem cells

intermediate host (Elmore et al., 2010). Humans primarily become infected via ingestion of contaminated meat or accidental ingestion of the oocyst stage shed from cats. Upon ingestion, parasites invade the intestinal epithelial cells and differentiate to the tachyzoite stage, a rapidly replicating form of the parasite that can infect monocytes and macrophages and disseminates the parasite throughout the body. Tachyzoites enter the brain within a week postinfection crossing the bloode brain barrier (BBB) either via transcytosis, paracellularly or trafficking across the BBB within white blood cells via a Trojan Horse mechanism (Mendez and Koshy, 2017; Barragan et al., 2005; Barragan and Sibley, 2003; Lambert and Barragan, 2010). Once in the brain parenchyma, tachyzoites initially replicate within microglia and astrocytes, but replication is limited due to effective IFN-g stimulated antimicrobial mechanisms in these cells (Halonen et al., 1998, 2001; Chao et al., 1993, 1994). Conversely in neurons, the tachyzoites differentiate to the bradyzoite stage, a slowly replicating form of the parasite that forms cysts within neurons (Fig. 8.2). The cysts containing bradyzoites are not cleared by the immune response and establish a lifelong persistent infection with cysts located in neurons. Cysts containing the bradyzoite form of the parasites are the dominant forms present in chronic infections that maintain the infection in the brain and are likely the cause of neurological effects during the chronic infection.

Bradyzoite and cyst biology Despite the key role played by the cyst and the bradyzoites in maintenance of the infection in the brain, the knowledge of cysts and bradyzoite biology remain poorly understood aspects of the parasites life cycle. Neurons are the host cell for cysts in chronic infection and appear to be specifically targeted by the parasite during the chronic infection although the preferential targeting of neuron host cell is not fully understood (Ferguson and Hutchison, 1987b; Cabral et al., 2016; Sims et al., 1989). Bradyzoites within cysts were long thought to differentiate to a nonreplicating bradyzoite, forming mature cysts that are dormant, relatively static entities that persist within neurons for the lifetime of the host (Ferguson & Hutchison, 1987a,b; Chew et al., 2012; Dubey et al., 1998). This view was challenged, however, by a recent studying finding that bradyzoites within mature cysts can undergo replication, either periodically in patches in the cysts or episodically in which all bradyzoites initiate replication synchronously, suggesting that cyst growth and expansion may occur during a chronic infection (Watts et al., 2015; Sinai et al., 2016). The significance of these findings is not fully understood but indicates that bradyzoites and cysts are dynamic as opposed to static entities within neurons in the brain. Bradyzoites within cysts also exhibit motility and have the ability to move intracellularly, suggesting that intracellular movement of bradyzoites between neurons could serve to disseminate the infection within the brain while avoiding the host immune response (Dzierszinski et al., 2004). Finally, a recent study analyzing cysts from in vivo infected neurons found that many intraneural cysts are located in dendritic processes in addition to the soma of the neuron (Koshy and Cabral, 2014). Overall these studies indicate that cysts

T. gondii: biology of chronic infection in the brain

FIGURE 8.2 Schematic of the infection of T. gondii of neurons in the central nervous system. Tachyzoites in the brain parenchyma infect neurons and convert to the slowly replicating bradyzoite stage within 12 h after infection, prior to parasite replication, with cyst wall material beginning to be expressed within this time period, as indicated by in vitro studies. Bradyzoites initially replicate slowly within cysts, differentiating to nonreplicating terminally differentiated bradyzoites (green) forming mature cysts that can develop near the soma or dendritic processes of neurons; some mature cysts contain nonreplicating terminal bradyzoites (green) in addition to replicating bradyzoites (dark gray), indicating that cysts are more dynamic entities (Sinai et al., 2016; Cabral et al., 2020).

are a heterogeneous population with distinct locations within neurons, with some cysts containing replicating as well as nonreplicating bradyzoites, and with bradyzoites possibly able to disseminate the infection intracellularly between neurons in the brain (Fig. 8.2). The significance of cyst heterogeneity, bradyzoite dynamic behaviors, and patterns of cyst growth are not understood but highlight the lack



CHAPTER 8 Use of human induced pluripotent stem cells

of understanding of the developmental biology of bradyzoites and cysts indicating that there also many aspects of host/parasite interactions of bradyzoites and cysts in neurons that remain poorly understood.

Pathogenesis of the chronic infection in the brain While the chronic infection was long considered asymptomatic in immunocompetent individuals, the association of the chronic toxoplasmosis with a wide range of neurological disorders, ranging from schizophrenia to epilepsy to mild behavioral and cognitive effects, indicates that the parasite does impact neurological function in the brain. The mechanism responsible for these parasite-induced neurological effects is not clear, but recent studies indicate that the intraneuronal cysts impair and alter the functions of infected neurons such as neurotransmission. For example, infection of dopaminergic neurons leads to an increase in dopamine secretion, due to parasite encoded tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine (Prandovszky et al., 2011; Wang et al., 2015). Other studies have found that infection causes altered CNS excitotoxicity either via increase in glutamate, the major excitatory neurotransmitter of the CNS or due to a disruption in reuptake of extracellular glutamate by astrocytes, or alternately via effects on g-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain (David et al., 2016; Brooks et al., 2015). In addition to these effects on neurotransmitters, infection with T. gondii has been found to have direct effects on neurons such as an alteration in dendritic arborization, spine number, and synaptic proteins, suggesting that the synaptic connections may be disrupted with possible neuronal losses (Parlog et al., 2014). Changes to neurotransmission either via impact on neurotransmitters and/or via structural changes all suggest mechanisms for parasite-induced behavioral changes. From the aforementioned studies, it is clear that the biology of the cysts and bradyzoite stages and the impact of infected neurons on neuronal functions are incompletely understood. Much of the basic understanding of bradyzoites and cysts in the brain and on brain function presented earlier has been generated from in vivo studies of chronically mice, which may not recapitulate the biology in human brain (Ferguson & Hutchison, 1987a,b; Ferguson et al., 1989). Furthermore, analysis of patterns of bradyzoite replication, cyst growth, and intraneuronal location of cysts are hard to discern from in vivo studies given the complex matrix of the brain and the difficulty of assessing temporal events from analysis of discrete time points from a chronic infection. Likewise, the effects on neurotransmission or structural effects on dendrites or synaptic connections are equally hard to address in vivo due to the structural complexities of the brain. A better understanding of the developmental biology of bradyzoites and cysts in the brain and the impact of the parasite on neuronal function is needed to understand the complex mechanisms leading to behavioral alterations of chronic toxoplasmosis and could lead to more effective drugs and treatment strategies for neurological symptoms resulting from chronic toxoplasmosis.

T. gondii: biology of chronic infection in the brain

Potential uses of 2D neuron monolayers to study T. gondii 2D neuronal models derived from hiPSCs offer many advantages to study T. gondii in neurons, such as the ability to create monolayers of relatively pure neurons that exhibit differentiated neuronal morphology that are conducive to single-cell or population-based assays and afford opportunities to address many of these outstanding questions about the developmental biology of bradyzoites and cysts, host/parasite interactions in neurons, and neuropathogenesis (Table 8.2). Some of the outstanding questions that can be addressed using 2D neuron monolayers are briefly addressed further.

Developmental biology of bradyzoites and cysts 2D neuron monolayers cultures could be used to conduct time course studies of bradyzoite differentiation and cyst maturation. For example, neuronal monolayers could be infected with tachyzoites and followed at various time points postinfection during bradyzoites differentiation and cyst maturation. These types of longitudinal studies of bradyzoite differentiation and cyst development would allow basic knowledge of temporal events that occur during bradyzoite differentiation and cyst maturation in human neurons to be obtained, studies that are currently not possible using nonhuman primary neurons or neuronal cell lines, which often cannot support long-term or mature cyst development. Additionally, live cell imaging studies could be done with 2D neuron monolayers allowing dynamic events of bradyzoite and cyst biology that occur such as patterns of cyst growth and bradyzoite replication to be studied. Dynamic aspects of bradyzoite biology such as intracystic bradyzoite motility or possible intracellular trafficking of bradyzoites between neurons could also be studied.

Host/parasite interactions Studies on host/parasite type studies could be conducted such as addressing the impacts of soma-associated cysts versus dendritic cysts on rate of cyst growth, cyst size, or cyst stability and the impacts of soma-associated cysts versus dendritic cysts on neuronal functions such as neurotransmission. Additionally, direct impact of parasite infection on other neuronal functions such as inducing neuronal apoptosis, functional silencing, and other mechanisms of direct impact of the parasite on neurons that have been proposed are to be addressed (Parlog et al., 2015; Alday and Doggett, 2017). These types of host/parasite studies often cannot be done with existing neuronal cell lines as they do not express mature neuronal characteristics and/or neuronal functions.

Neuropathogenesis The effects of bradyzoites/cyst on neurotransmitters could be assessed, using region-specific neurons to study the effects on dopaminergic, glutamatergic, and GABAergic neurotransmitters, respectively. Additionally, impacts of infection on neuronal structures such as dendrites and synapses could be assessed, questions



CHAPTER 8 Use of human induced pluripotent stem cells

that often cannot be addressed with existing neuronal cell lines as they do not express these neuronal morphological characteristics. Finally, the ability to culture bradyzoites and mature cysts via use of 2D neuron monolayer would afford the opportunity to screen drugs effective against bradyzoites and/or cysts, for which currently there are no effective treatments, as well as address the mechanism of action of any drugs found to be effective (Alday and Doggett, 2017). 3D neural organoids could potentially be used to screen drugs effective against mature cysts with replicating as well as nonreplicating bradyzoites, thus expanding the panel of potential drugs effective against bradyzoites and the cyst stage.

Previous studies using hiPSC-derived 2D neuronal culture The use of a hiPSC-derived 2D neuron monolayer to study T. gondii infection in human neurons was recently investigated (Tanaka et al., 2016). This study used an NSC cell line (NCRM-1, obtained from NIH), which was derived from a hiPSC cell created using an episomal vector containing Oct4, Sox2, c-Myc, and Klf4, as a reprogramming method and with cord blood CD34þ cells used as the starting somatic cell. The NSCs were subjected to neural induction/differentiation media that resulted in a relatively pure (>90% of cells), high-density monolayer of neurons. hiPSC-derived neurons were infected with T. gondii tachyzoites and development was followed for up to 2 weeks postinfection. Tachyzoites infected the neurons with conversion to the bradyzoite stage occurring within an hour of infection, with the majority of parasite vacuoles (over 90%) developing into cysts by 4 days postinfection. These intraneural cysts had defined cyst walls, contained parasites expressing bradyzoite-specific antigen and morphological characteristics indicative of bradyzoites, and developed into cysts of 25e30 mm in diameter, reflective of the cyst sizes that develop in vivo. Furthermore, cysts developed in both the soma and dendritic processes of neurons, similar to what has been observed in neurons in the brain, indicating that this in vitro derived human neuron culture model recapitulates many of the key aspects of cyst development that occur in vivo. Results from this hiPSC-derived 2D human neuron model indicate that this is a good culture model to address questions of developmental biology of bradyzoites and cysts, host/parasite interactions in neurons, and neuropathogenesis mechanisms. However, while this 2D human neuron model supported tachyzoiteebradyzoite conversion in neurons and spontaneous cyst development, bradyzoites and cysts could not be maintained for more than 7e10 days due to the conversion of bradyzoites to tachyzoites, the actively replicating form that results in lysis of the neuron. Thus, while hiPSC-derived neurons were conducive for study of cystogenesis and mature cyst formation, stable bradyzoite differentiation and maintenance of mature cysts could not be generated in this 2D monolayer, suggesting that the use of the more complex 3D cerebral organoids may be necessary for this biology to be recapitulated in vitro.

T. gondii: biology of chronic infection in the brain

Potential for uses of 3D cerebral organoids to study Toxoplasma gondii Cerebral organoids could be used to address some of the limitations of 2D neuron monolayer model and allow further exploration of host/parasite interactions and more complex mechanisms of neuropathogenesis of T. gondii to be addressed (Table 8.2). For example, 3D cerebral brain organoids contain ECM material and astrocytes, which allows for more mature phenotype of neurons to develop (Liu et al., 2018; Worsdorfer et al., 2020). As one of the disadvantages of the 2D human neuron monolayer for the study of T. gondii infection in neurons was the inability to maintain bradyzoite differentiation and cyst maturation longer than 7e10 days, the presence of astrocytes and interactions of neurons with ECM may allow for longer and more mature cyst development in neurons to occur, thus allowing studies addressing behavior of bradyzoites and biology of mature cysts, such as bradyzoite replication and patterns of cyst growth. For example, 3D cerebral organoids could be infected with tachyzoites, and conversion to bradyzoites and cyst maturation followed for 3 weeks, and the resulting mature cysts followed for a month or longer, thus more closely mimicking the time frame of cyst development in vivo where mature cysts are found in the brain at 3 weeks postinfection and maintained in the brain for up to a year, in murine models. Additionally, 3D cerebral organoids would allow more complex questions of neuropathogenesis of T. gondii in the brain to be addressed such as the effects of infection on neuronal networks and the interactions between infected neurons and astrocytes. As neuroneastrocyte interactions have been shown to be involved in the impact of the parasite on neurotransmission, the ability to address the role of astrocytes on neuropathogenesis of T. gondii is an attractive aspect of 3D cerebral organoid models (David et al., 2016). The use of 3D brain organoids has not been investigated for use with T. gondii, and numerous technical and experimental details would need to be overcome. For example, the best methods of introduction of the parasite into cerebral organoids would first need to be established. Additionally, imaging 3D structure and analysis of cellecell interactions and parasiteecell interactions would be challenging. In intact brain, such imaging has required serial sectioning and stitching of the tissue imaged by electron microscopy or confocal microscopy after immunostaining, or use of thick sections (160 mm) and tissue clearing techniques to identify and image whole cells that contain cysts, to allow 3D visualization and analysis of individual, infected neurons (Koshy and Cabral, 2014; Cabral et al., 2020). Similar techniques would be required with the use of 3D organoids, and while this would still be technically challenging, the small size of the organoid (w4 mm) would simplify the processes of imaging and analysis. Additionally, the ability to control the timing of infection and conduct longitudinal studies of a developmental process of the parasite (i.e., cyst development and maturation) would yield valuable new information and unique insights into these developmental processes and life histories of cysts, not



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possible via more simple analyses afforded by 2D neuron monolayers or attainable from analysis of in vivo generated cysts from fixed time points postinfection. Thus, while 3D organoids are technically challenging and time-consuming to produce and require complex analyses, the novel information gained would be significant and may be well worth the effort. A complementary approach of use of 3D cerebral organoid and 2D neuron monolayer culture may yield the most information. For example, 2D neuron monolayers would allow longitudinal studies and live cell imaging studies of bradyzoite and cysts during maturation, allowing temporal and dynamic events to be studied while 3D organoids, if they allow more mature cyst development and persistence of mature cysts in culture, would allow patterns of cyst growth and bradyzoite replication in mature cysts to be studied (Table 8.2). Likewise, 2D neuronal monolayers would allow for screening of potential antibradyzoite and cysts drugs with mechanism of drug action studies to be done, while 3D organoids could allow more robust testing of effective anti-bradyzoite/cyst drugs if mature and heterogeneous populations of cysts were able to be derived using brain organoids (Table 8.2).

Future trends and direction: use of hiPSC-derived 2D and 3D models to model human parasitic infections hiPSC-derived 2D neuronal monolayers and cerebral organoids have been used to study infection and neuropathogenesis of several neurotrophic viruses, such as West Nile Virus, Zika Virus, and Dengue Virus, illuminating virus-specific mechanisms responsible for disease caused by these viruses (Desole et al., 2019; Garcez et al., 2016). As discussed earlier, hiPSC-derived 2D and 3D cerebral organoids offer similar advantages for the study of host/parasite interactions and neuropathogenesis of T. gondii in the brain. Cerebral organoids may also be ideal systems to study the host/parasite interactions and pathogenesis of other neurotrophic parasites. A recent study, for example, used an hiPSC and 3D cortical organoid system to investigate heme-mediated brain injury associated with human cerebral malaria caused by Plasmodium falciparum, illustrating how these neuronal models may be useful to address pathogenesis mechanisms in other neuroparasitic infections (Harbuzariu et al., 2019). More generally, the use of organoid models affords a unique opportunity to model host/parasite interactions of other human parasites, which otherwise might not be cultured due to strict host/parasite specificity. Human intestinal organoids, for example, could be used to study host/parasite interactions of such protozoan parasites such as Cyclospora cayetanensis, Cryptosporidium spp., Giardia spp., and Entamoeba histolytica (Klotz et al., 2012). In sum, the advent of hiPSC technology has been useful for the study of neurobiological disorders where research has been complicated by lack of access of human neuronal tissues and the complex nature of many neurological disorders. The use of 2D and 3D human neuronal models provides similar opportunities to


address pathogenesis, disease mechanisms, and host/parasite interactions for chronic Toxoplasmosis, which has also been hindered by the lack of suitable human neuronal models. Use of these hiPSC-derived neuronal models has the potential to lead to more effective treatment strategies for neurological symptoms resulting from chronic infection with this parasite. More generally, the use of hiPSCderived 2D monolayers and 3D organoids similarly holds promise for disease modeling of host/parasite interactions and study of pathogenesis of other human parasitic infections.

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Induced pluripotent stem cells for modeling Chagas disease


Adriana Bozzi1, 2, David A. Stevens3, 4 1

Instituto Rene´ Rachou, FIOCRUZ, Belo Horizonte, Brazil; 2Departamento de Cieˆncias Biolo´gicas, Universidade Estadual de Santa Cruz, UESC, Ilhe´us, Brazil; 3California Institute for Medical Research, San Jose, CA, United States; 4Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA, United States

Chapter outline Cardiomyopathy ......................................................................................................240 Chagas disease ......................................................................................................240 Chagas disease pathogenesis ..................................................................................241 Clinical Chagas disease ..........................................................................................243 Immune response in Chagas disease........................................................................243 Role of therapy in Chagas disease and relation to immune response..........................244 New approaches to therapy .....................................................................................245 Models to study Chagas disease ..............................................................................246 iPSC for modeling Chagas disease ...........................................................................246 Acknowledgment.....................................................................................................248 References .............................................................................................................248 Abstract This chapter brings insights and perspectives on the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for modeling the pathogenic processes of Trypanosoma cruzi infection. This is a new tool for understanding Chagas disease mechanisms, as well as for screening therapeutic strategies to control this pathology. We give an overview of cardiomyopathy and a Chagas disease update focusing on pathogenesis, immune response, and therapeutic approaches. Finally, we discuss the technology of induced pluripotent stem cell (iPSC) as a platform to study human Chagas disease in vitro. Keywords: Cardiomyocytes; Cardiovascular disease; Chagas disease; Dilated cardiomyopathy; Heart disease; In vitro model; Induced pluripotent stem cells; iPSC; Trypanosoma cruzi.

iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00003-1 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 9 Induced pluripotent stem cells for modeling Chagas disease

Cardiomyopathy In the past decades, heart failure (HF) has emerged as a public health problem. Kannel, based on epidemiological studies obtained in the Framingham Heart Study, estimated that in the United States there are 5 million HF patients, with approximately 400,000 new cases per year (Kannel, 2000). The problem is presumed to be of a greater magnitude in Brazil (Kannel, 2000). Cardiovascular diseases (CVDs) are common and significant contributors to worldwide mortality and morbidity, with an estimated 17.3 million deaths per year (WHO). Age-specific death dominates prevalence and mortality, although that has fallen since 2005, mainly in high-income countries (Joseph et al., 2017). Most CVDs deaths are concentrated now in middle and lowincome countries (WHO; Roth et al., 2015). CVDs includes ischemic heart disease, cerebrovascular disease, hypertensive heart disease, cardiomyopathy and myocarditis, rheumatic heart disease, and others (WHO; Joseph et al., 2017). Cardiomyopathy is heart muscle disease with manifestations ranging from microscopic alterations in cardiac myocytes to fulminant HF, with the result of decreased blood supply to the body. The American Heart Association defines cardiomyopathy as a multifactorial disease, classified as primary (genetic, mixed, or acquired) or secondary (e.g., infiltrative, toxic, inflammatory) (Fig. 9.1), usually with inappropriate ventricular hypertrophy or dilatation (Maron et al., 2006). Dilated cardiomyopathy (DC), hypertrophic cardiomyopathy, restrictive cardiomyopathy, and arrhythmogenic right ventricular cardiomyopathy are the major types of cardiomyopathies (Wexler et al., 2009). DC affects 5 in 100,000 adults 20e60 years old and 0.57 per 100,000 children and is characterized by ventricle chamber enlargement and systolic dysfunction (Maron et al., 2006; Wexler et al., 2009). A broad range of factors can cause DC from infectious and parasitic agents such as coxsackievirus, adenovirus, HIV, mycobacteria, rickettsia, and Trypanosoma cruzi (T. cruzi), to toxins, alcohol, chemotherapeutic agents, and metals, among others (Maron et al., 2006). Frequently DC starts in the left ventricle (LV) and spreads to right ventricle and then to the atria, leads to progressive HF characterized by low LV contractile function, arrhythmias, conduction system abnormalities, and thromboembolism, perhaps resulting in sudden or HF-related death. It’s the third cause most frequent of HF and heart transplantation (Maron et al., 2006).

Chagas disease Chagas heart disease, caused by T. cruzi infection, is an important cause of HF in Latin America, with approximately 8 million chronically infected people. The increasing presence of Chagas disease in nonendemic areas (mostly owing to immigration, blood transfusion, and organ transplantation), as well as the resurgence of the disease in endemic countries, has been a major focus of attention in recent years. It has been

Chagas disease pathogenesis

FIGURE 9.1 Classification of cardiomyopathy according to American Heart Association.

estimated that over 300,000 people infected with T. cruzi currently live in the United States (Bern et al., 2011), and although relatively limited, epidemiological data from Europe has estimated 59,000e108,000 cases of Chagas disease, with higher numbers in Spain and Italy. In view of the current situation, the United States, France, Spain, and United Kingdom have instituted comprehensive blood bank and organ screening for T. cruzi (Anselmi, 2011; Basile et al., 2011). Moreover, there is now evidence for autochthonous transmission within areas of the United States (Garcia et al., 2015).

Chagas disease pathogenesis The invasion of the human host frequently (80%e90%) occurs by the passage through damaged skin or intact mucosa of metacyclic trypomastigotes released with the feces of infected triatomine insects after their blood meal. Other transmission forms of the disease can occur via organ transplantation, blood transfusion,



CHAPTER 9 Induced pluripotent stem cells for modeling Chagas disease

congenital transmission, and the oral route (ingestion of contaminated materials). Oral infection with T. cruzi currently represents the most frequently documented route of transmission in Brazil (Andrade et al., 2014). Trypomastigotes invade the cells and are initially confined to a parasitophorous vacuole. After escaping to the host cell cytoplasm, the parasites transform into amastigotes, the replicating forms. After several cycles of binary division, the amastigotes transform into mammalian-stage trypomastigotes. Upon host cell death, the trypomastigotes invade the adjacent uninfected cells or are carried by the blood and lymphatics to various organs. The replication of T. cruzi occurs within the cytoplasm of different cell types including macrophages, fibroblasts, skeletal and heart muscle cells, neuronal and epithelial cells (Fig. 9.2). During the early stages of infection, innate immunity is triggered by microbial-derived ligands on Toll-like receptors (TLRs) (Almeida and Gazzinelli, 2001; Medeiros et al., 2007). Immediately after parasitism, the recruitment of leukocytes to the tissue is triggered. T. cruzi induces antigen-presenting cells (APCs) to produce various endogenous mediators, including arachidonic acid (AA)-derived eicosanoids, nitric oxide (NO), cytokines, and chemokines.

FIGURE 9.2 Routes of T. cruzi infection and human stages.

Immune response in Chagas disease

Clinical Chagas disease The clinical course of Chagas disease comprises two distinct stages with intrinsic parasitological and immunological features: a long-lasting infection with a short acute phase, which is usually clinically nonapparent and progresses to a lifelong chronic phase characterized by distinct clinical forms known as indeterminate (IND), cardiac (CARD), and digestive (DIG). The infection has a self-limiting acute phase, with patent (or subpatent) parasitemia, which goes unnoticed in many infected individuals. At this stage, the parasites actively replicate in many different cell types, such as macrophages; smooth, striated, and cardiac muscle cells; adipocytes, and cells of the central nervous system (Bombeiro et al., 2012). Whereas many chronically infected individuals remain in the asymptomatic indeterminate phase, a sizable proportion (30%e35%) of patients develop the cardiac or digestive manifestations of chronic disease, which can be complicated by cardiac arrhythmias, HF, stroke, and sudden death (Moncayo and Silveira, 2009; Tanowitz et al., 2009). The parasite has a tropism to myocardial cells and forms nests, a pathological feature of the acute disease (Gutierrez et al., 2009). Chagas heart disease is characterized by intense myocarditis of a chronic inflammatory and progressive fibrotic process affecting the myocardium of both ventricles, which leads to myocardial remodeling, interstitial fibrosis, and changes in the extracellular matrix (ECM) (Nunes et al., 2012; Rocha et al., 2003). There is no doubt that chagasic cardiomyopathy (CC) is the result of an inflammatory process. Indeed, one of the pathologic hallmarks of CC is the presence of a large number of inflammatory cells in the myocardium. Such infiltration of immune cells can be a response to the cardiac tropism of the parasite or as a consequence of altered immunological tolerance; this remains controversial but likely depends on the genetic background of the host, as some people never develop heart disease despite infection (Gutierrez et al., 2009).

Immune response in Chagas disease Cytokines play key roles in regulating both parasite replication and immune responses in infected animals. It has been demonstrated that the cytokines IFN-g and TNF-a are involved in the protective response to T. cruzi (Aliberti et al., 2001; Castan˜os-Velez et al., 1998; Ho¨lscher, 1998; Silva et al., 1995; Vespa et al., 1994). IFN-g is synthesized shortly after infection, mainly by NK cells, in response to IL-12 and TNF-a (Aliberti et al., 1996). However, the CD4þ and CD8þ T cells also produce IFN-g during T. cruzi infection (Martins et al., 2004). In concert with TNF-a, IFN-g leads to the activation of inducible nitric oxide synthase (iNOS) (Vespa et al., 1994; Gazzinelli et al., 1992), the enzyme that catalyzes nitric oxide (NO) synthesis by macrophages and inhibits parasite replication (Vespa et al., 1994; Moncada and Higgs, 1991).



CHAPTER 9 Induced pluripotent stem cells for modeling Chagas disease

Although all the mechanisms that mediate parasite control have not yet been clarified in humans, it is believed that they rely greatly on the function of innate immune cells, such as natural killer (NK) cells, mainly CD16þCD56- phenotype, neutrophils, and macrophages, which seem to be activated by parasite surface molecules (Sathler-Avelar et al., 2003; Argibay et al., 2002). Of these molecules, particular attention is given to glycosylphosphatidylinositol mucins (GPI mucins), which are the most abundant T. cruzi surface molecules involved in parasite adherence to host tissues and critical in activating immune responses (Buscaglia et al., 2006). One of the most intriguing questions of human and experimental T. cruzi infection is why the immune system fails to totally eradicate the parasite. At the beginning of the infection (before development of the parasite-specific response), T. cruzi trypomastigotes escape lysis by the complement system, an evasion strategy that results from the presence of complement-regulatory molecules on the parasite surface (Joiner et al., 1988). In addition, internalized parasites of diverse T. cruzi strains escape the phagocytic vacuole of unprimed resident macrophages (Nogueira and Cohn, 1976), a strategy that relies on a variety of molecules with antioxidant properties (Piacenza et al., 2009).

Role of therapy in Chagas disease and relation to immune response Current drug therapies alleviate symptoms for only 50%e70% of CVD patients. The specific treatment of Chagas disease is based on the use of the nitro derivative benznidazole (N-benzyl-2-nitroimidazoleacetamide, Bz), which is known to reduce parasitism during the acute stage and early in the chronic infection (Fragata Filho et al., 1995; de Andrade et al., 1996). The effect of treatment during the chronic phase is more controversial, although there are reports in posttreatment follow-up studies showing that individuals treated with Bz and evaluated decades after the initial infection demonstrated significant protection from progression of heart pathology due to Chagas disease. However, despite the low cure rates observed in most patients receiving treatment during the chronic disease, several studies have suggested that the Bz treatment should be still recommended at late stages of Chagas disease to prevent disease progression, regardless of lack of complete parasite clearance (Viotti et al., 2006; Garcia et al., 2005; Sosa-Estani and Segura, 2006). It has been proposed that a relevant factor potentially influencing parasite clearance as well as morbidity control following the treatment for Chagas disease is the cooperative action between the drug effects and the host immune response (Michailowsky et al., 1998; Rassi et al., 1999; Sathler-Avelar et al., 2006, 2008, 2012). Sathler-Avelar and collaborators (Sathler-Avelar et al., 2006, 2008) have shown that soon after the end of Bz treatment, the NK-cells and CD8þT-lymphocytes are important sources of IFN-g and that IL-10 produced by CD4þT-cells and, with B lymphocytes, are putative key elements in the modulation of the immune response and control of tissue damage

New approaches to therapy

induced by the proinflammatory response. Moreover, in the IND patients, Bz therapy induces a change in cytokine patterns of peripheral blood monocytes, NK-cells, and CD8þT-cells toward a long-lasting proinflammatory-modulated profile that could be important to the maintenance of a nondeleterious immunological microenvironment (Sathler-Avelar et al., 2012). Studies support the hypothesis that Bz treatment causes a change in the host immune response leading to an immunomodulatory profile in IND and a broader change in CARD, eliciting a complex phenotypic functional network compatible with beneficial and protective immunological events (CampiAzevedo et al., 2015). After Bz therapy, an overall low immune activation was observed in individuals with the IND clinical form of Chagas disease that is reflected in the downregulated phagocytic capacity, decrease of the FCg-R (CD16, CD64) and TLR-4 expression in circulating leukocytes, and lower levels of NO and most cytokines evaluated. It is possible that in these patients, Bz therapy leads to changes in neutrophil and macrophage phagocytosis, and therefore the expression of molecules involved in antigen presentation, cytokines and NO production, therefore contributing to preventing disease progression (Campi-Azevedo et al., 2015). It also suggests that modulation of macrophage activity and of regulatory T cells producing IL-10 may maintain the balance between parasitism and tissue integrity in IND (Gomes et al., 2003; de Arau´jo et al., 2012). These findings reinforce the potential usefulness of Bz treatment and encourage further studies to elucidate the potential use of these immune mediators as biomarkers of disease progression and prognosis.

New approaches to therapy Despite the positive impact of new drugs (principally azoles) for the treatment of HF, the disease progresses and patient prognosis remains guarded, with reduced quality of life and survival. Thus, there is an enormous interest and need to seek newer therapies that may offer beneficial effects in the evolution of these patients. Of the new therapeutic procedures, the most promising seems to be stem cell therapy. Different cell types, such as bone marrow cells, mesenchymal stem cells (MSCs) from adipose tissue, and skeletal myoblasts, have been tested in basic and applied clinical studies (Larocca et al., 2013; Ribeiro Dos Santos et al., 2012; Guarita-Souza et al., 2006). In Brazil, preliminary studies including a limited number of patients have shown that the administration of stem cells from bone marrow is safe and potentially effective in patients with HF (Bocchi et al., 2010; Vilas-Boas et al., 2011). Using cardiac mesenchymal stem cells (CMSCs) that express endothelial cell and cardiomyocyte features upon defined stimulation culture conditions, Silva and collaborators (Silva et al., 2014) showed in a mouse model that the CMSCs exert a protective effect in chronic CC primarily through immunomodulation. Since chronic HF is commonly a progressive disease, in spite of intensive pharmacological treatment, in addition to conventional therapy, treatment regimens are needed that can improve quality of life and increase



CHAPTER 9 Induced pluripotent stem cells for modeling Chagas disease

ventricular performance and survival. Treatment with stem cells does not cure Chagas disease, but attempts to repair the damage that results from years or even decades of aggressive destruction of the myocardium.

Models to study Chagas disease Good insight into the role of cell-mediated immunity in control of Chagas disease pathogenesis as well as the search for new approaches to treatment has been provided from experimental models for T. cruzi infection. Experimental models have given important leads for research and are important for both basic and preclinical research, mainly due to ethical issues. It is difficult to initially evaluate the efficacy and toxicity of a new drug directly in human. As much as these models allow us to study a disease systematically, animals do not recapitulate the human system. In addition, other variables can complicate such a study, such as the choice of the animal model and strain, the dose of the pathogen, the route and timing of infection, maintenance of the animal, and control of exposure to other infectious agents, for example. Mice have been the most used animals for the study of Chagas disease, but the differences between these species may sometimes complicate directly moving to human application. For the study of any disease, without a doubt the best model would be human. Until recently, this was a reality far from being achieved. Due to the advent of hiPSC technology, today we are able to reproduce personalized human cells in vitro. Even though this does not replicate the entire human system, it is already a great advantage to conduct research on human cells, even if in vitro.

iPSC for modeling Chagas disease The iPSC are like embryonic stem cells, i.e., can differentiate into any cell type from ectoderm, mesoderm, and endoderm germ layer. The iPSC technology consists of reprogramming adult cells by introduction of four transcription factors (Oct3/ 4,Sox2, Klf4, c-Myc) (Takahashi and Yamanaka, 2006; Takahashi et al., 2007). Different cell types as well as different methods can be used to generate iPSC (Robinton and Daley, 2012; Oh et al., 2012a; Yamanaka, 2012). Among the cell types, fibroblasts or blood cells are the most studied for reprogramming, and retrovirus or lentiviruses are the common methods used to deliver the pluripotent factors. Although the iPSC could give rise to an entire organ without ethical problems, the major advantage of these cells is to generate personalized platforms (patientspecific) in modeling for in vitro research, patient-oriented drug discovery, study of the pathophysiology of disease, drug screening, and toxicology study (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Robinton and Daley, 2012; Yamanaka, 2012; Greenow and Clarke, 2012; Oh et al., 2012a, 2012b) (Fig. 9.3). In combination

iPSC for modeling Chagas disease

FIGURE 9.3 The iPSC technology platform approach.

with the “omics” field, the iPSC technology has provided dynamic and personalized genetic information that can be used for clinical management of the patient as well as insights in disease susceptibility (Matsa et al., 2016). The iPSC technology provides the generation of hiPSC-CMs that can accurately recapitulate the human cardiac pathophysiology. These cells are ideal for studying the mechanisms of heart diseases because they are nonimmortalized human cardiomyocytes that express relevant ion channels and sarcomeric proteins found in adult human cardiomyocytes (Sharma et al., 2014; Burridge et al., 2012; Yu et al., 2007). Although animal models can partially recapitulate human CVD phenotypes, they exhibit interspecies differences in heart rate, electrophysiology, and cardiogenesis (Lombardi et al., 2008). The iPSC technology allows modeling of the heart in vitro and opens new avenues to study cardiomyopathies associated with deterioration in myocardial function and linked to HF and cardiac sudden death. The development of an in vitro cardiovascular model is of interest in order to study the Chagas disease pathology. Recent studies have shown that the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) modeling



CHAPTER 9 Induced pluripotent stem cells for modeling Chagas disease

the pathogenic processes of T. cruzi infection provides a new tool for understanding of Chagas disease mechanisms as well for screening novel therapeutic strategies to control this pathology. Chagas disease was successfully modeled in vitro using the hiPSC-CMs technology (Bozzi et al., 2019). In this model, the human cardiomyocytes generated from blood cells showed receptors for T. cruzi invasion with differential expression during the culture days and were infected by T. cruzi strain I (Silvio, ATCC 50800) and T. cruzi strain II (TcY, ATCC 50832) (Bozzi et al., 2019; Sass et al., 2019a, 2019b). It is important to note that all parasite cycle stages were reproduced in hiPSC-CMs, i.e., infective stage, amastigotes form, and trypomastigotes bursting host cells. The hiPSC-CMs beat in synchrony in vitro and showed arrhythmias characterized by tachycardia, similar to that in patients with Chagas disease. iPSC-CMs showed significant changes in their gene expression profile, cell contractility, and distribution of key cardiac markers. Moreover, the infected iPSC-CMs exhibited a proinflammatory profile as indicated by significantly elevated cytokine levels and cell-trafficking regulators. This model is ideal for study of gene expression, the inflammatory network, contractility, cardiac markers, and drug screening, as we subsequently showed (Sass et al., 2019a, 2019b).

Acknowledgment We thank Luiz Felipe Bozzi da Silva for the figures.

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Induced pluripotent stemcell derived brain-like endothelial cells to study hostepathogen interactions with the bacterial pathogens Streptococcus agalactiae and Neisseria meningitidis

10 Brandon J. Kim

University of Alabama, Department of Biological Sciences, Tuscaloosa, AL, United States

Chapter outline Introduction ............................................................................................................256 Bloodebrain barrierdmeningeal bloodeCSF barrier ......................................256 Bacterial meningitis ................................................................................................257 Brain endothelial cell models and infection..............................................................258 Current iPSC models ...............................................................................................258 Group B Streptococcus ............................................................................................259 Neisseria meningitidis ................................................................................260 Bacterial interaction with iPSC-BECs........................................................................261 Group B Streptococcus ...............................................................................261 Neisseria meningitidis ................................................................................263 Current iPSC-BEC models and future outlook.............................................................265 iPSC based models and infections ...........................................................................266 References .............................................................................................................267 Abstract Bacterial meningitis is a devastating central nervous system (CNS) infection that occurs when certain bacteria are able to interact with and penetrate highly specialized CNS barriers and gain access to the brain. The bloodebrain barrier (BBB) and meningeal bloodecerebrospinal fluid barrier (mBCSFB) are primarily comprised of brain endothelial cells (BECs) that possess properties that contribute iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00006-7 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 10 Induced pluripotent stem-cell

to their barrier specialized barrier function. Recently, stem-cell-derived BEC-like cells have enabled researchers to investigate BECs in vitro that better mimic in vivo BECs than previous models. This chapter will explore the utilization of iPSC-BEC-like cells to study the hostepathogen interaction between BECs and two clinically relevant meningeal bacterial pathogens: Streptococcus agalactiae and Neisseria meningitidis. Keywords: Bacterial meningitis; Bloodebrain barrier; Brain endothelial cells; Group B Streptococcus; Hostepathogen; Hostepathogen interaction; In vitro models; iPSC-derived brain endothelial cells; Meningitis; Meningococcus; Neisseria meningitidis; Stem-cell-derived brain endothelial cells; Streptococcus agalactiae.

Introduction Bloodebrain barrierdmeningeal bloodeCSF barrier The brain is an extremely energy-demanding organ, and though it represents only approximately 2% of the total body’s mass, it requires about 20% of the body’s total glucose and oxygen (Raichle and Gusnard, 2002). The bloodebrain barrier (BBB) and the meningeal bloodecerebral spinal fluid barrier (mBCSFB) are critical cellular barriers that promote proper brain function (Kim et al., 2019a; Rua and McGavern, 2018). These highly specialized barriers are primarily composed of brain endothelial cells (BECs) that tightly regulate the passage of molecules in and out of the central nervous system (CNS) (Kim et al., 2019a; Rua and McGavern, 2018). The BBB consists of BECs that are supported by additional cell types such as pericytes, astrocytes, and neurons, which together form the neurovascular unit (NVU) (Abbott et al., 2010). These other cell types have been shown to contribute to BBB function and signal to BECs and vice versa (Abbott et al., 2010; Profaci et al., 2020). The mBCSFB possesses BECs with similar phenotypes to the BBB although without the support of the NVU. However, these endothelial cells possess similar phenotypes to the BBB and are found in the subarachnoid space of the meninges (Rua and McGavern, 2018). Together these important endothelial brain barriers maintain proper brain function by actively transporting nutrients into the brain while excluding harmful compounds (Abbott et al., 2010; Redzic, 2011; Engelhardt and Sorokin, 2009). BECs are unique when compared to most other endothelial cells in a number of ways. Compared to their peripheral endothelial cell counterparts, BECs possess a number of attributes that contribute to their specialized function. Namely, BECs lack fenestrations, express tight junctions to restrict paracellular passage, exhibit extremely low rates of endocytosis, and express an array of transporters that selectively import or restrict various substrates (Abbott et al., 2010; Profaci et al., 2020). BECs’ defining phenotypes promote their unique properties. Tight junctions are protein complexes that tightly hold adjacent cells together restricting the extracellular space observed between the cell membranes (Luissint et al., 2012). These complexes are largely comprised of occludin, zona-occludins (ZO), and claudins that are linked to the cytoskeleton. The tight junctions in BECs are more complex

Bacterial meningitis

and more cohesive than peripheral endothelial cells (Luissint et al., 2012). In particular, Claudin-5 has been shown to be critical as knockout mice present with a leaky BBB. Furthermore, Claudin-5 is the most abundant claudin found in BECs (Nitta et al., 2003; Greene et al., 2019). These junctions greatly increase the trans-endothelial electrical resistance (TEER) that has been estimated in vivo to be upward of 5000 U  cm2 (Butt et al., 1990). Endocytosis refers to a number of processes that cells use to take in material from the extracellular environment; however, in BECs, endocytosis is greatly restricted (Ben-Zvi et al., 2014; Siegenthaler et al., 2013). Recent work has demonstrated that early on in development, a gene termed Major Facilitator Superfamily Domain Containing 2A (MFSD2A) is upregulated specifically in BECs, and this results in the repression of endocytosis (Ben-Zvi et al., 2014). There are, however, exceptions; for example, the transferrin receptor is known to be actively taken up by endocytosis and transported from the luminal (blood side) to the apical (brain side) to deliver transferrin and iron to the CNS (Leitner and Connor, 2012). To transport nutrients such as sugars and carbon sources into the CNS, the BECs express nutrient importers such as GLUT1, which imports glucose (Profaci et al., 2020; Daneman and Prat, 2015). However, there are a number of solute and amino acid transporters that import these molecules into the CNS (Profaci et al., 2020; Daneman and Prat, 2015). Finally, to prevent passage of toxins and other xenobiotic compounds, BECs express an array of efflux transporters such as P-glycoprotein (P-gp), Breast Cancer Resistance Protein (BCRP), and Multidrug Resistance Proteins (MRPs) (Abbott et al., 2010; Profaci et al., 2020; Daneman and Prat, 2015). Together, these efflux transporters are thought to exclude a large percentage of known compounds (Lo¨scher and Potschka, 2005). These specialized phenotypes all contribute to the tight barrier phenotype exhibited by BECs.

Bacterial meningitis Bacterial meningitis is a serious life-threatening infection of the CNS, which when left untreated is uniformly fatal (Van der Flier et al., 2003). While modern medical interventions have transformed this once fatal disease into an often-curable one, survivors can still exhibit permanent neurological issues such as deafness, blindness, seizures, and stroke (Van der Flier et al., 2003; Doran et al., 2016). To cause meningitis, bacteria must interact with and penetrate the highly specialized BECs and activate the immune system (Kim et al., 2019a; Van der Flier et al., 2003; Doran et al., 2016; van Sorge and Doran, 2012). There are a number of bacteria that are capable of accomplishing this such as Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, E. coli K1 (Doran et al., 2016). Some bacteria such as Staphylococcus aureus, Listeria monocytogenes, Streptococcus suis, and Mycobacterium tuberculosis are not classically associated with bacterial meningitis, yet in certain cases can gain access to the CNS and cause disease (Doran et al., 2016; Chin, 2014). Bacteria classically associated with bacterial meningitis possess the common ability to colonize the host, survive in the blood stream, and interact with and penetrate the BECs to gain access to the CNS (Doran et al., 2016).



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Brain endothelial cell models and infection Researchers utilize many in vivo and in vitro models to examine BECs. In vivo models have the advantage of possessing physiologic conditions as well as an intact immune system. These include the mouse, rat, zebrafish, and rabbit; however, these models are inherently nonhuman and therefore suffer from the disadvantage of potential species differences (Lippmann et al., 2012; Warren et al., 2009; Syva¨nen et al., 2009). Rodent models have been used extensively and many have exploited direct CNS infection to examine leukocyte transmigration (Grandgirard et al., 2007; Koopmans et al., 2018; Koedel et al., 2001; Reib et al., 2011; Shapiro et al., 2000). To assess the bloodstream survival and BBB traversal, hematogenous routes of infection have been utilized by introducing pathogens via intraperitoneal, intravenous, or intracardiac injections (Ferrieri et al., 1980; Kim et al., 1992, 2015b; Tan et al., 1995; Hoffman et al., 2000; Wang and Kim, 2002; Liu et al., 2004; Doran et al., 2005; Banerjee et al., 2010). The selection of appropriate in vivo models ultimately relies on the biological question being asked as well as the facilities available to the researchers. In vitro models comprised of human BECs have been utilized to examine many different diseases including infection. Many of these cell-based models utilize human brain endothelial cells isolated and/or immortalized (Helms et al., 2015). These are easily scalable, and as they are of human origin, they solve the potential issues of species differences (Lippmann et al., 2012; Warren et al., 2009; Syva¨nen et al., 2009). Human primary cells have been isolated and utilized to study the BBB; however, in general they have issues with availability especially from healthy human tissue (Helms et al., 2015). Immortalized human brain endothelial cells, such as the hBMEC and CMEC/D3 lines, solve the issue of species differences and availability; however, many lack critical BBB phenotypes such as high TEER, barrier properties, and tight junction expression (Helms et al., 2015; Artus et al., 2014; Carl et al., 2010; Fo¨rster et al., 2008; Kim et al., 2017; Weksler et al., 2005; Paolinelli et al., 2013). Immortalized and primary brain endothelial cells have been used to study and interrogate the interactions with many pathogens including Streptococcus pneumoniae, Streptococcus agalactiae, Neisseria meningitidis, Escherichia coli K1, Listeria monocytogenes, and others (Kim et al., 2015b; Hoffman et al., 2000; Wang and Kim, 2002; Doran et al., 2005; Banerjee et al., 2010, 2011; Deng et al., 2018, 2019; Greiffenberg et al., 1998; Nizet et al., 1997; Spencer et al., 2019; Stins et al., 2001; Uchiyama et al., 2009; Badger et al., 1999; Bernard et al., 2014; Coureuil et al., 2010). Many mechanisms of bacterial interaction have been discovered using these models; however, interrogation of BEC physiology has been greatly limited partially due to some potentially lacking BBB properties.

Current iPSC models Induced pluripotent stem cells (iPSCs) have revolutionized tissue modeling in recent years. Human iPSCs have been able to generate tissues from a variety of organs.

Group B Streptococcus

Recently iPSCs have been utilized to differentiate into brain endothelial-like cells (Lippmann et al., 2012, 2014; Qian et al., 2017b; Stebbins et al., 2016; Hollmann et al., 2017; Neal et al., 2019). These iPSC-derived BECs (iPSC-BECs) express endothelial markers such as VE-cadherin and PECAM while lacking endothelial markers such as E-cadherin (Lippmann et al., 2014). Additionally they express BEC markers such as Glut-1, P-gp, BCRP, and Claudin-5 (Lippmann et al., 2012, 2014; Qian et al., 2017b; Stebbins et al., 2016; Hollmann et al., 2017; Neal et al., 2019). Functionally iPSC-BECs form a barrier of 2000þ ohms x cm2 bringing this model into the range of what has been estimated in vivo (Butt et al., 1990; Lippmann et al., 2012, 2014; Qian et al., 2017b; Stebbins et al., 2016; Hollmann et al., 2017; Neal et al., 2019). BECs recognize the neuro microenvironment and cues from other cell types of the NVU, and iPSC-BECs are able to respond to these NVU cells and increase their barrier properties (Lippmann et al., 2012; Hollmann et al., 2017; Appelt-Menzel et al., 2017; Canfield et al., 2017, 2019; Stebbins et al., 2019). In addition to BECs, protocols have been developed to derive other cell types of the NVU such as astrocytes, neurons, and pericytes (Canfield et al., 2017, 2019; Stebbins et al., 2019). A main advantage of these methodologies is that all of the cell types can be derived from the same isogenic source (Canfield et al., 2017, 2019; Stebbins et al., 2019). This iPSC-BEC model has been used to examine a number of neurological disorders such as AllaneHerndoneDudley syndrome, Huntington’s disease, ALS, Alzheimer’s disease, stroke, and others (Katt et al., 2019; Lee et al., 2018; Mantle and Lee, 2018; Rieker et al., 2019; Al-Ahmad et al., 2019; Lim et al., 2017; Vatine et al., 2016; Sances et al., 2018; Shin et al., 2019; Workman and Svendsen, 2020; Page et al., 2019; Mohamed et al., 2019; Qosa et al., 2016). Since iPSCs can be derived from patient fibroblasts that harbor the deleterious mutations, this can be a useful tool in disease modeling. In the context of infection, the iPSC-BEC model has been used in a few cases with viral, parasitic, and bacterial pathogens (Kim et al., 2017, 2019b; Alimonti et al., 2018; Gomes et al., 2019; Patel et al., 2018). Using the strengths of this model, infectious disease researchers are better able to elucidate mechanisms of BEC failure during disease. To date, two meningitis-causing bacteria have been examined for their interaction with iPSC-BECs: Streptococcus agalactiae and Neisseria meningitidis (Kim et al., 2017, 2019b; Gomes et al., 2019). The rest of this chapter will focus on the studies conducted with these bacteria and lessons learned from iPSC-BEC-based models.

Group B Streptococcus Streptococcus agalactiae (group B Streptococcus, GBS) is a Gram-positive commensal bacterium that can colonize various mucosal membranes of the human host (Doran et al., 2016; Doran and Nizet, 2004; Maisey et al., 2008; Patras and Nizet, 2018). GBS is the leading cause of neonatal meningitis and an emerging pathogen in susceptible adults. While modern medical interventions have reduced



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mortality, death still occurs in up to 10% of cases with 25%e50% of survivors experiencing permanent neurological damage (Doran et al., 2016; Doran and Nizet, 2004; Maisey et al., 2008; Patras and Nizet, 2018). A colonized mother can vertically transmit GBS to the neonate in utero or during the birthing process with upward of 50%e70% of newborns born to colonized mothers becoming colonized themselves (Doran and Nizet, 2004). Much work has been conducted to identify and characterize GBS virulence factors that contribute to interaction with BECs. Surface-expressed bacterial virulence factors such as the hypervirulent GBS adhesin (HvgA), pili, serine-rich repeat proteins (Srr), streptococcal fibronectin binding factor (SfbA), fibronectin-binding protein (FbsA), group B Streptococcus surface protein C (BspC), and the properly anchored lipoteichoic acid (LTA) have all been shown to contribute to interaction or invasion into BECs (Doran et al., 2005; Deng et al., 2019; Banerjee et al., 2011; Patras and Nizet, 2018; van Sorge et al., 2009; Maisey et al., 2007; Mu et al., 2014; Seo et al., 2013; Six et al., 2015; Tazi et al., 2010; Tenenbaum et al., 2005). GBS virulence is regulated by twocomponent signal transduction systems, and work has demonstrated that in GBS, the CovR/S, CiaR/H, and LtdR contribute to pathogenesis (Deng et al., 2018; Mu et al., 2016; Lembo et al., 2010). Interestingly, GBS meningitis isolates have been disproportionately represented by Type III strains of the multilocus sequenceetype (MLST) 17 and are considered hypervirulent strains. All of the identified Type III MLST-17 strains possess a specific adhesin HvgA (Tazi et al., 2010; Landwehr-Kenzel and Henneke, 2014). Currently there is no vaccine available to protect against GBS, and ongoing efforts target some of these identified bacterial virulence factors as antigens for potential vaccines (Carreras-Abad et al., 2020).

Neisseria meningitidis Neisseria meningitidis (Nm, meningococcus) is a Gram-negative bacterium that is a leading cause of bacterial meningitis worldwide (Doran et al., 2016; Virji, 2009). Nm can asymptomatically colonize the nasopharynx of 10%e40% of healthy adults (Pace and Pollard, 2012). However, under certain circumstances, Nm can penetrate the mucosal epithelium, enter the blood stream, and interact with and penetrate BECs gaining access to the CNS (Doran et al., 2016; Le Guennec et al., 2020). While being a global health threat, Nm is also the leading cause of epidemic meningitis in the “meningitis belt” of the sub-Saharan region of Africa and developing nations (Harrison et al., 2009). Nm possesses a number of virulence factors that allow bacterialeBEC interaction such as the Type IV pili (Tfp), Opc, ACP, and MspA (Doran et al., 2016; Bernard et al., 2014; Le Guennec et al., 2020; Coureuil et al., 2009; Hardy et al., 2000; Hung et al., 2013; Slanina et al., 2012; Turner et al., 2006). While there is a successful vaccine against many serotypes of Nm (A, C, W, and Y) and more recently against serotype B, Nm still represents a major cause of bacterial meningitis (Parikh et al., 2020). Studies to identify and characterize these virulence factors have been conducted mostly on primary and immortalized BEC lines (Le Guennec et al., 2020). Additionally, since Nm is a

Bacterial interaction with iPSC-BECs

human-specific pathogen, there is no robust in vivo model for the examination of NmeBEC interaction necessitating the need for the development of better in vitro models (Kim et al., 2019a; Kim & Schubert-unkmeir, 2019).

Bacterial interaction with iPSC-BECs Group B Streptococcus GBS is the leading cause of neonatal meningitis and can interact with and penetrate BECs. Until recently, studies on GBS primarily utilized immortalized cell lines and rodent models. The first studies using infectious agents and iPSC-BECs were accomplished using GBS as a model pathogen (Kim et al., 2017). It was found that GBS could adhere to and invade iPSC-BECs in a multiplicity of infectiondependent manner. GBS mutants that lacked surface expressed adhesins and invasins LTA, Srr, Pili, and SfbA all were observed to have attenuated phenotypes in their ability to attach to and enter iPSC-BECs, supporting previous observations in other models (Kim et al., 2017) (Fig. 10.1). Previous work had demonstrated that the BEC tight junctions were disrupted by GBS through the upregulation of a transcriptional repressor of junctional proteins known as Snail1 (Kim et al., 2015b). The upregulation of Snail1 was found to be necessary and sufficient for tight junction disruption (Kim et al., 2015b). Likewise, in the iPSC-BEC model, infection with GBS resulted in the upregulation of Snail1 and the downregulation of tight junction proteins (Kim et al., 2017) (Fig. 10.1). This observation was additionally supported by immunofluorescence where infected iPSC-BECs lost integrity of Occludin, Claudin-5, and ZO-1. Importantly, when compared to immortalized BECs, Claudin-5 levels could be observed in the iPSC-BEC human-based model, whereas immortalized BECs lack expression of this important claudin (Kim et al., 2017). Additionally, monitoring barrier integrity by TEER resulted in a loss of function after infection. Activation of BECs in response to GBS was shown to be partially pili-mediated via integrin-focal adhesion kinase (FAK) signaling through the MEK-Erk1/2 MAPK pathway (Banerjee et al., 2011; Shin et al., 2006). GBS-induced upregulation of neutrophilic cytokines and chemokines has been observed in immortalized cell models and in in vivo models (Banerjee et al., 2010; Deng et al., 2018, 2019; Mu et al., 2014; Tenenbaum et al., 2005; Kim et al., 2015a; Krishnan et al., 2013; Gendrin et al., 2015; Doran et al., 2003). Innate immune activation of iPSC-BECs by GBS was observed through the increased expression of interkeukin-8 (IL-8), CXCL1, CXCL2, and CCL20 (Kim et al., 2017) (Fig. 10.1). It was demonstrated for the first time that iPSC-BECs could be activated by proinflammatory insults from bacteria. BBB efflux transporters are important components of the barrier’s function and serve to restrict the diffusion of drugs and toxins (Abbott et al., 2010; Profaci et al., 2020; Daneman and Prat, 2015). Until recently, BEC efflux transporter function had not been examined during bacterial infection. P-gp is a well-studied transporter in



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FIGURE 10.1 Schematic of GBSeiPSCeBEC interaction. GBS virulence factors Pili, LTA, Srr, and SfbA contribute to adherence and invasion of iPSC-BECs (top cut out). GBS is able to adhere and invade iPSC-BECs. Infection results in Snail1-mediated tight junction disruption and upregulation of chemokines and cytokines. GBS disrupts P-gp efflux function and reduces P-gp expression and abundance in iPSC-BECs. Factors shown in yellow were not directly elaborated on iPSC models.

BECs and has been described to have numerous substrates from small lipophilic molecules to proteins such as Ab (Wang et al., 2016; Schinkel, 1999). The iPSCBEC model is particularly useful for interrogating efflux function as it expresses an array of functional transporters (Lippmann et al., 2012, 2014; Qian et al., 2017b; Hollmann et al., 2017; Neal et al., 2019). GBS was recently found to inhibit P-gp function as measured by a substrate exclusion assay (Kim et al., 2019b). Interestingly, GBS mutants that are less capable of interacting with BECs seem to inhibit P-gp at similar levels suggesting that the bacterial factor responsible for the diminished efflux function is yet undetermined (Kim et al., 2019b). After GBS infection, it appears that P-gp expression and abundance are reduced

Bacterial interaction with iPSC-BECs

(Kim et al., 2019b) (Fig. 10.1). While it is tempting to speculate that this would be the mechanism of decreased function, it is presently unclear if this observation is the cause of P-gp function failure. Further work is needed to determine the exact mechanism of efflux transporter dysfunction and to determine the bacterial factor responsible for P-gp inhibition.

Neisseria meningitidis Nm is a human-specific pathogen, thus modeling of relevant hostepathogen interactions is quite challenging. iPSC-BECs offer advantages as they are human-derived and possess robust BBB phenotypes compared to other in vitro models (Le Guennec et al., 2020). Using iPSC-BECs it was found that Nm has the ability to attach and invade BECs in a similar fashion to other models (Gomes et al., 2019). Tfp was demonstrated to promote Nm attachment to endothelial cells (Bernard et al., 2014; Coureuil et al., 2010; Hardy et al., 2000). In the iPSC-BEC model, a mutant Nm that overexpresses Tfp was demonstrated to be hyperadherent (Gomes et al., 2019) (Fig. 10.2). Interaction with endothelial cells and bacteria often results in a cellular response. In the case of Nm interaction with iPSC-BECs, it was determined that the bacteria cause recruitment of the cellular receptor CD147 (Bernard et al., 2014). These initial findings demonstrate that the stem-cell-derived model is able to recapitulate previous findings using immortalized lines (Gomes et al., 2019). As immortalized lines tend to lack robust barrier properties, barrier function was examined using iPSC-BECs. Barrier function as measured by TEER was greatly reduced after infection with Nm suggesting a destruction of tight junctions (Gomes et al., 2019) (Fig. 10.2). Tight junction disruption was confirmed through immunostaining for the junctional components ZO-1, Occludin, and Claudin-5. This barrier destruction also correlated with bacterial transmigration of the BEC barrier (Gomes et al., 2019) (Fig. 10.2). By using iPSC-BEC models, barrier defects such as these are readily observable. These types of studies have historically been more difficult to monitor in other in vitro models. Host cell signaling in response to Nm has been elucidated in a number of nonstem-cell-based models, which show upregulation of cytokines and rearrangement of junctional proteins following bacterial exposure. Integrin-focal adhesion kinase (FAK) signaling activated by OpceFibronectin interaction was required for Nm invasion into BECs (Slanina et al., 2012). Additionally, Tfp-mediated CD147 interaction has been suggested to promote the formation of cortical plaques on the cell surface and subsequent rearrangement of tight junction and adherence junctional components (Bernard et al., 2014; Coureuil et al., 2010). However, besides the localization of CD147 on iPSC-BECs, the other signaling pathways activated during infection have yet to be explored in the iPSC-derived model. The induction of proinflammatory cytokines is an important step in disease progression that results in the influx of leukocytes, primarily neutrophils. Infection of iPSC-BECs with Nm resulted in an increase in the expression of proinflammatory cytokines and



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FIGURE 10.2 Schematic of NmeiPSCeBEC interaction. Nm virulence factors Opc and Type IV pili promote attachment to iPSC-BECs, and Nm attachment colocalizes with the host CD147 receptor (top cut out). Nm can attach and invade iPSC-BECs and results in the upregulation of proinflammatory cytokines and chemokines coupled with secretion of IFN-gamma and RANTES. Tight junctions are disrupted and expression of Claudin-5 and ZO-1 is decreased in coordination with an upregulation of Snail1. Junctional disruption is correlated with increased barrier permeability to soluble molecules. RNAseq analysis of the host transcriptome revealed upregulation of VEGF, TNFAIP2, and stress pathways such as hypoxic response. Factors shown in yellow were not directly elaborated on iPSC models.

chemokines (Gomes et al., 2019) (Fig. 10.2). The cytokines most notably expressed were the neutrophilic cytokines and chemoattractants interleukin-8 (IL-8), CXCL1, CXCL2, and IL-6 (Gomes et al., 2019). Additionally detectable increases of RANTES and Interferon gamma were secreted (Gomes et al., 2019) (Fig. 10.2). This finding supports the previous observation that infection of iPSC-BECs can result in an activation of the innate immune response.

Current iPSC-BEC models and future outlook

Current iPSC-BEC models and future outlook Modeling hostepathogen interaction using iPSC-derived BECs is still in its infancy. To date, only a few studies using bacterial pathogens have been conducted and even fewer with viral pathogens and fungal toxins (Kim et al., 2017, 2019b; Alimonti et al., 2018; Gomes et al., 2019; Patel et al., 2018). At present, the iPSC-BEC model has some distinct advantages over immortalized and animal systems. Importantly the iPSC-BECs create an intact barrier that can be measured by TEER. In the cases for both GBS and Nm, it appears that TEER is disrupted following infection (Kim et al., 2017; Gomes et al., 2019). Additionally, until now a human-based in vitro model that localizes the tight junction proteins ZO-1, Claudin-5, and Occludin had yet to be utilized (Kim et al., 2017). During GBS infection, it was shown that all three of these junctional components are lost, and after infection with Nm, the Occludin staining becomes discontinuous (Kim et al., 2017; Gomes et al., 2019). In contrast, infection with the viral pathogen Zika virus leaves the barrier intact and is able to transmigrate across the iPSC-BECs without disrupting TEER (Alimonti et al., 2018). Furthermore, a secreted toxin produced by the fungal pathogen Aspergillus fumigatus is able to lower TEER without destroying tight junctions (Patel et al., 2018). iPSC-BECs are particularly useful in examining tight junctions and barrier function and will be a useful tool to further determine the mechanisms of barrier failure during infection. Activation of the innate immune system is important for disease progression leading to the upregulation and production of cytokines that recruit neutrophils to the meninges during meningitis (Doran et al., 2016; Page et al., 2019; Le Guennec et al., 2020; Coureuil et al., 2017). In response to bacterial infection, iPSC-BECs upregulate cytokines and chemokines that have been classically demonstrated to activate and recruit neutrophils (Kim et al., 2017; Gomes et al., 2019). Interestingly, however, while upregulation was easily detected using qPCR, ELISA-based detection of the protein product was unsuccessful (Kim et al., 2017; Gomes et al., 2019). Furthermore, in response to TNF-alpha, iPSC-BECs have been demonstrated to upregulate leukocyte adhesion molecules such as ICAM-1 and VCAM-1 (Linville et al., 2019). All models have their distinct advantages and disadvantages, and the iPSC-BECs may need to be optimized or modified to produce cytokines that can be secreted. While iPSC-BECs have been used for disease modeling in a variety of disorders, some potential pitfalls still exist. Through RNAseq analysis it was found that iPSCBECs tend to cluster with isolated brain endothelial cells when compared to iPSCs, mesoderm, endoderm, and ectoderm (Qian et al., 2017b). However, when iPSCBECs are compared only with other endothelial cells either primary or immortalized, they are notably distinct with certain epithelial signatures remaining (Workman and Svendsen, 2020; Vatine et al., 2019). Further expansion and differentiation protocols will likely be developed in the future that better mimic in vivo BECs, although a true benchmark for comparison remains to be delineated (Workman and Svendsen, 2020). These improved models will likely be able to further our understanding of



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hostepathogen interaction as the differentiation process becomes more in vivo-like. Finally, iPSC-BECs have been put into several other systems using microfluidics that can better replicate sheer stress and the brain capillary architecture (Vatine et al., 2019; Campisi et al., 2018; DeStefano et al., 2017; Fabre et al., 2019; Jagadeesan et al., 2020). Implementation of sheer stress would be an important step for understanding bacterial interaction with BECs, and it has been demonstrated that Nm can resist sheer stress after adhesion (Maı¨ssa et al., 2017). Further development of these capillary geometries can be useful as most experiments conducted on bacterialeBEC interaction are in a static condition. It is understood that iPSC-BECs present an exciting tool for the discovery of novel hostepathogen interactions between BECs and bacterial pathogens (Kim et al., 2017, 2019b; Gomes et al., 2019). iPSC-BECs possess attributes that make them particularly attractive such as being of human origin and forming a tight barrier. No doubt that future efforts improving these models will allow for the study of other aspects of bacterial meningitis such as the secretion of cytokines, trafficking of leukocytes, impact of sheer stress in a capillary, and roles for other CNS cell types in BEC function/dysfunction. Use of iPSC-BECs in infectious disease is presently in its infancy, and it will be exciting to uncover novel mechanisms of BBB failure during disease.

iPSC based models and infections The advent of iPSC technologies has dramatically altered the ability to model infectious diseases. Various tissue types derived from iPSCs have enabled scientists to examine a number of diseases from metabolic disorders to genetic abnormalities. In addition, the understanding of infectious disease has greatly benefitted from the use of iPSC-derived models. Viral infections have widely utilized iPSC-derived tissue models to study hostepathogen interactions. In particular, the derivation of hepatocytes or hepatocyte-like cells from iPSCs hasgreatly informed on the pathogenesis of Hepatitis B and Hepatitis C viruses (Sa-ngiamsuntorn et al., 2016; Sakurai et al., 2017; Kaneko et al., 2016; Yoshida et al., 2011). Additional models using various viruses include epithelial cells, neural progenitor cells, neuronal cells, and cardiomyocytes (D’Aiuto et al., 2012; D’Aiuto et al., 2018; Desole et al., 2019; Lanko et al., 2017; Sharma et al., 2014; Sato et al., 2019; Lafaille et al., 2012; Qian et al., 2017a). Furthermore, iPSC-derived macrophages and neutrophils were useful in examining bacterialehost interactions (Trump et al., 2019; Yeung et al., 2017; Hale et al., 2015). Organoids are self-organized cell cultures that can be derived from iPSCs and could better mimic tissues in three-dimensional space. iPSC organoids have the same advantages of iPSC-derived cell types with the added advantage of potentially incorporating 3D structure to the particular tissues being modeled. Various iPSC organoids from mimicking intestinal epithelium and brain organoids have informed on the study of Zika virus, Parainfluenza virus, cytomegalovirus, Salmonella, and E. coli infection (Porotto et al., 2019; Lees et al., 2019;


Brown et al., 2019; Karve et al., 2017; Forbester et al., 2015). Recently, brain organoids have begun to incorporate BECs, pericytes, and astrocytes to better replicate the NVU in 3D and more recently using iPSC (Logan et al., 2019; Bergmann et al., 2018). Future research will likely incorporate other NVU cell types and some of these novel 3D techniques for the examination of bacterial pathogens with the BBB.

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Sato, S., Hisaie, K., Kurokawa, S., Suzuki, A., Sakon, N., Uchida, Y., et al., 2019. Human norovirus propagation in human induced pluripotent stem cellederived intestinal epithelial cells. CMGH 7 (3), 686e688. Schinkel, A.H., 1999. P-Glycoprotein, a gatekeeper in the blood-brain barrier. Adv. Drug Deliv. Rev. 36 (2e3), 179e194. Seo, H.S., Minasov, G., Seepersaud, R., Doran, K.S., Dubrovska, I., Shuvalova, L., et al., 2013. Characterization of fibrinogen binding by glycoproteins Srr1 and Srr2 of Streptococcus agalactiae. J. Biol. Chem. 288 (50), 35982e35996. Shapiro, M.A., Donovan, K.D., Gage, J.W., 2000. Comparative therapeutic efficacy of clinafloxacin in a pneumococcal meningitis mouse model. J. Antimicrob. Chemother. 45 (4), 498e492. Sharma, A., Marceau, C., Hamaguchi, R., Burridge, P.W., Rajarajan, K., Churko, J.M., et al., 2014. Human induced pluripotent stem cell-derived cardiomyocytes as an in vitro model for coxsackievirus B3-induced myocarditis and antiviral drug screening platform. Circ. Res. 115 (6), 556e566. Shin, S., Maneesh, P.S., Lee, J.S., Romer, L.H., Kim, K.S., 2006. Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells. Microb. Pathog. 41 (4e5), 168e173. Shin, Y., Choi, S.H., Kim, E., Bylykbashi, E., Kim, J.A., Chung, S., et al., 2019. Bloodebrain barrier dysfunction in a 3D in vitro model of Alzheimer’s disease. Adv. Sci. 6 (20). Siegenthaler, J.A., Sohet, F., Daneman, R., 2013. “Sealing off the CNS”: cellular and molecular regulation of blood-brain barriergenesis. Curr. Opin. Neurobiol. 23 (6), 1057e1064. Six, A., Bellais, S., Bouaboud, A., Fouet, A., Gabriel, C., Tazi, A., et al., 2015. Srr2, a multifaceted adhesin expressed by ST-17 hypervirulent Group B Streptococcus involved in binding to both fibrinogen and plasminogen. Mol. Microbiol. 97 (6), 1209e1222. Slanina, H., Hebling, S., Hauck, C.R., Schubert-Unkmeir, A., 2012. Cell invasion by neisseria meningitidis requires a functional interplay between the focal adhesion kinase, Src and cortactin. PloS One 7 (6). Spencer, B.L., Deng, L., Patras, K.A., Burcham, Z.M., Sanches, G.F., Nagao, P.E., et al., 2019. Cas9 contributes to group b streptococcal colonization and disease. Front. Microbiol. 10, 1930. Stebbins, M.J., Wilson, H.K., Canfield, S.G., Qian, T., Palecek, S.P., Shusta, E.V., 2016. Differentiation and characterization of human pluripotent stem cell-derived brain microvascular endothelial cells. Methods 101, 93e102. Stebbins, M.J., Gastfriend, B.D., Canfield, S.G., Lee, M.S., Richards, D., Faubion, M.G., et al., 2019. Human pluripotent stem cellederived brain pericyteelike cells induce blood-brain barrier properties. Sci. Adv. 5 (3). Stins, M.F., Badger, J., Sik Kim, K., 2001. Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells. Microb. Pathog. 30 (1), 19e28. ¨ ., Palner, M., Kornum, B.R., Rahman, O., La˚ngstro¨m, B., et al., 2009. Syva¨nen, S., Lindhe, O Species differences in blood-brain barrier transport of three positron emission tomography radioligands with emphasis on P-glycoprotein transport. Drug Metab. Dispos. 37 (3), 635e643. Tan, T.Q., Smith, C.W., Hawkins, E.P., Mason, E.O., Kaplan, S.L., 1995. Hematogenous bacterial meningitis in an intercellular adhesion molecule-1-deficient infant mouse model. J. Infect. Dis. 171 (2), 342e349.


Tazi, A., Disson, O., Bellais, S., Bouaboud, A., Dmytruk, N., Dramsi, S., et al., 2010. The surface protein HvgA mediates group B streptococcus hypervirulence and meningeal tropism in neonates. J. Exp. Med. 207 (11), 2313e2322 [Internet] Available from: http://www. ncbi.nlm.nih.gov/pubmed/20956545%5Cnhttp://www.pubmedcentral.nih.gov/article render.fcgi?artid¼PMC2964583. Tenenbaum, T., Bloier, C., Adam, R., Reinscheid, D.J., Schroten, H., 2005. Adherence to and invasion of human brain microvascular endothelial cells are promoted by fibrinogenbinding protein FbsA of Streptococcus agalactiae. Infect. Immun. 73 (7), 4404e4409. Trump, L.R., Nayak, R.C., Singh, A.K., Emberesh, S., Wellendorf, A.M., Lutzko, C.M., et al., 2019. Neutrophils derived from genetically modified human induced pluripotent stem cells circulate and phagocytose bacteria in vivo. Stem Cell. Transl. Med. 8 (6), 557e567. Turner, D.P.J., Marietou, A.G., Johnston, L., Ho, K.K.L., Rogers, A.J., Wooldridge, K.G., et al., 2006. Characterization of MspA, an immunogenic autotransporter protein that mediates adhesion to epithelial and endothelial cells in Neisseria meningitidis. Infect. Immun. 74 (5), 2957e2964. Uchiyama, S., Carlin, A.F., Khosravi, A., Weiman, S., Banerjee, A., Quach, D., et al., 2009. The surface-anchored NanA protein promotes pneumococcal brain endothelial cell invasion. J. Exp. Med. 206 (9), 1845e1852 [Internet] Available from: http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid¼2737157&tool¼pmcentrez&rendertype¼ abstract. Van der Flier, M., Geelen, S.P.M., Kimpen, J.L.L., Hoepelman, I.M., Tuomanen, E.I., 2003. Reprogramming the host response in bacterial meningitis: how best to improve outcome? Clin. Microbiol. Rev. 16 (3), 415e429. van Sorge, N.M., Doran, K.S., 2012. Defense at the border: the blood-brain barrier versus bacterial foreigners. Future Microbiol. 7 (3), 383e394 [Internet] Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid¼3589978&tool¼pmcentrez& rendertype¼abstract. van Sorge, N.M., Quach, D., Gurney, M a, Sullam, P.M., Nizet, V., Doran, K.S., 2009. The group B streptococcal serine-rich repeat 1 glycoprotein mediates penetration of the blood-brain barrier. J. Infect. Dis. Immun. 199 (10), 1479e1487. Available from: http:// www.pubmedcentral.nih.gov/articlerender.fcgi?artid¼2674616&tool¼pmcentrez&render type¼abstract. Vatine, G.D., Al-Ahmad, A., Barriga, B.K., Svendsen, S., Salim, A., Garcia, L., et al., 2016. Modeling psychomotor retardation using iPSCs from MCT8-deficient patients indicates a prominent role for the blood-brain barrier. Cell Stem Cell 20 (6), 831e843. Vatine, G.D., Barrile, R., Workman, M.J., Sances, S., Barriga, B.K., Rahnama, M., et al., 2019. Human iPSC-derived blood-brain barrier chips enable disease modeling and personalized medicine applications. Cell Stem Cell 24 (6), 995e1005. Virji, M., 2009. Pathogenic neisseriae: surface modulation, pathogenesis and infection control. Nat. Rev. Microbiol. Wang, Y., Kim, K.S., 2002. Role of OmpA and IbeB in Escherichia coli K1 invasion of brain microvascular endothelial cells in Vitro and in Vivo. Pediatr. Res. 51 63 (5), 559e563. Wang, W., M. Bodles-Brakhop, A., W. Barger, S., 2016. A role for P-glycoprotein in clearance of alzheimer Amyloid ? -Peptide from the brain. Curr. Alzheimer Res. 13 (6), 615e620. Warren, M.S., Zerangue, N., Woodford, K., Roberts, L.M., Tate, E.H., Feng, B., et al., 2009. Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human. Pharmacol. Res. 59 (6), 404e413.



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Weksler, B.B., Subileau, E a, Perrie`re, N., Charneau, P., Holloway, K., Leveque, M., et al., 2005. Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J. 19 (13), 1872e1874 [Internet] Available from: http://www.ncbi.nlm.nih. gov/pubmed/16141364. Workman, M.J., Svendsen, C.N., 2020. Recent advances in human iPSC-derived models of the blood-brain barrier. Fluid. Barrier. CNS 17 (30). Yeung, A.T.Y., Hale, C., Lee, A.H., Gill, E.E., Bushell, W., Parry-Smith, D., et al., 2017. Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis. Nat. Commun. 8, 15013. Yoshida, T., Takayama, K., Kondoh, M., Sakurai, F., Tani, H., Sakamoto, N., et al., 2011. Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection. Biochem. Biophys. Res. Commun. 416 (1e2), 119e124.


Human induced pluripotent stem cells for modeling of Salmonella infection

11 Jessica L. Forbester1, 2


Division of Infection and Immunity/Systems Immunity University Research Institute, Cardiff University, Cardiff, United Kingdom; 2MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom

Chapter outline Introduction ............................................................................................................278 iPSCs: bridging the gap between human and animal research....................................280 Establishing iPSC-derived cellular systems as a model for Salmonella infection.........282 Differentiation of iPSCs to other Salmonella infection-relevant cell types ...................285 Using iPSCs to investigate the role of host genotype on Salmonella response phenotype ................................................................................................286 Using iPSCs for modeling the molecular consequences of human genetic variants .....289 Future trends and directions ....................................................................................291 Conclusions............................................................................................................295 Acknowledgments ...................................................................................................296 References .............................................................................................................296 Abstract Salmonellae are a group of gram-negative bacteria that cause infections in humans after invading the gastrointestinal mucosa, contributing significant morbidity and mortality in human populations. Models to study this pathogen are limited, in part due to the host-restricted nature of some Salmonella serovars. Induced pluripotent stem cells (iPSCs) are beginning to provide a tool to facilitate improved Salmonella pathogenesis modeling in an in vitro setting. iPSCs provide an unlimited resource of cells that can be differentiated down multiple cellular lineages, while retaining the genome of the donor. Therefore, iPSCs offer the exciting opportunity to apply patient-specific modeling to Salmonella research, providing an invaluable tool for probing how host genotype affects responses to infection. Although currently only in the early phases, iPSC-based modeling has already revealed novel mechanisms that human cells use to restrict Salmonella, highlighting the exciting potential for iPSC use in future hostepathogen research. iPSCs for Studying Infectious Diseases, Volume 8. https://doi.org/10.1016/B978-0-12-823808-0.00009-2 Copyright © 2021 Elsevier Inc. All rights reserved.



CHAPTER 11 Human induced pluripotent

Keywords: Differentiation; Enteric fever; Gastroenteritis; Gene editing; Genetic variants; Host restriction; Host specificity; Infection; Innate immunity; iPSCs; Macrophages; Modeling; Organoids; Pathogenesis; Patient specificity; Salmonella.

Introduction Salmonellae are a group of anaerobic, facultative bacilli that have the capacity to invade through the intestinal mucosa, causing infections in humans and animals, which can be classified broadly into two outcomes, self-limiting gastroenteritis or enteric (typhoid) fever, which can spread systemically and be fatal in human hosts, with each of these outcomes associated with distinct serovars. Nomenclature for Salmonella has been revised several times, but it is now generally accepted that there are three species within the Salmonella genus: S. bongori, S. enterica, and S. subterranean. Most human disease is associated with a single subspecies S. enterica subsp. Enterica, which includes the serovars that cause typhoid and paratyphoid in humans, S. Typhi and S. Paratyphi, respectively, as well as over 2000 gastroenteritis-causing serovars (Ravindran and McSorley, 2005). During infection, after passage through the intestinal epithelial cells (IECs), Salmonella can colonize the lamina propria and Peyer’s patches (PP), infecting host cells such as macrophages. If the host intestinal immune cells cannot limit further dissemination, Salmonella can spread systemically to the mesenteric lymph nodes, spleen, and liver (Andrews-Polymenis et al., 2010). Although infection with nontyphoidal serovars (NTS) is normally limited to the gastrointestinal tract, there has also been an increased incidence of invasive NTS (iNTS), which can cause invasive enteric disease in immune-compromised individuals and results in >600,000 deaths annually (Ao et al., 2015). As an estimated 94 million cases of NTS are expected to occur each year, salmonellosis still has a huge global infectious burden, with large economic burdens associated with the disease, as well as illness and death (Petrovska et al., 2016). Some Salmonella serovars maintain a broad host range, whereas others, such as S. Typhi, have become hostrestricted (Dougan and Baker, 2014). S. Typhi rapidly acquires resistance to the antimicrobials that are being used in the community, but can also lose resistance once these drugs are withdrawn. From these observations, it seems likely that antimicrobial resistance will emerge in areas endemic for typhoid, leading to treatment failure, changes in antimicrobial policy, and further resistance developing in S. Typhi isolates and other gram-negative bacteria (Britto et al., 2018). The rise of antibiotic resistance and the inability to treat these antibiotic resistant strains have resulted in high morbidity and mortality, combined with substantial economic losses; for example, it is estimated that 25,000 people die and V1.5 billion is spent annually due to antibiotic-resistant infections in Europe (Gut et al., 2018). Furthermore, the current licensed typhoid vaccines, including the attenuated oral vaccine Ty21a, are not considered to be optimal in terms of their efficacy. This lack of efficacious vaccine, combined with the growing antimicrobial resistance challenges faced


globally, and the increased incidence of iNTS strains, means that there is a need for development of new methods for controlling Salmonella infections (Bajracharya et al., 2014). Furthermore, fundamental knowledge regarding some aspects of Salmonella pathogenesis is lacking; for example, the precise role of typhoid toxin in severe typhoid disease and establishment of bacterial carriage in humans remains to be established (Gibani et al., 2019). Current enteric pathogen research has been limited by suitable model systems. Reliance on immortalized cell lines generated from human cancers for in vitro studies, which have multiple limitations including that they are often karyotypically abnormal and lack key features of the primary cells they are being used to model, has hindered progress (Langerholc et al., 2011; Pan et al., 2009). In vivo models, while extremely useful for defining systemic effects of infection and determining specific gene function in Salmonella immunity, have been hindered by species-specific differences in Salmonella serovar susceptibility. Mice are the most commonly used laboratory animal model for infection studies because of their small size, short gestation periods, ease of housing and handling, and the ability to control their genetics through breeding and manipulation of their genomes via conditional and inducible transgenic and gene knockout strains (Masopust et al., 2017). S. Typhi does not infect mice, and oral challenge of mice with S. Typhimurium does not provide a relevant model for Salmonella-derived enterocolitis, with minimal invasion into epithelial cells and little inflammation, although pretreating mice with the antibiotic streptomycin prior to infection results in an infection phenotype more similar to that observed in humans (Barthel et al., 2003). Limited human typhoid challenge studies have been conducted to study the pathophysiology of S. Typhi in human hosts, providing more information on hostepathogen interactions, which potentially can aid in improving S. Typhi vaccines, diagnostics, and therapeutics, which are currently imperfect (Waddington et al., 2014). However, these human challenge studies are difficult to establish, requiring large resources and the support of clinical teams; therefore, the development of additional laboratory systems to study Salmonellaehost interactions in a human-specific context is also necessary. In 2007, Takahashi et al. demonstrated that pluripotent stem cells could be generated from adult somatic cells by transduction with four transcription factors (TFs). These induced pluripotent stem cells (iPSCs) were shown to be morphologically similar to human embryonic stem (ES) cells and capable of differentiation to the three germ layers, endoderm, ectoderm, and mesoderm, in vitro and in teratomas (Takahashi et al., 2007). This major advance provided an exciting opportunity to drive advances in disease modeling, drug discovery, and regenerative medicine, because these systems allow for the recapitulation of human “disease in a dish,” allowing the growth of unlimited quantities of a specific individual’s cells and the differentiation of these cells into multiple cell and tissue types (Shi et al., 2017). Furthermore, iPSCs allow for exploration of the effects of human genotype on cell phenotype, allowing a combinatory approach to human genetics and the mechanisms of human cell biology, which previously had been distinct disciplines due to limited techniques to combine the two (Warren and Cowan, 2018). Specifically, for



CHAPTER 11 Human induced pluripotent

pathogenesis research, iPSCs allow for the development of patient-specific iPSCderived cellular platforms, which are ethically sourced, versatile, and noninvasive to acquire, allowing for analysis of the innate immune response to pathogens and the testing of new therapies (Trevisan et al., 2015). New methods for editing DNA further enhance the utility of iPSCs, providing methods to inactivate genes of interest, and also more recently to create isogenic control lines, whereby disease-causing mutations can be precisely edited into or repaired in iPSC lines (Kang et al., 2015; Sanjurjo-Soriano et al., 2020; Yusa et al., 2011). This review will focus on the potential of new iPSC technologies to supplement current models for Salmonella infection, focusing mainly on two well-studied host serovars that cause significant amounts of human disease, S. Typhi and S. Typhimurium. Insights from the first batch of studies to use this iPSC technology will be discussed, followed by suggestions for future directions that can be taken with iPSC systems to help enhance current knowledge of Salmonella pathogenesis.

iPSCs: bridging the gap between human and animal research During the course of Salmonella infection, multiple cell types may be infected by this bacterium, with multiple cell types also contributing to the establishment of an effective immune response (Kaur and Jain, 2012). Therefore, it is prudent to be able to study the interactions with, and the responses of, these different cell subsets to fully understand Salmonella pathogenesis. Although animal models, including mice, pigs, and cows, have proven useful in dissecting the interaction of this pathogen with its host, these models all have their limitations, including exhibiting different susceptibilities to Salmonella serovars, and it being challenging to study the effects of human genetic variation on pathogenesis within these models. For some primary human cell types, access to material can be difficult, for example, to acquire human primary intestinal tissue, a biopsy sample must be harvested via endoscopy (Kraiczy et al., 2019a). Generation of innate immune cells such as macrophages and dendritic cells (DCs) from human blood monocytes is a fairly routine procedure used by many laboratories, but these myeloid cells exhibit differences from their in vivo counterparts (Osugi et al., 2002) and may not accurately represent the innate immune cells that pathogens encounter during an infection. Furthermore, due to ethical restrictions, it may not always be possible to receive the genomic sequence of donors, limiting phenotypeegenotype conclusions from experiments. In addition, it is logistically difficult to generate multiple different primary cell types from one donor, limiting the ability to conduct assays on different cell types with the same genetic background. iPSCs are beginning to provide a very useful tool for bridging the gap between animal and human studies, in particular where human primary cell models are not available or are difficult to acquire in large numbers (Xu and Zhong, 2013), providing the opportunity to derive different cell subsets from the same individual.

iPSCs: bridging the gap between human and animal research

Furthermore, there are also now large-scale iPSC cell banks available to researchers, which provide access to multiple genomic, transcriptomic, and proteomic data sets corresponding to each banked iPSC line (Streeter et al., 2017). Robust differentiation protocols exist to differentiate iPSCs into multiple different cell types, with established protocols generating cell types relevant to Salmonella infection summarized in Table 11.1. Table 11.1 Differentiation of iPSCs to diverse cell types. By using signals such as small molecules, growth factors/cytokines, matrices, and other cells, iPSCs can be directed to differentiate down specific cellular lineages. Cell type

Differentiation protocol summary


Generation of embryoid bodies, harvest of monocytes, and adherent culture with serum and M-CSF to generate macrophages

Dendritic cells (DCs)

Generation of embryoid bodies, harvest of pre-DCs, and culture with interleukin (IL)-4 and GM-CSF on CellBind plates

Intestinal epithelial cells (IECs)

Generation of endoderm, followed by hindgut, and embedding of hindgut in a matrix-based culture system supplemented with prointestinal growth factors such as Noggin, R-Spondin-1, and Wnt3a Derivation of iPSCs from mature adult T cells and coculture of these T-cellderived iPSCs with OP9/DLL1 stromal cells, generating CD3þCD8þ T cells with the original antigen-specific TCR Formation of embryoid bodies, culture of embryoid bodies with IL-3 and G-CSF, and formation of stroma, harvest of progenitors, and culture with G-CSF Differentiation of iPSCs to endoderm, then foregut, and then hepatic endoderm, followed by hepatocyte maturation with hepatocyte growth factor (HGF) and oncostatin M (OSM) Generation of embryoid bodies, followed by culture of embryoid bodies with IL-3, IL-7, IL-15, stem cell factor (SCF), and fms-like tyrosine kinase receptor-3 ligand (FLT3L) to generate NK cells

T cells



NK cells

References for differentiation PLoS one (Wilgenburg et al., 2013) Methods in Molecular Biology (Mukherjee et al., 2018) Frontiers in Immunology (Sachamitr et al., 2018) Stem Cells (Sontag et al., 2017) Methods in Molecular Biology (Forbester et al., 2016) Nature (Spence et al., 2011) Cell Stem Cell (Vizcardo et al., 2013)

Stem Cells and Translational Medicine (Trump et al., 2019) Nature Protocols (Hannan et al., 2013b)

Stem Cells Translational Medicine (Knorr et al., 2013)



CHAPTER 11 Human induced pluripotent

Establishing iPSC-derived cellular systems as a model for Salmonella infection After surviving passage through the stomach and duodenum, Salmonella enters the host through the intestinal epithelium. Although in murine models microfold (M-) cells, which are IECs specialized in sampling luminal microbes and antigens found mainly overlying the mucosal lymphoid sites (Dillon and Lo, 2019), have been demonstrated to constitute the major entry sites for Salmonella to the underlying lamina propria, in human explants and human intestinal cell models, Salmonella pathogenicity island-1 (SPI-1)-dependent entry has also been observed. Here, S. Typhimurium has been shown to use the type III secretion system (T3SS) encoded on SPI-1 to inject effector proteins into the host cell. These effectors modulate the host cell cytoskeleton and induce “membrane ruffles,” which result in engulfment of the bacterium into the host cell in a modified endosome, termed a Salmonella containing vacuole (SCV) (Ly and Casanova, 2007). In addition, Salmonella may also move from the gut lumen to the lamina propria via uptake by CX3CR1þ DCs, or via destruction of tight junctions, allowing Salmonella residing in the lumen to move between epithelial cells into the lamina propria (Niess, 2005; Watson and Holden, 2010). IECs are the first cellular barrier Salmonella encounters. Although IECs were initially thought to provide no more than a static barrier to pathogen entry, it is now widely accepted that these cells play varied and important roles in host defense (Allaire et al., 2018). Interactions between Salmonellae and host IECs have typically been modeled using immortalized cell lines such as Caco-2 cells (Finlay and Falkow, 1990). However, these cell lines lack the full diversity of adsorptive and secretory cells found in the intestinal epithelium and fail to model the complex structure of crypt and villus regions. A large advance in the IEC field was made by Clevers and colleagues, who discovered that Lgr5þ intestinal stem cells (ISCs) could be isolated from primary intestinal tissue and seeded into a supporting matrix overlaid with media supplemented with growth factors promoting proliferation and differentiation of those ISCs, resulting in the formation of spheroids of intestinal tissue, containing crypt and villus-like structures (Sato et al., 2009). Subsequently, Spence et al. found that after the directed differentiation of iPSCs to endoderm and hindgut, this iPSC-derived hindgut could be harvested and placed into a similar culture system to primary LGR5þ ISCs, resulting in similar structures of intestinal epithelium to that generated from primary ISCs after a period of maturation (Spence et al., 2011). These intestinal human organoids (iHOs) display features of intestinal epithelium (summarized in Fig. 11.1) that are lacking in many immortalized cell lines (Forbester et al., 2018; Hannan et al., 2013a; Spence et al., 2011), and it has been shown that microinjection is an efficient method by which to deliver enteric pathogens such as S. Typhimurium to the apical side of these cells (Fig. 11.1C) (Forbester et al., 2016; Lees et al., 2019a). In addition, the development of monolayer culture for stem-cell-derived IECs (Thorne et al., 2018) may provide a simpler method for

Establishing iPSC-derived cellular systems

FIGURE 11.1 Features of intestinal human organoids derived from iPSCs. (A) iHOs comprise a hollow luminal cavity (L) surrounded by a polarized epithelium of IECs, which can arrange into crypt and villus-like structures. (B) iHOs display features of mature intestinal epithelium that can be observed by transmission electron microscopy (TEM) such as microvilli structures on enterocytes (MV), goblet cells (GC), Paneth cells at the bottom of crypt-like structures (shown in immunofluorescence images marked by Paneth cell marker lysozyme; LYZ); and other rare secretory cell types such as enteroendocrine cells (shown in immunofluorescence images marked by enteroendocrine cell marker chromogranin A; CHGA). (C) iHOs can be microinjected with pathogens such as S. Typhimurium, demonstrating characteristics of infection with this pathogen including large-scale cytoskeletal rearrangements (shown in immunofluorescence image, F-actin, red, S. Typhimurium SL1344 (SL1344), green; DAPI, blue) and formation of the SCV (TEM image).

Salmonella challenge of IECs and could make quantification of multiplicity of infection (MOI) easier, as this is complex in a 3D structure. Although the use of iPSCs for modeling Salmonellaehost interactions is in its infancy, due, in part, to these technologies being relatively new, several early studies



CHAPTER 11 Human induced pluripotent

have revealed the promise of these systems for future Salmonella pathogenesis research. The interaction of Salmonella with iHOs has been investigated. Here, iHOs were exposed to S. Typhimurium either basally by addition to the iHO culture media or apically via microinjection, and the transcriptional response of iHOs was measured by RNA-sequencing, demonstrating upregulation of components of the IEC innate immune response to Salmonella, including inflammatory cytokines and components of mucus. Distinct transcriptional profiles were generated based on delivery mechanism of S. Typhimurium, suggesting variation in pathogen sensing at apical versus basal surfaces. Furthermore, when S. Typhimurium was delivered apically via microinjection into the luminal cavity of the iHO, S. Typhimurium internalized into the IECs, recapitulating well-described features of S. Typhimurium infection such as triggering large-scale cytoskeletal rearrangements and formation of the SCV (visualized in Fig. 11.1C). Use of an invasion-deficient S. Typhimurium strain, which was significantly less able into internalize into iHO IECs, suggests that the iHO system could also be used for determining the pathogenesis of different Salmonella mutants (Forbester et al., 2015). The main site of infection for Salmonella in the gastrointestinal tract is thought to be the ileum (Carter and Collins, 1974), although this is based mainly on observations in animal models. Due to the presence of Paneth cells within iHO structures, it was initially reported that iHOs may be more similar to human small intestine (Forbester et al., 2015); however, in a study using DNA methylation analysis and RNA sequencing to compare regions of the intestinal tract to iHOs, it was found that iHOs grouped most closely with pediatric sigmoid colon epithelium. However, as previously observed, iHOs also expressed key small intestinal markers for Paneth cells, suggesting that iHOs may not be fully regionalized during differentiation and may display markers of both small and large intestines (Kraiczy et al., 2019b). Moreover, the development of M-cells in murine intestinal organoids has been shown to be dependent on NF-kB activation by RANK signaling, suggesting that it may be possible to hijack these pathways to drive the development of M-cells in iHOs (Kanaya et al., 2018). Salmonella can replicate within host macrophages and neutrophils, facilitating evasion of the host innate immune response by aiding protection from extracellular host defenses such as antimicrobial peptides (AMPs) and serum complement, as well as providing an intracellular niche without nutrient competition from microbiota, and also helping to facilitate systemic spread (Ellis et al., 2019). Macrophages are now relatively routinely derived from iPSCs by many laboratories (Buchrieser et al., 2017; Wilgenburg et al., 2013), and these iPSDMs have been shown to express various macrophage markers including CD11c, CD14, CD16, CD44, CD64, CD54, and CD200R, as well as responding to interleukin (IL)-4 or IFN-g to polarize toward alternative or classical activation states, respectively, upregulating either the classical marker HLA-DR or the alternative marker CD206. These macrophages can also phagocytose inactivated bacterial particles and produce cytokines in response to TLR ligation (Hale et al., 2015), providing a promising model for dissecting hostepathogen interactions.

Differentiation of iPSCs to other Salmonella infection-relevant cell types

The application of iPSDMs to cancer therapy (Senju et al., 2014) and for modeling rare genetic defects that may impact macrophage function (Jiang et al., 2014; Panicker et al., 2014) had previously been investigated; however, Hale et al. were the first to use these iPSDMs as a model to explore hostepathogen interactions. When iPSDMs were generated from healthy control iPSCs and infected with S. Typhimurium and S. Typhi, responses of iPSDMs varied dependent on which serovar they were exposed to, with serovar-dependent cytokine expression patterns, bacterial uptake, and bacterial killing observed (Hale et al., 2015), suggesting that iPSDMs provide a suitable system for dissecting responses to different Salmonella serovars. Furthermore, iPSDMs derived from four iPSC lines from individual donors have been compared to human monocyte-derived macrophages (MDMs) derived from five different individuals, pre- and post-lipopolysaccharide (LPS) stimulation. Broadly, transcriptional profiles were shown to be similarly conserved between MDMs and iPSDMs; however, there were some differences in expression of gene sets involved in antigen presentation and tissue remodeling, suggesting that the iPSDMs possibly resembled tissue-resident macrophages more closely than the MDMs, or, as described for other iPSC systems, resembled a more fetal-like macrophage (Alasoo et al., 2015). Thus far, iPSC-derived macrophages and IECs have been used for dissecting Salmonellaehost interactions, but with advances in iPSC differentiation techniques, the interaction of Salmonella with other host cell types is an avenue open to exploration.

Differentiation of iPSCs to other Salmonella infection-relevant cell types During infection with NTS serovars, DCs are important for establishing adaptive immunity to Salmonella by presentation of bacterial antigen to T cells, but DCs can also provide an additional mechanism for pathogen dissemination, and Salmonella has acquired mechanisms for manipulating DC function, for example, inducing the upregulation of CCR7 receptors on the DC surface, driving the migration of the DC to secondary lymphoid tissues such as the mesenteric lymph nodes (MLNs) from the PP, while also restricting antigen presentation on the DC surface (Swart and Hensel, 2012). DC sensing of Salmonella is extremely important in initiating the IL-12 axis and the IL-23 axis, whereby antigen presenting cell (APC)-produced IL-23 acts on T cells to induce IL-22 and IL-23A production, and also maintains IL-17A producing TH17 cells, and IL-12 acts on T cells to produce IFN-g, which in turn activates pathways in APCs such as macrophages, driving the elimination of intracellular pathogens (Godinez et al., 2011). However, SalmonellaeDC interactions are still not well understood, for example, understanding why different S. enterica strains survive and proliferate at different rates inside host DCs, the mechanisms underlying interference with antigen presentation in DCs, and how Salmonella eventually escapes from the DC are all open questions



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(Swart and Hensel, 2012). Using monocyte-derived DCs (MoDCs) and single-cell sequencing, Aulicino et al. demonstrated that infection of DCs with a highly invasive NTS strain ST313 resulted in higher expression of IL10 and MARCH1 and lower expression of CD83 by MoDCs, which the authors concluded helps ST313 to evade adaptive immune detection, helping to dissect one of the mechanisms this Salmonella strain has exploited to facilitate its own dissemination in vivo (Aulicino et al., 2018). Despite important studies like this dissecting some of the underlying mechanisms explaining important factors in Salmonella pathogenesis such as strain-specific differences in immune evasion, SalmonellaeDC interaction research has been majorly limited by the lack of a DC immortalized cell line, the heterogeneity of DCs obtained from primary precursors, and the difficultly of tracking infected DCs in in vivo systems (Swart and Hensel, 2012). DCs resembling human CD1cþ and CD141þ DCs can be generated from iPSCs, and these iPS-DCs have been shown to be capable of T cell activation (Sachamitr et al., 2018; Senju et al., 2011), so it is possible that these iPS-DCs may provide a useful tool for probing SalmonellaeDC interactions, although thus far, to my knowledge, there have been no studies of this nature, leaving this avenue open to exploration. When Salmonella spreads systemically, it has the capacity to invade a diverse range of cell types, such as hepatocytes in the liver (Conlan and North, 1992). The recent discovery of alternative entry mechanisms Salmonella uses to gain access to host cells other than its well-described T3SS, such as the use of the outer membrane protein resistance to complement killing (Rck) to induce internalization (Rosselin et al., 2010), is important as it is documented that Salmonella strains lacking the T3SS encoded on Salmonella pathogenicity island-1 (SPI-1) can still cause disease outbreaks and can adhere to and invade various cell types in vitro. However, it is not fully understood how and when Salmonella uses these various invasion mechanisms. The host cell receptor for Rck is thought to be epidermal growth factor receptor (EGFR), which is also highly expressed on hepatocytes. Rck/EGFR interactions may explain how Salmonella is able to colonize the liver, although this hypothesis remains to be fully tested (Mambu et al., 2017). As the generation of hepatocytes from iPSCs is already possible (Hannan et al., 2013b), iPSCs have the potential to provide a suitable model for addressing hypotheses such as these.

Using iPSCs to investigate the role of host genotype on Salmonella response phenotype As discussed earlier, iPSC-derived cellular systems have been shown to resemble features of human primary cells and, with the cell types explored, to recapitulate known Salmonella infection phenotypes, suggesting that iPSC-derived cells can provide a useful tool for pathogenesis research. However, the ability to incorporate a combination of host and pathogen genetic variation within a tractable laboratory system is, in my opinion, the most exciting and promising feature of iPSCs for advancing the hostepathogen research field.

Using iPSCs to investigate the role of host genotype

Susceptibility to infectious diseases shows significant interindividual variation, with a proportion of this susceptibility heritable. Data from candidate gene studies and patients with primary immunodeficiencies has helped to elucidate various host genetic determinants of iNTS disease and human enteric fever (Gilchrist et al., 2015). For example, in patients with a rare primary immunodeficiency, Mendelian Susceptibility to Mycobacterial Diseases (MSMD), where patients exhibit deleterious mutations in one of eight autosomal genes, with these mutations disrupting IL-12-dependent, IFN-g-mediated immunity, iNTS disease occurs in half of these patients, suggesting a fundamental role for this pathway in disseminated bacterial control (Averbuch et al., 2011; Prando et al., 2013). iPSCs have already been generated from a patient with complete autosomal recessive IFNgR1 deficiency, with macrophages derived from these iPSCs shown to have impaired MHC II expression after activation and reduced intracellular killing of Bacillus Calmettee Guerin (Neehus et al., 2018), highlighting the potential to use these systems to further probe the relevance of this pathway to Salmonella control in human cells. Furthermore, it is likely that there are other genetic loci that contribute to Salmonella susceptibility in humans that remain as yet unexplored. Two recent studies using iPSCs generated from an inflammatory bowel disease (IBD) patient with a homozygous mutation in IL10RB, predicted to introduce a premature stop codon resulting in a nonfunctional IL10R2 protein, highlighted the potential of using iPSCs generated from patients with monogenic disorders to further advances in establishing the protective mechanisms host cells apply against Salmonella. In these studies, the authors elucidated two novel mechanisms for Salmonella control by cytokine pathways utilizing the IL10R2 receptor in macrophages and IECs (summarized in Fig. 11.2). iPSDMs and iHOs derived from patient iPSCs were comparable morphologically to iPSDMs and iHOs generated from multiple healthy control iPSC lines, suggesting that the patient’s IL10RB mutation did not affect the differentiation capacity of iPSCs (Forbester et al., 2018; Mukhopadhyay et al., 2020). IL10R2 acts as the coreceptor for several cytokines, including IL-10, for which it forms a heterodimer with IL10R1, and IL-22, heterodimerizing with IL22R1. IL-22 has previously been demonstrated to be play a nonprotective role in S. Typhimurium infection in mice by reducing colonization resistance, allowing S. Typhimurium to outcompete the intestinal microbiota through acquired mechanisms allowing for evasion of host AMPs (Behnsen et al., 2014). However, in iHOs, IL-22 was shown to prime IECs to be less susceptible to S. Typhimurium colonization, via induction of increased phagolysosomal fusion after bacterial internalization, which was shown, by the use of S100A9/ iPSCs, to be Calgranulin B (S100A9)-dependent (Forbester et al., 2018). In iPSDMs generated from iPSCs from the same IBD patient, a reciprocal regulatory axis between the prostaglandin E2 (PGE2) and the IL-10 pathways was described, and this interaction was demonstrated to regulate macrophage activation. In patient iPSDMs, where IL-10 signaling was inhibited, there was microbial hyperresponsiveness and overproduction of PGE2, which limited the ability of these macrophages to kill internalized S. Typhimurium (Mukhopadhyay et al., 2020).



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FIGURE 11.2 Generation of iPSC-derived iHOs and iPSDMs from an IL10R2-deficient patient revealed novel antimicrobial restriction pathways regulated by IL-22 and IL-10 after S. Typhimurium challenge. iPSCs were generated from a patient harboring a homozygous mutation in the IL10RB gene that inactivates both the IL-10 and IL-22 receptor. iPSDMs were shown to be nonresponsive to IL-10 treatment, hyperactivated and to overproduce Prostaglandin E2 (PGE2), which directly correlated with reduced intracellular killing of S. Typhimurium (Mukhopadhyay et al., 2020). iHOs generated from the same iPSCs were nonresponsive to IL-22, demonstrating reduced production of components associated with a mucosal barrier phenotype, and exhibiting reduced phagolysosomal fusion within enterocytes, resulting in increased intracellular survival of S. Typhimurium (Forbester et al., 2018).

Furthermore, both studies made use of a “corrected” version of the IBD patient iPSCs, whereby a copy of the IL10RB gene had been inserted into the adenoassociated virus integration site 1 (AAVS1) locus in the patient iPSC genome, under the control of a constitutive promotor. In iPSDMs and iHOs derived from these corrected iPSCs, restoration of IL10R2 expression at both the mRNA and protein level was observed, comparable to healthy control levels, resulting in restoration of functional responsiveness to IL-10 and IL-22 and rescuing the pathogen control phenotypes initiated by IL-10 and IL-22 priming (Forbester et al., 2018; Mukhopadhyay et al., 2020).

Using iPSCs for modeling the molecular consequences

The expression and function of LRRK2 in human macrophages and microglia generated from iPSCs have recently been characterized, with macrophages/microglia generated from human LRRK2 knockout iPSCs; patient iPSCs with a heterozygous LRRK2 mutation G2019S, the most common identified genetic determinant of Parkinson’s disease (PD); and a repaired version of this line, in which G2019S has been replaced with WT sequence. Here, LRRK2 was demonstrated to be recruited to late phagosomes containing S. Typhimurium, colocalizing with late phagosomal markers RAB9 and LAMP1. The authors concluded that this suggests a possible role for LRRK2 in rerouting and recycling phagocytosed membrane, receptors, and contents (Lee et al., 2020). As LRRK2 is highly expressed in immune cells, there is a possibility that microbial infection could trigger PD in patients with a predisposed genetic background, with one of the main challenges for LRRK2 research defining the physiological activators of this signaling in immune cells (Herbst and Gutierrez, 2019), highlighting the potential for iPSCs to be used as a tool for interdisciplinary research. S. Typhi is rare among Salmonella serovars, causing disease solely in humans. This is in comparison to the majority of Salmonella serovars, which are not host-restricted. One possible explanation came from various studies in mice, which suggested that this host specificity may be explained by the activity of the RAB32/BLOC-3 pathway; S. Typhimurium has mechanisms to subvert this pathway in murine macrophages, which S. Typhi lacks (Spano and Galan, 2012). Therefore, it was suggested by Baldassarre et al. that either RAB32/BLOC-3 are not part of the host defense against Salmonellae in humans or that S. Typhi has evolved specific molecular strategies to evade the RAB32/BLOC-3 in humans. By differentiating iPSCs with a knockout of HPS4, one of the two BLOC-3 subunits, to iPSDMs and infecting these iPSDMs with S. Typhi, BLOC-3 knockout iPSDMs were demonstrated to have increased bacterial intracellular replication, suggesting this pathway is important for S. Typhi control in human macrophages, and therefore indicating that S. Typhi has instead evolved mechanisms to evade this defensive pathway (Baldassarre et al., 2021). In conclusion, initial data suggest that iPSCs provide a promising and useful model for dissecting further the mechanisms human cells use to protect against different Salmonella serovars. By generating iPSCs from patients with genetic variants, which are predicted to affect protein function, combined with the ability to use CRISPR/Cas9 to directly inactivate specific genes in iPSCs, the role of specific host genes in Salmonella pathogenesis can be fully elucidated.

Using iPSCs for modeling the molecular consequences of human genetic variants Individuals have a unique set of genetic variants that, along with environmental factors, contribute to the susceptibility of the individual to various pathologies. Monogenic disorders, where susceptibility is caused by inheritance of a single defective



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gene, are rare in populations (Hamazaki et al., 2017; Vannberg et al., 2011). However, common genetic variants have also been associated with susceptibility to various pathogens and disease outcome (Everitt et al., 2012; Lees et al., 2019b; Thye et al., 2012), and identifying more of these genetic variants will help to determine novel gene associations that will advance the understanding of the molecular pathways involved in the pathogenesis of specific infectious diseases. Genetic-wide association studies (GWAS) have revealed single nucleotide polymorphisms (SNPs) that associate with health risks, drug outcomes, and disease susceptibilities; however, to fully establish a precision method approach to medicine, where therapies are tailored to individuals based on their genome, it is important to further dissect how these variants contribute to the phenotypes described. iPSCs can provide a system with which to probe the molecular mechanisms underlying variant-driven phenotypes, helping to drive the precision medicine field (Hamazaki et al., 2017). Multiple established iPSC banks already exist and can act as a resource for researchers looking to explore host responses in diverse genetic backgrounds (Huang et al., 2019). iPSC-based cellular modeling systems provide the opportunity for researchers to perform large-scale studies analyzing diversity in host response to various stimuli, including pathogens. The GWAS field has highlighted the importance of regulatory polymorphisms in driving human phenotypic variation, leading to studies combining genetic mapping with characterization of molecular and cellular traits, such as cellular mRNA levels (Gaffney, 2013). Genetic differences in immune cells are known to profoundly alter individual cell response to environmental stimuli, manifesting at a molecular level as expression quantitative trait loci (eQTLs), altering the magnitude of gene expression changes poststimulation, and potentially altering the risk for complex immunologically mediated disorders. eQTLs alter the magnitude of gene-expression changes after stimulation (response eQTLs) possibly by disrupting the binding of stimulus-specific TFs, as eQTLs have also been shown to alter chromatin accessibility. Alasoo et al. used iPSCs to investigate enhancer “priming,” the idea that cell-type-specific TFs bind enhancers without directly affecting target gene expression, and then, after cellular activation by certain stimuli, response-specific TFs subsequently facilitate activation, producing a cell-type-specific response. In this study 86 iPSDM lines were treated with IFNg for 18 h; infected for 5 h with S. Typhimurium; or pretreated for 18 h with IFNg and then infected for 5 h with S. Typhimurium, producing ATAC-Seq and RNA-Seq data sets, and demonstrating that pre-existing genetic effects on chromatin propagate gene expression during immune activation, furthering the understanding of the molecular architecture of disease-associated variants (Alasoo et al., 2018). Although in their infancy, studies like that of Gaffney and colleagues have laid the groundwork for the use of iPSCs in large-scale studies dissecting how genetic variants may alter responses to pathogens such as Salmonella, with iPSCs facilitating a move toward dynamic studies of gene regulation in cell types relevant to the disease of interest, hopefully elucidating mechanisms underlying complex disease, which were previously difficult to study (Banovich et al., 2018).

Future trends and directions

Future trends and directions Currently, the use of iPSCs in Salmonella research has been limited to a small number of studies. It is important to remember that it is only relatively recently that iPSCs have become available to researchers, with the first method generating these cells published in 2007 (Takahashi et al., 2007). Furthermore, tools that allow for efficient genetic editing in iPSCs are even more recent, with CRISPR/Cas9 technologies first demonstrated to function in mammalian cells in 2013 (Cong et al., 2013; Mali et al., 2013) and then in iPSCs later in the same year (Hou et al., 2013). iPSCs are currently expensive to culture and technically more challenging to work with than immortalized cell lines. The growth factors required to differentiate these cells are also financially constraining; however, new protocols are being developed to use more cost-effective, small-molecule signals to direct the differentiation process (Du et al., 2018). In some iHO differentiation systems, the requirement for Wnt3a has been bypassed by the use of a GSK3 inhibitor, CHIR99021, whereby inhibition of GSK3 drives activation of the Wnt pathway (Forbester et al., 2016; Hannan et al., 2013a). Despite these limitations, iPSC use has expanded in other fields (Singh et al., 2015), and iPSC-derived cellular systems have also now been used to probe hostepathogen interactions for a large range of other pathogens (Ciancanelli et al., 2015; Leslie et al., 2015; Muffat et al., 2018; Ng et al., 2015). As these systems become more prevalent in multiple fields, it is likely technological advances in cell culture will reduce the cost and complexity of adopting this model. iPSCs offer some significant benefits in comparison to human primary cells including that iPSCs are capable of proliferation and self-renewal, allowing researchers access to unlimited numbers of cells; the ability to differentiate iPSCs into different cell types, particularly those that are difficult to isolate from human patients such as intestinal epithelium; and the relative amenability of iPSCs to genetic editing (Forbester et al., 2015; Trevisan et al., 2015; Yeung et al., 2017). It is clear that iPSCs offer an invaluable tool to pathogen researchers to probe how host genotype can alter the response of human cells to infection, tools that were previously limited within pathogenesis laboratories. Although the mouse has provided a huge number of insights into Salmonella pathogenesis, some of these findings have failed to translate into human cells. For example, Nramp1 was identified as important in controlling systemic infection to S. Typhimurium in mice, with this infection used as a model for S. Typhi infection in humans (Roy and Malo, 2002). However, there has been no correlation observed between susceptibility to S. Typhi infection in humans and Nramp1 alleles, indicating that this mechanism may not be important in human disease, and suggesting caution in interpreting findings directly from murine studies without probing human systems (Hurley et al., 2014). S. Typhi is humanrestricted, so has been particularly challenging to study within a laboratory setting, and although human challenge models offer an opportunity to study S. Typhi interaction in human hosts (Gibani et al., 2019), the scope of these studies is limited by ethical considerations. iPSCs offer the exciting opportunity to further probe insights from recent human challenge studies in a tractable, human, laboratory system.



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Human genetic variation at various loci has been linked to increased susceptibility to Salmonella infection. For example, an SNP in VAC14, which encodes a scaffold protein that is a component of the PIKfyve protein kinase complex, was identified as being associated with increased cellular susceptibility to S. Typhi invasion (Alvarez et al., 2017). In GWAS performed in Kenyan and Malawian children, a locus in STAT4, rs13390936, a context-specific eQTL for STAT4 mRNA expression, was identified as associated with NTS bacteraemia. Individuals carrying the NTSrisk genotype demonstrated decreased IFN-g production in stimulated natural killer (NK) cells and decreased circulating IFN-g during acute NTS bacteraemia (Gilchrist et al., 2015). A hugely attractive feature of iPSC technology is that it allows researchers the opportunity to derive cells from patients that carry all the genetic alterations that may be associated with a specific disease phenotype, giving the opportunity to study pathogenesis of a disease in vitro and potentially help to drive developments in therapeutics (Hockemeyer and Jaenisch, 2016). At a population level, most people exhibiting increased susceptibility to a pathogen do not have identified Mendelian disorders, with disease outcome resulting from interactions between host genetic variants, environmental factors, and the genome of the pathogen, therefore identifying genetic variants that predispose individuals to infectious diseases may highlight novel pathways that are important for controlling disease, identify new disease mechanisms, and also potentially give new vaccine and drug candidates (Newport and Finan, 2011). Furthermore, in the future, we may be able to prospectively stratify patients for therapies tailored to their individual genomes (Warren and Cowan, 2018). This could be applied to understanding individual risk to infection by pathogens such as Salmonella. iPSCs provide a system for storing an individual’s genetic information within laboratories, with the flexibility and potential to expand these cells and differentiate into the cell type relevant for the disease of interest. For other diseases such as cancer and cystic fibrosis, screens are already being performed on iPSC-derived cells generated from specific individuals (Gandre-Babbe et al., 2013; Merkert et al., 2019), potentially providing information to direct clinicians toward patient-specific treatment plans. iPSCs offer the opportunity to further probe how these genetic variations alter cell phenotype and ultimately molecular mechanisms that regulate pathogenesis. Moreover, improved methods of genome editing in iPSCs render it possible to generate isogenic control lines with correction of the disease-associated SNP, so that the specific effect of SNPs can be compared in iPSC lines of the same genetic background, making it easier to dissect the specific effects of the SNP on abnormal phenotype from the background genetic variation. Precise genome editing has been achieved in iPSCs using TALENs and zinc-finger nucleases (Merkert et al., 2017; Soldner et al., 2011; Ye et al., 2014), but it is likely that as CRISPR/Cas9 systems improve precise editing using this tool will become more common, and studies have already shown successful use of CRISPR/Cas9 for generating isogenic control iPSC lines (Zhang et al., 2017). Furthermore, the advent of CRISPR/Cas9-based genome-wide screening gives the opportunity to inactive large numbers of genes in human cells, helping to decipher which of those genes are important for processes

Future trends and directions

such as controlling Salmonella entry/replication (Yeung et al., 2019). Currently, these genome-wide screens have mainly been performed in cancer cell lines and have not translated well to iPSCs, but novel methods to develop improved, inducible Cas9 systems, which function more efficiently in iPSCs, may allow for these types of screens to be conducted in the future in iPSCs and their derivatives (Ihry et al., 2019). One limitation of the iPSC system is that the cells harvested after current differentiation protocols fail to recapitulate the full phenotype of mature, adult cells, leading to some urging caution in interpretation of iPSC-based study data (Rowe and Daley, 2019). Despite several studies suggesting that iPSC-derived cells retain a fetal-like phenotype (Baxter et al., 2015), a recent transcriptomic and DNA methylation study suggested that iHOs derived from iPSCs were most similar to pediatric sigmoid colon and demonstrated a morphology similar to mucosal-derived primary organoids, in addition grouping separately from fetal intestinal tissue. However, expression of certain innate defense genes was significantly lower in iHOs in comparison to primary tissue samples and primary intestinal organoids, suggesting that, indeed, iHOs may not reach the same level of differentiation as primary tissue. During the iHO differentiation protocol, iHOs are not exposed to microbial or immune cell signals, and therefore it is not surprising that iHOs may not reach the fully mature, regionalized phenotype of adult intestinal epithelium. However, this leaves open the opportunity to differentiate these cells further into different regions of the intestinal tract (Kraiczy et al., 2019b). As can also be applied to other iPSC-derived cell types discussed in this review, application of the expansion in knowledge regarding the processes driving cellular differentiation can be applied to refine iPSC differentiation protocols, which may lead to the generation of cells more similar to adult primary cells and provide the opportunity to generate more specialized cell types. For example, iPSDMs are thought to potentially resemble embryonic macrophages, and as embryonic macrophages are seeded early in development (Ginhoux et al., 2010; Schulz et al., 2012), it is possible that iPSDMs could provide a future source of tissue-resident human macrophages, which are currently very difficult to isolate from donors in significant numbers (Hale et al., 2015). Like other cellular models used in an in vitro setting, iPSCs provide a reductionist system; however, recent advances in various aspects of iPSC culture may allow for increased complexity of disease modeling. As discussed earlier, iHOs are already providing useful models for enteric pathogens, recapitulating more of the features of the intestinal epithelium than was previously possible in an in vitro setting, and displaying a variety of different cell types (Fig. 11.1B). The intestinal microbiota is key for driving the development and function of the IECs (Lee et al., 2018; Troll et al., 2018), so addition of microbiota to the iHO system may increase iHO differentiation. Coculture of iHOs with microbiota derived from intestine (Fig. 11.3A) would allow for the incorporation of colonization resistance into the iHO model, which is a key component to the ability of different Salmonella strains to establish infections in the intestine. One such study using a highthroughput microinjection system to introduce complex microbial communities to



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FIGURE 11.3 Technological advances to increase complexity of iPSC-derived cellular systems. Cells derived from iPSCs provide a reductionist system for studying hostepathogen interactions. However, there are multiple ways that the complexity of these systems may be enhanced, to increase relevance to in vivo infection. Some of these include: introduction of microbiota to iHO structures (A); coculture of iHOs with immune cells derived from iPSCs generated from the same individual (B); the use of transwells to culture monolayers of epithelium derived from iPSCs, making access to the apical surface less challenging (C); the generation of immune cells from iPSCs, and introduction to humanized mouse models, containing cells with disease-causing mutations (D).

the lumen of primary colonoids demonstrated that diverse bacteria could survive and grow within the colonoid lumen (Williamson et al., 2018). Cross talk between IECs and the intestinal immune cell population is fundamental for maintaining gastrointestinal homeostasis, and disruption of this cross talk has been linked to various gut diseases (Wittkopf et al., 2014). Therefore, coculture of intestinal immune cells and IECs is an attractive prospect (Fig. 11.3B), and iPSCs provide the opportunity to easily derive immune cells and IECs from the same individual. Successful coculture methods have been established for iPSC-derived microglia and cortical neurons (Haenseler et al., 2017), indicating that this may be possible for other iPSCderived cell types. The growth of the bioengineering field is also aiding the development of technologies for increasing the complexity of iPSC-derived cellular models. For example, the use of hydrogels to investigate cell behavior in 3D; microfluidics to


control exposure to soluble factors; and patterned surfaces to generate spatially controlled cocultures for investigation of cell-to-cell interactions are all developments that may help to recreate a cell-microenvironment more reminiscent of the in vivo situation and could be used for more faithful disease modeling (Kwon et al., 2018; Wheeldon et al., 2010). Methods have been developed for monolayer culture of primary intestinal epithelium on transwells (Fig. 11.3C), which can help increase accessibility to the apical side of IECs, make high-throughput screening simpler, and standardize comparisons between different cell lines (Kozuka et al., 2017). Furthermore, 3D coculture models using a transwell approach have been used to mimic S. Typhimurium interaction with the intestinal barrier during the acute phase of infection, combining a decellularized porcine gut matrix with human IECs, microvascular endothelial cells, and peripheral blood leukocytes (Schulte et al., 2020). There is also the possibility for iPSCs to be used in an in vivo situation. Humanized mice, which are immunodeficient mice repopulated with a functional human immune system by cotransplantation of human fetal thymus/liver tissues and CD34þ hematopoietic stem/progenitor cells, provide a method for probing the human immune system in vivo (Lan et al., 2006). It has also been demonstrated that iPSCs generated from patients with b-thalassemia, and subsequently engineered using CRISPR/Cas9 to correct b-thalassemia mutations, can be differentiated to hematopoietic stem cells (HSCs) and injected into sublethally irradiated NOD-scidIL2Rg/ mice, with these implanted HSCs surviving and differentiating, without causing tumors (Ou et al., 2016). Humanized mice have been previously shown to support S. Typhi replication and persistent infection (Song et al., 2010), and therefore the combination of this model and iPSCs (Fig. 11.3D), along with genetic editing via tools such as CRISPR/Cas9, may provide a really powerful tool in the future for dissecting the role of host genotype on Salmonella pathogenesis in vivo, helping to probe new therapeutics and vaccine candidates. Generation of iPSCs expressing stable fluorescent reporters may also provide a further useful tool for coculture studies and in vivo tracking, further enhancing the utility of these new technologies (Lopez-Yrigoyen et al., 2018).

Conclusions In conclusion, iPSCs have already started to provide a powerful, human-specific, in vitro tool for modeling hosteSalmonella interactions in multiple different cell types. As we still do not fully understand the mechanisms by which pathogens such as Salmonella subvert the host immune system, and with the rise in novel strains and antibiotic resistance in Salmonella populations, it is essential that we develop new models for studying Salmonella pathogenesis within a laboratory setting. iPSC-based systems offer the flexibility and scalability to be used as platforms for new antibacterial therapies, but also the potential for studying patientspecific bacterial disease outcome in these systems could lead to extensive advances in our understanding of Salmonella pathogenesis in human hosts.



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Acknowledgments I would like to thank Mr. David Goulding for performing the transmission electron microscopy (TEM) for the iHO phenotyping presented in Fig. 11.1. Dr. Jessica L Forbester is funded on a Wellcome Trust senior fellowship grant awarded to Dr. Ian Humphreys (207503/Z/17/Z). The figures in this chapter were exported with a paid subscription and created in Biorender.com.

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Index Note: ‘Page numbers followed by “f” indicate figures and “t” indicate tables.’

A Acquired immunodeficiency syndrome (AIDS), 17 Acyclovir (ACV), 80e81 Adenoassociated virus integration site 1 (AAVS1), 287e288 American Type Culture collection (ATCC), 4 Antigen presenting cell (APC), 241e242, 285e286 Antiherpetic drug screening, 80e84 Antiinflammatory cytokine, 39 Antimicrobial peptides (AMPs), 284 Aspergillus fumigatus, 265

B Bacterial meningitis blood-brain barrier, 256e257 brain endothelial cells (BECs), 256, 258 group B Streptococcus, 259e261 iPSC based models, 264f, 266e267 iPSC-BECs future outlook, 265e266 group B Streptococcus, 261e263 Neisseria meningitidis, 263e264 iPSC models, 258e259 meningeal blood-cerebral spinal fluid barrier (mBCSFB), 256 Neisseria meningitidis, 260e261 neurovascular unit (NVU), 256 trans-endothelial electrical resistance (TEER), 256e257 zona-occludins (ZO), 256e257 Blood-brain barrier, 256e257 Bradyzoites, 227 Brain-derived neurotrophic factor (BDNF), 33e34 Brain endothelial cell models, 258

C Caliciviridae, 9e10 Canine distemper virus (CDV), 132 Cardiac mesenchymal stem cells (CMSCs), 245e246 Cardiomyocyte differentiation, 103e104 Cardiomyocytes, 266e267 Cardiomyopathy, 240, 241f Cardiovascular diseases (CVDs), 240

Cell biology, 3e4 Central nervous system (CNS), 32e33, 71 Chagas disease cardiomyopathy, 240, 241f cardiovascular diseases (CVDs), 240 clinical disease, 243 definition, 240e241 heart failure (HF), 240 immune response, 243e244 iPSC modeling, 246e248, 247f models to study, 246 pathogenesis, 241e242, 242f therapy, 244e245 new approaches to, 245e246 Chagasic cardiomyopathy (CC), 243 Chloroquine (CQ), 81e82 Colony-stimulating factor 1 receptor (CSF1R), 34e35 Complex neural organoids, 222e223 Congenital Zika syndrome (CZS), 11e12 Coronaviridae, 10e11 Coronavirus antiinflammatory cytokine, 39 brain-derived neurotrophic factor (BDNF), 33e34 central nervous system (CNS), 32 colony-stimulating factor 1 receptor (CSF1R), 34e35 cytotoxic T cells (CTLs), 34e35 demyelination, 34e36 disease-modifying therapies (DMTs), 32 embryoidbody (EB), 37 encephalomyelitis (EAE), 33 human neural precursor cell transplantation, 37e39 Huntington’s disease (HD), 33e34 neural precursor-like cells (NPLCs), 39 neural progenitor cells (NPCs), 33e34 neural stem cells (NSCs), 33e34 neuroinflammation, 34e36 neurotropic coronavirus, 34e36 oligodendrocyte precursor cells (OPCs), 32e33 stem/progenitor cell remyelination, 39e41 Coxsackievirus B3, 16 Coxsackievirus infection biology of, 96e98 cardiomyocyte differentiation, 103e104




Coxsackievirus infection (Continued) disease in humans, 98e99, 98f enhanced green fluorescent protein (EGFP), 104 experimental models, 99e101 future perspectives, 106 hand-foot-and-mouth disease (HFMD), 98e99 induced pluripotent stem cells (iPSCs), 101 interferon (IFN), 102 iPSC modeling, 101e104 toll like-receptor 3 (TLR3), 103e104 two-dimensional (2D) monolayer differentiation protocol, 102e103 untranslated regions (UTRs), 96 vesicular stomatitis virus (VSV), 103e104 Cysts, 227 Cytotoxic T cells (CTLs), 34e35

D Demyelination, 34e36 Dendritic cells (DCs), 280 Dilated cardiomyopathy (DC), 240 Disease-modifying therapies (DMTs), 32 Dorsolateral prefrontal cortex (DLPFC), 74e75

E Electron microscopy, 70e71 Embryoid bodies (EBs), 37, 59e60, 218e219 Embryonic stem cell (hESCs), 5e6, 73, 279e280 Encephalomyelitis (EAE), 33 Endocytosis, 256e257 Enhanced green fluorescent protein (EGFP), 75e77, 104 Enterovirus D68, 16 Expression quantitative trait loci (eQTLs), 290 Extracellular matrix (ECM), 243 Extracellular vesicles (EVs), 128

F Flaviviridae, 11e13 Flow cytometry (FC), 84 Fluorescent in situ hybridization (FISH), 82e83

G Glial fibrillary acidic protein (GFAP), 74e75, 80 Glial subtypes, 219e220 Glycoprotein C (gC), 75e77 Group B Streptococcus, 259e263

H Hand-foot-and-mouth disease (HFMD), 98e99 Hayflick limit, 4 Heart failure (HF), 240

Hepadnaviridae, 13 Hepatitis A virus (HAV), 154e158 knowledge, 156e157 open questions, 158 in vitro models, 157e158 in vivo models, 157e158 Hepatitis B virus (HBV), 13, 158e161 knowledge, 158e159 open questions, 161 in vitro models, 159e160 in vivo models, 159e160 Hepatitis C virus (HCV), 12e13, 161e164 knowledge, 162 open questions, 164 in vitro models, 163e164 in vivo models, 163e164 Hepatitis delta virus (HDV) knowledge, 165e166 open questions, 166e167 in vitro models, 166 in vivo models, 166 Hepatitis E virus (HEV), 167e170 knowledge, 168e169 open questions, 170 in vitro models, 169 in vivo models, 169 Hepatitis viruses, 154e170, 155t Hepatocyte-like cells (HLCs), 154 Hepatotropic pathogen infections future directions, 181e182 hepatitis A virus (HAV), 154e158 knowledge, 156e157 open questions, 158 in vitro models, 157e158 in vivo models, 157e158 hepatitis B virus (HBV), 158e161 knowledge, 158e159 open questions, 161 in vitro models, 159e160 in vivo models, 159e160 hepatitis C virus (HCV), 161e164 knowledge, 162 open questions, 164 in vitro models, 163e164 in vivo models, 163e164 hepatitis delta virus (HDV) knowledge, 165e166 open questions, 166e167 in vitro models, 166 in vivo models, 166 hepatitis E virus (HEV), 167e170 knowledge, 168e169


open questions, 170 in vitro models, 169 in vivo models, 169 hepatitis viruses, 154e170, 155t hepatocyte-like cells (HLCs), 154 LDL receptor (LDLR), 180e181 limitations, 181e182 liver, 150e154, 151f pathogen-associated molecular patterns (PAMPs), 152 personalized modeling, 180e181 plasmodium liver stage, 170e173 current state of, 171 open questions, 173 in vitro model systems, 172e173 in vivo model systems, 172e173 Hepeviridae, 13 Herpes simplex encephalitis (HSE), 74 Herpes simplex virus, type 1 (HSV-1) infection central nervous system (CNS), 71 electron microscopy, 70e71 embryonic stem cell (hESCs), 73 herpes simplex encephalitis (HSE), 74 human induced pluripotent stem cells (hiPSCs), 74e79 Acyclovir (ACV), 80e81 antiherpetic drug screening, 80e84 chloroquine (CQ), 81e82 dorsolateral prefrontal cortex (DLPFC), 74e75 enhanced green fluorescentprotein (EGFP), 75e77 Flow cytometry (FC), 84 Fluorescent in situ hybridization (FISH), 82e83 glial fibrillary acidic protein (GFAP), 74e75, 80 glycoprotein C (gC), 75e77 herpesvirus entry mediator (HVEM), 71e72 human CNS-like neurons, 74e79 monomeric red fluorescent protein (mRFP), 75e77 neural progenitor cells (NPCs), 80 subventricular zone (SVZ), 80 tyrosine hydroxylase (TH), 77e79 Valacyclovir (VAL), 80e81 latency-associated transcripts (LATs), 71e72 varicella zoster virus (VZV), 70e71 Herpesviridae, 13e14 Herpesvirus entry mediator (HVEM), 71e72 HiPSC-derived 2D neuronal culture, 228 Host cell signaling, 263e264 Human alphaherpesvirus, 14

Human betaherpesvirus, 14 Human CNS-like neurons, 74e79 Human genetic variants, 289e290 Human herpesvirus 6 (HHV-6), 133e134 Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) modeling, 247e248 Human induced pluripotent stem cells (hiPSCs) advantages, 220 bradyzoites, 227 brain, chronic infection in, 226 brain-region-specific organoids, 222 complex neural organoids, 222e223 cysts, 227 directed differentiation, 217e218 direct reprogramming, 217e218 2D monolayers, 220 embryoid bodies (EBs), 218e219 glial subtypes, 219e220 hiPSC-derived 2D neuronal culture, 228 host/parasite interactions, 227 limitations, 220 methods, 217e223 neural organoids, 222 neuropathogenesis, 227e228 organoid culture, future developments in, 222e223 organoids, 221e222 principles, 217e223 protocols, 219 three-dimensional (3D) neural organoid models, 220e223, 221t Toxoplasma gondii, 217 bradyzoite, 224e226 brain, chronic infection in, 223e230 cyst biology, 224e226 2D neuron monolayers, 227e228 two dimensional (2D) monolayer cultures, 216e217 Human induced pluripotent stem cells (hiPSCs), 49e50, 74e79 Human neural precursor cell transplantation, 37e39 Huntington’s disease (HD), 33e34

I Induced cell death, 49e55 Induced pluripotent stem cells (iPSCs), 101, 258e259 American Type Culture collection (ATCC), 4 applications, 6e9, 7te9t Caliciviridae, 9e10




Induced pluripotent stem cells (iPSCs) (Continued) cell biology, 3e4 Coronaviridae, 10e11 embryonic stem cells, 5e6 Flaviviridae, 11e13 future directions, 18 Hayflick limit, 4 Hepadnaviridae, 13 Hepeviridae, 13 Herpesviridae, 13e14 normal cells, 4 Orthomyxoviridae, 15 Paramyxoviridae, 15 Picornaviridae, 16 Polyomaviridae, 16 Retroviridae, 17 stem cells, 5e6 Togaviridae, 17 virology, 2, 3f viruses, 2e3 as obligate parasites, 2e3 Inflammatory bowel disease (IBD), 287e288 Influenza A virus (IAV) differentiation potentials, 55e60 embryoid bodies (EBs), 59e60 human induced pluripotent stem cells (hiPSCs), 49e50 induced cell death, 49e55 interferon-induced transmembrane protein (IFITM3), 60e62 interferon regulatoryfactor 5 (IRF5), 60e62 interferon regulatory factor 7 (IRF7), 60e62 neural progenitor cells (NPCs), 55 neuraminidase (NA), 48 pluripotency, 57e59 pluripotent stem cells (PSCs), 49 SOMAScan proteomic screening, 59e60 trophoblasts stem cells (TSCs), 52e55 Innate immune system, 265 Integrin-focal adhesion kinase (FAK) signaling, 263e264 Interferon (IFN), 102 Interferon-induced transmembrane protein (IFITM3), 60e62 Interferon regulatoryfactor 5 (IRF5), 60e62 Interferon regulatory factor 7 (IRF7), 60e62 Intestinal human organoids (iHOs), 282e283, 283f Intestinal stem cells (ISCs), 282e283 Invasive NTS (iNTS), 278e279 In vivo models, 279 iPSC-derived cellular systems, 282e285 iPSC modeling, 246e248, 247f

J John Cunningham virus (JCV), 129e130 John Howard Mueller (JHM), 136

L Latency-associated transcripts (LATs), 71e72 LDL receptor (LDLR), 180e181 Left ventricle (LV), 240

M Macrophages, 284 Mendelian Susceptibility to Mycobacterial Diseases (MSMD), 287 Meningeal blood-cerebral spinal fluid barrier (mBCSFB), 256 Merkel cell polyomavirus, 16 Mesenchymal stem cells (MSCs), 245e246 Monocyte-derived DCs (MoDCs), 285e286 Monocyte-derived macrophages (MDMs), 285 Monomeric red fluorescent protein (mRFP), 75e77 Mouse hepatitis virus (MHV), 130e131 Multiple-sclerosis-associated retrovirus (MSRV), 132 Multiplicity of infection (MOI), 282e283 Murine leukemia virus (MLV), 131e132 Myelin, 122 Myelin-associated glycoprotein (MAG), 122 Myelin basicprotein (MBP), 122

N Natural killer (NK) cells, 244 Neisseria meningitidis, 260e261, 263e264 Neural cell adhesion molecule (NCAM), 134e135 Neural organoids, 222 Neural precursor-like cells (NPLCs), 39 Neural progenitor cells (NPCs), 33e34, 55, 80 Neural stem cells (NSCs), 33e34 Neuraminidase (NA), 48 Neuroinflammation, 34e36 Neuropathogenesis, 227e228 Neurotropic coronavirus, 34e36 Neurovascular unit (NVU), 256 Newcastle disease virus, 15 Nontyphoidal serovars (NTS), 278e279

O Oligodendrocyte precursor cells (OPCs), 32e33, 124 canine distemper virus (CDV), 132


challenges with, 134e135 disruption, 126e134 future perspectives, 137e138 HHV-6, 133e134 induced pluripotent stem cells (iPSCs), 134e136 advantages, 135e136 brain organoids, 136 viral CNS infection, 136 John Cunningham virus (JCV), 129e130 maturation, 124e125, 125f migration, 124e125, 125f Mouse hepatitis virus (MHV), 130e131 murine leukemia virus (MLV), 131e132 myelin, 122 myelin-associated glycoprotein (MAG), 122 myelin basicprotein (MBP), 122 proliferation, 124e125, 125f Semliki Forest virus (SFV), 134 Theiler’s murine encephalomyelitis virus (TMEV), 126e129 virus-induced demyelination, 123 zika virus, 132e133 Organoids, 221e223, 266e267 Orthomyxoviridae, 15

P Paramyxoviridae, 15 Parkinson’s disease (PD), 289 Pathogen-associated molecular patterns (PAMPs), 152 Picornaviridae, 16 Plasmodium liver stage, 170e173 current state of, 171 open questions, 173 in vitro model systems, 172e173 in vivo model systems, 172e173 Pluripotency, 57e59 Pluripotent stem cells (PSCs), 49, 129 Polyomaviridae, 16 Progressive multifocal leukoencephalopathy (PML), 129e130 Proteolipid protein (PLP), 122

R Remyelination, 128 Resistance to complement killing (Rck), 286 Retroviridae, 17 Rubella virus, 17

S Salmonella containing vacuole (SCV), 282 Salmonellae

adenoassociated virus integration site 1 (AAVS1), 287e288 antigen presenting cell (APC), 285e286 antimicrobial peptides (AMPs), 284 dendritic cells (DCs), 280 embryonic stem (ES) cells, 279e280 expression quantitative trait loci (eQTLs), 290 future trends, 291e295 host genotype, 286e289 human genetic variants, 289e290 inflammatory bowel disease (IBD), 287e288 intestinal human organoids (iHOs), 282e283, 283f intestinal stem cells (ISCs), 282e283 invasive NTS (iNTS), 278e279 iPSC-derived cellular systems, 282e285 macrophages, 284 Mendelian Susceptibility to Mycobacterial Diseases (MSMD), 287 monocyte-derived DCs (MoDCs), 285e286 monocyte-derived macrophages (MDMs), 285 multiplicity of infection (MOI), 282e283 nontyphoidal serovars (NTS), 278e279 Parkinson’s disease (PD), 289 resistance to complement killing (Rck), 286 Salmonella containing vacuole (SCV), 282 Salmonella pathogenicity island-1 (SPI-1), 286 transcription factors (TFs), 279e280 type III secretion system (T3SS), 282 in vivo models, 279 Salmonella pathogenicity island-1 (SPI-1), 286 Semliki Forest virus (SFV), 134 Shadow plaques, 32e33 SOMAScan proteomic screening, 59e60 Sox10, 124 Stem cells, 5e6 Stem/progenitor cell remyelination, 39e41 Streptococcus agalactiae, 259e260 Subventricular zone (SVZ), 80

T Theiler’s murine encephalomyelitis virus (TMEV), 126e129 Three-dimensional (3D) neural organoid models, 220e223, 221t Togaviridae, 17 Toll-like receptors (TLRs), 103e104, 241e242 Toxoplasma gondii, 217 bradyzoite, 224e226 brain, chronic infection in, 223e230 cyst biology, 224e226 2D neuron monolayers, 227e228




Transcription factors (TFs), 279e280 Trans-endothelial electrical resistance (TEER), 256e257 Trophoblasts stem cells (TSCs), 52e55 Two dimensional (2D) monolayer, 102e103, 216e217 Type III secretion system (T3SS), 282 Tyrosine hydroxylase (TH), 77e79

U Untranslated regions (UTRs), 96

V Valacyclovir (VAL), 80e81 Varicella zoster virus (VZV), 70e71 Vesicular stomatitis virus (VSV), 103e104 Virus-induced demyelination, 123

Z Zika virus (ZIKV), 6e9, 132e133 Zona-occludins (ZO), 256e257