Stem Cell Innovation in Health and Disease: The Intestine [2] 0128212691, 9780128212691

Citation preview

The Intestine

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Stem Cell Innovation in Health and Disease

The Intestine Volume 2

Edited by

Ahmed H.K. El-Hashash The University of Edinburgh (UK)-Zhejiang International Campus, (UoE-ZJU Institute), Haining, P.R. China Centre of Stem Cell and Regenerative Medicine, Zhejiang University Schools of Medicine and Basic Medicine, Hangzhou, P.R. China

Eiman Abdel Meguid Centre for Biomedical Sciences Education, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, Belfast, United Kingdom

Series Editor

Ahmed H.K. El-Hashash

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. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-821269-1 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: Samantha Allard Production Project Manager: Sreejith Viswanathan Cover Designer: Miles Hitchen Typeset by MPS Limited, Chennai, India

Dedication This book is dedicated to: Dr. Magdy Elhefnawy. My gratitude is the fairest blossom which springs from my soul to you. Thanks for lighting up our lives. My great parents, my brilliant wife Heba and her parents, and my children Hoor, Noor, and Lien. In memory of Dr. Gamal Madkour. Your inspiration, support and word to me shan’t be forgotten. Ahmed H.K. El-Hashash, PhD This book is dedicated in honor of my mother, Heba Nasr, who is a source of unyielding support and devotion. In memory of my father, Mr. M. Abdel Meguid who served as a constant source of inspiration to me and to my sons, Hossam Amr, Omar Amr, and daughter-in-law, Salam Baleed, who made my life worthwhile. Thank you for your love and support. Eiman Abdel Meguid, PhD

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“If learning the truth is the scientist’s goal, then he must make himself the enemy of all that he reads.” “The seeker after the truth is not one who studies the writings of the ancients and, following his natural disposition, puts his trust in them, but rather the one who suspects his faith in them and questions what he gathers from them, the one who submits to argument and demonstration, and not to the sayings of a human being whose nature is fraught with all kinds of imperfection and deficiency. Thus the duty of the man who investigates the writings of scientists, if learning the truth is his goal, is to make himself an enemy of all that he reads, and, applying his mind to the core and margins of its content, attack it from every side. He should also suspect himself as he performs his critical examination of it, so that he may avoid falling into either prejudice or leniency” (Alhazen [Ibn al-Haytham], 965 1040 AD)

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Contents List of contributors ..................................................................................................xv About the editors .................................................................................................. xvii Foreword .................................................................................................................xix Foreword .................................................................................................................xxi Preface ................................................................................................................. xxiii Acknowledgments ................................................................................................ xvii

CHAPTER 1 Cutting-edge tools and approaches for stem cell research and application in intestinal diseases......... 1 Ahmed El-Hashash 1.1 Introduction ....................................................................................1 References...................................................................................... 3

CHAPTER 2 Organotypic intestinal cell culture as a new modality for intestinal function and cellular processes ...................................................................... 5 Taylor Broda and Magdalena Kasendra 2.1 Defining organotypic intestinal cell culture models .....................5 2.2 Intestinal structure and functions...................................................7 2.3 Modeling of intestinal functions using organotypic cell culture models ..............................................................................12 2.4 Toward emulation of organ-level functions ................................16 2.4.1 Co-development ................................................................ 18 2.4.2 Co-culture.......................................................................... 19 2.4.3 Bioengineering .................................................................. 20 Acknowledgment ......................................................................... 22 References.................................................................................... 22

CHAPTER 3 Role of human gastrointestinal organoids in discovery and translational medicine........................ 29 Alexandra Calor, Mirjam van Weissenbruch and Stefania Senger 3.1 Introduction ..................................................................................29 3.1.1 Nomenclature and distinction: organoids enteroids colonoids tumoroids ................... 29 3.1.2 Advantage of organoids in basic and applied research of human GI diseases ......................................... 30

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3.2 Current application and perspective use of organoids in precision and personalized medicine ...........................................32 3.2.1 Genetic disease.................................................................. 34 3.2.2 Gastrointestinal immune-related disorders ....................... 35 3.2.3 Enteral infections .............................................................. 39 3.2.4 Gastrointestinal cancer...................................................... 43 3.2.5 Transplant application....................................................... 47 3.3 Ethical perspective on organoids biobanks .................................48 References.................................................................................... 49

CHAPTER 4 Engineered stem cells combine stem cell and gene therapy approaches to move intestine therapy from bench to bed ......................................... 59 4.1 4.2 4.3 4.4

4.5

4.6

Mahmoud Shaaban Mohamed, Mahmoud I. Elbadry and Chao-Ling Yao Introduction ..................................................................................59 Genome editing ............................................................................60 Genome editing of stem cells ......................................................61 Therapeutic applications of gene-edited stem cells.....................61 4.4.1 Adult stem cells ................................................................ 61 4.4.2 Mesenchymal stem cells ................................................... 62 4.4.3 Embryonic stem cells........................................................ 63 4.4.4 Induced pluripotent stem cells.......................................... 63 Gene-edited stem cell therapy for intestinal diseases .................64 4.5.1 Intestinal diseases.............................................................. 64 4.5.2 Intestinal stem cell therapy for intestinal diseases........... 65 4.5.3 Gene editing in intestinal enteroids .................................. 66 4.5.4 Engineered organoids for colorectal cancer ..................... 68 4.5.5 Gastric cancer.................................................................... 69 Conclusion and future prospects..................................................70 References.................................................................................... 70

CHAPTER 5 Role of CRISPR/Cas9 and other gene editing/ engineering technology in intestine diseases........... 75 5.1 5.2 5.3 5.4 5.5

Yiyi Yang and Xiaowen Cheng Brief introduction of CRISPR-Cas9 ............................................75 CRISPR/Cas 9 application in colorectal cancers ........................77 CRISPR/Cas9 applications in inflammatory bowel disease........79 CRISPR/Cas9 applications in gut microbiota .............................81 Other gene editing tools ...............................................................81

Contents

5.6 Limitations and perspectives........................................................82 References.................................................................................... 83

CHAPTER 6 Application of new approaches for intestinal repair and regeneration via stem cell based tissue engineering ................................................................. 87 Ahmed El-Hashash 6.1 Structure and cellular components of the small intestine ...........87 6.2 Stem cells used in small intestine regeneration ..........................88 6.2.1 Pluripotent stem cell derived cells ................................... 88 6.2.2 Adult intestinal stem cells ................................................ 89 6.2.3 Mesenchymal stem cells ................................................... 90 6.3 Developing biological tissue-engineered grafts...........................90 6.3.1 Decellularization of tissues or organs .............................. 91 6.4 Tissue-engineered small intestine ................................................92 6.5 Role of stem cell based transplantation in intestinal regeneration ..................................................................................93 6.6 Conclusion and future directions .................................................93 References.................................................................................... 94

CHAPTER 7 Induced pluripotent stem cells in intestinal diseases .................................................................... 101 7.1 7.2 7.3 7.4

7.5 7.6

Adegbenro Omotuyi John Fakoya, Adekunle Ebenezer Omole, Nihal Satyadev and Cynthia Oghenekome Okaruefe Introduction ................................................................................101 iPSC-based disease modeling ....................................................102 Intestinal organoids ....................................................................103 iPSCs and organoids in intestinal diseases................................104 7.4.1 Colorectal cancer ............................................................ 104 7.4.2 Hirschsprung disease....................................................... 107 7.4.3 Inflammatory bowel disease ........................................... 108 7.4.4 Parasite ............................................................................ 110 7.4.5 Viruses............................................................................. 110 Clinical trials ..............................................................................111 Limitations..................................................................................112 References.................................................................................. 113

CHAPTER 8 Potential of embryonic stem cells for treating intestinal diseases.................................................... 123 Ahmed El-Hashash 8.1 Embryonic stem cells .................................................................123

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8.2 ESC-based therapy of the gastrointestinal diseases ..................124 8.2.1 ESC-based therapy for inflammatory bowel disease ..... 124 8.3 Conclusions ................................................................................127 References.................................................................................. 128

CHAPTER 9 Stem-cell therapy with bone marrow (hematopoietic) stem cells for intestinal diseases .................................................................... 131 9.1 9.2 9.3

9.4 9.5 9.6

9.7

Mahmoud Shaaban Mohamed, Mahmoud I. Elbadry and Chao-Ling Yao Introduction ................................................................................131 Bone-marrow HSC niche ...........................................................133 Clinical applications of HSCs....................................................134 9.3.1 Leukemia......................................................................... 134 9.3.2 Cancer.............................................................................. 136 Autologous hematopoietic stem cell transplantation for autoimmune diseases..................................................................136 HSCs for the treatment of genetic blood cell diseases .............138 HSCs for the treatment of intestinal diseases............................139 9.6.1 Inflammatory bowel diseases.......................................... 139 9.6.2 HSCs for mucosal healing .............................................. 141 Conclusion, challenges, and future directions ...........................142 References.................................................................................. 143

CHAPTER 10 Role of mesenchymal and other stem cell therapy in intestinal diseases .................................. 147 Jingwen Liu and Deming Jiang 10.1 Introduction ................................................................................147 10.2 Mesenchymal stem cells ............................................................148 10.2.1 Mechanism of MSC treatment of intestinal diseases......148 10.2.2 Application of MSCs in the treatment of IBD............. 150 10.2.3 Application of MSCs in the treatment of radiation enteritis .......................................................................... 153 10.2.4 Application of MSCs in the treatment of liver transplantation-related bowel disease ........................... 154 10.2.5 Application of MSCs in the treatment of intestinal tumor ............................................................................. 155 10.3 Hematopoietic stem cells ...........................................................155 10.4 Intestinal stem cells....................................................................156 10.5 Conclusions and prospects .........................................................158 References.................................................................................. 159

Contents

CHAPTER 11 General discussion, conclusion remarks, and future directions........................................................ 165 Ahmed El-Hashash 11.1 Advances of organotypic intestinal cell culture and gene editing/engineering in intestinal repair, regeneration, and diseases: current challenges and future prospectives ................166 11.1.1 Role of pluripotent stem cells in intestinal repair, regeneration, and diseases............................................. 168 References.................................................................................. 170 Index ......................................................................................................................173

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List of contributors Taylor Broda Center for Stem Cell and Organoid Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States; Division of Pediatric General and Thoracic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States Alexandra Calor Department of Pediatrics, Division of Neonatology, Amsterdam UMC, VU University Medical Center, Amsterdam, The Netherlands Xiaowen Cheng Department of Medical Biochemistry and Microbiology, SciLifeLab Uppsala, The Biomedical Center, University of Uppsala, Uppsala, Sweden Mahmoud I. Elbadry Internal Medicine Department, Division of Hematology, Faculty of Medicine, Sohag University, Sohag, Egypt Ahmed El-Hashash The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, P.R. China; Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, P.R. China Adegbenro Omotuyi John Fakoya University of Medicine and Health Sciences, Basseterre, St. Kitts and Nevis Deming Jiang Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou, P.R. China Magdalena Kasendra Center for Stem Cell and Organoid Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States; Division of Pediatric General and Thoracic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States Jingwen Liu Laboratory of Gastroenterology Department, Zhejiang University School of Medicine, Hangzhou, P.R. China Mahmoud Shaaban Mohamed Zoology Department, Faculty of Science, Assiut University, Assiut, Egypt Cynthia Oghenekome Okaruefe All Saints University School of Medicine, Roseau, Dominica Adekunle Ebenezer Omole American University of Antigua College of Medicine, St. John’s, Antigua Nihal Satyadev University of Medicine and Health Sciences, Basseterre, St. Kitts and Nevis

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Stefania Senger Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital, Boston, MA, United States; Harvard School of Medicine, Boston, MA, United States Mirjam van Weissenbruch Department of Pediatrics, Division of Neonatology, Amsterdam UMC, VU University Medical Center, Amsterdam, The Netherlands Yiyi Yang Department of Experimental Medical Science, Experimental Neuroinflammation Laboratory, Lund University, Lund, Sweden Chao-Ling Yao Department of Chemical Engineering, National Cheng Kung University, No. 1, University Road, Tainan, Taiwan

About the editors Professor Ahmed H.K. El-Hashash has completed his PhD from Manchester University, United Kingdom. He is a fellow of the California Institute of Regenerative Medicine (CIRM) and New York University Medical School (MSSM), United States. He worked as a senior biomedical research scientist at the Mount Sinai School of Medicine of New York University and Children’s Hospital Los Angeles. He was an assistant professor and the principal investigator of Stem Cell and Regenerative Medicine at the Keck School of Medicine and Ostrow School of Dentistry of The University of Southern California, United States. Professor Ahmed El-Hashash has joined the University of EdinburghZhejiang International Campus, as the tenure track associate professor of Biomedicine, Stem Cell and Regenerative Medicine. He is also an adjunct professor at the School of Basic Medical Science and School of Medicine, Zhejiang University. He acts as the discussion leader at the prestigious Gordon Research Seminar/Conference in United States, and a peer reviewer/international extramural review for The Medical Research Council (MRC) grant applications, London, United Kingdom. He has been invited to cochair, coorganize, and/or present his research at several international conferences in the United States, Spain, Greece, Egypt, Germany, and China. He is the Editor-in-Chief of two research journals, and the editor or author of several books on stem cell and regenerative medicine that were published by the prestigious publishers, Elsevier, Springer-Nature, and Imperial College London Press. Dr. Eiman Abdel Meguid, MBChB, PhD, PGCHET, SFHEA, is a senior lecturer at the Centre for Biomedical Sciences Education, School of Medicine, Dentistry and Biomedical Sciences, Queen’s University Belfast, United Kingdom. Dr. Abdel Meguid obtained her bachelor’s degree in Medicine and Surgery and her PhD in Anatomy and Embryology from Alexandria University. She completed her PhD thesis at Eberhard-Karls Universita¨t Tu¨bingen, Germany. Dr. Abdel Meguid is an innovative educator and researcher. She has published multiple scientific works in leading journals, and she is a reviewer and a member of the editorial board of a number of journals. She taught gross anatomy to medical, dental, human biology students, and students enrolled in MSc in Clinical Anatomy. She is a member of the Risk Management Group Committee of the Royal College of Physicans and Surgeons and a member of the Court of Examiners for the Royal College of Physicians and Surgeons, Glasgow. She is also a member of the Career Development Committee of the American Association of Clinical Anatomist and a local Ambassador for the British Association of Clinical Anatomists.

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Foreword It is with great pleasure that I pen this foreword to the Stem Cell Innovation in Health & Disease. The field of stem cell biology and application in human diseases is moving extremely rapidly as the concept and potential practical applications have moved from theoretical concepts into human clinical trials with often outstanding results and have thus entered the mainstream. Despite this worldwide intensity and diversity of endeavor, there remains a smaller number of book volumes that are focused almost entirely on the Stem Cell Innovation in Health & Disease, and this volume is an example of this kind of books that also brings the novel scientific findings of most of the leaders of this research field together. The concept of stem and progenitor cells has been known for a long time. However, it was the progress toward embryonic stem (ES) cells, which truly leads the field of stem cell research and related field such as regenerative medicine. ES of the mouse cells originally came from many research studies that aimed at the identification of the mechanisms that control and progress embryonic differentiation. Despite being magnificent, the cell differentiation in culture was overshadowed experimentally by their use as a vector to the germ line and hence as a vehicle for experimental mammalian genetics. These studies led to research on targeted mutation in up to one-third of gene loci and an ongoing international program to provide mutations in every locus of the mouse gene. A positive outcome of these studies has been to greatly illuminate our understanding of human genetics. In addition, promising research studies that focus on discovering the equivalent human ES cells will certainly provide a universal source of a diversity of tissue-specific precursors, as a resource for tissue repair and regenerative medicine. Progress toward understanding various aspects of mesenchymal stem cell biology and development, including self-renewal, cellular differentiation, and pluripotentiality, that is fundamental developmental biology at the cell and molecular level, now stands as a gateway to major clinical applications both current and in the future. This book provides a timely, up-to-date state-of-the-art reference of stem cell roles in intestine health and disease. Understanding the function of endogenous tissue-specific stem and progenitor cells in various organs will greatly enhance the use of stem cells in tissue repair and regeneration and as a therapy for a wide range of human diseases. The rapid advances of different stem cell types, including the induced pluripotent stem cell research and potential clinical applications, point to the great possibilities of patient-specific ad hominem treatment. As Sir Martin Evans, who is credited with discovering ES cells and received the Nobel Prize for Physiology or Medicine in 2007, said, this patient-specific ad hominem treatment will help to open the door to personalized medicine, in which patients are stratified into different groups, and therapeutics are tailored based on the individual patient’s response. However, thus far the high costs associated with this technology may not allow it to be

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commercially viable, as Sir Evans said. Quite properly, this book concentrates on multidisciplinary cutting-edge research techniques and approaches that are currently the hottest topics in stem cell research, from which the solid clinical applications will arise for intestinal diseases. Professor Alexzander A. A. Asea University of Texas MD Anderson Cancer Center, Houston, TX, United States Prof. Alexzander A. A. Asea is currently a professor and the Director of Precision Therapeutics Proteogenomics Diagnostics Center at the University of Toledo College of Medicine, United States. He was a professor and consultant immunologist at the University of Texas MD Anderson Cancer Center (Houston, United States). He obtained his PhD from Gothenburg University (Sweden) where his studies formed the basis for clinical trials of combined histamine and interleukin2, a drug now known as Ceplene, currently prescribed for patients with metastatic melanoma and high-risk acute myelogenous leukemia. Prof. Asea is a highly innovative and accomplished world-renowned research scientist and visionary executive academic leader with exceptional executive leadership experience spearheading strategic planning, research, training, education, and commercialization initiatives. He has received numerous honors and awards and has received grant funding from the federal government, industry, private foundations, and local community groups. He currently has five pending patents, over 255 scientific publications, books, reviews, news headliners, and editorials in a wide range of medical disciplines including stem cell biotherapeutics, cancer, diabetes, obesity, neurosciences, cardiovascular disease, exercise immunophysiology, aging, nanotechnology, thermal therapy, medicinal plants, and biomarker discovery.

Foreword It is a great honor and privilege to participate in introducing a unique book and reference on a topic of great importance: Stem Cell Innovation in Health & Disease, which I consider a rich addition to the global scientific biomedical library. The editors and chapter authors have addressed the subject by reviewing the latest and most updated research that has taken place in major advanced centers that are specialized in this field around the world. Nowadays, we find ourselves facing a great mission in using stem cells of various types in the treatment of many diseases that were not able to be cured in the past. Dr. Magdy Elhefnawy Chief physician at Menshawi Hospital, Tanta University Medical School, Tanta, Egypt President of Gharbia Medical Syndicate, Egypt

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Preface The research on stem cells can be traced back to over 20 years when two British pioneer scientists, Sir Martin Evans and Matthew Kaufman, together with the American scientist, Gail Marin, were the first to culture and derive embryonic stem cells (ESCs) from mouse embryos in 1981 in a laboratory. This was followed by the important discovery of human ESCs in 1998. Stem cell research is a fast-growing field that has remarkably expanded as new research and experience which broaden our knowledge about different aspects of stem cell biology and applications, making stem cell research as one of the most exciting aspects of biomedical research. Both embryonic and adult stem cells are currently a remarkably fast-growing field of research, with an astonishing annual growth rate of 77% since 2008. The volume of research output, and thus publication, has therefore increased significantly in all areas of stem cell research. Research is currently underway in different laboratories worldwide to generate other functioning whole organs such as the lung, intestine, kidney, and other human body organs. Scientists worldwide continue to apply new stem cell based discoveries, to the betterment of human diseases that have clearly brought forth much hope for better human life. There is an increased number of publications and discoveries on stem cells using cutting edge in vitro and in vivo research tools in the last two decades from a wide range of research universities and institutes in the United States, Europe, Japan, Australia, Canada, China, and many other countries, which have established centers for stem cell research and regenerative medicine. More recent data have been accumulated on intestinal stem cell biology and the potential applications of stem cells in intestinal diseases that are facilitated by the recent development of a broad range of cutting edge in vitro and in vivo research tools and approaches, including mouse and human organoid cultures, genetic editing in vitro and in vivo, human induced pluripotent cell (iPC) models of disease, haploid cells for genetic as well as compound screening paradigms, genetically engineered mice, and stem cell transplantation to cure diseases. For example, a recent progress has been achieved in developing intestinal organoids that are derived from patient tissues and expanded in culture. These organoids offer unique insight into individual patient disease and are a potential route to personalized treatments of intestinal diseases (Angus et al., 2020). In addition, intestinal organoids were generated from human induced pluripotent stem cells (iPSCs; Tsuruta et al., 2020). Moreover, there is a recent progress in both the development of biological tissue-engineered grafts and tissue-engineered small intestines. Furthermore, the potential applications of embryonic stem cells in advancing cellular therapy, modeling of human diseases, and drug discovery in different organs, including the intestine have been reported. These rapid advances in stem cell study and application in the intestine using fast-growing cutting-edge

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research approaches need to be collected and reviewed, which is the main goal of this book. This book contains several chapters describing cutting-edge research on the intestinal stem cell functions, modeling intestinal functions and diseases, and applications of stem cells in intestinal repair, regeneration, and diseases. These chapters include insights ranging from using mouse and human organoid cultures, stem cell-based tissue engineering, and mesenchymal stem cells/iPCs both to study stem cell functions and model intestinal diseases, through the cutting-edge research aiming to bring stem cells from bench to bedside, including the potential application of these stem cells in the treatment of intestinal diseases. This book aims to provide an important updated resource for undergraduate students, graduate students, researchers, and clinicians in recent applications of stem cells in intestinal diseases using recently developed cutting edge in vitro and in vivo research tools and approaches. The book has 11 chapters, covering applications of stem cells in the intestine. Chapter 1, Cutting-Edge Tools and Approaches for Stem Cell Research and Application in Intestinal Diseases, introduces cutting-edge tools and approaches used for stem cell research and application in intestinal diseases, while Chapter 2, Organotypic Intestinal Cell Culture as a New Modality for Intestinal Function and Cellular Processes, describes how organotypic intestinal cell culture is a new modality for intestinal function and cellular processes. Chapter 3, Role of Human Gastrointestinal Organoids in Discovery and Translational Medicine, describes the role of human gastrointestinal organoids in discovery and translational medicine. Chapter 4, Engineered Stem Cells Combine Stem Cell and Gene Therapy Approaches to Move Intestine Therapy From Bench to Bed, and Chapter 5, Role of CRISPR/Cas9 and Other Gene Editing/Engineering Technology in Intestine Diseases, describe how engineered stem cells combine stem cell and gene therapy approaches to move intestine therapy from bench to bed and the role of gene editing/engineering technology in intestine diseases. Chapter 6, Application of New Approaches for Intestinal Repair and Regeneration via Stem Cell-Based Tissue Engineering, describes the application of new approaches for intestinal repair and regeneration via stem cell-based tissue engineering. Chapters 7 10 discuss cutting-edge research on stem cell applications in intestinal repair, regeneration, and diseases, including the role of iPSCs in the intestine (Chapter 7: Induced Pluripotent Stem Cells in Intestinal Diseases), the potential applications of embryonic stem cells in treating intestinal diseases (Chapter 8: Potential of Embryonic Stem Cells for Treating Intestinal Diseases), and the roles of hematopoietic stem cells (Chapter 9: Stem Cell Therapy With Bone Marrow (Hematopoietic) Stem Cells for Intestinal Diseases) and mesenchymal stem cells (Chapter 10: Role of Mesenchymal and Other Stem Cell Therapy in Intestinal Diseases) in intestinal diseases. Chapter 11, General Discussion, Concluding Remarks, and Future Directions, describes conclusions drawn from the cutting-edge research that are used to understand stem cell functions in intestinal repair, regeneration, and diseases, and discusses future directions in this field.

Preface

Although we could not hope to be comprehensive in the coverage of recent stem cell innovation in the intestine health and disease, our main goal in compiling this book was to bring together a selection of the current progress in the applications of stem cells in intestinal repair, regeneration, and diseases that are facilitated by the recent development of a broad range of cutting edge in vitro and in vivo research tools and approaches. In preparing this book, we aimed at making it accessible not only to those working in the stem cell and regenerative medicine field but also to nonexperts with a broad interest in stem cells, intestinal diseases, and regeneration biology and medicine. Our hope is that this book will be of value to all concerned with stem cell applications in pulmonary diseases, repair, and regeneration. We are indebted to our authors, who graciously accepted their assignments and who have infused the text with their energetic contributions. We are incredibly thankful to Dr. Elisabeth Brown, Dr. Patricia Gonzalez, and other staff at Elsevier, who published this book. Professor Ahmed H.K. El-Hashash, PhD Professor of Biomedicine, Stem Cell & Regenerative Medicine, The University of Edinburgh (UK)-Zhejiang International Campus, (UoE-ZJU Institute), Haining, P.R. China

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Acknowledgments Esam I. Agamy, PhD Professor, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates Joseph Allen, PhD School of Social Sciences, Education and Social Work, Queen’s University Belfast, United Kingdom Wadah Alhassan, BSc California State Polytechnic University, Pomona, Pomona, CA, United States Alexzander A. A. Asea, PhD Visiting Professor and Consultant Immunologist, Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States Mohamed Berika, PhD Assistant Professor of Anatomy, Faculty of Medicine, Mansoura University, Mansoura, Egypt Applied Medical Science College, King Saud University, Riyadh, Saudi Arabia Karen Ek, BSc California State University San Bernardino, San Bernardino, CA, United States Magdy Elhefnawy, MD, PhD President, Gharbia Medical Syndicate, Tanta University Medical School, Tanta, Egypt Thoria Roshdy Haggag, MD Former Chairman, Department of Internal Medicine, Mansoura University Hospital, Mansoura, Egypt Ali Kotab Hasan, PhD Professor, Faculty of Education, Tanta University, Tanta, Egypt Haifen Huang, BSc California State Polytechnic University, Pomona, Pomona, CA, United States Junfeng Ji, PhD Professor of Stem Cells and Regenerative Medicine, Chairman, Dr. Li Dak Sum & Yip Yio Chin Centre for Stem Cell and Regenerative Medicine, School of Medicine, Zhejiang University, Hangzhou, P.R. China John Ku, BSc California State Polytechnic University, Pomona, Pomona, CA, United States

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Karol Lu, BSc University of Southern California, University Park, Los Angeles, CA, United States Gamal Madkour, PhD Professor, Department of Zoology, Tanta University School of Science, Tanta University, Tanta, Egypt Moustafa Mahmoud, MD, PhD Professor, Department of Surgery, Tanta University School of Medicine, Tanta University, Tanta, Egypt Mohammed A. Motabagani, MHPEdu, PhD Professor and Chairman of Anatomy, College of Medicine, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia Estomih P. Mtui, MD, PhD Professor of Anatomy in Radiology, Weill Cornell Medical College, New York, NY, United States Mohamed Labib Salem, PhD Professor and Director of Cancer, Research Centre, Tanta, Egypt Medical University of South Carolina, Charleston, SC, United States Osama Saad Salama, MD, PhD Professor and Former Chairman, Department of Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt Mohammed Sharaf-Eldin, MD, PhD Professor, Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt Mohamed Hassan Wahdan, MD, PhD Professor of Anatomy, Faculty of Medicine, Cairo University, Cairo, Egypt

CHAPTER

Cutting-edge tools and approaches for stem cell research and application in intestinal diseases

1

Ahmed El-Hashash1,2 1

The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, P.R. China 2 Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, P.R. China

1.1 Introduction The stem cell research and therapy field has grown up so fast over the past two decades, with many new and astonishing discoveries, representing the most exciting aspects of biological and biomedical research. Remarkably, stem cell research filed is growing over twice as fast (7%) as the reported world average growth in research (i.e., 2.9%). The annual growth rate of one rapidly grown type of stem cells; induced pluripotent stem cells (iPSCs), which was awarded the Nobel Prize in Physiology/Medicine in 2012, is an astonishing 77% since 2008. A significant increase in the volume of research output and publication has been reported in all areas of stem cell research. Major advances have been recently achieved in stem cell research field by the generation of the first functioning whole organ, thymus, in the laboratory, and the first documented in vitro fertilized human baby girl who has children of her own. Stem cell and biomedical research is currently underway in different national and international laboratories with ambitious goals of generating several other functioning whole organs, including the intestine and kidney. Advances in stem cell biology have seen the rise of an exciting new fields of regenerative medicine and research. Regenerative medicine is a multidisciplinary branch of translational research in tissue engineering and molecular biology, which deals with the “process of replacing, engineering or regenerating human cells, tissues, or organs to restore or establish normal function.” Regenerative medicine, therefore, aims at repairing injured tissues to restore normal cellular function. This field holds the promise of engineering damaged tissues and organs via stimulating the body’s own repair mechanisms to functionally heal previously irreparable tissues and/or organs. The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00013-8 © 2021 Elsevier Inc. All rights reserved.

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CHAPTER 1 Cutting-edge tools and approaches

Understanding stem cell biology and function could eventually lead to the identification of more innovative solutions for the treatment of many severe human disorders or diseases, restoring normal morphogenesis and/or regeneration of body tissues and organs. Rapid advances are also likely in stem cell applications in both repairing and regeneration of injured organs after injury, as well as for treating major and severe human diseases. The current overall goal of much of the research published on stem cells in different body organs is to improve human health through both better understanding of stem cell function and development of methods to bridge the bench and bedside. To accomplish this goal, scientists have developed and deployed a broad range of cutting edge in vitro and in vivo research tools and approaches, including mouse and human organoid cultures, genetic editing in vitro and in vivo, human iPSC models of disease, haploid cells for genetic as well as compound screening paradigms, genetically engineered mice, as well as stem cell transplantation to cure diseases. These multidisciplinary cutting-edge research techniques and approaches are currently the hottest topics in stem cell research, which enable scientists to investigate stem cell function, model the complexity of human diseases, and use stem cells in the treatments of these diseases in different organs, including the intestine. Intestinal stem cells are well reported and used to investigate stem cell biology since they are easily accessible and renew rapidly. They are well maintained throughout the human life. Structurally, the intestinal epithelium in humans consists of crypts containing both intestinal stem cells and other cell types (the proliferative compartment), and villi that represent intestinal mucosa folds and contain the differentiation cell compartment. Multipotent intestinal stem cells can generate all cell types of the intestinal lineage that exist in the intestinal surface, including goblet cells, enterocytes, Paneth cells, and endocrine cells (Arrighi, 2018). The intestine is among the leading organs, in which several cutting edge in vitro and in vivo research tools and approaches are recently developed and used to investigate stem cell biology/function and the potential applications of stem cells in the treatment of intestinal diseases and in intestinal repair and regeneration. The recently developed cutting-edge approaches/tools include genetic editing in vivo and in vitro, murine and human organoid cultures, human iPSC models of diseases, genetically engineered mice, haploid cells for genetic as well as compound screening paradigms, and stem cell transplantation for treatments of human diseases. For example, gut epithelial organoid culture is an emerging powerful technique for investigating the intestinal cellular/molecular biology, physiology, and pathology (Sato et al., 2011; Yui et al., 2012) and was most recently derived from iPSCs that will facilitate high-throughput screening of pathogenic factors, drug discovery, and candidate treatments for gastrointestinal diseases (Takahashi et al., 2018; Workman et al., 2018). In addition, a recent progress has been achieved in generating gut organoids to investigate intestinal physiology and diseases and model the gastrointestinal tract (Almeqdadi et al., 2019;

References

Min et al., 2020). Moreover, human iPSC-derived intestinal organoids were recently generated for drug testing (Yoshida et al., 2020). iPSCs were also used to model intestinal diseases (Liu et al., 2015; Rowe and Daley, 2019). Furthermore, human iPSC-derived intestinal tissues are successfully engendered to model intestinal diseases (Workman et al., 2017; Fair et al., 2018). Thus advanced technologies such as iPSCs and intestine-Chip are recently used to produce human intestinal lining that re-creates living tissue inside organ-chip, opening the door to personalized testing of drug treatments of inflammatory gastrointestinal diseases (Workman et al, 2017). Remarkably, the first ever attempt at gene editing inside the body was made by scientists in 2017, and this new technique could give a major boost to the fledgling field of gene therapy for different organs, including the intestine. Interestingly, stem cell transplant was shown in recent clinical trials as a promising modality of treatment for complicated inflammatory bowel diseases (Salem and Selby 2017) and chronic intestinal diseases such as Crohnaˆhts disease at Nottingham University, United Kingdom (in 2018). This book reviews recent advances in stem cell study and application in the intestine using fast-growing and multidisciplinary cutting-edge research techniques and approaches. The book is structured into two major sections describing cutting-edge research for understanding stem cell functions in the intestine, and for developing methods to bring stem cells from bench to bedside, respectively. Each section includes insights ranging from using mouse and human organoid cultures, genetic editing in vitro and in vivo, and human iPSCs to study intestinal stem cell functions and model intestinal diseases, through the cutting-edge research aiming to bring stem cells from bench to bedside, including the potential application of iPSCs, embryonic stem cells (ESCs), and other stem cells (stem cell transplants) in the treatment of intestinal diseases. This book, therefore, discusses the fact-based promise of stem cells and regenerative medicine in the intestine in the real world.

References Almeqdadi, M., Mana, M.D., Roper, J., Yilmaz, O.H., 2019. Gut organoids: mini-tissues in culture to study intestinal physiology and disease. Am. J. Physiol.-Cell Physiol. 317 (3), C405 C419. Arrighi, N., 2018. Definition and classification of stem cells. In: Arrighi, N. (Ed.), Stem Cells: Therapeutic Innovations Under Control. Elsevier, pp. 1 45. Fair, K.L., Colquhoun, J., Hannan, N.R.F., 2018. Intestinal organoids for modelling intestinal development and disease. Phil. Trans. R. Soc. B 373, 1 10. Liu, J., Shi, B., Shi, K., Zhang, H., 2015. Applications of induced pluripotent stem cells in the modeling of human inflammatory bowel diseases. Curr. Stem Cell Res. Ther. 10 (3), 228 235. Min, S., Kim, S., Cho, S., 2020. Gastrointestinal tract modeling using organoids engineered with cellular and microbiota niches. Exp. Mol. Med. 52, 227 237.

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Rowe, R.G., Daley, G.Q., 2019. Induced pluripotent stem cells in disease modelling and drug discovery. Nat. Rev. Genet. 20, 377 388. Salem, G.A., Selby, G.B., 2017. Stem cell transplant in inflammatory bowel disease: a promising modality of treatment for a complicated disease course. Stem Cell Investig. 4, 95. Sato, T., et al., 2011. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141, 1762 1772. Takahashi, Y., Sato, S., Kurashima, Y., Yamamoto, T., Kurokawa, S., Yuki, Y., et al., 2018. A refined culture system for human induced pluripotent stem cell-derived intestinal epithelial organoids. Stem Cell Rep. 10, 314 328. Workman, M.J., Mahe, M.M., Trisno, S., Poling, H.M., Watson, C.L., Sundaram, N., et al., 2017. Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system. Nat. Med. 23, 49 59. Workman, M.J., Gleeson, J.P., Troisi, E.J., Estrada, H.Q., Kerns, S.J., Hinojosa, C.D., et al., 2018. Enhanced utilization of induced pluripotent stem cell-derived human intestinal organoids using microengineered chips. Cell. Mol. Gastroenterol. Hepatol. 5, 669 677. e662. Yoshida, S., Miwa, H., Kawachi, T., et al., 2020. Generation of intestinal organoids derived from human pluripotent stem cells for drug testing. Sci. Rep. 10, 5989. Yui, S., Nakamura, T., Sato, T., et al., 2012. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr51 stem cell. Nat. Med. 18, 618 623.

CHAPTER

Organotypic intestinal cell culture as a new modality for intestinal function and cellular processes

2

Taylor Broda1,2 and Magdalena Kasendra1,2 1

Center for Stem Cell and Organoid Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States 2 Division of Pediatric General and Thoracic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, United States

2.1 Defining organotypic intestinal cell culture models The term “organotypic model” originally referred to explanted tissue that retains the same or highly similar functions with its in vivo counterpart (Dedhia et al., 2016; Randall et al., 2011). In the last decade, this definition continued to evolve to include various ex vivo and in vitro cell culture systems emerging in the field of organ development and tissue engineering. Consequently, the umbrella term “organotypic” now encompasses: short-term cultures of explants, long-term cultures of primary tissue derived cells, and the de novo differentiated organ-like structures from human pluripotent stem cells (hPSCs) (Dedhia et al., 2016; Randall et al., 2011; Shamir and Ewald, 2014). In essence, an organotypic model is a miniature organ or tissue grown in vitro from three-dimensional (3D) clusters of stem cell derived cells that selforganize and exhibit similar functionality as the tissue of origin (Wallach and Bayrer, 2017; Fair et al., 2018). Here, we focus on the organotypic models of human intestine, which can be divided into two categories based on the type of the stem cells they originate from, including human adult stem cells (hASCs)- or hPSCs-derived models. hASCs-derived organotypic cell cultures are established from population of intestinal stem cells contained within crypts isolated from endoscopic biopsy or surgically resected tissue, while directed differentiation of hPSCs into intestinal tissue in vitro leads to the development of hPSCs-derived models. Importantly, hPSCs can give rise to a broad array of cells—including epithelium and mesenchyme—by contrast, the differentiation potential of multipotent hASCs is limited to intestinal epithelial cell types (Wallach and Bayrer, 2017; Finkbeiner and Spence, 2013; Spence et al., 2011; Sato et al., 2011; Munera et al., 2017; Singh et al., 2020). Therefore building upon the guidelines proposed in 2012 by intestinal stem cell consortium (Stelzner et al., 2012), we will use terms: “enteroids” and “colonoids” when referring The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00004-7 © 2021 Elsevier Inc. All rights reserved.

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to purely epithelial cultures established from primary tissue of small or large intestine, respectively. While the term “organoids” will be used when referring to cultures that originate from hPSCs and are composed of both epithelial and mesenchymal components. Consequently, human intestinal organoids (HIOs) and human colonic organoids (HCOs) refer to hPSCs-derived models representing small and large intestine, respectively. All of the above mentioned organotypic intestinal cell culture models share common features, including their stem cell (multipotent or pluripotent) origin and their requirement for the presence of minimum stem cell niche components (extracellular matrix and growth factors-rich media) for their long-term survival in vitro. Further, they share the capability to self-renew and self-organize into 3D structures composed of intestinal epithelium (with or without surrounding mesenchymal niche) by undergoing multilineage differentiation (Wallach and Bayrer, 2017; Fair et al., 2018; Spence et al., 2011; Sato et al., 2011; Munera et al., 2017; Singh et al., 2020; Zachos et al., 2016). However, it is critical to recognize that hASCs- and hPSCs-derived organotypic intestinal cultures represent two very different systems in terms of the methodology used to generate them, structural complexity, maturation level, as well as modeling capabilities (Wallach and Bayrer, 2017; Fair et al., 2018; Spence et al., 2011; Sato et al., 2011; Munera et al., 2017; Singh et al., 2020; Zachos et al., 2016). Thus when choosing the appropriate system for their studies, researchers should be cognizant of these differences, as they pertain to particular advantages and disadvantages, and more importantly, to the ability of these models to recapitulate the structure and physiology of human intestinal tissue in vitro. Enteroids and colonoids, established from intestinal biopsy or tissue resection, retain in vitro their original lineage specification, regional identity, and maturation level of the tissue of origin (Wallach and Bayrer, 2017; Sato et al., 2011; Singh et al., 2020). They grow embedded in a extracellular matrix and form relatively simple, purely epithelial structures representing human duodenum, jejunum, ileum, or colon dependently on the source of the hASCs (Wallach and Bayrer, 2017; Sato et al., 2011; Singh et al., 2020). Enteroids and colonoids can be expanded without any apparent limit or genetic harm and are amendable to cryopreservation, aiding the development of living biobanks of intestinal tissues from healthy or diseased individuals (Sato et al., 2011; Singh et al., 2020; Mahe et al., 2015). The establishment of enteroids or colonoids requires a minimal time investment in comparison to hPSCs-derived models. However, it may be challenged by limited access to human primary tissue, especially from healthy individuals or regions of the gastrointestinal track which are sampled through endoscopy procedure less frequently (Spence et al., 2011; Sato et al., 2011; Munera et al., 2017; Singh et al., 2020). Conversely, HIOs and HCOs are generated from hPSCs via a multistep differentiation protocols that mimic subsequent stages of embryonic development (Spence et al., 2011; Munera et al., 2017; Singh et al., 2020; McCracken et al., 2011). Notably, establishment of organoid cultures does not rely on the availability of human intestinal tissue, as hPSCs can be derived from donated human blastocysts

2.2 Intestinal structure and functions

(human embryonic stem cells, hESCs) or reprogrammed from various types of somatic cells, such as blood cells or skin fibroblasts (induced pluripotent stem cells, iPSCs). In contrast to enteroids and colonoids, these organoid cultures possess mesenchymal cells, which interact closely with intestinal epithelium and that support its growth, differentiation, and functionality, similarly to what is seen in vivo. In addition, the proximal to distal regional identity of hPSCs-derived cultures can be manipulated during the differentiation process to create organoids that resemble human duodenum, ileum, or colon (Spence et al., 2011; Munera et al., 2017; Singh et al., 2020; Tsai et al., 2017). Despite the increased level of versatility and cellular complexity in comparison to hASCs-derived models, HIOs and HCOs display a fetal-like phenotype and fail to mature in vitro regardless of culture duration (Spence et al., 2011; Munera et al., 2017; Singh et al., 2020; Finkbeiner et al., 2015b). Transplantation of organoids into immunocompromised mammalian hosts enhances their maturation and is commonly used to improve functionality of the in vitro engineered hPSCs-derived intestinal tissues (Munera et al., 2017; Singh et al., 2020; Watson et al., 2014; Mahe et al., 2017). Detailed comparison of the hASCs- and hPSCs-derived intestinal cell culture models is provided in Fig. 2.1. In summary, organotypic intestinal cell cultures represent significant advancements in modeling human intestinal development, composition, and function. However, despite their numerous strengths, they still suffer from several shortcomings. First and foremost, the presence of fully enclosed internal lumen, which limits the access to the apical surface of cells, and poses a challenge for the use of organotypic intestinal models in studies of absorption, transport, and host microbe interactions (Spence et al., 2011; Sato et al., 2011; Munera et al., 2017). They often lack numerous biological, topological, and mechanical cues present in the native human intestine, including interactions with neural, endothelial, immune cells, proper crypt villi axial microstructure, and mechanical forces, such as fluid flow or stretch associated with peristalsis (Spence et al., 2011; Sato et al., 2011; Munera et al., 2017). The reductionist nature of hASCs- and hPSCsderived models may be beneficial for the mechanistic explorations of cellular processes, as it offers the possibility to perform studies in the absence of confounding influences from the surrounding tissues or local tissue microenvironment (Dedhia et al., 2016). However, to increase the appeal of this systems to study more complex intestinal functions it is critical to ensure they faithfully recapitulate the in vivo tissue counterpart. The convergence of organotypic intestinal cell cultures with engineering technology offers unprecedented platforms for accurate in vitro modeling of 3D intestinal tissue architecture and physiology.

2.2 Intestinal structure and functions The human intestine is a highly complex and dynamic organ that performs a myriad of vital functions encompassing digestion and absorption of nutrients, motility

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FIGURE 2.1 Generation methods and characteristics of organotypic intestinal models. Schematic overview of the methods used for the establishment of human adult stem cells derived (left) and human pluripotent stem cells derived (right) organotypic intestinal models and table summarizing main points of similarity and difference between the two types of cell cultures.

(peristalsis), immune surveillance, and protection from potentially harmful luminal content and pathogens. It is made of multiple layers of specialized tissues, including epithelium, connective tissues with blood vessels, nerves and smooth muscles, which collectively form a hollow tube surrounding a central lumen. The human intestine is subdivided into two sections: the small intestine and the large intestine, also known as colon, which composition and structure are shown in Fig. 2.2 (Zorn and Wells, 2009; Sancho et al., 2004; Rubin, 2007; Shaw et al., 2012). The major functions of the small intestine are chemical digestion of food and the absorption of nutrients and minerals into the bloodstream. These functions are

2.2 Intestinal structure and functions

FIGURE 2.2 Composition and structure of small intestine and colon. Simplified schematic cross-sectional representation of small intestine and colon depicting key morphological features and cellular components of both tissues.

supported by (1) division of the small intestine into three functionally distinct segments, the duodenum, jejunum, and ileum; (2) presence of intricate morphological features, including circular folds, villi, and microvilli, which increase the total surface area available for absorption; and (3) synchronized contraction of muscles (peristalsis) that enables movement of the food along the digestive tract (Fair et al., 2018; Shaw et al., 2012; Karasov and Douglas, 2013). The strikingly folded epithelial lining of small intestine consists of numerous finger-like luminal projections, known as villi and submucosal invaginations, termed crypts (Sancho et al., 2004). Crypts constitute a niche for the population of vigorously proliferating intestinal hASCs that fuel the active self-renewal of intestinal epithelium (Fair et al., 2018; Sancho et al., 2004; Gjorevski and Ordonez-Moran, 2017). The large intestine, or colon, is responsible for processing of undigested luminal content after most nutrients are absorbed in the small intestine. It is composed of four segments: the ascending, transverse, descending, and sigmoid colon (Fair et al., 2018; Abe et al., 2018). The colonic epithelium is folded into crypts, similarly to small intestinal tissue, but does not display villi (Sancho et al., 2004). It performs essential roles of absorbing water and electrolytes, producing and

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assimilating vitamins and metabolizing undigested polysaccharides to short-chain fatty acids (SCFAs) (used as energy source) (Cummings, 1984; Topping and Clifton, 2001; Said and Mohammed, 2006). In addition, the colon is inhabited by a myriad of commensal bacteria that are critical for the maintenance of normal gut health. These colonic microbes are known to aid in digestion, improve intestinal barrier function, enhance resistance against pathogenic infection, and promote the development of host immune responses (Pickard et al., 2017; Rolhion and Chassaing, 2016). The intestinal epithelium is the most rapidly self-renewing tissues in the human body. Within an estimated turnover time of around 5 days, new epithelial cells are generated at the bottom of the crypts, migrate rapidly upward to the villi tips (in the small intestine) while undergoing multilineage differentiation, to finally be shed into the lumen upon reaching the top of the villi (van der Flier and Clevers, 2009). This rapid self-renewal is sustained by a population of highly proliferative LGR5 1 intestinal stem cells, which reside at the base of the crypts and give rise to transit-amplifying (TA) daughter cells, which differentiate as they migrate from the crypt toward villi (van der Flier and Clevers, 2009). TA cells undergo four or five rounds of cell division before they cross the crypt villus boundary and initiate to differentiate into multiple epithelial cell types required for normal intestinal functions (van Es et al., 2012; Noah et al., 2011). These include absorptive cell types (termed enterocytes in the small intestine and colonocytes in the colon) and secretory cell lineages—comprising of goblet, Paneth, enteroendocrine, and tuft cells, in addition to microfold (M) cells responsible for sampling luminal antigens for mucosal immune surveillance (van der Flier and Clevers, 2009; Noah et al., 2011). Enterocytes and colonocytes collectively are the most abundant intestinal cell types. They are noted for the presence of dense microvilli on their apical cell surface. These microscopic protrusions of cell membrane form a continuous brush border rich in membrane-associated molecules, which include various enzymes and transporters. Obtaining nutrition and energy from food relies on the presence of brush border enzymes and transporters as they digest and absorb nutrients from intestinal lumen into systemic circulation (van der Flier and Clevers, 2009; Noah et al., 2011). Goblet cells are the major secretory cells distributed along the entire length of the human intestine. Their prevalence increases from around 10% 15% of all epithelial cells in the small intestine to approximately 50% in the colon (Noah et al., 2011). Goblet cells are primarily responsible for the production and secretion of mucins and trefoil factors. Mucins form a crucial physical and chemical barrier protecting intestinal epithelium from potentially harmful luminal agents such as pathogenic bacteria, environmental toxins, noxious dietary components, whereas trefoil factors contribute to the mucosal repair (van der Flier and Clevers, 2009; Noah et al., 2011). Enteroendocrine cells (EECs) are scattered throughout the small and large intestine and comprise about 1% of the epithelial cell population. At least 15 distinct subtypes of EECs have been identified, which secrete a wide range of hormones involved in regulation of satiety, energy

2.2 Intestinal structure and functions

metabolism, and gastrointestinal motility (van der Flier and Clevers, 2009; Noah et al., 2011). Paneth cells represent an epithelial cell lineage, which under normal homeostatic conditions remains unique to the small intestine. However, in the presence of inflammation or chronic stress their ectopic appearance can be detected in the colonic tissue (van der Flier and Clevers, 2009; Noah et al., 2011). Paneth cells are located at the base of the crypts and support the growth and survival of stem cells via the expression of critical growth factors and cytokines (Clevers and Bevins, 2013). In addition, they play an important role in innate immune response through the secretion antimicrobial peptides (AMPs), defensins, and lysozymes into the gut lumen (Keshav, 2006; Steenwinckel et al., 2009). These effectors protect the host from enteric pathogens, help shape the composition of the colonizing microbiota, and act as the safeguards from bacterial translocation across the epithelium (Bevins and Salzman, 2011). Aside of these four well-characterized intestinal epithelial cell types, there are two less-studied cell types: tuft cells and M cells. Tuft cells, also known as brush cells, represent relatively rare and short-lived chemosensory cells, which are present in both small intestine and colon. They are characterized by the presence of long microvilli projecting from their apical cell surface into the gut lumen (Noah et al., 2011; Gerbe et al., 2011). Tuft cells orchestrate parasite type 2 immunity in the gut through the release of interleukin-25 that activates innate lymphoid cells responsible for the control of parasitic worm infections (Howitt et al., 2016; Gerbe and Jay, 2016). Another specialized intestinal epithelial cell type is represented by the M cells, which are located within lymphoid follicle-associated epithelial overlying Peyer’s patches of the small intestine (Noah et al., 2011; Mabbott et al., 2013). M cells are easily distinguishable thank to their irregular brush border and reduced microvilli. The presence of immune-surveillance receptors on the apical cell surface along with the ability of these cells to transcytose antigens is crucial for the induction of an efficient immune response or creation of tolerance to various luminal antigens (Mabbott et al., 2013). The intestinal epithelium works in concert with other specialized tissue types, including mesenchyme, endothelium, immune cells, and the enteric nervous system (ENS) to perform more complex organ-level functions (Allaire et al., 2018; Fung and Vanden Berghe, 2020; Heidemann et al., 2006; Sanders et al., 2012). For example, rhythmic, peristaltic waves of muscular contraction along the gut wall result from the highly integrated and coordinated activity of smooth muscle cells, enteric neurons, and specialized gut pacemaker cells called interstitial cells of Cajal (Sanders et al., 2012). The unique architecture of the gastrointestinal tract facilitates these interactions, as its various components organize into closely connected layers joined together by connective tissue as well as by the neural and vascular elements (Zorn and Wells, 2009; Sancho et al., 2004; Rubin, 2007). The presence of extensive network of enteric neurons and glia along the gastrointestinal tract is critical for regulating gut motility, secretion, and blood flow (Fung and Vanden Berghe, 2020). In turn, ENS is essential for a variety of intestinal functions raging from digestion and absorption, passage of luminal content, as

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well as maintenance of body fluid homeostasis. The intestinal vasculature is crucial for the systemic uptake of digested nutrients, as a barrier against microbes, and for the recruitment and conditioning of immune cells (Heidemann et al., 2006; Gentile and King, 2018). Finally, the numerous tissue-resident immune cells present within the thin layer of loose connective tissue that underlie the intestinal epithelium, play a central role in the regulation of barrier integrity, participate in host’s defense against pathogens, promote mucosal healing, and shape the composition of intestinal microbiome (Hooper et al., 2012; Agace and McCoy, 2017). Resident macrophages are a dominant and highly phagocytic immune cell type present in the gut tissue responsible for clearing apoptotic and senescent epithelial cells and acting as sentinels for pathogen recognition and elimination (Bain and Schridde, 2018).

2.3 Modeling of intestinal functions using organotypic cell culture models Although the majority of our knowledge on intestinal development and function stems from mouse studies, organotypic intestinal cell culture models provided a new window into a better understanding of these processes in humans. Both, hASCs- and hPSCs-derived models have proven to mimic regenerative capacity and differentiation of native intestine and to be useful for studies of intestinal absorption and transport, tissue permeability, secretion of mucins and intestinal hormones, as well as for modeling of intestinal responses to microbiota (Zachos et al., 2016). Coordinated activity of nutrient and ion transporters is necessary to support the primary function of human intestine, which is absorption of nutrients. Absorption of glucose occurs via sodium-glucose cotransporter 1 (SGLT1) and is energized by inwardly directed Na1 gradient (Wright et al., 2011). While the influx of small peptides into intestinal epithelial cells is mediated by intestinal peptide transporter 1 (PEPT1) and is driven by H1 gradient (Chen et al., 2010). These electrochemical gradients support balance of fluids and electrolytes across the intestinal membrane and are maintained by apical ion transporters, including sodium-hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane receptor (CFTR). Human enteroids recapitulated in vitro physiologically relevant transport of electrolytes, including absorption of neutral Na1 and stimulated secretion of fluid and anions under both basal and pathophysiological conditions (Foulke-Abel et al., 2016). Radiometric pH-sensitive fluorophore SNARF-4F and multiphoton microscopy was used to measure NHE3 function through detection of changes occurring in the intracellular pH of individual enteroids as a result of Na1-dependent realkalization. Basal activity of NHE3 showed to be similar in duodenal, jejunal, and ileal enteroids and were susceptible to second messengers (i.e., cyclic nucleotides) and bacterial enterotoxins (i.e., cholera toxin). This functionality was

2.3 Modeling of intestinal functions using organotypic cell

further used to mimic pathogenic infection as well as pharmacological modulation by known inhibitors (S3226 and EIPA) (Foulke-Abel et al., 2016). Evaluation of the CFTR function using a relatively easy and fast assay for forskolin-induced swelling confirmed the utility of duodenal organoids to study secretion of electrolytes and fluids in human intestine. Activation of CFTR resulted in a rapid uptake of chloride ions and water into the lumen of enteroids and their subsequent swelling (Foulke-Abel et al., 2016; Dekkers et al., 2013). The presence of an active transport of ions (Na 1 , K1, and Cl2) was also demonstrated in human colonoids grown as monolayers on Transwell inserts, as determined by the use of ion chromatography detection system (Li et al., 2017). Similar to hASCs-derived cultures, also intestinal organoids have been successfully applied in studies of intestinal transport. Most of the functional findings in this context come from the assays performed on epithelial only cultures derived from post transplanted HIOs, which we will refer to as epithelial organoids. While functionally more mature, these cultures have simplified structure facilitating the microscopy-based assessment. Epithelial organoids recapitulated in vitro absorption of glucose and di-peptides as confirmed by the uptake of fluorescent analog of glucose 6-NBDG and fluorescently labeled peptides β-Ala-Lys-AMCA and FITC-Gly-Sar (Spence et al., 2011; McCauley et al., 2020). Moreover, they have demonstrated the presence of functional NHE3 and CFTR as measured by Na1-dependent intracellular pH recovery after acidic challenge and VIP-induced swelling assay (McCauley et al., 2020). Intestinal epithelium acts as a selectively permeable barrier allowing for the absorption of nutrients, electrolytes, and water, while simultaneous preventing the entrance of intraluminal toxins, antigens, pathogenic, and commensal bacteria into the underlying tissue and the bloodstream (Chelakkot et al., 2018). Despite the presence of fully enclosed lumen that poses a challenge for in vitro assessment of tissue permeability, both hASCs- and hPSCs-derived models have proven useful for emulation of intestinal barrier function in health and disease. It was demonstrated through the exposure of the organotypic intestinal cell cultures to fluorescently labeled dextran and monitoring of its diffusion across the epithelial barrier via imaging. Fluorescent tracer was either added into the media surrounding 3D organotypic cultures or introduced into the lumen through microinjection (Leslie et al., 2015; Hill et al., 2017b). This methodology enabled detection of significant changes in permeability of hASCs-derived models treated with EGTA or IFNγ (Han et al., 2019; Wosen et al., 2019) and HIOs exposed to bacteria and their toxins (Leslie et al., 2015; Hill et al., 2017a,b; Karve et al., 2017; Pradhan et al., 2020). Additionally, Ha et al. have demonstrated a positive, protective influence of the probiotic strain of Lactobacillus rhamnosus GG on intestinal barrier function of human enteroids and colonoids (Han et al., 2019). Further confirmation and complementation of these findings come from the use hASCs- and hPSCs-derived organotypic intestinal cultures grown as two-dimensional cultures (monolayers) on Transwell inserts, which enables an easy assessment of tissue permeability to FITC-dextran and measurement of transepithelial electrical

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resistance (Roodsant et al., 2020; Kozuka et al., 2017; Kauffman et al., 2013). Several studies adopted this technique to demonstrate the capability of enteroids to model improved integrity of intestinal epithelium in the presence of bacterial metabolites such as SCFAs as well as disruption of intestinal barrier function in response to pathogenic infection (Roodsant et al., 2020; Pearce et al., 2020). The human intestinal mucosal barrier, in addition to its epithelial layer, is comprised of various molecules which are secreted into the lumen. These include mucins, which are produced by the goblet cells and that reinforce barrier function, facilitate passage of the luminal contents along the gastrointestinal tract, protects the epithelial cells from digestive enzymes, and prevents the direct contact of microorganisms with the epithelial cell layer (Johansson and Hansson, 2016). VanDussen et al. demonstrated that enteroid- and colonoid-derived epithelial monolayers can be used for the in vitro assessment of mucus thickness (VanDussen et al., 2015). Fluorescent beads were added to the cell culture media in the apical compartment and allowed to sediment. Then, the thickness of the mucus layer was determined based on the 3D Z-stack reconstruction of confocal images. The distance between the beads and the apical surface of the GFP-labeled epithelial cells corresponded to the mucus thickness. Ileal and rectal monolayers showed to accumulate approximately 26 and 36 μm thick mucus layers, respectively (VanDussen et al., 2015), which are significantly thinner than observed in vivo. Reported mucus thickness values in human small intestine range from 150 to 300 μm and about 700 μm in large intestine (Corfield, 2018). Studies of others demonstrated the protective role of mucins secreted by colonoid-derived monolayers against colonization with pathogens (Liu et al., 2020; In et al., 2016). Decreased mucus thickness showed to correlate with increased colonization of colonic epithelia with pathogenic strains of Escherichia coli (enterohemorrhagic and enteroaggregative E. coli) (Liu et al., 2020; In et al., 2016). Secretion of mucins by the goblet cells and its changes in the context of bacterial infection were also confirmed in HIOs (Munera et al., 2017; Hill et al., 2017a). Notably, luminal microinjection of HIOs with nonpathogenic E. coli showed to promote formation of robust mucin layer at the epithelial cell surface (Hill et al., 2017a). Taken together, these results demonstrated that hASCs- and hPSCs-derived organotypic intestinal models can recapitulate in vitro secretion of mucins by the goblet cells, enabling the use of these systems for studies of host microbiota interactions. However, neither of these cultures can reproduce the thickness or physiologically relevant structure of human mucin layer suggesting that further progress is needed to faithfully reproduce human mucus in vitro. Secretion of hormones by EECs represents another critical function of the human intestine as they exert a wide range of endocrine and paracrine actions. These include regulation of energy intake and metabolism, gut motility, contraction of the gallbladder, pancreatic enzyme secretion, gastric emptying, and gastric acid secretion (Gribble and Reimann, 2016). Demonstrated presence of EECs in both hASCs- and hPSCs-derived models suggests their potential applicability into the studies of gut hormone secretion in response to luminal stimuli. Indeed,

2.3 Modeling of intestinal functions using organotypic cell

human enteroids have shown to respond to SCFA stimulation by an enhanced development of a specific subtype of EECs, L-cells, followed by the increased release of GLP-1 under both basal and glucose-stimulated conditions (Petersen et al., 2014). Additionally, monolayer cultures established from fragmented enteroids have shown to secrete increased levels of PYY and ghrelin into the basal cell culture media upon stimulation with butyrate, a specific type of SCFA. Further confirmation comes from the studies of Kozuka et al. (2017) and Goldspink et al. (2020), who demonstrated the release of intestinal hormones (i.e., GLP-1) from ileal enteroid derived monolayers upon exposure to a set of pharmacologically active compounds, including forskolin (a potent activator of adenylyl cyclase) and a range of GPCR agonists. A slightly different approach into the studies of the EEC function has been taken in the case of HIOs. Sinagoga et al. employed the EEC-inducing property of the transcription factors NEUROG3 to firstly enrich for the presence of these cells in HIOs in vitro, and subsequently evaluate their response to nutrient stimulation. Exposure of NEUROG3-induced HIOs to luminal glucose resulted in stimulated secretion of gastric inhibitory polypeptide (GIP) and reduced production of ghrelin. These results demonstrate that HIOs are able to mount an incretin and satiety response analogous to one observed in vivo (Sinagoga et al., 2018). Epithelial antimicrobial defense of the human intestine is supported by the presence of Paneth cells, which reside in the intestinal crypts of the small intestine (Gassler, 2017). These specialized cells sense and respond to pathogens through the production and secretion of several types of AMPs. AMPs inhibit microbial growth through the direct lysis of the bacterial cell wall and modulation of bacterial metabolism. Experimental evaluation of the Paneth cells function in in vitro grown human enteroids and organoids has been reported recently (Hill et al., 2017a; Pearce et al., 2020; Ferrer-Picon et al., 2020). In human enteroids, the production of AMPs was assessed under the exposure to SCFA. A significant increase in the expression of REG3β, REG3γ, and β-defensin 1(DEFB1) was observed in 3D cultures treated with butyrate (Pearce et al., 2020; Ferrer-Picon et al., 2020). While in the HIOs a highly upregulated gene expression and secretion of human β-defensin 2 peptide was reported following their luminal microinjection with E. coli. Notably, levels of β-defensin 2 produced by infected HIOs were sufficient to suppress microbial growth (Hill et al., 2017a). Another specialized intestinal cell type involved in mounting protective immune response to pathogens in addition to the creation of tolerance to commensal microbiota is called microfold (M) cell (Mabbott et al., 2013). M cells sample microorganisms or molecules from the gut lumen, transport them across the epithelial cell layer and deliver to professional immune cells to stimulate a protective immune response. Although under the standard growth conditions hASCs- and hPSCs-derived are devoid of the M cells, addition of the exogenous RANKL into cell culture media showed to be sufficient to induce functional M cell development in enteroid system (de Lau et al., 2012; Kanaya et al., 2018; Wood et al., 2016; Rouch et al., 2016). M cell enriched human enteroids showed to exhibit

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an increased uptake of microparticles and bacteria (Salmonella typhimurium) consistent with the crucial role of M cells in antigen sampling and transcytosis (Rouch et al., 2016). To date, however, there have been no published reports demonstrating development of functional M cells in HIOs grown in vitro. Although it is now clear that the transplantation into animal host improves the maturation of the M cells in hPSCs-derived intestinal tissue (Yu et al., 2020), the functional evaluation of these cells is currently lacking. Recently, the relatively rare population of tuft cells has begun to be explored using organotypic intestinal cell cultures. Parasite sensing capability of these cells was demonstrated in mouse enteroids, as assessed by the increase of intracellular calcium levels and tuft cell depolarization upon exposure to small intestinal helminth Trichinella spiralis (Faniyi et al., 2020). Administration of IL-13, a cytokine secreted by innate lymphoid cells in the context of helminth infection, showed to promote tuft cell hyperplasia analogous to what is observed in vivo (Howitt et al., 2016; von Moltke et al., 2016). However, further studies are needed to confirm these findings in hASCs- and hPSCs-derived systems. In summary, remarkable progress has been made toward emulation of human intestinal cell and tissue-level functions using human organotypic intestinal cell cultures. However, these models do not fully recapitulate the complexity and organ-level physiology of the native human intestine, as they lack cellular components (i.e., enteric neurons, immune cells and vasculature), mechanical forces (i.e., shear and stretch), and appropriate geometry of extracellular microenvironment. Thus a number of recent studies have focused on improving the complexity of human organotypic intestinal cell cultures through the use of co-development and co-culture methods as well as various engineering-based approaches.

2.4 Toward emulation of organ-level functions The importance of the in vivo microenvironment in intestinal tissue development was revealed by the transplantation of immature intestinal organoids into animal hosts, which resulted in their increased morphological and functional resemblance to the native human tissue (Watson et al., 2014; Finkbeiner et al., 2015a; Cortez et al., 2018). HIOs engrafted underneath the kidney capsule of immunocompromised mice or into the intestinal mesentery displayed in vivo-like crypt/villus architecture of epithelium as well as appropriate differentiation and distribution of smooth muscle cells and subepithelial myofibroblasts. Additionally, the transplanted intestinal tissues demonstrated digestive functions as observed by improved permeability and uptake of peptides. These studies suggested that additional cues from the in vivo environment, which are currently absent in the static culture of HIOs, are required to support their maturation. In recent years, significant strides have been made to improve the complexity of organotypic cell culture models to recapitulate more closely the appreciable

2.4 Toward emulation of organ-level functions

intricacy of in vivo small and large intestine. These efforts were focused on incorporation of missing cellular components through their direct co-development or co-culture and integration of various environmental cues by the use of bioengineering methods shown in Fig. 2.3 (Holloway et al., 2019; Takebe et al., 2017; Takebe and Wells, 2019). The co-development approach is unique to hPSCs, which can be differentiated simultaneously, from a single population of pluripotent stem cells, toward multiple different cells types within the same culture. For example, a co-development procedure has been recently established for the concurrent differentiation of intestinal endothelium alongside HIOs (Holloway et al., 2020). While the co-culture approach can used in both hPSCs- and hASCsderived models and denotes incorporation of independently derived or

FIGURE 2.3 Strategies for enhancing organotypic intestinal models to enable emulation of organ-level complexity and functions. Co-development and co-culture methods help to achieve increased cellular complexity in human organotypic intestinal cell cultures. Transplantation into animal host provides in vivo cues, which are critical for maturation of human pluripotent stem cell derived tissues. Engineering-based approaches, including micropatterning, microfluidics, and three-dimensional bioprinting, allow for incorporation of physical and/mechanical cues into organotypic intestinal models to recapitulate native microenvironment of intestinal tissue. Collectively, the use of these methods in conjunction with organotypic intestinal cell cultures enables improved modeling of intestinal tissue architecture and physiological functions in vitro.

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differentiated cell populations into one in vitro culture system. For instance, HIOs and precursors of enteric neurons can be differentiated in parallel from two independent sources or even one single source of hPSCs, and then subsequently combined into one culture to create innervated organoids (Workman et al., 2017). Similarly, enteroids derived from hASCs can be co-cultured with macrophages differentiated from peripheral blood monocytes to create immune enhanced organotypic intestinal cell culture system (Noel et al., 2017). Increased cellular diversity and modularity enables modeling of the continuous cellular crosstalk, which occurs between various components of intestinal tissue in vivo and that is critical for accurate reconstitution of more complex organ-level function at the in vitro level. Nevertheless, the dynamic and complex microenvironment of the native intestinal tissue consist of not only cells, but also topological, mechanical, and biophysical cues that contributes to the establishment and maintenance of normal organ physiology (Vining and Mooney, 2017). These microenvironmental cues, including shear stress induced by the blood flow, stretch associated with peristalsis, and appropriate geometry of extracellular milieu can be incorporated into organotypic intestinal cell culture models using engineering-based techniques. For example, biomimetic collagen scaffold has been created and seeded with enteroids-derived intestinal stem cells to recapitulate the intrinsic crypt villus architecture of the small intestinal tissue and proper distribution of proliferative and differentiated cells (Wang et al., 2017). In the following, we will discuss examples of how co-development and co-cultures methods as well as various bioengineering approaches have been used to improve the physiological relevance of organotypic intestinal models and their capability to model more complex organlevel functions.

2.4.1 Co-development Until recently, transplantation was required to achieve vascularization of organotypic intestinal models relying on indispensable support from the physical environment of the animal host’s tissue. Interestingly, organoids transplantation into highly vascularized regions of immunocompromised mice resulted in improved architecture and maturation of engrafted tissue and its closer resemblance to the native intestine suggesting a critical role of endothelium in intestinal development (Watson et al., 2014; Cortez et al., 2018). Moreover, in vitro co-culture of endothelial cells with other hPSCs-derived systems such as liver organoids showed to significantly improve their function (Camp et al., 2017; Takebe et al., 2013). Nevertheless, co-development or co-culture methods enabling development of vascularized HIOs were missing. Holloway et al. established modified HIOs culture conditions in which an early developing and quickly fleeting population of endogenous endothelial cells can be maintained alongside epithelium and mesenchyme for prolonged periods of time. Vascularized HIOs showed to possess appropriately patterned intestine-specific vasculature in vitro leading to the improved complexity and biological resemblance of this model to native

2.4 Toward emulation of organ-level functions

developing intestine. However, it is still unknown whether the presence of endothelial cell population has any impact on the functional maturation of intestinal organoids (Holloway et al., 2020).

2.4.2 Co-culture Other groups have used co-culture techniques to increase complexity of organotypic cell culture models and to improve their capability to accurately recapitulate intestinal tissue composition and function. Using this strategy a successful integration of hPSC-derived endothelial cells into HCOs has recently been reported (Park et al., 2020). However, also in this case, further studies are warranted in order to better understand the impact of increased cellular complexity of the resulting tissue construct on its ability to model intestinal functions. On the other hand, a significant progress has been made toward a functional integration of ENS into organotypic intestinal cell culture models. The presence of ENS components in vivo is essential for a variety of intestinal functions including digestion and absorption, peristalsis, as well as maintenance of body fluid homeostasis (Fung and Vanden Berghe, 2020). Several different groups established co-culture systems, which combine the use of hPSCs-derived organotypic models with precursors of enteric neurons (vagal neural crest cells) leading to improved modeling of functional ENS and neuroepithelial interactions (Workman et al., 2017; Park et al., 2020; Fattahi et al., 2016; Lau et al., 2019). Incorporation of vagal neural crest cells into HIOs resulted in development of neurons and glia, cell lineages which are absent in organoids cultured independently (Workman et al., 2017). Importantly, differentiated enteric neurons exhibited rhythmic waves of calcium transients confirming their functionality in vitro. In addition, innervated HIOs when transplanted into animal host gained the ability to undergo peristaltic-like contractions in response to electrical-field stimulation. Similar level of functional integration of ENS has also been confirmed in colonic organoids (Lau et al., 2019). Notwithstanding, none of these co-culture models were able to recapitulate connection between ENS and EECs of the epithelium, which is important for conveying nutrient sensing information (Bohorquez et al., 2015). Recently, this has been accomplished by the use of enteric neural crest cell (ENCC) spheres—a 3D progenitor rich culture competent to differentiate into ENS lineages (Fattahi et al., 2016; Schlieve et al., 2017). HIOs seeded with ENCC spheres onto a scaffold where transplanted into the intestinal mesentery of immunodeficient host for maturation in vivo. A 3D confocal microscopy showed the presence of axonal projections to EEC bodies in the innervated tissue constructs suggesting the establishment of neuroepithelial synaptic connections. Nevertheless the functionality of these connections and the level of neuroepithelial interactions has yet to be fully explored (Schlieve et al., 2017). Collectively, HIOs ENS co-cultures resulted in improved complexity and function of the organotypic intestinal cell culture models allowing for investigation of intestinal motility and neuroepithelial functions.

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The highly integrated and coordinated crosstalk between intestinal epithelium and the underlying immune system is vital for the maintenance of intestinal homeostasis (Allaire et al., 2018). Thus significant efforts have been made to develop strategies to co-culture hASCs- and hPSCs-derived models with immune cells and increase their physiological relevance. A co-culture of enteroids grown as monolayers on a Transwell insert with macrophages showed to improve maturation and barrier integrity of intestinal epithelium (Noel et al., 2017). Moreover, the apical exposure of this system to enterotoxigenic and enteropathogenic E. coli, has led to the increased number of intraepithelial macrophage projections, followed by efficient phagocytosis and stabilization of barrier function—a highly coordinated in vivo-like response (Noel et al., 2017). Innate immune functions were also recapitulated in hPSCs-derived models by co-culturing HIOs with neutrophils followed by their exposure to Shiga toxin-producing E. coli O157:H7 (Karve et al., 2017). Pathogenic infection was associated with increased release of proinflammatory cytokines and reactive oxygen species, loss of epithelial integrity and recruitment of neutrophils into the infected HIO tissue (Karve et al., 2017). Interestingly, Jung et al. has recently shown that the need for in vivo maturation of HIOs, which is required to fully recapitulate the adult intestine, can be circumvent by their co-culture with T lymphocytes (Jung et al., 2018). They have demonstrated that IL-2, as a mediator of the immune system, promotes in vitro maturation of HIOs. Increased expression of mature intestinal markers and enhanced functionality, including improved activity of a drug efflux transporter P-glycoprotein (P-gp), ion transporter CFTR and elevated secretion of GIP, was observed in IL-2 treated HIOs (Jung et al., 2018). These studies have further highlighted the importance of increasing the cellular complexity of the existing organotypic intestinal cell culture systems to appropriately recapitulate the physiological and pathophysiological functions of human intestine.

2.4.3 Bioengineering A steadily accumulating body of evidence has led to recognition that the topological properties of the tissue and mechanical cues have the power to direct a variety of cell functions, including cell migration, proliferation, and differentiation (Nelson et al., 2005; Hannezo and Heisenberg, 2019; Baptista et al., 2019) Thus to bridge the gap between in vitro organ models and their in vivo counterparts, a number of microenvironmental factors, including appropriate tissue topology, mechanical forces and biophysical cues must be incorporated into organotypic cell culture systems. The convergence of stem cell biology with engineering have recently offered an unprecedented control over the design and complexity of ex vivo cell microenvironments and greatly aid investigations of intestinal cell functions in vitro (Takebe et al., 2017). Microfabrication techniques have been used to develop 3D biomimetic cell culture scaffolds that recapitulate the intrinsic crypt villi axial microarchitecture of the small intestinal tissue (Wang et al., 2017; Nikolaev et al., 2020). In the

2.4 Toward emulation of organ-level functions

native human intestine, stem cells which reside at the bottom of the crypts divide, differentiate and migrate continuously toward the top of the villi where they replace lost and damage cells, ensuring the maintenance of proper tissue integrity and functions (van der Flier and Clevers, 2009). The topography of intestinal tissue, which guides spatial cell compartmentalization, influences the magnitude of the forces experienced by the cells at various locations along crypt villus axis and their access to essential morphogens (growth factors and cytokines). In consequence, it possesses a great impact on the maintenance of intestinal homeostasis (van der Flier and Clevers, 2009). Wang et al. have demonstrated that the use of 3D biomimetic collagen scaffold, which emulates surface topography of human small intestine allows for a spatial control of intestinal cell fate and differentiation in vitro (Wang et al., 2017). hASCs-derived monolayers cultured on biomimetic 3D substrates on Transwell inserts were exposed to distinct culture media (either enriched in growth factors that maintain the stem cell niche, or inhibitors that stimulate epithelial differentiation) from the bottom or top compartments, respectively. Established in this manner, gradient of morphogens led to appropriate patterning of developing tissue along crypt villi axis, including formation of stem/ progenitor cell zones at the crypt bottom and migration of differentiating cells toward the top of the villi. Moreover, this study demonstrated that engineeringbased approach can complement organotypic cell culture systems by incorporating topological cues to enable spatiotemporal control of stem cells self-renewal and self-organization. More recently, the convergence of organotypic intestinal cell culture models and microfluidics resulted in the creation of Intestine-on-a-chip technology (also known as Intestine-Chip, Gut-Chip) (Kasendra et al., 2018; Workman et al., 2018). This approach enabled a precise control of mechanical forces, including fluid flow and stretch, which increased the physiological relevance and physicomechanical complexity of microengineered intestinal tissues. In addition, it allowed development of higher order 3D cell culture systems, which cellular complexity can be fully controlled and fine-tuned by incorporation of multiple microscale compartments into a single chip device (Ronaldson-Bouchard and VunjakNovakovic, 2018). Each of these compartments may contain cells of a different type, including epithelial, endothelial, immune, and stromal cells that interact with each other through the direct contact or secreted endocrine or paracrine factors. Using this engineering approach complex in vitro organ models can be built in a stepwise manner, adding additional cell types, exogenous factors, and microenvironmental parameters, until a relevant level of complexity is recreated in vitro (Ronaldson-Bouchard and Vunjak-Novakovic, 2018). hASCs- and hPSCsderived organotypic intestinal cell cultures were successfully incorporated in the chip device allowing for the development of 3D microengineered tissues that recapitulate the cellular architecture, multilineage differentiation, and physiological functions of native human intestine, including digestion and absorption of nutrients, secretion of mucins, activity of drug metabolism enzymes, and response to inflammation (Kasendra et al., 2018; Workman et al., 2018; Sontheimer-Phelps

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et al., 2020; Kasendra et al., 2020; Tovaglieri et al., 2019; Sunuwar et al., 2020). Moreover, the ability to precisely manipulate and monitor the oxygen gradient inside of the microfluidic Intestine-on-a-chip enabled the long-term co-culture of multispecies human gut microbiome with hASCs-derived intestinal cells (JaliliFiroozinezhad et al., 2019). Taken together, the integration between organotypic culture models and microfluidics have opened the doors for studying more complex physiological and pathophysiological processes in vitro. Nikolaev et al. has recently proved that all three powerful technologies, including organotypic cell cultures, microfabrication of biomimetic scaffold, and microfluidics can be combined into one system (Nikolaev et al., 2020). Resulting mini-gut tubes were able to recapitulate in vitro some of the critical aspects of the native cellular milieu associated with intestinal tissue development and regeneration, including luminal flow, physiologically relevant geometry of extracellular matrix, constant cell turnover, enabling the biological fidelity, and levels of control which were not achievable in the past. This new bioengineered organotypic cell culture model showed to emulate intestinal tissue homeostasis and regenerative responses following injury and to support month-long studies of parasite host interactions. In summary, various biology- and engineering-based approaches have been exploited to develop organotypic culture systems that more holistically recapitulate the cellular complexity and dynamic nature of native human intestine, and therefore more accurately model complex organ-level functions. Moving forward, in parallel to the continued advancement of these models, research efforts should focus on improving their fidelity, reproducibility, and scalability. Adoption and democratization of these advanced in vitro tools across the research community will undoubtedly enhance our understanding of human biology in the contexts of intestinal development, homeostasis, and disease.

Acknowledgment We thank Rachael Kiniyalocts for the creation of figures using BioRender.com.

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CHAPTER

Role of human gastrointestinal organoids in discovery and translational medicine

3

Alexandra Calor1, Mirjam van Weissenbruch1 and Stefania Senger2,3 1

Department of Pediatrics, Division of Neonatology, Amsterdam UMC, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital, Boston, MA, United States 3 Harvard School of Medicine, Boston, MA, United States

3.1 Introduction 3.1.1 Nomenclature and distinction: organoids–enteroids– colonoids–tumoroids The term “organoid” was originally introduced to describe three-dimensional (3D) biological structures generated from cells of different sources including tissue segments (Levy et al., 2009; Sambuy and De Angelis, 1986) and transformed cell lines (Salerno-Goncalves et al., 2016) when cultivated embedded in an extracellular-like matrix. With the discovery of adult stem cells (ASCs) and factors required for their propagation, together with methodologies for the derivation of organoids from induced pluripotent stem cells (iPSCs), the term has been mostly referring to the result of proliferation from these two kinds of stem cells (Sato et al., 2009, 2011; Senger et al., 2018; Spence et al., 2011). Organoids as intended today, must express the following characteristics: (1) to grow in a 3D conformation that mimics the tissue of origin; (2) to differentiate into multiple cell types and in equivalent proportions as in the organ or tissue; (3) to express functions that belong to the organ or tissue of origin; and (4) to selfrenew by maintaining a pool of progenitor cells without requiring in vitro transformation. Significant differences exist between organoids from iPSC and ASC. The iPSC-derived cultures can originate both epithelial and mesenchymal cell lineages (Spence et al., 2011), while ASC organoids develop exclusively the epithelial lineage of the gastrointestinal (GI) tract (Sato et al., 2009). For this reasons, the name “organoids” was originally suggested for iPSC-derived culture, whereas 3D The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00010-2 © 2021 Elsevier Inc. All rights reserved.

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cultures from ASC were given the name of enteroids and colonoids if originated, respectively, from small and large intestine (Stelzner et al., 2012). Nonetheless, there are no established rules to name organoids from other GI regions like stomach, esophagus, liver, gallbladder to mention a few. As a consequence, the scientific community uses “organoids” as a generic term and has conceived numerous names and acronyms for newly established cultures. In this chapter, we will adopt the noun “organoids” as a generic term and will state origin and names as provided in the original studies. It must be mentioned here a third kind of organoid derived from cancer, also known as tumoroid. Culturing conditions have been established for colon carcinoma, adenoma, head and neck squamous cell carcinoma (HNSCC), Barrett’s epithelium, and liver and pancreas cancer (Sato et al., 2011; Broutier et al., 2017; Driehuis et al., 2019c; Kijima et al., 2019; Seino et al., 2018). Tumoroids provide a model with intermediate complexity between standard two-dimensional cell culture and tumors in vivo or xenografts (Sato et al., 2011). Similar to other organoids, tumoroids exhibit higher structural complexity in terms of cell–cell and cell–matrix interactions. These, in turn, reflect on the establishment of a significant in vivo-like tumor microenvironment (TME) that influences the communication with other cells like immune and stromal cells (Nair et al., 2017).

3.1.2 Advantage of organoids in basic and applied research of human GI diseases Cell lines, experimental animals, and ex vivo human tissue (Hyun et al., 1732) have been instrumental in scientific and medical research advancements over the past half century. The 3D organoid technology has been only recently developed (Sato et al., 2009) and in a few years has already proven to be a relevant research tool over the conventionally used models, thanks to significant advantages. Specifically, cell lines are derived from tumors or genetically modified (immortalized) cells. For this reason, they do not express all cell types representative of a normal tissue and have lost many inhibitory checkpoints of primary cells. On the contrary, organoids develop into polarized single layer of cells (monolayer) with apical microvilli and basal side, express virtually all cellular types derived from the common stem cell, respond to stimuli that halt proliferation, promote differentiation, and activate programmed cell death, as in normal tissue. Furthermore, cell biology techniques originally developed for cell lines, including cryopreservation, in vitro manipulation, lentiviral transduction, and gene editing, can be swiftly applied to both iPSC-derived and ASC-derived organoids (Fig. 3.1). When compared to multicellular organisms, such as invertebrates, mice, or primates, organoids appear a rather reductive system. However, animals do not faithfully represent the human biology, expressing significant differences in metabolism and immune responses, which are especially important in understanding the pathophysiology of many GI diseases (Verma, Senger, et al., 2020).

3.1 Introduction

FIGURE 3.1 Modality to generate gastrointestinal (GI) organoids and applications. Organoids can be generated starting from GI biopsies containing adult stem cells (ASC) or from induced pluripotent stem cells (iPSCs). In the first approach, ASCs are harvested from biopsies of the GI segment of interest. Next, ASCs are plated and expanded in vitro using growth factors and morphogens that mimic the “adult stem cell niche.” As a result, organoids that are generated from ASC proliferation functionally and morphologically mirror features of the tissue of origin. Organoids from several parts of the GI tract have been generated in this way from the mouth to the rectum. To generate GI organoids from iPSC, researchers must first revert somatic cells, like fibroblasts or keratinocytes harvested from a skin biopsy, into pluripotent stem cells. Based on the specific cell lineage to be generated, iPSC must be programmed to differentiate in one of the tree embryonal sheets. To obtain GI organoids, iPSC will be differentiated into the endoderm lineage before undergoing further differentiation. The iPSC differentiation in GI organoids will mimic GI ontogenesis. Both iPSC and ASC organoids can be employed for drug discovery and to model GI diseases. GI organoids derived from ASC faithfully recapitulate the tissue of origin, including the expression of epigenetic modification, for this reason, they are particularly relevant to be employed in precision and personalized medicine. Organoids are also amenable to be cryopreserved for long-term storage, allowing the generation of valuable patients-derived organoids biobanks. Genetic changes can be introduced in stem cells from both iPSC and ASC origin and will be stably inherited by their progeny of organoids. In the future, we foresee that organoids could be transplanted into patients to fix genetic mutations. Created with BioRender.com.

To overcome this limitation, research laboratories are actively working on engineering complex coculture systems keeping into account the interaction between tissues and the environment to reproduce biological responses as physiological as possible (Salerno-Goncalves et al., 2016; Duell et al., 2011; Lei et al., 2014; Walimbe et al., 2017). Ideally, organotypic culture should be able to mimic the

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complexity of the GI more closely, as seen in biopsies or resection from human donors, without the limitations of the ex vivo culture, being this last short-living and with greater experimental variability when compared to in vitro culture. Besides expanding the capabilities of basic science research, organoid technology holds great promise for translational research with particular regard to shed light on the contribution of the epithelium in the pathogenesis of intestinal diseases. Induced PSC can be generated from minimally invasive skin biopsies and are particularly useful for the study of genetic diseases. However, it takes a lengthy (few weeks), complex, and expensive protocol to generate “mature” intestinal organoids from this source (Spence et al., 2011; Huang et al., 2019). On the contrary, enteroids can be readily generated from ASCs and can grow 3D structure with villus-like and crypt-like domains in only 1 week. Organoids from GI stem cells can be employed for disease modeling as they express both the genetic and epigenetic makeup of the tissue of origin, allowing functional testing of new drugs, relevant for patient stratification and precision medicine and to evaluate subject-to-subject variability in response to treatments for cancer (de Winter-de Groot et al., 2020), enterobacteria, viral infections (Verma, Senger, et al., 2020; Nickerson et al., 2018; VanDussen et al., 2015), and chronic diseases (Senger et al., 2018; Dotti et al., 2017; Freire et al., 2019) (Table 3.1; Fig. 3.1). Furthermore, organoids from gut, liver, and kidney can also be employed in drug toxicity studies complementing the use of ex vivo human tissues and animal testing (Clevers, 2016). Finally, proof-of-concept studies have demonstrated that organoids can be used for transplantation into animals (Fordham et al., 2013; Sui et al., 2020), opening the venue for organoids-based regenerative medicine and gene therapy (Clevers, 2016) (Fig. 3.1). In the next sections, we will address the advancement in the use of human organoids in modeling GI diseases and will present exemplary studies with relevant translational applications in precision and personalized medicine.

3.2 Current application and perspective use of organoids in precision and personalized medicine Over the past several years, precision medicine has been moving from a mere idea to a tangible reality of the medical practice. It is often used as a synonym for personalized medicine; however, distinctions can be made. In precision medicine, patients can be grouped or stratified based on genetic backgrounds that would benefit from a purposely customized therapy. As an example, carcinomas can have a very similar clinical appearance, but they can be the result of mutations in different genes, which in turn will be more sensitive to certain treatments rather than others (Tran et al., 2015). Personalized medicine or personalized care refers to a treatment designed only for one patient and

3.2 Current application and perspective use of organoids

Table 3.1 Use of organoids in the treatment of gastrointestinal (GI) diseases. GI region modeled

Disease/application

Origin (iPSC/ASC)

References

Mouth

Cancer/drug design

Patients biopsies

Salivary glands

Postradiation effect treatment/regenerative medicine Cancer/drug design

Salivary gland tissue (ASC)

Kijima et al. (2019), Tanaka et al. (2018b), Driehuis et al. (2019a) Driehuis et al. (2019a), Tanaka et al. (2018a),

Pharynx

Esophagus Stomach

Small intestine Region: duodenum

Barrett’s esophagus/ drug design Helicobacter pylori gastritis, gastric cancer/drug design, and host–pathogen interaction Celiac disease/model validation Cystic fibrosis/model validation and drug design

T. gondii/host– pathogen interaction and model validation. Enteropathogen infections: EPEC, EHEC NEC/model validation Region: ileum Colon Rectum Liver

Gallbladder

Salmonella Typhi infection/host– pathogen interaction IBD[UC]/model validation Cystic fibrosis/drug design Liver cancer/model validation/drug design Cholangiopathies including cholangiocarcinoma/ model validation

Biopsies from patients with head and neck cancer Patients biopsies (ASC) Gastric organoids (ASC and iPSC)

Patients duodenal biopsies (ASC) Patients duodenal biopsies (ASC) and iPSC-derived intestinal organoids of CF patients Intestinal biopsies (ASC)

Kijima et al. (2019), Tanaka et al. (2018b) Sato et al. (2011), Kijima et al. (2019) McCracken et al. (2014), Bartfeld et al. (2015), Huang et al. (2015b), Seidlitz et al. (2019) Freire et al. (2019), Dieterich et al. (2020) Dekkers et al. (2013), Fleischer et al. (2020), Schwank et al. (2013)

VanDussen et al. (2015), Delgado Betancourt et al. (2019)

iPSCASC from fetal small intestine Terminal ileum biopsies (ASC)

Senger et al. (2018), Finkbeiner et al. (2015) Nickerson et al. (2018)

Colon resections (ASC) Rectum biopsies from CF Patients Biopsies of primary liver cancer patients/ iPSC Biopsies of patients primary sclerosing cholangitis (ASC)

Dotti and Salas (2018) Berkers et al. (2019), Dekkers et al. (2016) Broutier et al. (2017), Takebe et al. (2013), Broutier et al. (2016) Soroka et al. (2019)

(Continued)

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Table 3.1 Use of organoids in the treatment of gastrointestinal (GI) diseases. Continued GI region modeled Pancreas

Disease/application

Origin (iPSC/ASC)

References

Pancreatic ductal adenocarcinoma/model validation/drug design

Pancreatic spheres from pancreatic ductal and acinar cells, iPSC, and ASC

Driehuis et al. (2019c), Seino et al. (2018), Dantes et al. (2020), Huang et al. (2015a), Kumar et al. (2016)

considers the influence of individuals’ genes, environment, and lifestyle to tailor specific interventions. Examples of diseases that would benefit from personalized care strategies include cystic fibrosis (CF), advanced and metastatic cancer (Dekkers et al., 2013; Howell et al., 2018; Pauli et al., 2017; van de Wetering et al., 2015), and inflammatory bowel disease (IBD) (McGovern, 2014).

3.2.1 Genetic disease 3.2.1.1 Cystic fibrosis (CF) CF is a genetic autosomal recessive disease in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Being autosomal recessive, the presence of two mutated alleles can cause symptoms. Mutations in CFTR disrupt the regulation of the chloride channel (Bartfeld and Clevers, 2017; Rafeeq and Murad, 2017), and, as a consequence, limit the efflux of water from tissues to the mucosa surface, which is necessary for keeping the mucus thin and fluid. Although CF has the most obvious symptoms in the airways, other organs can be affected, including GI, pancreas, and reproductive organs. Complications such as bacterial infections, malnourishment, and inflammation are linked to CFTR loss of function due to mucus buildup (Dekkers et al., 2013; Bartfeld and Clevers, 2017). The most frequent CFTR mutation is the deletion of phenylalanine 508 (F508), causing impairment in the folding of the protein. Nonetheless, more than 1900 CFTR mutations have been identified so far (Dekkers et al., 2013; Bartfeld and Clevers, 2017) and classified based on their outcomes (Griesenbach and Alton, 2015; Wilschanski et al., 1995) in six classes: (1) defective protein production mutations, that are associated with the most severe form of CF; (2) mutations including F508, that interfere with the protein folding and posttranslational processing; (3) mutations affecting CFTR responsiveness to its regulators; (4) mutations that hamper CFTR functionality, like ion selectivity; (5) mutations altering the correct protein synthesis; and (6) mutations affecting protein retention at the cell surface. The pairing of different mutant CFTR alleles is also frequent, presenting a wide range of symptoms and severity, adding complexity to CF’s

3.2.2 Gastrointestinal immune-related disorders

management (Griesenbach and Alton, 2015). Although CFTR-specific drugs are under intensive clinical studies, limitations in the restoring capabilities of the current therapeutics under investigation (Rabeh et al., 2012; Van Goor et al., 2009; Welch et al., 2007) and the observed subject-to-subject variability to respond to therapies suggest that the development of new compounds together with patientcentered strategies would significantly improve the management of the disease. In a seminal study, Dekker and colleagues generated organoids from CF patients’ intestine and employed them to establish an in vitro assay to rapidly test the CFTR functionality (Dekkers et al., 2013). The assay makes use of forskolin, an activator of CFTR. Upon forskolin stimulation, normal organoids undergo significant fluid accumulation in their lumen, causing the organoids to swell. Because of mutations in the CFTR gene, less swelling occurs in CF organoids (Dekkers et al., 2013). The change in volume correlates with the severity of the CFTR genotypes. As proof of concept, the authors used this test to evaluate the effect of multiple drugs and their synergistic combinations to restore CFTR activity. As a result, the study provided relevant data in support of the approval of two compounds (VX-809 and VX-770) for the treatment of CF caused by F508 mutations (Dekkers et al., 2013). Another important finding of the study was to establish that the response to drug treatments varied greatly among patients’ organoids despite similar genotypes, suggesting that other factors might be at play and further supporting the introduction of in vitro drug screening on patients’ organoids for tailored, personalized treatments. Current studies are exploiting the forskolininduced swelling assay to screen potentially effective drugs on organoid from CF patients with rare mutations and to identify clinical responders and nonresponders to treatments (Rossi et al., 2018). Further significant progress in the care of CF has been made by genetically engineering organoids to correct the CFTR gene as an alternative approach to drug treatments. Proof-of-concept studies have shown that this can be done by editing the CFTR locus in iPSC (Fleischer et al., 2020) and ASC from patients (Schwank et al., 2013), resulting in organoids with functional CFTR gene. Prospectively, these genetically modified organoids could be transplanted into the patients to rescue the genetic defect.

3.2.2 Gastrointestinal immune-related disorders 3.2.2.1 Celiac disease (CeD) Recent development of organoids from patients affected with celiac disease (CeD) has shown their relevance in better understanding some features of this condition and could potentially be employed to develop novel treatments for its care and prevention. CeD is an autoimmune disease that affects about 1% of the general population (Catassi et al., 2014). It is triggered by dietary gluten in genetically predisposed people (HLA DQ2+ and DQ8+) and causes significant

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inflammation of the small intestine leading to tissue damage, such as atrophy of the villi and crypt hyperplasia (Dieterich et al., 2020), and development of tissue autoantibodies (Caja et al., 2011). The only treatment available for these patients is rigorous and lifelong withdraw of gluten from the diet. Upon gluten-free diet (GFD) implementation, many patients fully recover from the GI injury. Nonetheless, about 10%–40% show persistent tissue damage (Lebwohl et al., 2014). The intestinal epithelium is the “target” of the inflammatory insult, but evidence suggests that it might also be involved in the earlier steps leading to the onset of the disease. Changes in intestinal permeability and antigen trafficking (Zuo et al., 2020), together with intestinal dysbiosis, have been hypothesized to be responsible for the rise of CeD and other autoimmune diseases in the past 50 years (Manfredo Vieira et al., 2018; Olivares et al., 2018). Understanding the early events occurring before CeD onset might help shed light on the activation of the adaptive immune response and identify approaches to prevent it. To explore whether organoids could help the study of CeD, our group has generated organoids from duodenal biopsies obtained from patients during the acute phase (exposure to gluten) (Freire et al., 2019). Global gene expression analysis has revealed that celiac organoids retain a gene expression profile consistent with the acute inflammatory status when compared to control organoids (Freire et al., 2019). Functional and molecular features previously identified in CeD intestinal biopsies are indeed observed in celiac organoids, including dysregulation of barrier- (Pizzuti et al., 2004; Schumann et al., 2012), innate immunity- (Forsberg et al., 2004; Levy et al., 2017; Serena et al., 2019), and stem cell-gene expression (Senger et al., 2015), suggesting that CeD organoids embody a valid model of a celiac intestine. Our observations were also corroborated by a subsequent study conducted on organoids derived from patients with CeD in remission (on a GFD) (Dieterich et al., 2020). To further validate the use of celiac enteroids, we have performed functional analysis on organoidderived 2D cultures (monolayers), providing, for the first time, evidence on the response of celiac intestine to gliadin, one of the main immunogenic component of gluten. Compared to previous work done in cell lines (Drago et al., 2006), we showed that celiac epithelium, but not healthy, builds a significant immune response to gluten. Noticeably, our study demonstrated how microbiota-derived metabolites modulate these responses (Freire et al., 2019). Finally, we have shown that the CeD epithelium alters the response of macrophages to gliadin (Serena et al., 2019), further supporting the use of patient’s derived organoids to model GI cross-talk occurring between epithelium, environment, like food antigens and microbiota, and immune cells.

3.2.2.2 Inflammatory bowel diseases (IBD) IBD is a collective term that describes GI chronic inflammatory diseases like Crohn’s disease (CD) and ulcerative colitis (UC). IBD affects about 8 million people globally (GBD 2017 Inflammatory Bowel Disease Collaborators, 2020). It

3.2.2 Gastrointestinal immune-related disorders

is characterized by chronic relapsing-remitting or continuously active inflammation of the bowel, causing fistulas (in CD) or ulcers (in both), abdominal pain, bloating, and diarrhea, with rectal bleeding in UC, sometimes accompanied by extraintestinal symptoms in liver, skin, joints or eyes. IBD frequently leads to long-term disability, surgical interventions, and high healthcare costs. Moreover, because of its common early-onset, typically between 15 and 35 years of age, significantly increases the risk of developing colorectal cancer (CRC) (Nadeem et al., 2020) and further emphasize the importance of prompt appropriate treatments. The pathogenesis of IBD involves a dysregulated immune response to dysbiotic gut microbiota (McDowell et al., 2020; Su et al., 2019). Genome-wide association studies have shown that IBD has a strong genetic component with more than 200 loci found altered in patients (Momozawa et al., 2018). While the immune system is key to its pathogenesis, the epithelium plays a pivotal role in its onset and relapse. Studies have shown that the intestinal epithelial barrier is altered in CD patients (Wyatt et al., 1993) and increased intestinal permeability can be used as an earlier indicator of clinical relapses in CD (Wyatt et al., 1993). Consistent with increased permeability, the epithelial tight junctions are defective in IBD patients (Landy et al., 2016). Tight junctions prevent free movement of bacteria and foreign antigens into the underlying lamina propria and act as gatekeepers by sealing the intercellular space between adjacent cells. Additional protective mechanisms also deficient in IBD epithelium include inefficient mucus production by the goblet cells and reduced secretion of protective antimicrobial peptides by Paneth cells (Stange and Schroeder, 2019), both contributing to epithelial barrier failure. Intestinal organoids from patients could help to understand the contribution of the epithelium to IBD pathophysiology (Schulte et al., 2019). As previously emphasized, the organoids from iPSCs are of great interest to study the effect of genetic mutation, whereas organoids from intestinal ASCs carry both genetic and epigenetic alteration relative to the site of origin (Dotti et al., 2017; Howell et al., 2018), which can be relevant in generating appropriate drug treatments for precision and personalized medicine. An example of the usefulness of organoids in IBD research are two recent discoveries of rare mutations in the genes caspase 8 (Lehle et al., 2019) and NOX1 (Schwerd et al., 2018) in young patients’ with IBD. Both studies reported that those newly identified mutations occurred in subjects no-responding to standard treatments. Organoids from the GI of patients were employed to correlate genetic mutations with reduced activity in respectively the apoptotic responses (Lehle et al., 2019) and radical oxygen species clearance (Schwerd et al., 2018). Taken together, these data suggest that organoids can be adopted as a clinically relevant tool to study the mechanism of action of newly identified genetic mutations falling into the IBD spectrum and allow the development of targeted treatments for rare forms of the disease.

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3.2.2.3 Necrotizing enterocolitis Necrotizing enterocolitis (NEC) is one of the most common life-threatening conditions affecting premature babies in developed countries. NEC has the highest incidence among very premature (28th gestational week or less) and very low birth-weight infants (Heida et al., 2017; Schindler et al., 2017), which have a 10% risk of developing the disease and high mortality rate (Neu, 2014; Neu and Walker, 2011). Despite a greater understanding of its pathophysiology, modest progress has been made over the past several decades in terms of its prevention. Evidence indicates that the premature intestine cannot handle the colonization of the microbiota occurring at birth (Nair et al., 2018), leading to abnormal immune reaction, inflammation and ultimately to intestinal necrosis. The involvement of the intestinal epithelium seems to be key as data suggest that the premature intestine does not have a fully competent intestinal barrier, leading to bacteria or bacterial antigens translocation into lamina propria, where resident immune cells build an inappropriate immune response (Neu and Walker, 2011; Hunter et al., 2008). There has been considerable effort to generate valid animal and in vitro models to study NEC pathogenesis (Lu et al., 2014). Recently, scientists have turned to develop organoids from iPSC (Finkbeiner et al., 2015) and human fetal gut (Senger et al., 2018; Fordham et al., 2013) to further investigate the connection between immature epithelium and NEC. Human intestinal organoids (HIO) from iPSC develop into intestinal epithelium with immature (fetal) characteristics. Finkbeiner and colleagues provided evidence that genes involved in the development of the digestive tract are upregulated in HIOs compared to adult tissue, similar to what is seen in fetal intestine (Finkbeiner et al., 2015), whereas genes related to digestive function and host defense are expressed at higher levels in the adult intestine and less in HIO. The authors showed that HIO develops with associated mesenchymal cells and requires implantation into a mouse host to promote maturation of the epithelium. Our laboratory has recently generated fetal enterospheres (FEnS) organoids from therapeutic aborts spanning from 11 to 22.5 weeks gestational ages (Senger et al., 2018). Global gene expression analysis has shown that FEnS from closer developmental stages express a similar gene signature. Furthermore, FEnS data sets matched the gene expression of actual intestinal fetal mucosa, validating the fetal organoids as a faithful proxy of a developing human intestine. To study the immune responses of the immature intestine to colonizing bacteria, we have exposed FEnS to nonpathogenic bacteria and their endotoxins. Our study has provided evidence that fetal organoids are not functionally equal, being only organoids from the late second trimester, a developmental age compatible with live birth, capable of responding to bacterial cues (Senger et al., 2018). In summary, these two models show significant differences and complemental advantages. Both can help in understanding the process underlying the maturation of the fetal intestinal mucosa. FEnS faithfully reproduces the characteristics of the mucosa of origin, in line with observations of other organoids derived from

3.2.3 Enteral infections

ASC and can be employed in translational applications to develop adjuvant therapies for the very premature intestine. However, because derived from therapeutic aborts can carry genetic mutations that might affect functional responses and bias conclusions. On the contrary, HIO has intrinsic more variability in its composition as it also expresses mesenchymal cells and, by requiring implantation into mouse to mature, needs more complex and expensive protocols and has less controlled growth conditions compared to in vitro systems. Nonetheless, HIO is ethically less challenging as it is not generated from aborts. More recently, Li and colleagues have generated organoids from NEC resections and age-matched controls (Li et al., 2019). The authors of this study have shown that organoids from NEC had reduced regenerative capabilities and identified some potential therapeutic use of the morphogen WNT-7 that rescued the observed defect. Taken together, these data show the relevance of using fetal organoids for designing strategies to promote normal mucosa maturation and NEC prevention, whereas NEC organoids can help in designing therapies for the treatment of the disease.

3.2.3 Enteral infections The role of human intestinal epithelial cells in the pathogenesis of many enteral infections is not entirely understood. Organoids can provide evidence on the mechanism of action underlying the initial colonization of the pathogens and how the epithelium and specific individual cell types are involved in the process. Human organoids are particularly relevant to study human restricted pathogens, like Salmonella Typhi (S. Typhi) (Nickerson et al., 2018), from which only limited models are available. Organoids can help to study emerging new viruses like SAR-CoV-2 (Lamers et al., 2020) and to design alternatives to antibiotics for the treatment of bacterial agents that have developed significant antibiotic-resistant strains (Llanos-Chea et al., 2019). Findings relative to the use of organoids in the study of exemplary enteropathogens are presented in this paragraph.

3.2.3.1 Helicobacter pylori Helicobacter pylori infection is a common global chronic condition affecting millions of patients. If left untreated, it can lead to comorbidities such as peptic ulcers, chronic gastritis, gastric cancer and gastric mucosa associated lymphoid tissue lymphoma (Kim et al., 2011). Because H. pylori is associated with such severe gastric diseases, great efforts have been devoted to elucidate the elements that drive its colonization of the gastric mucosa, including bacterial and host genetics, and environmental factors. Since its discovery, scientists have tried to develop and validate models to study its pathophysiology, and this quest has been challenging (Fiorentino et al., 2013;

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Graham et al., 2004; Taylor and Fox, 2012). Recently, researchers have turned to primary cell models to generate human gastric organoid (hGO) from iPSC (McCracken et al., 2014) and ASC (Bartfeld et al., 2015). These studies have provided evidence on the role of the virulence factor CagA in promoting bacterial colonization of gastric cells (McCracken et al., 2014) and activation of the proliferative program in gastric stem cells (Bartfeld et al., 2015). In a third study, hGO was employed to establish that H. pylori can sense urea produced by the gastric cells and use it as a chemoattractant to guide the pathogen to begin colonization (Huang et al., 2015b). Together, these studies provide evidence that hGO is a robust in vitro system for elucidating the mechanisms of H. pylori–host interaction. Expanding the study to multiple donors’ organoids might help to identify underling potential host-specific genetic loci that lead to peptic ulcer disease upon H. pylori colonization (Miftahussurur and Yamaoka, 2015). Furthermore, hGO, being validated as a physiologically relevant model for gastric mucosa can be employed for the study of human H. pylori isolates carrying antibiotic resistance.

3.2.3.2 Salmonella enterica serovar Typhi Enteric typhoid fever is a significant global health burden, with approximately 22 million cases per year and, 200,000 deaths occurring mostly in developing countries (Crump et al., 2015). It predominantly affects children and older adults and, if not treated, can reach a rate of fatality in the range of 10%–30% (Buckle et al., 2012). S. Typhi ingested with contaminated water or food colonizes the small intestine causing GI symptoms. If the pathogen escapes the immune surveillance, it can cross the gut barrier and convert into a systemic multiorgan infection causing high fever, sepsis, and ultimately death. It can also develop into a chronic infection, increasing the risks of gallbladder carcinoma (Koshiol et al., 2016). Antibiotic therapy is the first line of treatment for S. Typhi infection. However, the emergence of multidrug resistance is significantly reducing the therapeutic options for this life-threatening disease. Because S. Typhi is a human restricted pathogen, it has been difficult to study and most of the conclusions have been drawn from research done on Salmonella enterica, serovar Typhimurium, that causes similar systemic infection and Typhoid fever symptoms in mice. Studies with ex vivo biopsies and human volunteers have been also done. The limitations of ex vivo models have already been addressed, whereas research with human subjects, although ideal, has been ethically challenging. Given such reasons, the use of human organoids appears particularly relevant for the study of S. Typhi. In a proof-of-concept research, ASC-derived organoids were used to shed light on the interaction between S. enterica serovars and the epithelium. S. Typhi infection in human tissue biopsies and human intestinal enteroid-monolayers provided evidence that S. Typhi caused cytoskeletal rearrangements, microvilli destruction and encapsulation of bacteria in cytoplasmic vesicles in both models (Nickerson et al., 2018). The study also demonstrated divergence in infection modalities between serovars. As opposite to Typhimurium, Typhi did not disrupt the

3.2.3 Enteral infections

intestinal barrier and host cell viability. Furthermore, S. Typhi did not associate with microfold cells (M cells), as previously proposed by studying S. Typhimurium invasion in mice. Taken together, this study has highlighted the significance of the use of human organoids to compare species-specific responses of each Salmonella serovar, which is relevant for developing unique therapeutic strategies for their treatments. Organoids from patients were also used to explore how specific genes affect Typhi infectivity and ability to induce inflammation in the host (Verma, Prescott et al., 2020). Furthermore, recent studies made use of organoids to elucidate how the microbiota is modulating the host–pathogen interaction (Min et al., 2020). Although human organoids were not used in this last research, we foresee that more reports will focus on these relevant findings.

3.2.3.3 SARS-CoV-2 (COVID-19) The novel strain of coronavirus named “Severe Acute Respiratory Syndrome Coronavirus 2” (SARS-CoV-2), also known as COVID-19, was originally discovered in Wuhan, China, in late December 2019, as the etiological agent causing atypical respiratory distress triggered in infected people. The disease can rapidly evolve into pneumonia characterized by lung interstitial inflammation and edema and can be lethal in vulnerable people like the elderly or with underlying conditions such as obesity, diabetes, hypertension, and cardiovascular disease. In March 2020, WHO declared COVID-19 a pandemic after more than 100,000 cases were diagnosed in more than 110 countries and territories around the world (WHO, 2020). Since SARS-CoV-2 is a newly emerged disease, there is still limited understanding of the modality of spreading, which makes its circulation more difficult to control. The mechanism of action driving SARS-CoV-2 entry into cells, requires the interaction between the viral spike protein with the host angiotensin-converting enzyme 2 (ACE2) receptor and the activity of the serine protease TMPRSS2 (Hoffmann et al., 2020). While the research efforts have initially focused on the respiratory symptoms, it is now clear that SARS-CoV-2 can affect multiple organs (Wang et al., 2020). In fact, ACE2 is abundantly expressed on many cell types including airways cells (Yao et al., 2020), pericyte (Chen et al., 2020), kidney (Hassanein et al., 2020), and enterocytes (Aplcwrjas, 2020). About one in five patients that contract SARS-CoV-2 suffers with GI manifestation, like nausea or vomiting and diarrhea (Aplcwrjas, 2020). Importantly, SARSCoV-2 viral RNA has been found in fecal samples, even 2 weeks after the patient was negative to COVID-19 by nasal swabs (Wu et al., 2020), suggesting survival of the virus in the GI long after the infection of the airways has been resolved. Based on evidence that SARS-CoV-2 affects the GI, a recent study has employed human small intestinal organoids from both ileum and duodenum to investigate the modality of infection and replication of the virus in the GI tract (Lamers et al., 2020). The organoids were grown under four different culturing conditions to enrich the culture with progenitor/stem cells, secretory, or absorptive cells to

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evaluate the expression of ACE2 mRNA in these cells (Lamers et al., 2020). The study found that ACE2 was prevalently expressed in absorptive lineage rather than secretory cells. Consistently with this observation, and similar to SARS-CoV activity, SARS-CoV-2 was efficiently infecting enterocyte cells, whereas not noticeable infection of secretory cells was observed. The study also demonstrated that the intestinal epithelium permits SARS-CoV-2 replication (Lamers et al., 2020). Specifically, new viral particles were observed already 24 hours after infection, whereas the enterocytes start dying by apoptosis after 60 hours (Lamers et al., 2020). The scientists also showed that the SARS-CoV-2 infected cells activated significant type I and III interferon responses. By providing novel and insightful new data on SARS-COV-2 etiology, the study has provided evidence that SARS-COV-2 can also spread via the GI tract. The study also implies that human organoids represent faithful experimental models to study the biology of all coronaviruses in the GI tract.

3.2.3.4 Toxoplasma gondii Toxoplasma gondii is a protozoan parasite that can cause infection in humans and other mammals via the ingestion of contaminated water/food with its oocysts/ cysts. It is estimated that about 30% of the human population has been exposed to T. gondii (Flegr et al., 2014). Because of its complex life cycle and worldwide diffusion, T. gondii has been difficult to eradicate. At the present day, there is not drug able to cure toxoplasmosis. The symptoms of the infection can vary greatly, from mild to severe illness with the parasite infecting multiple organs, including muscles, brain, placenta, mammary glands, gonads, and liver. In immunocompetent hosts a robust cell– mediated immune response controls but does not clear the protozoa, thus enabling long-term parasite persistence in brain and muscle tissues. In immunecompromised people, T. gondii can reactivate and can develop into cerebral toxoplasmosis. Additionally, T. gondii infection has been linked to granuloma and necrosis of the liver as well as liver cirrhosis (Alvarado-Esquivel et al., 2011) and, when acquired during pregnancy, can lead to miscarriage, stillbirth, or damage to the baby’s nervous system and other organs (Harker et al., 2015). While we have acquired significant knowledge of the immune responses to toxoplasmosis, there is still limited data about the early events occurring in the gut tissue after oral exposure. Because T. gondii can cross every epithelial barrier, including gut and nonpermissive blood–brain barrier and placenta, great efforts have been invested to gain more insight on the modalities driving T. gondii movements across epithelia. Important insights have been acquired by studying T. gondii infection using intestinal epithelial cell lines. Barragan et al. (2005) have shown that the parasite uses the cell adhesion molecules of the immunoglobulin (Ig) superfamily to dock at intercellular boundaries of the cells, suggesting a paracellular route of transmigration across the epithelium. However, the transcellular route has also been suggested as T. gondii can parasite neutrophils encountered in

3.2.4 Gastrointestinal cancer

the gut lumen and use them as a carrier to spread to other parts of the body (Coombes et al., 2013). Although relevant, these studies have not addressed the contribution of each epithelial cells type involved in T. gondii initial colonization and modality of breaching the intestinal barrier and species-specific mechanism of dissemination. Intestinal organoids from human and animal origin could help to further shed light on these processes (Delgado Betancourt et al., 2019).

3.2.4 Gastrointestinal cancer In recent years, substantial progress has been made in terms of diagnosis and treatment of cancer patients, resulting in better survival rates and improved quality of life. Unfortunately, this does not apply to all malignances. Among GI tumors, esophageal, gallbladder and, pancreatic cancer have a grim prognosis of 5-year survival rate below 20% in the United States (Aberle et al., 2018). The pharmaceutical industry faces many challenges in developing effective and safe drug treatments. It has been estimated that a new drug requires about 10 years from initial discovery to market, high costs, and bares low success rate, as only about 12% of all the drugs entering clinical development (Phase I) will be approved (Prasad and Mailankody, 2017). As a result, new anticancer drugs have the lowest approval rate by the US Food and Drug Administration (FDA) agency (Nass et al., 2018). A disconnect between preclinical data and clinical results have been blamed a possible culprit of high failure rate, as animal and in vitro models do not provide relevant features of the TME and immune responses as occurring in patients. Another challenge is posed by the observed intra- and interpatients cancer heterogeneity caused by the high mutational rate of primary tumors and the occurrence of mutations upon repeated chemotherapy in relapsing cancers. The emerging field of precision oncology might provide some solutions to the mentioned challenges. By analyzing whole-exome data from tumors, precision oncology aims at identifying treatment designed for specific cancer’s genetic makeup. However, even when targetable genomic alterations are discovered, patients do not always respond to therapy (Pauli et al., 2017). Strategies to confirm therapeutic efficacy and/or to identify additional alternative therapeutic options before treatments’ administration would be beneficial to both clinicians and patients. In this context, patient-derived tumoroids may help to improve delivering more effective personalized anticancer therapies (Aberle et al., 2018). The following subsections will provide some exemplary studies regarding GI tumoroids characterization and their potential employment in personalized anticancer therapies.

3.2.4.1 Tumoroids in precision medicine for gastrointestinal cancer According to WHO estimates, CRC is in the top three most common cancer worldwide (WHO, 2021). Scientists have identified a handful of pathways

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including RAS-MAPK, WNT, P13K, P53, and TGF-B that, if altered, can lead to the onset of CRC and have shown proof of concept how organoids from CRC can help design personalized drug screening (van de Wetering et al., 2015). A remarkable validation of this approach has been provided by a recent study conducted on a large collection of patient-derived tumoroids from advanced cancers, including colon/rectum, esophagus, pancreas, small intestine, and many more that were not responding to first-line treatments (Pauli et al., 2017). The authors of the study implemented a workflow that combined genomic analysis of the tumor to identify significant mutations and gene copy number alterations together with high-throughput drug screening on tumoroids. Genomic analysis served to guide the selection of treatments. The study showed that colorectal tumoroid from a subject carrying mutation in KRAS was resistant to all chemotherapeutics but resulted responsive to multiple drugs already approved for CRC treatment, when cotreated with MEK inhibitors, whereas tumoroids from CRC bearing nonmutated (wildtype) KRAS alleles responded to EGFR inhibitors treatment, in combination with histone deacetylases and IGF-1R inhibitors (Pauli et al., 2017). By incorporating genomic analysis and tumoroids in the drug screening, the authors of the study concluded that single-agent treatment is not effective for the care of advanced cancers. Furthermore, tumoroids appeared to be useful to establish cancer sensitivity even when the genetic profiling of the tumor did not provide any potential target (Pauli et al., 2017). In a similar study, patient-derived organoids (PDO) were incorporated in a coclinical trial platform. The study aimed at evaluating the use of PDO from heavily pretreated metastatic CRC and gastroesophageal cancers in establishing whether tumoroids can be used as an effective predictive tool for treatment outcomes. The study compared the responses to chemotherapy in 21 patients and matched in vitro PDO. Confirming the findings of the previous study, the authors observed that PDO had a significant predictive value anticipating the results observed in the clinical setting for each enrolled subject (Vlachogiannis et al., 2018). Another remarkable finding of this study was the discovery that gastric cancer (GC) tumoroids overexpressing the gene ErbB2 exhibited strong sensitivity to lapatinib, a drug that is currently approved in combination with capecitabine to treat metastatic breast cancer but not used for GC. In addition, a GC organoid line with amplified AKT1 was the only organoid that responded significantly to two anti-AKT antibodies treatment (Vlachogiannis et al., 2018). Tumoroids derived from HNSCC have also been validated as a tool for the selection of personalized treatments (Tanaka et al., 2018b; Driehuis et al., 2019a,b). A panel of 31 HNSCC-derived organoid lines was established from the epithelium of the mouth, larynx, and pharynx, and employed to screen a set of 56 cancer-drugs and in vitro radiotherapy. Genomic analysis showed that alterations in the organoids matched the actual tumor of origin. The studies showed that HNSCC tumoroids responded to treatments used for other GI cancers targeting same mutations

3.2.4 Gastrointestinal cancer

identified in HNSCC tumoroids. Importantly, the study from Driehuis and colleagues provided evidence that HNSCC organoids were also useful to predict which tumor would benefit of radiotherapy treatment following chemotherapy (Driehuis et al., 2019a). We want to conclude this section discussing few progress on the use of organoids for the care of primary liver carcinoma (PLC) and pancreatic cancer, that are currently among the most difficult to treat tumors (Tanaka et al., 2018b; Valery et al., 2018). PLC is reported to be the sixth most common cancer worldwide, and the second most common cause of death by cancer (Valery et al., 2018; Bosch et al., 2004). Hepatocellular carcinoma (HCC) accounts for 75%–85% of PLC cases and intrahepatic cholangiocarcinoma (ICC) for 10%–15% (Valery et al., 2018). Importantly the burden of PLC is hypothesized to increase in the next 15 years globally (Valery et al., 2018). Treatment options are limited for PLC patients, and as seen for other cancers, tumoroids from PLC could be useful for testing the efficacy of much needed new drugs. Developing organoids from liver biopsies has been challenging. Primary hepatocytes have a limited proliferative capacity in vitro; they dedifferentiate easily during culture and lose their viability and function upon cryopreservation. Nonetheless, thanks to the combined efforts of multiple laboratories, it has been possible to identify the right conditions to grow hepatic organoids from normal tissue (Huch et al., 2013), iPSC (Takebe et al., 2013), and PLC, including HCC (Broutier et al., 2017; Huch et al., 2015) and ICC (Broutier et al., 2017). In a proof-of-concept study, liver tumoroids have been employed successfully for disease modeling and drug screening (Broutier et al., 2017). In line with our previous examples, the study concluded that a combined approach of tumor genotyping and high-throughput drug screening using tumoroids appear to be a valuable approach for personalized treatment of PLC. Relatively to pancreatic carcinoma, PDOs have been recently derived from pancreatic ductal adenocarcinoma (PDAC) (Driehuis et al., 2019c; Seino et al., 2018). The organoids from 30 patients have been employed to screen a panel of 76 therapeutic agents, including drugs not routinely used to treat PDAC (Driehuis et al., 2019c). The study first validated PDO by comparing the molecular signature of organoids and tissue of origin, then established the efficacy of chemotherapeutics that could effectively kill PDO. As seen for other similar drug screenings, this approach has identified multiple compounds that showed efficacy against PDO. Of note, there was no single agent that resulted effective for all PDOs, indicating that a “personalized” approach might be beneficial for the treatment of PDAC (Driehuis et al., 2019c). Based on the many examples provided in this paragraph, we can conclude that tumoroids are a robust tool to be adopted in precision and personalized oncology protocols for the cure of many kinds of GI cancers, particularly for the treatment of advanced and metastatic ones. The use of tumoroids in drug screening can improve standard clinical protocols as the efficacy of drugs are readily tested to offer alternative care when standard clinical options have already been exhausted and can support label expansion of drugs approved for the treatment of other

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cancers. Furthermore, tumoroids can provide novel insights on the biology of cancer including how individual tumors adapt to therapies. Finally, by generating larger database of drug sensitivity screening paired with tumor genetics, it will be possible to identify potential therapeutic strategies even when only genomic data are available.

3.2.4.2 Tumoroids in immuno-oncology Immuno-oncology aims at developing treatments that take advantage of the body’s immune system to fight cancer. Different approaches go under the name of immuno-oncology, including the development of blocking antibodies, genetically engineered tumor-specific T cells (CAR-T), and bulk tumor-infiltrating lymphocyte therapy (Rosenberg and Restifo, 2015). The immune system is the first line of defense against tumor growth. Nonetheless, cancer cells can escape the immune surveillance by different mechanisms. They can become “invisible” to the immune system by no longer expressing surface antigens. They can alter the expression of components involved in cancer cells apoptosis and resist to the attack of immune cells (Steven and Seliger, 2018). They can express immune checkpoints inhibitors, preventing the activation of immune cells (Pardoll, 2012). Therapies that block the immune checkpoints inhibition have been the most successful strategies in the recent times in immuno-oncology. The first successful immune checkpoint antiblocking therapy was developed against the gene products CTLA-4 for the treatment of patients with metastatic melanoma (Weber et al., 2008), followed by the development of inhibitory anti-PD-1 and anti-PD-L1 antibodies (Sahni et al., 2018). AntiPD-1/PD-L1 and anti-CTLA-4 therapies are used, respectively, alone as first line therapy or in combination with other treatments for certain CRCs (Andre et al., 2021; Casak et al., 2021). We have already mentioned that organoids express only the epithelial cell lineage if derived from ASC and epithelial and mesenchymal cells if derived from iPSC, both lacking the immune component. However, organoids technology can be successfully employed to help developing immunebased anticancer strategies. Both tumor and immune cell are very plastic and are influenced by the surrounding microenvironment. For this reason, it is fundamental to study how they interact in complex micromilieu, referred as TME. A novel colon tumoroid culture system that includes epithelial, stromal, and immune cells has been developed to explore the response to oncotherapy in relationship of TME (Finnberg et al., 2017). This system is named ALI organoids, where ALI stands for “air–liquid interface,” based on the culturing modality. ALI organoids could maintain primary tumor cells and associated TME for up to 44 days upon isolation from tissue. However, the culture could not be frozen. The authors of this research demonstrated retention of immune cells in the ALI-culture during the study, although significant changes in the type of immune cells phenotype were observed when compared to original tumor. As proof of concept, the scientists investigated the response of the tumoroid to known biologically active drugs,

3.2.5 Transplant application

demonstrating the value of this in vitro model. A similar study used a different approach to achieve a complex tumoroid culture, including organoids together with fibroblasts and peripheral blood mononuclear cells (PBMC) (Seino et al., 2018; Tsai et al., 2018). However, this approach has limitations as the use of PBMC and isolated stromal cells might not necessarily reflect the original TME. More research is needed to validate this last model.

3.2.5 Transplant application Latest developments in regenerative therapy support stem cell transplantation for the treatment of various diseases (Sun et al., 2014; Atala, 2015; Tanaka et al., 2018a). With regard of GI pathologies, there is a significant interest in studying stem cell use for the treatment of IBD patients. The Autologous Stem Cell Transplantation International Crohn’s Disease (ASTEC) clinical trial has provided evidence on the effect of conventional therapy versus a combined therapy with hematopoietic stem cell transplantation (HSCT) (Lindsay et al., 2017). The autologous HSCT treatment has been so far considered only for highly refractory CD patients when a surgical procedure is not possible. The results from the clinical trial have shown that the administration of HSCT led to both clinical and endoscopic improvements in about 44% of the treated cases. A clear mechanism of action on how HSCT helps IBD patients has not been proven. However, it is hypothesized that HSCs can reset the immune system stopping inflammation and restoring immune tolerance. Another effective approach in stem cell therapy employs mesenchymal stem cells derived from adipose tissue or bone marrow for the care of refractory perianal fistulas in patients with CD (Ciccocioppo et al., 2011; Garcia-Olmo et al., 2009). Although encouraging, the stem cell-based therapies harbor intrinsic dangers. Some patients have developed mucositis, whereas others had their symptoms worsened or did not see any improvement (Murayama et al., 1995). Furthermore, these protocols do not employ intestinal epithelial stem cells or organoids to promote the healing of the mucosa. There are no published studies attempting the engraftment of intestinal organoids to cure human GI disease. Nonetheless, successful transplantation of intestinal stem cells (ISCs) has been accomplished in mouse models of experimental colitis, demonstrating technical feasibility (Fordham et al., 2013; Fukuda et al., 2014; Yui et al., 2012). In a proof-ofconcept study, organoids were expanded in vitro and, to promote engraftment, were instilled in the intestinal lumen embedded in a collagen-based matrix (Fordham et al., 2013). With this approach, the study team could observe that organoids attached stably to damaged regions of the colon after only 3 hours. This evidence suggests that ISC transplantation could be an appealing therapeutic approach to reestablish the epithelial barrier in IBD damaged intestine.

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A relevant effort has also been devoted to the generation of transplantable functional salivary glands for patients that lost functional glands upon radiotherapy for the treatment of head and neck cancer. Salivary glands were grown in vitro from human biopsies (Yi et al., 2016). Orthotropic transplant of the gland primordia was succesfully attempted in mice; where they developed in fully functional salivary glands, demonstrating the feasibility of this approach (Sui et al., 2020; Shin et al., 2018). While promising, more research is needed to evaluate the safety and stability of organoids before clinical studies for regenerative purposes are launched in humans.

3.3 Ethical perspective on organoids biobanks We have discussed in length the great potential of organoids for the biomedical field. Based on what has been discussed, it is likely that, in the near future, organoid technology will become a more prominent tool in research and development, if not an alternative to animal models. However, the creation of organoids biobanks poses some ethical challenges. While all around the world, more biobanks of everlasting organoids are created, the ethical solutions still lag on important issues, leaving the research community without clear guidelines and creating a potential disconnect with the society at large. Organoids from patients raise ethical issues related to research subjects consent for the procurement of biomaterial with the purpose of stem cells isolation, organoids long-term storage, future use, ownership, organoids sharing, retracting consent, and recontacting the donor or family members if novel clinically relevant findings arise (Bredenoord et al., 2017). Nonetheless, organoids might offer also solutions to old ethical issues related to animal testing. The use of animals for research purposes is common practice, though it is not considered ethically neutral (Bredenoord et al., 2017). A set of values established by Russell and Burch in 1959 (Russell and Burch, 1959), described as the principle of the “3 Rs” (replacement, reduction, and refinement), has been internationally accepted to balance bioscience need for in vivo experimentation and animal’s protection. The principles propose to replace and/or reduce experimental animals with alternative models whenever possible and refining procedures to decrease animals’ pain and distress (Fernandes and Pedroso, 2017). Although commonly accepted, these principles have been applied often in conflicting ways, leading to ethical controversies (Fernandes and Pedroso, 2017). In line with these common sentiments, large institutions like the Wellcome Sanger Institute have already developed a program to promote the use of organoids to partially reduce the burden of drug testing on animals. The controversy surrounding in vivo experimentation is not limited to the scientific community but involves the society at large, with many organizations and activists fighting the use of animal testing. To align expectations among stakeholders, it will be relevant to understand how recent guidelines released by the International Society for Stem Cell Research

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(ISSCR) are perceived by both the general public and biomedical researchers (Daley et al., 2016). Prospective surveys are needed to understand how society relates to organoids-based research and how it perceives the risk and benefits of organoids’ use compared to other models (animal and human embryo). We foresee that the establishment of internationally harmonized regulatory procedural framework for living biobanks among countries and/or institutions bound by common ethical standards, will help to guard donor’s sensitive data protection and will facilitate material sharing across the international scientific community, hopefully, accelerating the development of much needed regulatory guidelines in this field of research.

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CHAPTER

Engineered stem cells combine stem cell and gene therapy approaches to move intestine therapy from bench to bed

4

Mahmoud Shaaban Mohamed1, Mahmoud I. Elbadry2 and Chao-Ling Yao3 1

Zoology Department, Faculty of Science, Assiut University, Assiut, Egypt Internal Medicine Department, Division of Hematology, Faculty of Medicine, Sohag University, Sohag, Egypt 3 Department of Chemical Engineering, National Cheng Kung University, No. 1, University Road, Tainan, Taiwan

2

4.1 Introduction Recently, stem cells have gained our attention for their potential use in regenerative medicine and engineering of tissues and organs. Stem cell properties were described by Till and McCulloch (1961). These features are the ability to “self-renew” or divide to produce more stem cells lineages and the ability to differentiate to give rise to several specialized cells. These potential features make stem cells an attractive cell source for clinical applications. The potency, or potential, of different stem cell types refers to their ability to give rise to specialized cells and can be organized as a hierarchy. Stem cells have direct clinical applications as a good substrate from which to derive regenerative cell therapies. Although the biological characterization of various nonhematopoietic stem cells (HSCs) is still in its early stages in the laboratory, therapeutic experience with HSCs suggests that other stem cell types will likely have successful clinical applications (Garate et al., 2015). The HSC has the ability to establish normal, healthy blood cells in patients following transplantation of normal blood stem cells from analog individuals treating disorders of the blood system including immune deficiency, thalassemia, and leukemia. Engineered HSC gene therapy has a potential promise for the treatment of several hematopoietic disorders, because genetic engineering of HSCs could lead to long-term correction of blood cells while avoiding the immune system response complications resulting from nonmatched transplantation. Gene therapy is a novel therapeutic tool that has unique properties compared with traditional chemical or small molecule drugs (Garate et al., 2015). The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00006-0 © 2021 Elsevier Inc. All rights reserved.

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While chemical drugs are most often targeted at modulation of the activities of proteins that are crucial in the development and progression of diseases, gene therapy can directly transfer therapeutic genes to the areas affected by disease via various vector systems and can promote expression of desired proteins using endogenous transcription and translational systems (Joo and Lee, 2018). Stem cell engineering requires the introduction of new genetic material into a cell, which is called stem cell genetic engineering (SCGE). Stem cells can be another way delivering therapeutic genes. Stem cells have been considered as novel sources that can provide exogenous additional regeneration potential to damaged tissues in patients because they have self-renewing abilities and can differentiate into various functional cell types. One specific type of stem cell engineering is reprogramming by conversion of somatic cells into induced pluripotent stem cells (iPSCs). The conventional method to introduce a therapeutic gene into stem cells involves the use of a vector derived from a certain class of virus called retrovirus. SCGE technology gives promising scientific results with genetically modified stem cells (Hacein-Bey-Abina et al., 2003; Bago et al., 2016). A recent significant progress in the stem cell field has demonstrated ability to transform many human and mouse cell types—including adult skin cells—into pluripotent stem cells.

4.2 Genome editing In general, genome editing strategies include DNA modifications in living organisms; these involve “beneficial” deletions, changes via gene replacement, insertions, and corresponding tools have been established for achieving these modifications in stem cells during the recent years (Chen and Goncalves, 2018). Genome modification technologies based on common engineered nuclease-based platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, and clustered regularly interspaced short palindrome repeats (CRISPR) in combination with Cas9 RNA-guided endonucleases, use the endogenous DNA repair system to easily target any genomic location (Li et al., 2019). Gene editing involves the insertion or deletion of DNA sequences to the entire original gene. This involves the delivery of DNA, modified DNA sequences, or linear single-stranded vectors that are homologous to the target gene sequence with the exception of the base or bases intended for alteration. Genome editing is recently used in stem cell basic biomedical research and therapy of several disorders, for the determination of potential therapeutic targets, and for the development of promising therapeutic agents (Qi, 2017). Successful use of stem cell gene therapy requires that modified genes delivered to stem cells persevere in the self-renewing stem cells, and in their mature and differentiated lineage. This requirement for transgene perseverance is significant because: (1) transgenes present only temporally in stem cells undergoing

4.4 Therapeutic applications of gene-edited stem cells

self-renewing proliferation will be of short-term therapeutic use; and (2) expression of the reformative gene likely extends its effect in the differentiated lineage, which may be huge number of cell divisions downstream of the genetically modified stem cell. Transgene perseverance can be completed, either by integration in to one of the existing chromosomes, or by combination of the transgene in an artificial human microchromosome (Garate et al., 2015).

4.3 Genome editing of stem cells Stem cells are a suitable platform for genome editing strategies due to their selfrenewal capability and ability to secrete endogenous proteins, such as coagulation factor IX, VEGF, FGF-b, Ang1, and sRAGE (Hoke et al., 2012; Lee et al., 2019). However, it has been reported that high passage number affects the self-renewal ability of stem cells (Crisostomo et al., 2006). Thus it is critical to develop therapeutic genome-edited stem cell lines that have most of the safety requirements. Furthermore, these cells should have high efficacy, quality, and reproducibility. Human iPSCs have been another considerable improvement for genetic research in human cells. Their ability of self-renewing allows them to be gene targeted, cloned, genotyped, and expanded. Successfully targeted iPSC clones can then be differentiated into several cell types to determine the effects of the induced mutations. The ability to easily alter human iPSCs also holds enormous clinical promise for generating synthetic organs and safer gene-based therapies. However, while immortalized human cancer cell lines have been modified with almost high efficiency (Fu et al., 2014), much lower success rates have been achieved in human iPSCs (Mali et al., 2013).

4.4 Therapeutic applications of gene-edited stem cells Currently, several types of gene-edited stem cells are being used for therapeutic applications. Stem cells can be divided into three major groups based on their origin: adult stem cells, embryonic stem cells (ESCs), and iPSCs (Leventhal et al., 2012; Lee et al., 2020). Among these, HSCs, amniotic mesenchymal stem cells (MSCs), iPSCs and human ESCs, and cardiomyocytes, neural, skin, and muscle cells derived from iPSCs and human ESCs have been heavily studied as therapeutic tools.

4.4.1 Adult stem cells Adult stem cells are lineage-limited tissue-specific stem cells, with a limited capability to differentiate into different functional cell types. Because they are stem cells, they spontaneously tend to have high self-renewal ability. Due to these

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properties, several types of adult stem cells have been involved in gene editing based therapeutic studies (Lee et al., 2020). HSCs and progenitor cells (HSPCs): Recently, several studies aimed to provide evidence that gene-edited HSPCs have high therapeutic potential in the area of blood and immune system diseases in mice. Most of the therapeutic studies using gene-edited adult stem cells are depending on the use of HSPCs, as they can differentiate into all mature blood cell types and can be harvested. Besides, HSPCs play a critical role in the production of various types of blood cells with different functions during hematopoiesis (Rieger and Schroeder, 2012). Meanwhile, it has been supposed that genetic modifications, such as mutations and epigenetic alternations, are responsible for the pathogenesis of malignant hematopoietic disorders, such as myelodysplastic syndrome, myeloproliferative neoplasms, and leukemia (Goldman et al., 2019). However, the precise mechanism how these genes coordinate normal or abnormal hematopoiesis is not completely reviled. Therefore current advances in genome editing tools can increase our knowledge of the functional effects of gene loss. For example, using the Cas9-sgRNA RNP system, genes encoding EED, SUZ12, and DNMT3A were successfully depleted in mouse and human HSPCs. The disordering rate was greater than 60% in mouse and at least 75% in human HSPCs; this led to the clarification of the role played by the genes of interest in disease development (Gundry et al., 2016). Gene editing based stem cell therapy is significantly fit to other diseases caused by single base pair mutations, such as sickle cell disease (SCD) and β-thalassemia. SCD is one of the most popular genetic disorders that cause anemia and multiorgan defects because it affects hemoglobin, which delivers oxygen throughout the body. SCD is initiated by a single point mutation at codon 6 of the human β-globin-coding gene, in which adenine is replaced by thymine, causing alternations in the amino acid sequence of hemoglobin (Inusa et al., 2019).

4.4.2 Mesenchymal stem cells MSCs represent a major adult stem cell population of diverse origin inside the human body and play critical roles in stem cell based therapies due to their abundance, low oncogenic features, moderate potential of differentiation into other cell types, and paracrine effects (Fitzsimmons et al., 2018). Despite randomized controlled clinical trials have not yet determined their safety and efficacy, wild-type MSCs have been widely used for treating tissue injury or inflammationrelated diseases, such as myocardial infarction, graft versus host disease, liver cirrhosis, osteoarthritis, or rheumatoid arthritis (Wei et al., 2013). MSC-based stem cell therapy has been developed for several decades, and some efficacy has been demonstrated if the MSCs are allowed to differentiate into an apposite cell type or applied to a relevant disease. Clinical trials using MSC-based therapy are limited, and most have failed due to the limited viability of these cells in pathological lesions and to safety issues (e.g., arrhythmias). For example, only 1% of

4.4 Therapeutic applications of gene-edited stem cells

MSCs were observed in inflamed hearts 4 days after transplantation (Toma et al., 2002). Therefore it is critical to evolve a strategy that can afford the survival of the transplanted cells and lead to efficient MSC-based therapies.

4.4.3 Embryonic stem cells ESCs are pluripotent stem cells with indefinite self-renewal capacity and the capability to differentiate into any cell type in the body. According to the NIH database, approximately 60 ESC-related clinical trials are either ongoing or have been completed. Though the importance of ESCs, the number of trials is extremely low because of the ethical problems raised in many countries (Ismail, 2015). Thus ESC-related research now aims to identify more efficient tools for the development of stem cells from human embryos (Shand et al., 2012). Previous study conducted a study to develop an imaging reporters and suicide genes (human somatostatin receptor type 2 and human sodium iodide symporter) for the of observation cell survival during transplantation of ESC-derived cardiomyocytes (Collins and Gottlieb, 2018). However, in this study, while human ESCs harboring a radionuclide imaging reporter gene was created using ZFN tools, no improvement in myocardial infarction was observed. However, this study contributed to the clinical translation of stem cell reporter gene-based imaging and our understanding of the safety transition mechanism regulating undesired teratoma development during cell therapy. The combination of the eminent pluripotency and elevated proliferation of ESCs with the specificity of genome editing can lead to highly efficient stem cell therapies for many incurable diseases once ethical and safety issues are override.

4.4.4 Induced pluripotent stem cells iPSCs are pluripotent stem cells derived from the reprogramming of adult somatic cells using specific pluripotent genes, such as OCT4, SOX2, c-MYC, and KlF4 (Takahashi and Yamanaka, 2006). iPSCs are eligible substitutes to replace ESCs in terms of pluripotency and differentiation abilities. Besides, iPSCs are potential cell sources for disease model development, drug development, and cytotoxicity research as they may originate from patients (Takahashi and Yamanaka, 2006; Omole and Fakoya, 2018). In addition, iPSCs are more frequently being used in biomedicine field as they have the same properties as ESCs, while their use is ethically acceptable. Until now, more than 100 iPSC-based therapies have been successfully recorded in clinical trials and yielded positive outcomes in the preclinical animal studies. Progress in iPSC and programmable nuclease-based methods have synergistically improved the power of gene editing based stem cell therapies and hold the promise for big discoveries. In several studies, CRISPR methods have been used to alter iPSCs. This involves a therapeutic study on hemophilia A (classical hemophilia) using

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genetically modified iPSCs. Hemophilia A is considered as an X-linked genetic and bleeding disease caused by the lack of functional coagulation factor VIII. Repeated injection of recombinant factor VIII is the current therapy for patients with hemophilia A. However, this is not the best treatment because some of the patients develop anti-FVIII neutralizing antibodies after receiving the injections. Besides, the need for repeated injections and the correlating high cost of the therapy are economic and quality of life load for the patients. On the other side, gene-edited stem cells can result in lifelong change. Subsequently, FVIII geneedited stem cell therapy provides the potential for significant enhancement over the conventional methods for curing hemophilia A (Park et al., 2015). Moreover, the metabolic disease mucopolysaccharidosis has been targeted using CRISPRedited iPSC therapy. In this study, they reported iPSC illation and ex vivo gene editing using a mouse model of mucopolysaccharidosis type 1. In this context, they edited the gene encoding α-L iduronidase in deficient iPSCs using the CRISPR/Cas9 system. The authors used CRISPR to deplete the neomycinresistant gene cassette in exon VI of the α-L iduronidase gene of deficient iPSCs and repaired the gene with donor DNA template without any indel mutation (Miki et al., 2019). The pluripotency and differentiation capability of iPSCs offer them major players for drug discovery studies.

4.5 Gene-edited stem cell therapy for intestinal diseases 4.5.1 Intestinal diseases Intestinal failure (IF) could be a rare clinical case that ends up in patients’ failure to afford normal growth and nutritional and hydration status without the use of parenteral nutrition (PN) (Wang et al., 2006; Vargas, 2013). The patients with IF can be categorized according to the small bowel length. Short bowel syndrome (SBS) is the most known reason of IF, and it usually results from inclusive bowel resection, considerably due to necrotizing enterocolitis, congenital anomaly (e.g., gastroschisis or intestinal atresia), and ischemia from malrotation and pathology (Vargas, 2013). In adults and older children, SBS is usually linked to Crohn’s disease, leading to acute transmural inflammation that requires surgical resection. Therefore SBS-associated IF occurs across the age spectrum and is mostly related to prematurity and polygenetic diseases. On the other hand, patients with nonSBS type of IF have normal or lower small bowel surface area in spite of a normal-length bowel. Besides, a growing number of congenital diseases of the gut epithelial tissue result in a generalized loss of nutrient absorption ability. Amongst the diseases that change the villus crypt axis and result in a decrease in surface area are microvillus inclusion disease (Canani et al., 2015) and congenital tufting enteropathy (Salomon et al., 2011; Sivagnanam et al., 2008). Moreover, many diseases that change intestinal endocrine cells differentiation or function can cause IF despite of a normal surface area (Goulet and Ruemmele, 2006;

4.5 Gene-edited stem cell therapy for intestinal diseases

Ruemmele et al., 2006). Patients with nonepithelial types of IF also include a wide group of those with severe dysmotility diseases, such as Hirschsprung’s disease and chronic intestinal pseudo-obstruction syndrome that have diverse abnormalities in smooth muscle and enteric neuronal cell differentiation (Ruemmele et al., 2006). Correspondingly, an extending number of immunologic diseases, such as IPEX (immune dysfunction, polyendocrinopathy, enteropathy, X-linked), can result in IF, and immunomodulatory treatment or bone marrow transplantation are effective in a limited number of patients (Canani et al., 2015). Most non-SBS types of IF are caused by a monogenic disorder, which may be applicable to stem cell-based therapy. The current management of patients with IF includes close dietary amendment of PN and enteral feeds, and surgical interference. PN is the cornerstone of treatment for patients with IF; however, its long-term use is linked to several considerable limitations, including IF-associated liver disease (Lacaille et al., 2015), catheter-related infections (Raad et al., 2007), and central venous thrombosis (Vidal et al., 2014). Furthermore, in some patients with specific lineaments that may limit survival, allogeneic intestinal and liver transplantation is significantly a life-saving therapeutic alternative option (Kubal et al., 2015). However, intestinal transplantation is associated with a high mortality; more than half the patients die awaiting transplantation and the 5-year survival of patients undergoing transplantation is approximately 60% (Smith et al., 2014). To relieve the risk for allograft rejection, lifelong potent immunomodulatory agents with parochial therapeutic index are required; thence, these therapies not suddenly lead patients to severe recurrent infections and the risk for various posttransplant malignancies (Collu et al., 2012; Sasai, 2013). Therefore the development of alternative cures is needed to improve the long-term management strategies in patients with IF. Intestinal stem cell (ISC) research has made potential progress in improving the understanding of the unique cell populations involved in self-renewal and differentiation during homeostatic and stressed conditions. Different methods can be utilized to isolate and expand both human somatic ISCs and iPSCs in intestinal organoids that recapitulate the native epithelium and can be grown on various extracellular environments in a two-dimensional (2D) or three-dimensional (3D) culture systems (Mohamed et al., 2014; Jabaji et al., 2014).

4.5.2 Intestinal stem cell therapy for intestinal diseases By supplying the requirements for the maintenance of ISCs, intestinal crypts can build marvelous structures under 3D culture systems, which consist of a lumen surrounded by a villus-like epithelial layer with budding crypt-like domains (Sato et al., 2009). These intestinal epithelial tissue-like structures are predominantly called budding organoids (BOs) and maintained in long-term culture by sequential passaging with self-renewing cell divisions of ISCs, differentiation into different functional cells on the villi, and expulsion of apoptotic cells into the central lumen (Sato et al., 2009). Moreover, these BOs have potential for reconstitution

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of intestinal crypt villus units after transplantation into the injured colon (Fukuda et al., 2014). Similar to crypts from the adult intestine, fetal intestinederived progenitor cells (FIPCs) are able to form single-layered epithelial tissuelike structures under 3D culture conditions. However, FIPCs form spherical organoids (SOs) without crypt-like budding structures, even under the culture conditions established for BOs with epidermal growth factor (EGF), Noggin, and R-spondin1 (designated ENR) (Fordham et al., 2013). To promote the generation of BOs from FIPCs, exogenous Wnt stimulation is required during passaging, although its subsequent exclusion further accelerates BO formation and allows continuous generation and maintenance of BOs in cultures with ENR. FIPCderived SOs were reported to contribute to regeneration of the adult colonic epithelium after transplantation in an induced colonic injury model (Fordham et al., 2013). Thus the intestinal organoid culture system can be a feasible method for investigating and treating intestinal diseases. However, it is not easy to collect the intestinal tissue fragments required as sources of BOs and SOs from living adult donors and unborn children, respectively. Stem cell therapy is a favorable alternative tool to suppress the current limitations of IF treatments, but genetic alternations of ISCs will be indispensable to fully achieve their potential. Despite the use of recent culture systems, gene editing of ISCs is boosted by the highly amenable and reproducible culture of intestinal epithelium. The clonogenic capacity and rapid expansion of spheroids should allow culture of genetically identical populations of intestinal epithelium. However, optimal strategies to expand human ISCs, remove native in situ ISCs while maintaining a receptive niche, and optimally engraft genetically manipulated ISCs have yet to be described. If successful engraftment of modified ISCs into the removed small bowel niche of IF patients is possible, autologous ISC transplantation will be a favorable treatment choice.

4.5.3 Gene editing in intestinal enteroids Viral and nonviral gene-editing systems have been demonstrated. However, optimization has not been demonstrated for the use of human ISCs in gene-based therapy (Nayerossadat et al., 2012). Genome-integrating viral vectors are usually featured by highly efficient and long-term transgene expression. Meanwhile, safety issues do exist, and current lentiviral constructs have a reduced frequency of integration into sites that display to cancer (Koo et al., 2011). Spheroids can be transduced by lentiviruses, which allow the manipulation of genes within these cells, but the transduction of enteroids is confined (Van Lidth de Jeude et al., 2015). Although nonintegrating viruses, such as adenovirus, are also highly efficacious in transduction and safer, they can provide only a limited period of transgene expression (Engelhardt et al., 1993). Previous studies have showed that the reprogramming technology can allow the conversion of differentiated somatic cells into other differentiated and functional cell types, including cardiomyocytes, neurons, and hepatocytes (Sekiya and Suzuki, 2011).

4.5 Gene-edited stem cell therapy for intestinal diseases

Previous study showed that four transcription factors, Hnf4α, Foxa3, Gata6, and Cdx2, can directly reprogram mouse fibroblasts to be converted into FIPCs. These induced FIPCs (iFIPCs) form could SOs that develop into adult BOs containing cells with ISC features. The resulting stem cells produce all intestinal epithelial cell lineages and can proceed into self-renewing cell divisions. After transplantation, the induced spherical and BOs can reconstitute colonic and intestinal epithelial tissues, respectively (Fig. 4.1) (Dutta et al., 2017). Furthermore, the same integration of four defined transcription factors can also induce human iFIPCs. Therefore this developed system may be applicable for disease analysis and therapy development (Miura and Suzuki 2017). Genome editing with specific nucleases to edit DNA facilitated gene knockouts, generated tissue-specific cell lineage reporters, overexpressed genes from defined loci, and introduced point mutations in stem cells (Hockemeyer et al., 2009). The CRISPR/Cas9 site-specific nuclease technology for genome engineering has significantly improved. CRISPR/Cas9 editing was used to alter the CFTR locus by homologous recombination in cultured ISCs from cystic fibrosis patients. The altered allele is expressed and completely functional, as measured in the expanded enteroids. This study provides an evidence for gene editing by

FIGURE 4.1 Engineered intestinal organoids. Schematic diagram of organoid-based gene therapy. Recent genome editing techniques including CRISPR/Cas9 editing have been successfully used to repair disease sites in cultured intestinal stem cells from human patients with a genetic defect. The current progress establishes the organoid culture system as a talented tool for adult stem cell based gene therapy to treat human diseases in the intestinal tract. Source: Created with BioRender.com.

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homologous recombination in ISCs derived from patients with a monogenic abnormality. However, two main limitations remain for the application of genetically edited intestinal enteroids in clinical application: the development of ISC removal and engraftment methods and the settlement of the safety concerns (Schwank et al., 2013). The homeobox gene Cdx2, a homolog of the Drosophila gene caudal, has been involved in the control of differentiation in the intestinal epithelium. Previous study showed that mice with inactivated allele of the Cdx2 gene developed multiple intestinal polyp-like lesions that did not express Cdx2 and that contained areas of squamous metaplasia in the form of keratinizing stratified squamous epithelium, similar to that exist in the mouse esophagus and forestomach. Colonic lesions from 98 Cdx2 1 / 2 mice have been observed and reported that the lesions are composed of heterotopic stomach and small intestinal mucosa. Therefore Cdx2 guides endodermal differentiation toward a caudal phenotype and those inadequate levels of expression in the developing distal intestine resulting in homeotic transformation to a more rostral endodermal phenotype, such as forestomach epithelium that does not express Cdx2 during normal development (Beck et al., 1999).

4.5.4 Engineered organoids for colorectal cancer Intestinal organoids derived from tumor and matched normal epithelial tissues can suitable research tools for tumor biology. Therefore this is one of the most highlighted improvements in organoid research and the capacity to manipulate the genomes, transcriptomes, and epigenomes of normal epithelial organoids to study the role of specific changes in the progress of tumorigenesis. Murine organoid culture system was first used to track the early stages of tumorigenesis. Li et al. (2014) adopted the air-liquid interface (ALI) culture system combined with genetically modified mouse model and the retrovirus-mediated delivery of shRNA constructs, to simulate a multistep tumorigenesis in organoids derived from digestive tract, including the colon, stomach, and pancreas. In addition, pancreatic and gastric organoids displayed dysplasia as a result of expression of KrasG12D, p53 loss or both. Meanwhile, colonic organoids needed Apc, p53, KrasG12D, and Smad4 mutations for malignant transformation to invasive adenocarcinoma-like phenotype. All engineered organoids exhibited histological characteristics similar to adenocarcinoma after subcutaneous implantation was performed to immunocompromised mice (Drost et al., 2015). Recent study aimed to introduce a paradigm of multistep tumorigenesis of conventional CRC, which is characterized by chromosomal instability (CIN). Drost and colleagues (Matano et al., 2015) demonstrated CRISPR-mediated knock-out of the tumor suppressors APC, TP53, and SMAD4, conjugated with CRISPR-mediated knock-in of the oncogenes KRASG12D to model multistep tumorigenesis. Subsequently, after the selection based on niche factors in the culture media, cultures of organs were successfully built with complex oncogenic multigene modules that contain up to four

4.5 Gene-edited stem cell therapy for intestinal diseases

contemporaneous changes. Organoids which express AKST factors (APC, KRASG12D, SMAD4, and TP53) could grow without stem cell niche factors such as Wnt-3, R-spondin-1, and EGF. Moreover, AKST organoids were capable to produce tumors with features of invasive carcinoma upon subcutaneous implantation into immunocompromised mice. Recent study (De Sousa e Melo et al., 2017) applied a similar way to model tumorigenesis by inserting an additive CRISPR-mediated knock-in of the oncogene PIK3CAE545, together with AKST. Both studies demonstrated that organoids with APC and TP53 mutations exhibited significant aneuploidy, which is the hallmark of the CIN pathway. The application of xenotransplantation of engineered colorectal tumor organoids facilitated the study of cancer stem cells in vivo (Fumagalli et al., 2017) and results in metastatic disorders, making organoids a potential research tool for metastasis deep studies (Cancer Genome Atlas Research Network, 2014). De Sousa e Melo et al. (2017) involved CRC mouse with the Lgr5DTR/eGFP allele which lead to new animal models carrying Apc and KrasG12D. In addition, these models express a diphtheria toxin receptor fused to an eGFP under the endogenous regulatory region of Lgr5, which allowed specific abstraction and visualization of Lgr5-positive stem cells. The deletion of cancer stem cells resulted in the significant reduction of liver metastases. Furthermore, primary tumors did not recede, indicating that Lgr5-positive cancer stem cells are indensible for metastasis. In addition, an animal study which used (Nanki et al., 2018) orthotopically transplanted CRISPR-mediated KRAS, APC, TP53, and SMAD4 comutated human colon organoids into mice, determined that metastases to the liver and lungs occurred in 44% of the mice. Almost no metastasis occurred when organoids carrying mutations in only three of these four genes were transplanted to mice; however, the absence of the fourth mutation could be overcome by providing the niche factor upstream of the absent mutation.

4.5.5 Gastric cancer Gastric cancer is classified into four molecular subtypes according to deep sequencing: Epstein-Barr virus positive, MSI, CIN, and genomically stable (Nanki et al., 2018). Recent report (Seidlitz et al., 2019) used organoids to describe the genotype-phenotype linkage in gastric cancer. Phenotype analyses of organoids derived from gastric cancer patients indicated multiple genetic and epigenetic ways to give Wnt and R-spondin niche independency. They showed that induction of RNF43 and ZNRF43 mutations was enough for gastric cancer organoids to gain R-spondin independency. The phenomenon was then advocated in CRISPR-Cas9 engineered gastric organoids. In addition, another study (Seidlitz et al., 2019) generated human and murine gastric cancer organoids which restated the typical features and altered pathways of each of four molecular subtypes of gastric cancer. The combination of organoids and CRISPR-Cas9 technologies promotes research on the molecular mechanisms of gastric cancer tumorigenesis and

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progression, thereby accelerating the development of preclinical gastric cancer models for novel drug development and personalized medicine.

4.6 Conclusion and future prospects The use of PN and allogeneic intestinal transplants improves the survival rates in patients with IF. However, ongoing treatments have serious long-term drawbacks. The current evolution of new methods for expansion of human ISCs and iPSCs facilitated the in vitro culture and reconstruction of cellular structures that resemble the native bowel. Furthermore, when intestinal enteroids were transplanted into appropriate donor settings, they can be developed into an intact mucosal epithelium with all functional cell types, and the proper ECM facilitates the growth capacity of the mucosal epithelium. Conclusively, the evolution of innovative procedure to genetically modify stem cells, including CRISPR/Cas9, has made the use of genetic engineering in stem cell field possible and provides new openings to accomplish autologous stem cell transplantation for IF cases with severe monogenic enteropathies. Although the current procedures used to eliminate abnormal mucosal epithelium, dosing and optimal expansion of ISCs, the substitution of abnormal epithelium with rebuked epithelium emerging from gene editing of autologous stem cells is a possible therapeutic advance. Furthermore, genetically engineered stem cells based therapy opens a new hope for the IF patients.

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Role of CRISPR/Cas9 and other gene editing/ engineering technology in intestine diseases

5

Yiyi Yang1, and Xiaowen Cheng2, 1

Department of Experimental Medical Science, Experimental Neuroinflammation Laboratory, Lund University, Lund, Sweden 2 Department of Medical Biochemistry and Microbiology, SciLifeLab Uppsala, The Biomedical Center, University of Uppsala, Uppsala, Sweden

5.1 Brief introduction of CRISPR-Cas9 A tremendous effort has been made over decades to develop technology to edit the genome. Genomes are composed of billions of DNA bases and contain all of the genetic information from organisms. The possibility to change DNA bases at precise sites sheds light on many perspectives, not only molecular biology but also applications in medicine and agriculture. Genome editing tools have come to the revolutionary point when CRISPR-Cas9 was discovered and developed by several pioneers. It is known as a system consisting of a short guide RNA (sgRNA) which could lead Cas9 protein to specific locations for editing the genome. It becomes a robust, simple and widely used approach in the scientific community. Compared with the other tools, zinc finger nucleases (ZFNs), and transcription activator-like effector (TALEN) proteins, CRISPR-Cas9 is the easiest and the most flexible tool to apply for different species (Adli, 2018). And the editing efficiency is the same or higher than the other methods. CRISPR is short for clustered regularly interspaced short palindromic repeats. These repeats were first described in 1987 by Nakata and colleagues in Escherichia coli (Ishino et al., 1987). However, the importance of these repeats has not been revealed until 2005 when several remarkable findings were published. First, CRISPRs were found consistently conserved in bacteria and archaea (Mojica et al., 2000). Second, the nonrepeating DNAs between these repeats belong to viruses and other mobile genetic elements (Bolotin et al., 2005; Pourcel et al., 2005; Mojica et al., 2005). Later CRISPR-associated (Cas) genes were found to encode proteins with putative nuclease and helicase 

Contributed equally to this chapter.

The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00011-4 © 2021 Elsevier Inc. All rights reserved.

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domains (Jansen et al., 2002). Following scientists proved the CRISPR-Cas system as a mediator in the adaptive immune system to fight against the virus in Streptococcus thermophilus (Barrangou et al., 2007). The mechanism of CRISPR-Cas9 has been investigated by several groups. Working CRISPR-Cas loci have three main components: (1) a CRISPR array of identical repeats, (2) invader DNA-targeting spacers that encode the CRISPR RNAs (crRNAs), and (3) an operon of Cas genes encoding the Cas protein components (Fig. 5.1A). Interestingly, the identical repeats are inserted between invader DNAtargeting spacers, which can be inspected by viruses to find their hosts in nature (Andersson and Banfield, 2008; Sun et al., 2013). In bacteria, adaptive immunity takes place when a short sequence of component II is introduced into component I. Afterward transcription of precursor crRNA is under a process of maturation to generate individual crRNAs. Each of these has a repeat part and an invader targeting part. Matured crRNA will then direct Cas proteins to the sites complementary to the crRNA spacer sequence and cleave foreign nucleic acid. This is a fighting strategy of bacteria to generate a lethal cause for invaders.

FIGURE 5.1 CRISPR-Cas9 system. (A) The Cas gene operon with tracrRNA and the CRISPR array. (B) The pathway of antiviral defense is associated with Cas9 and the tracrRNA:crRNA complex, RNA coprocessing by ribonuclease III. (C) DNA cleavage by guided RNA (short guided-RNA). Modified from Doudna, J.A., Charpentier, E., 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346 (6213), 1258096.

5.2 CRISPR/Cas 9 application in colorectal cancers

More studies have been conducted to further understand the CRISPR system. Some essential findings have paved the way for it to become a game-changing genome editing tool. One of the critical findings is the protospacer adjacent motif (PAM). PAMs on the invading DNA were found at regions adjacent to the crRNA-targeted sequence and that this sequence is required to work together with the base-paired crRNA sequence in the process of DNA target recognition (Jinek et al., 2012; Gasiunas et al., 2012). There are two classes of CRISPR/Cas systems using diverse molecular mechanisms to succeed in tasks of nucleic acid recognition and cleavage. Class 1 is widely found in Archaea including type I and type III CRISPR systems. Class 2 contains type II, IV, V, and VI (Andersson and Banfield, 2008). However, the type II system is the simplest one using only one protein Cas9 to guide recognition and cleavage, rather than a large complex of Cas (Garneau et al., 2010). Another important finding is the critical role of transactivating (tracrRNA) in the maturation of crRNA found in S. thermophilus published by Charpentier and her colleagues in 2011 (Deltcheva et al., 2011). This small RNA is localized upstream of the type II CRISPR-Cas locus and mediates in a sequence-specific manner with ribonuclease III and Cas9 for activation of crRNA maturation (Fig. 5.1B). In the following year, the CRISPR/Cas9 was shown as a programmable dual-RNA-guided DNA endonuclease to cleave DNA using a duplex of tracrRNA:crRNA (Jinek et al., 2012). In addition, people found this system is transferable to use in other organisms and a fusion of tracrRNA and crRNA, called sgRNA was assigned to guide cleavage of CRISPR-Cas9 at target sites (Fig. 5.1C) (Jansen et al., 2002). This tool has grown rapidly during these years and provides a large potential for various genome-editing purposes. Different CRISPR systems are under investigation and beyond genome editing, it could also regulate target genes, play a role in epigenetics and chromatin manipulation. Using this powerful application, one also needs to pay considerable attention to the safety and ethical issues to approach this technology to the whole society. The impact of CRISPR-Cas9 is huge and a new generation of it is on its way. The transformation of research results to clinical and other fields will be beneficial to all humankind.

5.2 CRISPR/Cas 9 application in colorectal cancers Colorectal cancer (CRC) is the third most deadly and fourth most commonly diagnosed cancer worldwide, with approximately two million new cases and one million mortality in 2018, according to GLOBOCAN 2018 data (Bray et al., 2018). CRCs have been considered as the transformation of normal epithelium to benign adenoma and ultimately to an invasive and metastatic tumor. The initiation and development of CRCs involve multiple steps accumulation of gene mutation. For example, over 80% of CRCs contain mutations in the gene adenomatous polyposis coli (APC), which is a tumor suppressor by negatively regulating the

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Wnt pathway (Cancer Genome Atlas Network, 2012). In addition, mutations in driver genes such as kirsten rat sarcoma 2 viral oncogene homolog (KRAS), ß-Raf proto-oncogene serine/threonine-protein kinase (BRAF), SMAD4, and TP53 also contribute to the invasion and metastasis of CRC (Eklo¨f et al., 2013; Fleming et al., 2013). It is essential for CRC research to identify such mutations, and assess the results of modification on these mutation. CRISPR/Cas9 becomes a powerful functional genomics tool to target and modify the genomic sequence, therefore providing insight into this technology in CRC oncogenic research, in vitro and in vivo, will be beneficial. This gene editing technique has been applied in many CRC cell lines (Blanas et al., 2018; Li et al., 2016; Wenzel et al., 2020). Using the de novo gene expression system called CRISPR/dCas9-VPR, Blanas et al. have transcriptionally activated the fucosyltransferase 4 (Fut4) and Fut9 gene in the MC38 cell line (a murine CRC cell line lacking expression of the Fut4 and Fut9 enzymes) (Blanas et al., 2018). They reported induction of these two genes results in specific neoexpression of functional Lewis-antigen on the cell surface. The Lewis expression further influences the core fucosylation, sialylation, and antennarity, as well as the subtypes of N-glycans on MC38-glycovariants. Another group, Li et al., utilized the CRISPR/Cas9 system to generate and target partitioning defective 3-like protein (Par3L) mutations in Caco-2 cells (a human CRC cell line) (Li et al., 2016). They observed that the depletion of Par3L led to suppressing Lkb1/AMPK signaling cascade causing higher sensitivity to chemotherapies and irradiation. Moreover, Wenzel and his colleagues utilized the CRISPR/Cas9 system to inactive transcription factor 7 like 2 (TCF7L2) in different CRC cell lines including HT29, HCT116, and LoVo (Wenzel et al., 2020). TCF7L2 is an essential transcription factor involved in Wnt/ ß-Catenin signaling pathway. And it is one of the most frequently mutated genes in CRC. Loss-of-function of TCF7L2 showed morphological alteration, increased migration, and invasion in a cell-line-specific manner and may thereby increase malignancy. Furthermore, a group in New York has developed a better CRISPR/Cas9 system with higher transfection efficiency in mutations of CTNNB1 gene in DLD1 CRC cells, which bypass the sensitivity of cell lines against the combination of MEK and Tankyrase inhibitors (Zafra et al., 2018). They have reconstructed the Cas9 sequence together with modifications of two different positions on the N-terminal of nuclear localization signal in Cas9 protein to improve the gene targeting efficiency in mammalian cell lines. The mutated DLD1 cell line has successfully activated Wnt signaling by editing CTNNB1 and enabled it to be resistant to the suppression of Wnt signaling upon Tankyrase inhibitors. Although cell lines have been widely applied to study tumor biology, the gut organoids offer many advantages for CRC research. These three-dimensional “mini-gut” cultures are derived from intestinal stem cells that maintain many structural and functional features of the intestine (Clevers, 2016). The organoids allow for long-term in vitro expansion and remain genetically stable over a long period. The combination of organoid and CRISPR/Cas9 has been applied in

5.3 CRISPR/Cas9 applications in inflammatory bowel disease

several studies. As early as 2013, Schwank et al. have successfully adapted CRISPR/Cas9 technology in mouse and human-derived intestinal organoids (Schwank et al., 2013). In 2015 two important research groups (Drost et al., 2015; Matano et al., 2015) published papers based on using the CRISPR/Cas9 system in human intestinal organoids. They introduced mutations into commonly mutated tumor suppressor genes (APC, SMAD4, and TP53) and oncogenes (KRAS and/or PI3K) in the organoids, and these mutant organoids have the ability to grow independently of some stem-cell-niche factors. Both studies showed the mutation accelerated missegregation on an extensive aneuploid, which is a hallmark of the progression of CRC (Drost et al., 2015; Matano et al., 2015). The mutant organoids can form into tumors after xenotransplantation into mice. In addition, Drost and his group modeled mismatch repair-deficient CRC in human colon organoids by deleting key DNA repair genes. They observed that the mutation profiles of deficient in a mismatch repair gene MutL homolog 1 (MLH1) were consistent with the observation in CRC patient cohorts who have similar defects (Drost et al., 2017). In in vivo studies, the first genetically engineered mouse models (GEMMs) of CRC were developed by mutagen ethylnitrosourea (Enu) treatment and subsequently intercross breeding to get the multiple intestinal neoplasia (Min) model (Moser et al., 1990). Unlike many other cancer types, it is not easy to get the CRC GEMMs that accurately recapitulate advanced stage disease in the correct anatomical location. This is because traditional GEMMs are frequently developed through extensive intercrossing or de novo generation of mice with desired genetic mutations. Some GEMMs harboring mutations (e.g., germline APC) could easily induce lesions in the small intestine instead of the large intestine. And the overall tumor burden limits the time whereby malignant progression may occur. The development of gene editing organoid has been provided insight into CRC animal models (O’Rourke et al., 2017; Drost et al., 2015; Matano et al., 2015; Fumagalli et al., 2017). The models closely mimic key histopathological features of human cancer in animals (Golovko et al., 2015). Roper et al. have optimized the organoid transplantation method, by injecting the organoid into colon mucosa under colonoscopy-guide. This allows us to model a more rapid and accurate adenoma-carcinoma-metastasis sequence of tumor progression.

5.3 CRISPR/Cas9 applications in inflammatory bowel disease Inflammatory bowel disease (IBD) is a lifelong condition characterized by chronic inflammation and mucosal destruction in the intestine. Crohn’s disease (CD) and ulcerative colitis (UC) are the two main forms of IBD. The former can affect the entire gastrointestinal (GI) tract, but usually in the colon and terminal ileum, while the later in the rectum and colon. Clinical syndrome of IBD includes

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severe abdominal pain, rectal bleeding, and diarrhea. There are 2.5 3 million people suffering from IBD with a direct healthcare cost of 4.6 5.6 billion Euros per year in Europe alone (Burisch et al., 2013). The exact pathogen of IBD is still unclear. It has been established that genetic predisposition for the development of IBD. Genomic researchers have found that over 200 potential IBD susceptibility genes (e.g., NOD2) (Ye and McGovern, 2016). CRISPR/Cas provides a more effective way to further analyze various roles of these genes in IBD. Beurler et al. have identified the link between glycine amidinotransferase (GATM) gene and IBD (Turer et al., 2017). GATM activation is necessary for rapid replenishment of intestinal mucosa via promoting the synthesis of creatine. CRISPR/Cas9 targeted GATM mutated mice displayed increased cell death and metabolic stress in response to injury. However, a genetic predisposition to IBD is not sufficient to explain the majority situation of IBD cases. Environmental factors may act as foreign antigens and trigger the immune responses in the intestine. An influx of inflammatory cells in the intestine can damage intestinal mucosa and promote the progress of IBD. Thus much attention has been paid to the immune responses of IBD. One research has applied the CRISPR/Cas9 system to knockout Interferon-γ (IFN-γ) in mice lacking nuclear factor κB (NF-κB) essential modulator (NEMO) (Eftychi et al., 2019). The deficit of IFN-γ blocks both epithelial damage and leukocyte infiltration, as well as mitigation of pro-inflammatory response in colon tissue, compared to the mice only lacking NEMO. In another study, researchers aim to examine the role of microRNA (miR)-125a in the progression of IBD. miR-125a knockout mice were generated using CRISPR/Cas9. They found more severe colitis and the levels of inflammatory genes (such as Interleukin [IL]-17A, IFN-γ, tumor narcosis factor-α [TNF-α], and IL-6) were remarkably increased in miR125a KO mice than WT (Ge et al., 2019). Another microRNA, miR-146b, also has been demonstrated to have an important role in intestinal mucosal repair in a mouse model of UC and colitis-associated cancer (Deng et al., 2019). They applied CRISPR/Cas9 to knockout miR-146b in mice and utilized nanoparticles loading with miR-146b to target specifically intestinal macrophages for mucosal repair in miR-146b deficient mice with UC. miR-146b strongly inhibited macrophage activation by suppressing the Toll-like receptor 4 (TLR4). The mice with UC were rescued by using nanoparticles carrying miR-146b delivered to macrophages in the intestine by reduction of pro-inflammatory cytokines including TNF-α, IL-6, and IL-1 (Deng et al., 2019). Furthermore, it has been reported that mutation takes place at single nucleotide polymorphisms (SNPs) of IL-2 receptor A (IL-2RA) and is able to cause dysfunction of activation of T cells resulting in IBD and autoimmune disorders (Limanskiy et al., 2019). These SNPs with point mutations are potential candidates for CRISPR/Cas9 editing. Given the evidence that the failure of clinical trials for patients who were at active mild/moderate stage of CD receiving human recombinant IL-10, the multiple proinflammatory cytokines are required to modulate instead of using IL-10 alone. At the late stage of disease, abnormal regulation of different pro-inflammatory cytokines

5.5 Other gene editing tools

occurs, so that single target impact is subtle. With the CRISPR-Cas9 tool, it is possible for us to edit multiple genes at one time. This will help researchers to give patients optimal intervention at proper timing.

5.4 CRISPR/Cas9 applications in gut microbiota The gut microbiome is the microorganisms colonized within the GI tract. It contains trillions of microbial communities, including bacteria, yeast, and viruses. In the healthy human body, the microflora remains a beneficial symbiotic relationship with their host. Disorders of gut microbiota contribute to many diseases such as obesity, IBD, CRC, and even Parkinson’s disease (Cho and Blaser, 2012; Gorecki et al., 2019). Therefore microbiota has been attracted much attention to diseases interventions. Administration of engineered modified Escherichia coli (E. coli) to mice can reduce their food intake and obesity (Chen et al., 2014). Attenuated strains of Salmonella spp. and Listeria monocytogenes can be used as a delivery vehicle for anticancer drugs (Nemunaitis et al., 2003; Rothman and Paterson, 2013). Genetically modified Lactococcus lactis secreting IL10 provides a therapeutic method for IBD (Steidler et al., 2003). The gut microbes carry a large pool of genetic materials, approximately 100 times as many as in the human genome. CRISPR/Cas system can be introduced into target bacteria. They can kill, genome editing, or modulation of gene expression in the target bacteria without touching the rest of the microbiome (reviewed by Ramachandran and Bikard, 2019). Several studies have utilized CRISPR/Cas9 to carry phagemids to selectively get rid of the clinically relevant bacterial pathogens E. coli and Staphylococcus aureus (Bikard et al., 2014; Citorik et al., 2014). They used phagemid transduction to deliver CRISPR/Cas9 constructs on plasmids. The constructs were primarily programmed to target antimicrobial resistance genes in bacteria. They have effectively performed this modified technology in gut bacteria. The other group showed sequence-specific depletion of bacteria containing virulence genes using a similar system, thus effectively changing the sensitivity of bacteria back to antibiotics. Overall, these studies have confirmed the big potential of CRISPR/Cas9 in the application of effectively changing the composition of certain groups of microbials in the gut.

5.5 Other gene editing tools Before CRISPR/Cas9 has been discovered, researchers were dependent on other more complicated editing tools, such as ZFNs and TALEN. Levy and his colleges have applied the ZFN system to abolish the function of cystic fibrosis transmembrane conductance regulator (CFTR) gene in Caco-2/15, a human CRC cell line (Kleme et al., 2016). They found increased protein expression of pro-inflammatory cytokines,

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including TNF-α, IL-6, and cyclooxygenase-2 (COX-2) with elevated lipid peroxidation in the knockout model in vitro (Kleme et al., 2016). This evidence suggests the important role of the CFTR gene in cystic fibrosis-related intestine disorder. The other tool called TALEN is also widely utilized in the field. H3 lysine 79 methylation (H3K79me) is one of the histone modifications, which is known involved in gene transcription and DNA damage repair. It has been implicated in CRC and other cancer diseases. Disruptor of telomeric silencing 1-like methyltransferase (DOT1L) is the only methyltransferase associated with three stages of H3K79me. Due to the low amount of DOT1L and poor quality of chromatin immunoprecipitation DOT1L antibody, it was difficult to investigate its functionality. Therefore researchers developed a knock-in method to tag it using TALEN to follow DOT1L in the process of methylation (An et al., 2017). Another group has established a TALEN tool to target the promoter of IL-12B gene (Chen et al., 2020). IL-12B is a shared subunit of IL-12 and IL-23 released by macrophages, a common immune response found in IBD. Thus suppression of IL12-B could be a potential therapeutic target for IBD patients. The consequences of targeting the selective region of IL-12B promoter by TALEN are loci-specific DNA methylation and downregulation of IL-12B (Chen et al., 2020). In addition, it is implied that gastrins have important roles in the progression of GI cancer. Gastrins are peptides hormones promoting the secretion of gastric acid and growth of the gastric mucosa. The group has previously shown that zinc ion is able to activate gastrins promoter. Therefore TALEN system was used to engineer a reporter construct targeting the endogens human gastrin gene promoter in SW480 colon cancer cells (Marshall et al., 2015). They found that the gastrin promoter is activated by zinc ion in a concentration-dependent fashion in vivo and in vitro, probably via triggering the MAPK pathway (Marshall et al., 2015).

5.6 Limitations and perspectives We have witnessed a stunning step in the development of novel technology in science society. Due to the weakness of TALEN and ZFN gene editing tools are mostly resolved by CRISPR-Cas9, here we have focused mainly on the challenges of the CRISPR-Cas9 system. The application of this new technology has been published in a significant number of papers in different fields, including agriculture, pharmaceutical development, and epigenetic modulation. CRISPR-based technology is an unprecedented tool and will favor us in many breakthrough discoveries in science in the future. It has drawn such great attention from the public and shined a light not only on various cancers but also on some genetic diseases. However, CRISPR-based tools also bring many technical challenges and limitations as well as ethical issues. One of the limitations is the low percentage of genome sequences available for editing by CRISPR-based tools. This is due to the target for original Cas9 is

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Application of new approaches for intestinal repair and regeneration via stem cell based tissue engineering

6

Ahmed El-Hashash1,2 1

The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, P.R. China 2 Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, P.R. China

Acute or chronic intestinal diseases affect small or large intestine and include a wide range of diseases such as diverticular disease, constipation, and irritable bowel syndrome. Autoimmune, infectious and physiological states can affect both large and small intestines. Intestinal diseases have major health and economic impacts and affect different countries worldwide.

6.1 Structure and cellular components of the small intestine The small intestine consists of the duodenum, jejunum, and ileum. The wall of the small intestine contains a mucosa with simple columnar epithelial cells, then both submucosa and smooth muscle that has distinct circular and longitudinal muscular layers, and finally the serosa layer. There is a characteristic crypt villus architecture in the small intestinal epithelial layer in humans. Notably, crypts are characterized by proliferative cells, while differentiated cells reside in the villi. Both the crypt and villus contain many different cell types (Noah et al., 2011). While the crypt villus units that are self-renewing consist of columnar epithelial cells, the differentiated cells, including secretory cells (goblet cells, enteroendocrine cells, and Paneth cells) and enterocytes absorptive cells dominate the villi (Clevers, 2013; Wells and Spence, 2014). The crypt contains undifferentiated and highly proliferative cells, such as intestinal stem and progenitor cells and transit amplifying cells, which undergo differentiation after migration toward the crypt The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00009-6 © 2021 Elsevier Inc. All rights reserved.

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top (Clevers, 2013; Qi et al., 2020). While Laminin, proteoglycans, fibronectin, and collagen are the major extracellular matrix (ECM) components, collagens type IV and VI are important in the regulation of crypt cell matrix interactions in the small intestine (Beaulieu et al., 1994; Tibbitt and Anseth, 2009; Groulx et al., 2011; Eweida and Marei, 2015).

6.2 Stem cells used in small intestine regeneration Several types of stem cells can be used in the regeneration of small intestine in vivo.

6.2.1 Pluripotent stem cell derived cells 6.2.1.1 Embryonic stem cells Embryonic stem cells (ESCs) are unique pluripotent cells that can self-renew and differentiate (Coraux et al., 2005). ESCs are derived from the inner cell mass of human or animal embryos at the blastocyst stage of embryonic development. They possess the ability of indefinite replication in vitro and, therefore, can selfrenew in an undifferenced state, with retaining the differentiation ability into a variety of cell types that belong to all three germinal layers of the body in vivo and in culture (Coraux et al., 2005). ESCs have, therefore, great regenerative and therapeutic potentials for injured or diseases tissues (Baharvand and Hassani, 2013). For example, ESCs can be induced to differentiate into many specialized cell types, including lung, heart, renal, neurons, and insulin-producing cells that can be used for tissue repair and regeneration. Despite their importance, there are several problems in the application of ESCs in intestinal diseases, including the immunologic barrier, the risk tumor formation such as teratomas that form when applying ESCs in vivo, and difficulties of ESC production (Qi et al., 2020) The differentiation of ESCs into both intestinal organoids (Watson et al., 2014) and intestinal lineages, including goblet cells, enterocytes, and Paneth cells (Ogaki et al., 2013), was reported. Intestinal organoid cultures can be developed from stem cells or organ-specific progenitor cells via a self-organization process (Lancaster and Knoblich, 2014; Clevers, 2016), which leads to the formation of complex 3D structures with similar architecture and function to intestinal tissues (Rossi et al., 2018).

6.2.1.2 Induced pluripotent stem cells Induced pluripotent stem cells (iPSCs) are adult cells that were reprogrammed to resemble ESCs and could be created from either adult somatic and/or blood cells (Takahashi and Yamanaka, 2006; Khademhosseini and Langer, 2016). This reprogramming occurs by in vitro by introducing certain ESC-maintenance factors and genes Oct3/4, Klf4, Sox2, and cMyc. iPSCs have similar ESC characteristics and

6.2 Stem cells used in small intestine regeneration

regenerative properties (Takahashi and Yamanaka, 2006). Since they have similar abilities and characteristics to ESCS, iPSCs can give rise to many cell types, including ectodermal, endodermal, and mesodermal cells (Devineni et al., 2016; Gomes et al., 2017). Interestingly, human iPSCs can differentiate into enterocytes, simple columnar epithelial cells (Kodama et al., 2016; Kabeya et al., 2017). In addition, iPSCs could form human intestinal organoids (McCracken et al., 2011; Spence et al., 2011; Flores et al., 2015). iPSCs have several other advantages that include minimizing the immune rejection risks and allowing donor-derived pluripotent cells as well as diminishing ethical conflicts (Gomes et al., 2017). However, iPSCs and their derived cells may show both some immunogenicity (Tan et al., 2014; Lancaster and Knoblich, 2014) and tumorigenic potentials (Lee et al., 2013). Therefore more attention should be paid to iPSCs-based clinical applications.

6.2.2 Adult intestinal stem cells Adult intestinal stem cells (ISCs) are multipotent and essential for self-renewing the epithelial cell compartment in the intestine. Active ISCs are usually localized to the base of crypts and characterized by expression of Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5; Barker et al., 2007; Hou et al., 2017), while the B lymphoma Mo-MLV insertion region 1 (Bmi1) is the quiescent intestinal stem cell putative biomarker (Sangiorgi and Capecchi, 2008). In addition, Bmi1 is a biomarker of early secretory progenitors (Yan et al., 2012; Buczacki et al., 2013). Interestingly, the revert of Bmi1 positive cells to Lgr5 positive stem cells is reported in response to intestinal injury and repair and important for the maintenance of intestinal homeostasis (Tian et al., 2011). ISCs were successfully used to establish intestinal organoid cultures that are recently developed tools for mimicking the physiological processes and pathophysiology of the small intestine (Sato et al., 2009; Spence et al., 2011; Wang et al., 2015; Gjorevski et al., 2016; Rossi et al., 2018). These organoid cultures are also useful tools for the potential treatments of intestinal diseases. For example, transplanted colonic organoids onto the damaged mucosa in the dextran sodium sulfate-colitis model can integrate into the recipient intestinal epithelial tissue, leading to the repair of the damaged mucosal layers (Yui et al., 2012). Similarly, the in vivo transplantation of both fetal intestinal organoid and small intestinal organoid cultures onto damaged mucosal layers was also reported (Fordham et al., 2013; Fukuda et al., 2014). Interestingly, a study by Baimakhanov et al. (2016) successfully created an engineered intestinal epithelial tissue from Lgr5-positive stem cells. This engineered epithelial cells showed positive immunostaining for Paneth cells, Ki67, enteroendocrine cells, goblet cells, and the absorptive enterocytes’ microvilli (Baimakhanov et al., 2016). More intestinal stem cell based therapies are still under development. These therapies were tested in preclinical animal models for both treating some gastrointestinal

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diseases, including Inflammatory Bowel Disease (IBD), and repairing intestinal mucosa’s damages (Okamoto and Watanabe, 2015).

6.2.3 Mesenchymal stem cells Mesenchymal stem cells (MSCs) are multipotent cells derived from adult tissues, including the bone marrow, placenta, peripheral blood, and adipose tissue (Li et al., 2017a,b). MSCs can differentiate into three germ layers and, therefore, give rise to ectodermal lineage (e.g., neurocytes), mesodermal lineage (e.g., osteoblast, adipocyte, chondrocyte), and endodermal (e.g., hepatocytes) lineage (Ullah et al., 2015; Wang et al., 2017). Human MSCs are mostly derived from the adipose tissue and bone marrow (Pittenger et al., 2019), able to both promote tissue repair and angiogenesis and produce growth factors, and show other properties, including immunosuppression, antiapoptotic, antiinflammatory, and nerve protection properties (Bhardwaj and Kundu, 2012; Wang et al., 2017; Chen et al., 2018; Tasli et al., 2018). MSCs derived from the bone marrow can be used in the treatment of colitis in animal models of the disease (Hayashi et al., 2008), and their safety and effect were validated in clinical trials of human Crohn’s disease (Duijvestein et al., 2010; Forbes et al., 2014). In addition, they can help with the repair of injured intestinal tissues by both producing cytokines and growth factors, which could promote intestinal stem cell proliferation and differentiation, and transdifferentiation into intestinal stem-like cells (Chen et al., 2013). Moreover, bone marrow-derived MSCs could facilitate the regeneration of tissues by reducing both inflammatory and autoimmune responses (Chen et al., 2013; Linard et al., 2013; Flores et al., 2015). This was supported by MSC suppression effects on rejection that occurs in heterotopic small intestine transplant animal models by downregulating the expression levels of CD68, PDGF, and TGF-β1 factors (Li et al., 2017a,b). Notably, bone marrow derived MSCs can regulate immune cell activities, including regulatory B cell induction, and this mechanism could lead to the attenuation of IBD in animal model (Chen et al., 2019). Similarly, MSCs derived from the adipose tissue can affect the IBD since infused MSCs may lead to a clinical remission in the animal model of severe IBD (Pe´rez-Merino et al., 2015). Furthermore, human gingiva derived MSCs show immunomodulatory effects that could ameliorate tissue damages in colitis animal models (Zhang et al., 2009).

6.3 Developing biological tissue-engineered grafts Common approaches for the development of biological tissue-engineered grafts include biocompatible scaffold recellularizations. This engineering/fabrication approach uses grafts that have proper and characteristic cell adhesion and

6.3 Developing biological tissue-engineered grafts

mechanical properties, which allow the promotion of both cellular proliferation and reorganization into organ-like structures (Langer and Vacanti, 1993; Hutmacher, 2001). Synthetic or naturally occurring biodegradable polymers could be used to prepare the engineered scaffolds (Ou and Hosseinkhani, 2014). These scaffolds can be also of biological origin (Ou and Hosseinkhani, 2014). In the small intestine, there are several fabrication technologies that could be used to make bioengineered scaffolds, including decellularization of tissues or organs, bioprinting, biotextile, and electrospinning technologies and organ-on-achip (Qi et al., 2020). Both intestinal structure and function could be promoted in culture when specialized cell types are effectively combined with the bioengineered scaffolds that could facilitate the in vivo regeneration of the small intestine in the future (Qi et al., 2020). The most common fabrication technology used to make bioengineered scaffolds, decellularization of tissues or organs, will be described in the following section.

6.3.1 Decellularization of tissues or organs Decellularization of tissues or organs is one of the most common method for the fabrication of engineered scaffolds for the small intestine regeneration since it could be associated with the retention of both the architecture and biological components from the native intestinal tissue such as the vasculature structure and cell proliferation factors, respectively (Totonelli et al., 2012). Both enzymatic/chemical and mechanical methods are involved in the decellularization of tissues or organs (Gilpin and Yang, 2017). However, the enzymatic/chemical methods are the most common and involve the use of several reagents, including trypsin enzyme or detergents, to remove the tissue cells that result in the formation of 3D bioengineered scaffold with insoluble matrix components (Stabler et al., 2015). Despite preserving the anisotropic tissue characteristics (Porzionato et al., 2018), most decellularization techniques can change scaffolds’ mechanical properties and performance by badly affecting ECM structural proteins, including laminin, elastin, collagen, and fibronectin (Gilpin and Yang, 2017). They can also lead to the loss of glycosaminoglycans and proteoglycans in the cell matrix depending on used reagent type and concentration and treatment time during decellularization (Gilpin and Yang, 2017). The in vivo feasibility and applicability of the decellularization/recellularization techniques were well shown in the engineering of rat tissues (Stabler et al., 2015). A recently developed decellularized small intestine submucosa ECM is commonly used in regenerative medicine due to its characteristic features, including its support of intestinal cell differentiation and proliferation, 3D architecture that facilitates the ingrowth of host cells, and immunogenicity limitations (Mosala Nezhad et al., 2016; Ropcke et al., 2017). The detergent-enzymatic treatment method was used for the decellularization of rat intestine, followed by repopulation with stem cells derived from the amniotic fluid (Totonelli et al., 2012). Other studies demonstrated that decellularized porcine small intestine could support the

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proliferation of MSCs derived from human adipose tissue and be implanted in the underlying abdominal muscles of animal models without immunoreaction signs (Nowocin et al., 2016; Grandi et al., 2018). In the small intestine, digested decellularized small intestine matrix could form hydrogel (gel-like) materials, which are injectable and show several comparable properties to Matrigel or collagen for cell culture (Saldin et al., 2017). Interestingly, the gels of decellularized small intestine submucosa ECM have a similar composition to endodermal-derived tissues (Giobbe et al., 2019) and could support the formation of intestinal organoids that express crypt markers such as SMOC2, OLFM4, and LYZ. Decellularized small intestine is, therefore, a highly promising scaffold that could be used in the small intestine regeneration and repair in vivo (Totonelli et al., 2012; Hussey et al., 2018).

6.4 Tissue-engineered small intestine Tissue-engineered small intestine, normally mimics the structure of small intestine, is important for restoring normal intestinal functions and offers hope for treating a range of small intestinal diseases. Different cell sources, biomaterials, and methods could be used to produce proper tissue-engineered small intestines. If properly produced from the patient’s own cells, tissue-engineered small intestine can restore normal functions of the intestine through autologous transplantation (Spurrier and Grikscheit, 2013; Dosh et al., 2018). Structurally, a welldesigned engineered small intestine contains several tissue and cell types in the correct order/orientation, including epithelial, mesenchymal, blood vessel, smooth muscle, and neuronal cells (Clevers et al., 2019) and supports the complex reciprocal interactions, relationships, functions, and survival of these different types of cells (Spurrier and Grikscheit, 2013). Properly designed engineered intestine should, therefore, have similar structure and morphology to the native intestine, which include inner intestinal epithelial layer surrounded by outer muscular layers, as well as blood vessels, and both smooth muscle and nerve cells in the intestinal mesenchyme (Sala et al., 2009). For example, murine and human tissue engineered small intestines with welldifferentiated epithelial cells have been produced and showed similar ultrastructure, functional brush-border enzymes, and ion transporters to the native intestinal tissue (Grant et al., 2015). In addition, tissue-engineered small intestines were recently produced using human iPSC-derived intestinal endothelial and epithelial cells that repopulate decellularized rat intestinal matrix (Kitano et al., 2017). Interestingly, tissue-engineered small intestines could restore both intestinal functions and weight gain after implantation into Lewis rats with small bowel resection (Grikscheit et al., 2004). Moreover, implanted tissue-engineered small intestines could recapitulate native small intestinal functions (i.e., both absorptive and digestive functions) in genetically identical or immunodeficient host mice

6.6 Conclusion and future directions

(Grant et al., 2015). Tissue-engineered small intestines, therefore, offer hope for tissue replacement after intestinal resection and/or injury (Grant et al., 2015).

6.5 Role of stem cell based transplantation in intestinal regeneration There are currently some appropriate approaches for regenerating small intestine with small lesions, including stem cell based transplantations (without scaffolds; Qi et al., 2020). The most common stem cell types used in this approach are MSCs, hematopoietic stem cells (HPSCs), iPSCs, and ISCs (Ko et al., 2010; Clerici et al., 2011; Yui et al., 2012; Wagnerova and Gardlik, 2013; Fordham et al., 2013). MSCs have several features, including their immunosuppressive properties and homing ability to the inflammation/ injury areas after administration, which make them a highly promising therapy for IBD (Griffin et al., 2013). Moreover, transplanted MSCs into damaged intestinal tissues demonstrate several features such as proliferation, production of cytokines and growth factors, and trans-differentiation into ISCs (Chen et al., 2013). Similarly, systemically administrated MSCs could reduce both inflammatory and autoimmune responses and improve tissue regeneration in animal models of radiation-induced proctitis (Linard et al., 2013) and chemically induced colitis (Chen et al., 2013). In addition, recovery of the small intestinal mucosa was reported after MSC administration in induced intestinal inflammation murine models (Lykov et al., 2018). While systematically administrated stem cells may have some side effects and risks when used in intestinal repair/regeneration, scaffolds could significantly enhance this process by increasing stem cell functionality and viability, leading to improved stem cell engraftments in vivo (Qi et al., 2020). This was supported by the fabrication of hyaluronic acid-based hydrogel patch scaffolds that showed an adequate elastic modulus and a remarkable enhancement of tissue adhesiveness (Shin et al., 2019) and, therefore, could facilitate and improve the engraftments of MSC-based organoids onto murine intestinal surface in vivo (Qi et al., 2020). Organoid transplantations were further promoted by the recent development of both synthetic hydrogel biomaterials and naturally derived extracellular matrices to replace Matrigel matrix (Gjorevski et al., 2016; Gjorevski and Lutolf, 2017; Cruz-Acuna et al., 2017; Capeling et al., 2019). Both biomedical research and clinical application of other stem cells such as HPSCs in intestinal repair/regeneration are still limited.

6.6 Conclusion and future directions Despite recent progresses in the study of ISCs, organoid culture, and tissueengineered small intestine, there are many challenges that face the regeneration of

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small intestine probably due to its complex architecture and function. Therefore more research and development of novel methodology and technology are still needed to enhance small intestinal repair and regeneration, particularly using large animals with gastrointestinal system that is similar to humans to verify murine model study results. In addition, more studies are need on the functional regeneration of tissue-engineered small intestines, including secretory, absorptive, and immune functions. Since there is currently donor shortage and lifelong immunosuppression that limits intestinal transplantation, much attention has been paid to tissue-engineered small intestines since they could offer large constructs of small intestine for therapeutic applications. However, some reseeded tissue-engineered small intestines do not function well in vivo, despite having a very similar structure to native adult human small intestine (Finkbeiner et al., 2015). This may be because these matrices could prevent the infiltration of important cells, including vascular cells and the imitated vascularization of intestinal tissues can badly affect intestinal cell function, integration, and survival (Schneeberger et al., 2017). Generation of microchannels in these matrices using 3D bioprinting or similar techniques before repopulation with iPSC-derived endothelial cells and other vessel supporting cells could be one approach to solve this problem (Gao et al., 2015; Vecchione et al., 2016; Qi et al., 2020). Interestingly, the current availability of both iPSC-derived endothelial cells and vessel supporting cells such as smooth muscle cells can facilitate the generation of patient-specific human intestinal organoids using iPSCs from the same individual (Qi et al., 2020). Furthermore, the functions of tissue-engineered intestines, including secretion, contractility, and motility, could also be improved by incorporating enteric neurons that improve the innervation of these engineered intestine. Indeed, proper innervation is a key in small intestinal functions and innervation-related defects can lead to several intestinal diseases (Brookes et al., 2016; Mourad et al., 2017).

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Mourad, G.H., Barada, K.A., Saade, N.E., 2017. Impairment of small intestinal function in ulcerative colitis: role of enteric innervation. J. Crohns Colitis 11 (3), 369 377. Noah, T.K., Donahue, B., Shroyer, N.F., 2011. Intestinal development and differentiation. Exp. Cell Res. 317 (19), 2702 2710. Nowocin, A.K., Southgate, A., Gabe, S.M., Ansari, T., 2016. Biocompatibility and potential of decellularized porcine small intestine to support cellular attachment and growth. J. Tissue Eng. Regen. Med. 10 (1), E23 E33. Ogaki, S., Shiraki, N., Kume, K., Kume, S., 2013. Wnt and Notch signals guide embryonic stem cell differentiation into the intestinal lineages. Stem Cell 31 (6), 1086 1096. Okamoto, R., Watanabe, M., 2015. Perspectives for regenerative medicine in the treatment of inflammatory bowel diseases. Digestion 92 (2), 73 77. Ou, K.L., Hosseinkhani, H., 2014. Development of 3D in vitro technology for medical applications. Int. J. Mol. Sci. 15 (10), 17938 17962. Pe´rez-Merino, E.M., Uso´n-Casau´s, J.M., Zaragoza-Bayle, C., Duque-Carrasco, J., Marin˜asPardo, L., Hermida-Prieto, M., et al., 2015. Safety and efficacy of allogeneic adipose tissue-derived mesenchymal stem cells for treatment of dogs with inflammatory bowel disease: clinical and laboratory outcomes. Vet. J. 206 (3), 385 390. Pittenger, M.F., Discher, D.E., Peault, B.M., Phinney, D.G., Hare, J.M., Caplan, A.I., 2019. Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regen. Med. 4, 22. Porzionato, A., Stocco, E., Barbon, S., Grandi, F., Macchi, V., De, R., 2018. Caro, Tissue engineered grafts from human decellularized extracellular matrices: a systematic review and future perspectives. Int. J. Mol. Sci. 19 (12). Qi, D., Shi, W., Black, A.R., et al., 2020. Repair and regeneration of small intestine: a review of current engineering approaches. Biomaterials 240, 1 18. Ropcke, D.M., Ilkjaer, C., Tjornild, M.J., Skov, S.N., Ringgaard, S., Hjortdal, V.E., et al., 2017. Small intestinal submucosa tricuspid valve tube graft shows growth potential, remodelling and physiological valve function in a porcine model. Interact. Cardiovasc. Thoracic Surg. 24, 918 924. Rossi, G., Manfrin, A., Lutolf, M.P., 2018. Progress and potential in organoid research. Nat. Rev. Genet. 19 (11), 671 687. Sala, F.G., Kunisaki, S.M., Ochoa, E.R., Vacanti, J., Grikscheit, T.C., 2009. Tissueengineered small intestine and stomach form from autologous tissue in a preclinical large animal model. J. Surg. Res. 156 (2), 205 212. Saldin, L.T., Cramer, M.C., Velankar, S.S., White, L.J., Badylak, S.F., 2017. Extracellular matrix hydrogels from decellularized tissues: structure and function. Acta Biomater. 49, 1 15. Sangiorgi, E., Capecchi, M.R., 2008. Bmi1 is expressed in vivo in intestinal stem cells. Nat. Genet. 40 (7), 915 920. Sato, T., Vries, R.G., Snippert, H.J., van de Wetering, M., Barker, N., Stange, D.E., et al., 2009. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459 (7244), 262 265. Schneeberger, K., Spee, B., Costa, P., Sachs, N., Clevers, H., Malda, J., 2017. Converging biofabrication and organoid technologies: the next frontier in hepatic and intestinal tissue engineering? Biofabrication 9 (1), 013001. Shin, J., Choi, S., Kim, J.H., Cho, J.H., Jin, Y., Kim, S., et al., 2019. Tissue tapes-phenolic hyaluronic acid hydrogel patches for off-the-shelf therapy. Adv. Funct. Mater. 29 (49).

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Spence, J.R., Mayhew, C.N., Rankin, S.A., Kuhar, M.F., Vallance, J.E., Tolle, K., et al., 2011. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470 (7332), 105 U120. Spurrier, R.G., Grikscheit, T.C., 2013. Tissue engineering the small intestine. Clin. Gastroenterol. Hepatol. 11 (4), 354 358. Stabler, C.T., Lecht, S., Mondrinos, M.J., Goulart, E., Lazarovici, P., Lelkes, P.I., 2015. Revascularization of decellularized lung scaffolds: principles and progress. Am. J. Physiol. Lung Cell Mol. Physiol. 309 (11), L1273 L1285. Takahashi, K., Yamanaka, S., 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 (4), 663 676. Tan, Y., Ooi, S., Wang, L.S., 2014. Immunogenicity and tumorigenicity of pluripotent stem cells and their derivatives: genetic and epigenetic perspectives. Curr. Stem Cell Res. Ther. 9 (1), 63 72. Tasli, P.N., Bozkurt, B.T., Kirbas, O.K., Deniz-Hizli, A.A., Sahin, F., 2018. Immunomodulatory behavior of mesenchymal stem cells. Adv. Exp. Med. Biol. 1119, 73 84. Tian, H., Biehs, B., Warming, S., Leong, K.G., Rangell, L., Klein, O.D., et al., 2011. A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable. Nature 478 (7368), 255 259. Tibbitt, M.W., Anseth, K.S., 2009. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol. Bioeng. 103 (4), 655 663. Totonelli, G., Maghsoudlou, P., Garriboli, M., Riegler, J., Orlando, G., Burns, A.J., et al., 2012. A rat decellularized small bowel scaffold that preserves villus-crypt architecture for intestinal regeneration. Biomaterials 33 (12), 3401 3410. Ullah, I., Subbarao, R.B., Rho, G.J., 2015. Human mesenchymal stem cells - current trends and future prospective. Biosci. Rep. 35 (2). Vecchione, R., Pitingolo, G., Guarnieri, D., Falanga, A.P., Netti, P.A., 2016. From square to circular polymeric microchannels by spin coating technology: a low cost platform for endothelial cell culture. Biofabrication 8 (2), 025005. Wagnerova, A., Gardlik, R., 2013. In vivo reprogramming in inflammatory bowel disease. Gene Ther. 20 (12), 1111 1118. Wang, X., Yamamoto, Y., Wilson, L.H., Zhang, T., Howitt, B.E., Farrow, M.A., et al., 2015. Cloning and variation of ground state intestinal stem cells. Nature 522 (7555), 173 178. Wang, J., Cen, P., Chen, J., Fan, L., Li, J., Cao, H., et al., 2017. Role of mesenchymal stem cells, their derived factors, and extracellular vesicles in liver failure. Stem Cell Res. Ther. 8 (1), 137. Watson, C.L., Mahe, M.M., Munera, J., Howell, J.C., Sundaram, N., Poling, H.M., et al., 2014. An in vivo model of human small intestine using pluripotent stem cells. Nat. Med. 20 (11), 1310 1314. Wells, J.M., Spence, J.R., 2014. How to make an intestine. Development 141 (4), 752 760. Yan, K.S., Chia, L.A., Li, X., Ootani, A., Su, J., Lee, J.Y., et al., 2012. The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proceed. Nat. Acad. Sci. USA 109 (2), 466 471. Yui, S., Nakamura, T., Sato, T., Nemoto, Y., Mizutani, T., Zheng, X., et al., 2012. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5 (1) stem cell. Nat. Med. 18 (4), 618 623. Zhang, Q., Shi, S., Liu, Y., Uyanne, J., Shi, Y., Shi, S., et al., 2009. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis. J. Immunol. 183 (12), 7787 7798.

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7

Adegbenro Omotuyi John Fakoya1, Adekunle Ebenezer Omole2, Nihal Satyadev1 and Cynthia Oghenekome Okaruefe3 1

University of Medicine and Health Sciences, Basseterre, St. Kitts and Nevis 2 American University of Antigua College of Medicine, St. John’s, Antigua 3 All Saints University School of Medicine, Roseau, Dominica

7.1 Introduction More than a decade ago, the world of stem cell science witnessed a remarkable discovery, the birth of a new type of pluripotent stem cells termed “induced pluripotent stem cells” (iPSCs). In 2006 Dr. Shinya Yamanaka and his team of scientists pioneered the description of induction of pluripotency in mouse somatic skins cells by enforced expression of four genes, namely, Oct 3/4, Sox2, Klf4, and c-Myc (OSKM/Yamanaka factors) (Takahashi and Yamanaka, 2006). The same reprogramming method was used in 2007 by the same team of scientists to generate human iPSCs (hiPSCs) from human somatic cells (Takahashi et al., 2007). Dr. James Thomson’s team also independently reported the generation of hiPSCs using a different set of reprogramming factors in the same year 2007 (Yu et al., 2007). iPSCs have infinite proliferation in culture and pluripotent capabilities as they can differentiate into the three embryonic germ cell layers (ectoderm, mesoderm, and endoderm) and can generate all cells of an adult organism. Though embryonic stem cells (ESCs) which were derived 25 years earlier than iPSCs are also pluripotent with infinite proliferative capability, their generation from preimplantation embryos has always been fraught with strong ethical issues relating to embryo destruction, and this has strongly impeded their application clinically (Evans and Kaufman, 1981; Martin, 1981; Omole and Fakoya, 2018). iPSCs as an alternative source of pluripotent stem cells with the same degree of differentiation potential as ESCs bypass these ethical drawbacks, thus removing any hindrance to advancement in pluripotent stem cell research and their application clinically. Indeed, iPSCs hold great promise in the generation of patient-specific pluripotent stem cells for human disease modeling, drug development, and individualized cell-based therapy for various diseases (Omole and Fakoya, 2018). This chapter looks at how iPSCs have been used in studying some intestinal diseases.

The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00003-5 © 2021 Elsevier Inc. All rights reserved.

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7.2 iPSC-based disease modeling For the purpose of developing therapeutic strategies, it is essential to comprehend the molecular and pathological mechanisms of human diseases. Traditionally, we study human diseases using well-established animal models, which provide an in vivo setting for the investigation of pathological mechanisms. Throughout the years, animal models have proved invaluable in the pharmaceutical industry. However, substantial interspecies differences have hindered the adequate recapitulation of human disease phenotype in animal models. Consequently, therapeutic strategies developed using animal models often failed in clinical studies. Therefore there is a need for humanbased disease models to supplement the current nonhuman experimental models such as rodents. The capability to differentiate into any disease-relevant cell types, the easy accessibility, availability, and expandability, and their patient origin have empowered hiPSCs as the perfect system for the development of patient-based cellular models and patient-specific therapeutics compounds for personalized medicine. The “disease in a Petri dish” model can be realized by directly generating iPSCs with the diseasecausing mutation from patients with the disease of interest (Omole and Fakoya, 2018). With rapid progress in genome editing technology (like CRISPR/Cas9), we can now initiate genetic changes into iPSCs in a site-specific manner. By introducing a sitespecific mutation into nondiseased iPSCs, we can generate isogenic iPSCs lines that are genetically matched to patient iPSCs, which recapitulate the true pathology of the disease of concern, for hiPSC-based disease modeling (Omole and Fakoya, 2018). Consequently, iPSCs are an excellent model system for monogenic disorders. Phenotypically, iPSCs are young, hence they are perfect for the modeling of early onset disorders (Shi et al., 2020). Late-onset disorders are difficult to model by iPSCs since aging is an essential contributing factor. Induction of cellular aging in iPSCs is a fundamental criterion for iPSCs-based cellular modeling of late-onset disorders. So far, there have been multiple reports involving the use of iPSCs to model the pathological mechanisms of a variety of diseases, such as neurological disorders, cardiovascular diseases, cancer, and so on (Omole and Fakoya, 2018; Yoshida and Yamanaka, 2017; Wu and Hochedlinger, 2011; Chamberlain, 2016; Kumar et al., 2018; Kondo et al., 2013; Israel et al., 2012; Cooper et al., 2012; Devine et al., 2011). Initially, iPSC models were based on a single cell type of the disease of interest. Nowadays, iPSCs-based coculture models of more than one cell type are common. For example, amyotrophic lateral sclerosis and Alzheimer’s disease were modeled by cocultures of neurons and astrocytes (Nagai et al., 2007; Di Giorgio et al., 2007; Zhao et al., 2017). There has been a recent shift away from the conventional two-dimensional (2D) monolayer adherent in vitro cell cultures to the powerful “organoid” model, which is a 3D multicellular in vitro tissue construct that mimics its corresponding organ in vivo. The 2D monolayer culture lacks the tissue architecture, organization, and complexity evident in endogenous organs in vivo. This “miniorgan in a dish” organoid models recapitulate the cellular heterogeneity, architecture, structure, organization, functions, and

7.3 Intestinal organoids

cell cell interactions seen in organs in vivo, thus allowing us to model diseases more realistically (Corro` et al., 2020). Following the landmark study in 2009 from Sato et al., where intestinal organoids were generated from adult intestinal stem cells (ISCs), iPSCs and other stem cells have been used to develop organoids of various organs for disease modeling purposes (Sato et al., 2009; Wiegand and Banerjee, 2019; McCracken et al., 2014; Takebe et al., 2013; Sampaziotis et al., 2015; Ogawa et al., 2015; Dye et al., 2015; Spence et al., 2011; Takasato et al., 2016; Watson et al., 2014; Tucker et al., 2014; Camp et al., 2015; Mariani et al., 2015; Cugola et al., 2016; Qian et al., 2016; Garcez et al., 2016; Gabriel et al., 2016; Otani et al., 2016; Pa¸sca et al., 2015).

7.3 Intestinal organoids One of the important applications of iPSCs is their ability to differentiate into organoids, a collection of cells that closely resembles tissue. Intestinal tissue consists of mature cells such as enterocytes, goblet cells, and enteroendocrine cells as well as Paneth cells, which are specific to the small intestine (van Es and Clevers, 2014; Barker and Clevers, 2010). These mature cells renew every 4 5 days from ISCs, which can be further subdivided into fast-cycling Lgr5 1 cells and slowcycling Tert 1 cells (Kuratnik and Giardina, 2013; Sato et al., 2011; Barker and Clevers, 2007; Roth et al., 2012). The complexity of the intestinal cellular architecture and intercellular signaling make organoids a significantly better model to imitate in vivo conditions than monocultures of intestinal cells. Human intestinal organoids (HIOs) can be derived from either iPSCs, ESCs, or ISCs. The first protocol for their derivations from iPSCs was published by Spence et al. in 2011 and described a three-step process that imitated the development process in a human fetus (Spence et al., 2011; McCracken et al., 2011). iPSCs were cultured with activin A to form definitive endoderm. Then, these endodermal cells were exposed to fibroblast growth factor 4 (FGF4) and WNT3A to differentiate them into mid/hindgut bodies. Finally, these cells were further differentiated into intestinal epithelium by culturing with R-spondin 1, Noggin, and epidermal growth factor. This protocol generated organoids that more closely resembled the small intestine. The total time required for differentiation was reported to be 28 days. However, this more closely represents human fetal intestines, and mature phenotype requires culture for several months. At the moment, HIOs from iPSCs (HIO-iPSCs) are used significantly less for research purposes than HIOs from ISCs (HIO-ISCs). HIO-iPSCs are limited because they acquire genetic and epigenetic mutations while being cultured for longer periods of time (Liang and Zhang, 2013). In contrast, HIO-ISCs have been shown to be genetically, epigenetically, and phenotypically stable over long periods of culture duration (Sato et al., 2009; Dotti and Salas, 2018; Schwank et al., 2013). In addition, HIO-ISCs have been shown to maintain the properties of the region, age, and disease (e.g., ulcerative colitis, Crohn’s disease) from which the

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ISCs are initially derived (Middendorp et al., 2014; Kraiczy et al., 2019; Howell et al., 2018). Similar studies should be conducted using HIO-iPSCs. However, as methodologies for their maintenance and differentiation improve, HIO-iPSCs will likely surpass HIO-ISCs in their use in research settings due to their ability to differentiate into various tissue types and the minimally invasive biopsy needed to gather fibroblasts from patients.

7.4 iPSCs and organoids in intestinal diseases 7.4.1 Colorectal cancer Colorectal cancer (CRC) is the second leading cause of cancer-caused mortality worldwide (Ferlay et al., 2010). Cancer is described by pathologists as anaplasia, which is derived from the Greek terms “ana” meaning backward and “plasis” meaning “formation.” A hypothesis that malignant tumors occur when mature cells dedifferentiate (or “grow backward”) into cancer stem cells (CSCs) was proposed several decades ago. Growing evidence suggests that CSCs are required for tumor initiation in certain tumor types, however, the CSC theory is still controversial (Lapidot et al., 1994; Galli et al., 2004; Singh et al., 2004; Wang et al., 2009; Kelly et al., 2007; Quintana et al., 2008; Shackleton et al., 2009). As both somatic and cancer cells have been dedifferentiated into embryonic stem-cell like state and because evidence shows that reprogramming factors are overexpressed in CRC, there is a growing interest in understanding the corollaries between generation of iPSCs and CSCs in relation with CRC (Takahashi et al., 2007; Yu et al., 2007; Carette et al., 2010; Miyoshi et al., 2010; Saiki et al., 2009). The first effort to use hiPSCs for CRC therapeutic purposes was to administer them as a vaccine. Given that both stem cells and cancer cells express oncofetal antigens, the group hypothesized that administration of stem cells may generate a protective immune response. The ease of iPSC cell line generation as well as improvements in ESC cell lines were critical to the feasibility of the study. Using a mouse model, the study injected two different human ESC cell lines (H9 and CT2) and one hiPSC line (TZ1) as vaccines. Irradiated CT26 CRC tumor cell line was injected as a positive control and PBS was injected as a negative control. When injected 2 weeks before tumor challenge, H9 showed drastic reduction in tumor size and weight as compared to the PBS group. A significant size reduction was also seen upon immunization 5 weeks before tumor challenge but not as drastically. This indicated that H9 was able to generate an effective memory antitumor immune response. In addition, H9 injected mice demonstrate a Gr1 1 CD11b 1 myeloid derived suppressor cells, which are known to impede tumor immunity (Gabrilovich and Nagaraj, 2009). Similar findings were seen with CT2 injections, but not with TZ1 injections, which were unable to impede tumor growth. Interestingly, TZ1 vaccination was able to produce a significant expression of IFNγ and IL-4, indicating that tumor rejection relied on processes

7.4 iPSCs and organoids in intestinal diseases

beyond cell-mediated immunity (Li et al., 2009). While no further investigations on the use of iPSCs for vaccination in CRC have been done, the results from this group indicate that this therapeutic pursuit may not be promising. Several groups have studied the correlation between CRC and iPSC-related gene expression. In a retrospective study of 79 patients, Saiki et al. found that LIN28 expression was directly correlated with Duke stages, while KLF4 was inversely correlated. The study also reported SOX2 being overexpressed in Dukes D disease, indicating the gene may play a role in metastasis (Saiki et al., 2009). Another retrospective study of 67 patients compared genes expressed CRC cells to matched normal cells. CRC tumor cells expressed higher levels of OCT4 and LIN28 and lower levels of NANOG. A CRC genetic signature of OCT4, NANOG, and LIN28 expression was correlated with a tumor site in the rectum, a negative lymph node state, and an advanced Duke’s classification. CRC with a signature of OCT4, NANOG, LIN28, and SOX2 was correlated with reduced survival outcomes for those in Duke’s B or C stages (Liu et al., 2013). Further studies of a larger sample size should be conducted to confirm the findings from these two studies. In a similar methodology to Liu et al., Zhou et al. studied the prognostic value of large intergenic noncoding RNA ribonucleic acids-regulator of reprogramming (lincRNA-ROR) by analyzing matched CRC and normal human tissue (Zhou et al., 2016). LincRNAROR was first described in iPSCs and later found to be an important regulator of epithelial-to-mesenchymal transition in breast cancer (Loewer et al., 2010; Takahashi et al., 2014; Hou et al., 2014). Zhou et al. used the median value of lincRNA-ROR expression among the 60 patients in the study to define high and low expression groups. The high expression group was correlated with pT stage, pN stage, AJCC stage, and vascular tumor invasion. iPSC-related genes were also studied in animal models. One such gene, KL5, which was previously identified to be strongly expressed at the base of intestinal crypts, was further studied using a mice model (Friedman et al., 2009; McConnell et al., 2007). KLF5 knockout in Lgr5 1 stem cells suppressed their proliferation and was associated with nuclear localization of β-catenin. Findings from two mouse crosses, both positive for oncogenic β-catenin, but only one of which was a KLF5 knockout, suggest that KLF5 is a key regulator of intestinal oncogenesis as the KLF5 knockout mice showed near complete suppression of lethal adenomas and carcinomas. Tumor suppressive miRNAs (miR-451, miR-193b, miR125a-5p, let-7d, and let-7e) were also found to be upregulated in these knockout mice (Nakaya et al., 2014). A different group studied the effect of NANOG by injecting a NANOG-knockdown CRC line into mice and comparing it to a positive control. NANOG-knockdown mice showed slowed tumorigenesis, and these cells were more susceptible to 5-fluorouracil, a common chemotherapy agent. The same study also compared the expression of OCT4, SOX2, NANOG, KLF4, and C-MYC of several CRC cell lines to normal colonic epithelium. In most CRC cell lines, all of these factors were found to be overexpressed except for Ujjain studies. Taken together, these animal and human studies indicate the importance of understanding iPSC-related gene expression in CRC.

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One important research strategy for understanding CRC involves transforming cancer cells into iPSCs. However, as mentioned previously, low reprogramming efficiency is a key limitation (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Lowry et al., 2008; Huangfu et al., 2008). To address this issue, Hoshino et al. studied the effects of hypoxia and TP53 deficiency on conversion efficiency of wild-type HCT116 CRC cells into iPSCs. The group used a lentiviral-mediated transfer of murine retroviral receptors followed by retroviral-mediated gene transfer of Yamanaka factors as their induction protocol. When compared to normoxic conditions (21% O2), hypoxic conditions (5% O2) presented with a conversion efficiency approximately four times greater. From this finding, the group further elucidated that the hypoxia-inducing factor pathway may play a role in reprogramming. TP53-null cells also showed a significant improvement in conversion efficiency (Hoshino et al., 2012). Another limitation of IPSCs for clinical use is their high expression of oncogenes such as c-MYC and KLF4 (Okita et al., 2007). Miyazaki et al. introduced a novel reprogramming method using introduction of miRNAs via lipofectamine that target Yamanaka factors in CRC cells lines. As compared to the CRC cell lines, normal reprogramming methodologies increased c-Myc by 2.7 times, while miRNA-induced pluripotent cancer cells (miPCCs) significant reduced c-Myc levels in all three CRC cell lines that were used (DLD-1, RKO, HCT116). Further investigation on the miPCCs from the DLD-1 cell line showed increased expression of protective p16INK4A, p21WAF1, and miR-145. These miPCCs also showed decreased expression of various multidrug resistance proteins and hence may have improved response to chemotherapy. The group is currently developing a nanoparticle for safe in vivo delivery of mature miRNAs (Miyazaki et al., 2015). Finally, two groups have worked on colon organoids derived from familial adenomatous polyposis (FAP) patients. Crespo et al. derived two HIOs from fibroblasts of two FAP patients (FAP-HIO) and compared them to wild type HIOs for drug screening purposes. By using this methodology, the group was able to show that XAV939 and rapamycin affected cell proliferation in both wild type HIOs and FAP-HIOs, but geneticin only affected FAP-HIOs, indicating its superior therapeutic specificity (Crespo et al., 2017). While only a select few drugs were tested in this study, this methodology indicates the potential for personalized drug screening for CRC. Sommer et al. also derived FAP-HIOs from fibroblasts, but studied their genetic differences with wild type HIOs. Using RNA sequencing by digital gene expression, the group found FAP-HIOs to have abnormal expression of Cadherin-17, Isocitrate dehydrogenase 1, lactate dehydrogenase A, and vimentin, among several other genes. By using gene ontology, gene set enrichment analysis, and ingenuity pathway analysis, the group showed exactly which components of the PPAR signaling pathway were mutated. Interestingly, the study also screened more than 400 IPSC colonies and found that none had a homozygous APC mutation, suggesting that APC may be critical to maintenance of pluripotency (Sommer et al., 2018). The findings by these two groups hint at the numerous possibilities that HIO-iPSCs offer to better study and model CRC.

7.4 iPSCs and organoids in intestinal diseases

7.4.2 Hirschsprung disease Hirschsprung disease (HSCR) is a common intestinal motility disorder characterized by the absence of enteric ganglion cells in distal regions of the colon. HSCR presents in newborns with an incidence of 1 in 5000. HSCR is present as shortsegment disease in 80% 85% of cases (S-HSCR), long-segment in 20% of cases, and total colonic aganglionosis in 3% 8% of cases (Parisi, 2019). HSCR is a life-threatening disease that requires immediate surgical resection of the aganglionic segment. However, despite resection, gastrointestinal dysfunction of the remaining digestive tract often persists. In cases of total colonic aganglionosis there is a high rate of complications and repeat operations are often necessary. Given the limited therapeutic strategies for HSCR, pluripotent stem cell-based therapies offer opportunities for significant improvement of clinical outcomes. While neural crest stem cells (NCSCs) had been previously generated from hESCs, Lee et al. was the first to report a protocol for their derivation from hiPSCs (Pomp et al., 2005; Lee et al., 2007; Jiang et al., 2009; Hotta et al., 2009; Lee et al., 2010). Within 2 years, two more such protocols were reported with one group defining a one-step protocol and another showing the in vitro myelination ability of Schwann cells differentiated from the generated NCSCs (Menendez et al., 2011; Liu et al., 2012). The protocol defined by Menendez et al. is particularly promising because it not only achieved a high enrichment of NCSCs from hIPSCs but also established self-renewal capacity of NCSCs for over 30 passages. Notably, the activation of Wnt signaling and suppression of the Activin A/Nodal pathway are central to this protocol (Menendez et al., 2011). NCSCs derived from hiPSCs were later verified to have the same differentiation potential as those derived from hESCs (Li et al., 2018). The next critical step in hiPSC utility for HSCR was taken by Workman et al. as they reported developing an HIO with a functional enteric nervous system (HIOENS). After using a protocol that was previously reported to generate cranial neural crest cells, these cells were incorporated into spheroids by low-speed centrifugation. An abundance of βIII-tubulin positive neurons and S100β1 glia were found in the mesenchyme of HIOs-ENS. Furthermore, HIOs-ENS were reported to have a spatial relationship between the various cell types that closely resembled human fetal intestine (Workman et al., 2017). Given the various physiological differences between human and mice intestinal development and physiology, HIOs-ENS may be a significantly improved translational model to mice in the study of HSCR (Baetge and Gershon, 1989; Blaugrund et al., 1996; Anlauf et al., 2003). The same group further studied HIOs-ENS by engrafting them into mice and allowing several weeks for maturation. The transplanted HIOs-ENS stained positive for various markers including CALB1, TH, calretinin, CHAT, 5-HT, and most notably NOS1, which was not expressed in in vitro HIOs-ENS. By repeating the same protocol, but by using pluripotent stem cells that contained PHOX2B mutations, a known driver of complete aganglionosis, the authors were also able create a potential engrafting model for HSCR that had reduced neural count and

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poorly developed intestinal smooth muscle (Workman et al., 2017). In some contrast to protocol described in Workman et al., Chang et al. devised a two-step process to achieve mice implantation by first engrafting an HIO (without an ENS) and then, at a later time point, injecting enteric neural crest cells (ENCCs) derived from hiPSCs. Thorough immunohistochemical analysis revealed the matured intestines to be positive for TUJ1, S100, CKIT, SMA, ECAD, MUC2, CHGA, LYSO, CALB, CHAT, NOS1, 5-HT, PCNA, and LAMIN, verifying similar findings by Workman et al. (Workman et al., 2017; Chang et al., 2020). Notably, the neural cells were OCT4 negative, indicating reduced risk for teratoma formation. Another interesting use for hiPSCs for HSCR is their ability to assist in detection of genetic mutations. By harvesting fibroblasts from patients with S-HSCR, Lai et al. generated iPSCs and then further differentiated them into ENCCs. By comparing the transcriptomes of the fibroblasts, iPSCs, and ENCCs to their wild type counterparts, the vinculin gene (VCL) M209L mutation was found to be associated with S-HSCR. The author reports this to be the first known genetic association with S-HSCR (Lai et al., 2017). The findings described in the CRC section, along with this study, suggest a disease-agnostic use of iPSCs in combination with genomic and transcriptomic analysis to derive novel correlations between mutations and disease. Combining these findings with CRISPR-Cas9 sequence editing has powerful therapeutic implications. It is suggested that between 2 and 4 million ENCCs are needed to colonize a mouse colon (Fattahi et al., 2016). The extrapolation of this finding for human colons may suggest that enteric system colonization is an unrealistic feat. However, the positive results seen in various engraftment experiments and the potential for earlier detection of HSCR through novel genetic analysis suggest that further study of hiPSCs could result in therapeutic interventions for this disease.

7.4.3 Inflammatory bowel disease Inflammatory bowel diseases (IBD) are chronic disorders that have a relapseremitting pattern and their incidence is increasing worldwide (Holmberg et al., 2017; Khor et al., 2011). The two most common forms of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). While the specific causes of IBD is unknown, IBD is associated with hyperactivation of immune cells and secretion of inflammatory cytokines, such as TNF-α (Satsu et al., 2006; Okamoto and Watanabe, 2016). Intestinal fibrosis, which is caused by chronic inflammation, is often a secondary complication of IBD (Andres and Friedman, 1999). More recently, there has been growing evidence that implicates intestinal epithelial cell dysfunction in the pathogenesis and progression IBD, with specific reference to its role in integrity maintenance, generation of defense secretions, and bacterial detection (Dotti and Salas, 2018; McCole, 2014; Peterson and Artis, 2014; Pastorelli et al., 2013).

7.4 iPSCs and organoids in intestinal diseases

In specific to the study of IBD, HIOs have a significant advantage over cell lines, human fetal intestinal organ culture, and animal models (Yoo and Donowitz, 2019). Because cell lines are genetically transformed, they often contain genotypes that are markedly different from their cells of origination (Grabinger et al., 2014). In contrast, HIOs maintain their original in vivo characteristics and the genotype associated with their stem cells (Sato et al., 2009). In addition, HIOs maintain the structure of a crypt or villus and are able to generate all types of intestinal epithelial cells, allowing for the study of paracrine and autocrine signaling (Foulke-Abel et al., 2014). The understanding of the structure disintegration and intercellular signaling could be critical to better characterizing IBDs. Human fetal intestinal organ culture has been used to understand the pathogenesis of celiac disease and enterocolitis and does provide multiple cell types unlike cell lines (Nanthakumar et al., 2000; MacDonald and Spencer, 1988). However, this medium too falls short to HIOs because of its short viability and difficulties with real-time monitoring (Al-Lamki et al., 2017). HIOs can therefore allow for collection of greater datasets over a long-term period that would likely better imitate the progression of IBD. Finally, animal models, while replicating a truly systemic IBD, fall short to HIOs given their inability to imitate human phenotypes. The phenotypic and mechanistic differences between humans and animals may be contributing to the high failure rate of drugs in clinical trials (Foulke-Abel et al., 2014). This gap, in addition to the high-throughput in vitro drug screening capacity of HIOs, make HIOs a superior choice for initial testing of IBD drug candidates. One group generated HIO-iPSCs with small intestinal traits for the purposes of assessing their use as models for IBD drug screening. HIO-iPSCs were treated with TNF-α to imitate the pathogenesis of IBD. Using fluorescent material, the group showed TNF-α disrupted tight junctions. However, after administration of infliximab (IFX), a TNF-α inhibitor, tight junctions were restored. In addition, TNF-α altered levels of Villin 1, MUC2, interleukin 1β, and ISC marker olfactomedin. All of these alterations were significantly rescued by the administration of IFX. A second, similar experiment was also done using TGF-β, which is implicated in tissue fibrosis. When TNF-α and TGF-β were coadministered, levels of a-SMA, vimentin, collagen type-1, and fibronectin were all significantly altered. After administration of SB431542, a TGF-β inhibitor, all these alterations were significantly rescued. Taken in sum, these findings imply that HIO-iPSCs are capable of imitating the mucosal damage and tissue fibrosis characteristic of IBD and can be used for screening of IBD drug candidates (Onozato et al., 2020). HIOs-iPSCs are not yet perfect models for IBD. HIOs lack immune cells, which are associated with IBD pathogenesis and progression. By coculturing with group 3 innate lymphoid cells (ILC3s) and macrophages, two separate groups have created HIO models that address this limitation (Lindemans et al., 2015; Noel et al., 2017). In this coculture, Lindemans et al. was able to show that ILC3s are capable of producing IL-22. In turn, this IL-22 played a key role in activating ISCs to promote proliferation and increase the size of the HIOs

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(Lindemans et al., 2015). Noel et al. showed that macrophage coculture improved the HIO’s epithelial barrier function (Noel et al., 2017). Additional coculture experiments are needed to derive an organoid model that is more reflective of the role of the immune system in human intestinal tissue.

7.4.4 Parasite Culture of parasites is unique, because unlike bacteria, the parasites are dependent on their host organisms for sustenance. While there is literature that supports the in vitro culture of parasites, there are still significant limitations that remain (Taylor and Baker, 1987; Visvesvara and Garcia, 2002). First, some parasites such as Cyclospora cayetanensis cannot be cultured in vitro (Ortega and Sanchez, 2010). Other parasites such as Cryposporidium Spp. and Giardia duodenalis have limited culturing protocols (Arrowood, 2002; Be´ne´re´ et al., 2010; Karanis, 2018). While some parasites are cultured in axenic systems, these are extremely limited as they offer no insight into the relationship of the parasite with its biochemic dependencies (Clark and Diamond, 2002; Ahmed, 2014). Finally, parasites that are dependent on hosts to complete their life cycle or have hosts in specific species prove extremely difficult to study (Ahmed, 2014; Schuster, 2002; Sibley et al., 2002). Given that the most common intestinal parasites affect up to 20% 30% of the population worldwide, there is an extreme need for improved culture methodologies (World Health Organization, 1987; Kucik et al., 2004). HIOs offer opportunities for the improved study of habitat parasite interactions for G. duodenalis and Toxoplasma gondii (Klotz et al., 2012). G. duodenalis is easily cultured in vitro, but its pathogenicity in humans is still not understood. Two reviews suggest there is still a lack of understanding as to how apoptosis and loss of intestinal barrier function is driven by the parasite and which virulence factors cause this process (Ankarklev et al., 2010; Cotton et al., 2011). HIOs provide an ecosystem to study this interaction at the cellular and molecular levels. One limitation remains the lack of an immune system in HIOs, however, a recent NIH grant suggests this implementation is being studied [REF: https://grantome. com/grant/NIH/U19-AI116482-05-6235]. With Toxoplasma gondii, HIOs primarily offer the opportunity to test the virulence of various genotypes at a large scale (Rao et al., 2012; Wendte et al., 2011). As feline intestinal organoids are generated, there is a great to better characterize the mechanisms of the T. gondii sexual reproduction cycle, which occurs only in felines (Augustyniak et al., 2019). Finally, similarly to the discussion in the IBD section, HIOs offer an opportunity to screen for new antiparasitic drugs in a rapid and cost-effective manner (Ebert and Svendsen, 2010).

7.4.5 Viruses Similar to parasites, virus pathogenicity is heavily dependent on its interaction with its host. In fact, this dependency is even more closely intertwined as viruses

7.5 Clinical trials

use host cellular machinery in an effort to replicate in both lysogenic and lytic cycles (Ryu, 2017). Several viruses have intestinal involvement as diarrhea is a common symptom of viral disease. In fact, rotavirus is the leading cause of diarrhea in young children worldwide, and noroviruses cause nearly 50% of all foodborne diarrheal outbreaks in the United States (Anon., 2013, 2013; Hall et al., 2013). By combining the use of organoids and CRISPR/Cas9 technology, there have been new mechanistic discoveries in our understanding of rotaviruses, noroviruses, enteroviruses, and adenoviruses (Kim et al., 2019). In 2015 two groups used HIOs to characterize rotavirus. Yin et al. showed that IFN-α, anti-VP7 antibody, and ribavirin have virus-suppressive activity in HIOs (Yin et al., 2015). Separately, Saxena et al. reported the effects of NSP4 peptide on HIOs, by demonstrating its ability to cause water influx (Saxena et al., 2016). These findings, along with findings that shows the similarity of Rotavirus pathogenicity in humans and HIOs, suggest that HIOs are ideal models for the further study of rotavirus (Yin et al., 2015). Ettayebi et al. was the first to report successful culture of norovirus using HIOs. The group describes the role that human bile plays in activating viral replication. Using this model, they were able to identify the need for a functional fucosyltransferase 2 enzyme to be susceptible to GII.4 noroviruses, and hence providing a therapeutic target (Ettayebi et al., 2016). HIOs infected with enteroviruses were first reported in 2017. Drummond et al. described HIOs infected by various sub-families of enteroviruses including echovirus, coxsackievirus B, and enterovirus 71 (Drummond et al., 2017). This suggests the possibility of further study of these enteroviruses by using HIOs. iPSCs also play a role in the study of adenoviruses. Using mouse small intestine organoids, Wilson et al. was able to identify the role of Paneth cells, specifically through the production of α-defensin, in protecting intestines from the virus (Wilson et al., 2017). Later, the same group used HIOs to culture adenovirus and showed the functionality of interferon treatment. This study elucidated that adenovirus had the preference to infect goblet cells (Holly and Smith, 2018). The progress with adenoviruses and viral infectivity of HIOs at large suggest that HIOs allow for the advancement of our understanding in viral diseases in a manner that was not previously possible.

7.5 Clinical trials Comparing the findings from 2019 and 2020 reviews of the state of hiPSC-based clinical trials, we observe a 100% increase in the number of these trials over 13 months (Deinsberger et al., 2020; Yamanaka, 2020). However, for the pathologies presented in this chapter, only four notable trials in the United States that are ongoing as of December 2020. In one trial, the National Cancer Institute is recruiting cancer patients, including those with CRC, to further characterize the

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ability to generate hiPSC stem cell lines from tumor cells (Trial Registration Number: NCT03407040). These stem cell lines would in turn be used to create antigen specific T-cells (Vizcardo et al., 2013). Similarly for HSCR, another clinical trial is collecting whole blood from patients and their unaffected family members to better characterize hiPSCs for future treatments (Trial Registration Number: NCT04476225) (Finkbeiner et al., 2015). Of the aforementioned pathologies, only CRC has a therapeutic, FT500, that is being studied in two clinical trials (Trial Registration Numbers: NCT04106167 and NCT03841110). FT500 is an allogenic natural killer immunotherapy that is derived from a clonal master hiPSC cell line, and its effects are being studied as a combination therapeutic with checkpoint inhibitors and immunosuppressants (Hsu et al., 2018; Jenkins et al., 2018). These limited number of clinical trials represent the novelty of the field and suggest that the therapeutic potential of hiPSCs for GI diseases is yet to be unlocked.

7.6 Limitations Although all the progress mentioned in this chapter represents exciting opportunities, iPSCs, at the moment, still have important limitations that are worth reviewing. First, iPSCs maintain immunogenicity, meaning that allogeneic grafts threaten to be rejected due to human leukocyte antigen (HLA) mismatch (Xu et al., 2019; Williams et al., 2017). HLA are cell-surface molecules that regulate the immune system by presenting antigenic peptides to T-cells. While autologous grafts represent the ability to overcome this limitation, the high costs and extended timeline to administer an individualized treatment make this therapeutic strategy unlikely to be pursued in the next several years (Bravery, 2015). Recent advances in HLA cloaking, often using CRISPR-Cas9 technology, represent an opportunity to address allogeneic immunogenicity and should be further characterized in clinical trials (Xu et al., 2019; Meissner et al., 2015; Gornalusse et al., 2017; Deuse et al., 2019). Another important limitation of iPSCs is their heterogeneity. This remains a great concern when considering the use of iPSCs for disease modeling and regenerative cell therapy since this leads to poor reproducibility of research. Studies have suggested that genetic and epigenetic factors contributed largely to heterogeneity in iPSCs (Cahan and Daley, 2013; Choi et al., 2015; Nishizawa et al., 2016). Other reasons hypothesized for heterogeneity include culture conditions, donor cell type and “epigenetic memory,” and reprogramming methods (Hayashi et al., 2019). Some of these variation issues can be circumvented by using gene editing technologies like CRISPR/Cas9, some researchers have tried converting a “primed” state of iPSC into a “naı¨ve” state to address heterogeneity (Brons et al., 2007; Tesar et al., 2007). More studies are needed to identify the factors contributing toward the heterogeneity of iPSCs.

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Overall, the limitations of iPSCs have the potential to be overcome, and the tremendous progress in addressing GI diseases by using iPSC technologies in the past decade represents the potential for iPSC-based therapeutics to be a commonplace clinical application in the decades to come.

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Potential of embryonic stem cells for treating intestinal diseases

8

Ahmed El-Hashash The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, P.R. China Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, P.R. China

8.1 Embryonic stem cells Embryonic stem cells (ESCs) are unique pluripotent cells that can self-renew and differentiate (Coraux et al., 2005; Chagastelles and Nardi, 2011). ESCs are derived from the inner cell mass of human or animal embryos at the blastocyst stage of embryonic development. They possess the ability of indefinite replication in vitro and, therefore, can self-renew in an undifferenced state, with retaining the differentiation ability into a variety of cell types that belong to all three germinal layers of the body in vivo and in culture, and can also form functional airway epithelial cells (Coraux et al., 2005; McIntyre et al., 2014; Sadeghian Chaleshtori et al., 2016). ESCs have, therefore, great regenerative and therapeutic potentials for injured tissues or diseases (Baharvand and Hassani, 2013). For example, ESCs can be induced to differentiate into many specialized cell types, including lung, heart, renal, neurons, and insulin-producing cells that can be used for tissue repair and regeneration. In addition, stem cells have a major role in the maintenance of the integrity of adult tissues and have many applications in tissue repair and regeneration (reviewed recently by Ilic and Ogilvie, 2017). Human ESCs were first isolated in 1998 and attracted the attention of scientists worldwide who investigated the potential application of these ESCs in cell therapy. One potential application is the manipulation of some important genes in ESCs for correcting gene deficiencies before therapeutic implantation in different human diseases. Treatments of ESCs with the antidifferentiation cytokine leukemia inhibitory factor can make these cells indefinitely proliferate in vitro and retain their ability to give rise to different cell and tissue types; while adult stem cells can differentiate into cell types within a particular lineage and show a limited proliferation capacity. Many in vivo and in vitro research studies have uncovered the functions of several signaling pathways and extracellular signals in ESC differentiation (Quante and Wang, 2009). This has accelerated the success of ESC The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00001-1 © 2021 Elsevier Inc. All rights reserved.

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differentiation into mesodermal and ectodermal tissues. ESC differentiation into endodermal tissues, including the gastrointestinal organ systems and related organs has been also reported (Quante and Wang, 2009). The potential applications of human ESCs in advancing cellular therapy, modeling of human diseases, and drug discovery have been reviewed (Ilic and Ogilvie, 2017). A remarkable approach is to use somatic cell nuclear transfer to develop patient-specific ESCs, by reimplanting a nucleus from a donor somatic cell into an enucleated oocyte. This will lead to the generation of a cloned embryo (Dolly the sheep is a good example). Interestingly, combining the nuclear cloning with gene/ cell therapy has considered as a valid approach and strategy for the treatments of genetic disorders in animal studies (Quante and Wang, 2009).

8.2 ESC-based therapy of the gastrointestinal diseases 8.2.1 ESC-based therapy for inflammatory bowel disease Inflammatory bowel disease (IBD) refers to a group of intestinal disorders causing digestive tract inflammation, including damages of the immune system and intestinal tissues. These damaged intestinal tissues can be repaired by stem cell therapy that can also correct the associated immunological abnormalities. While adult SCs have remarkable therapeutic potentials for tissue reaper/regeneration and degenerative diseases’ treatment, the pluripotent potential of ESCs enables them to differentiate into intestinal epithelial and immune cells, allowing these cells to restore the intestinal epithelium and the immune balance in the murine model of colitis (Singh et al., 2010). Interestingly, the small intestine is a remarkable system to investigate stem cell biology in mammals, including their proliferation, behavior, and differentiation. The structural organization of mouse intestine includes an epithelial monolayer that lines the intestine and forms villus and crypt structures, which have distinct functions and morphology. Notably, the most rapidly proliferative adult tissues in mammals are both small intestine and colon epithelia that are renewed by stem cells residing in the crypt base every 3 5 days. After they are generated by stem cells, the transit-amplifying cells, reach the crypt villus junction before differentiation to give rise to the four terminally differentiated type of cells in the mucosa that are Paneth, goblet, enterocyte, and enteroendocrine cells (Barker et al., 2008). While Paneth cells are differentiated cells that could not migrate upward, the other three cell types show upward migration to the villi tips from the crypt (van der Flier and Clevers, 2009; Wang and Hou, 2010). In addition, highly proliferative and pluripotent stem cells reside in a zone near the crypt base and the villus may provide a stem cell niche that is important for stem cell proliferation, migration, and differentiation. The renewal of the entire intestinal epithelium occurs every 72 hours in murine intestine and 5 days in human intestine (Wright, 2000; van der Flier and Clevers, 2009; Wang and Hou, 2010). The

8.2 ESC-based therapy of the gastrointestinal diseases

interaction between stem cells and their niche is important for their behavior, proliferation, and differentiation (Singh and Hou, 2008; van der Flier and Clevers, 2009; Wang and Hou, 2010). Stem cells could be used in the treatment of IBD due to their homing properties to the injury sites and differentiation capacity into both lymphocyte and epithelial cells that modulate both immune response and damages of tissues (Singh et al., 2010). ESCs have a high potential for tissue repair and regeneration since they can differentiate into all cell types in the body and produce a clinically relevant number of defined cell populations. ESCs are hot topics in research aiming to find treatments for inflammatory diseases and to identify mechanisms by which acquired or congenital human diseases occur (Murry and Keller, 2008; Singh et al., 2010). Although ESC plasticity enables them to produce different type of cells, it may also make ESCs could not control their behavior. Interestingly, ESCs could ameliorate piroxicam-induced colitis in IL10 deficient mice (Srivastava et al., 2007). In addition, predifferentiated ESCs in culture could exclusively migrate and home to the small intestine, colon, and the liver; show a long-term engraftment; decrease both tissue damage and inflammation; and restore immune balance in IL10-/- mice (Srivastava et al., 2007). Furthermore, an improved protocol has been used to efficiently enrich mesenchymal stem cells (MSCs) from human ESCs by specifically inhibiting the SMAD2/3 pathway (Sa´nchez et al., 2011). These human ESC-derived MSCs show several features, including their potential for multilineage differentiation, and their potent antiinflammatory and immunosuppressive properties in vivo and in vitro that enable them to protect against experimental IBD (Sa´nchez et al., 2011).

8.2.1.1 IBD therapy using other stem cell types Other types of stem cells are also important for the treatment of IBD, including MSCs and hematopoietic stem cells (HSCs). These stem cells bring hope for transferring stem cell-based therapy/approach from in vivo animal models of IBD to human patients. For example, the characteristic immunosuppressive action of stem cells is important in their clinical applications on IBD patients. In addition, studies that are based on stem cells could eventually lead to developing novel drugs for the treatment of IBD (Singh et al., 2010). In addition, the function of MSCs derived from human gingival as an antiinflammatory and immunomodulatory component of the immune system was demonstrated in vivo (Zhang et al., 2009). They ameliorated inflammation-related tissue damages in experimental colitis and may be a promising source for IBD cell therapy (Zhang et al., 2009). The bone marrow derived stem cells (BM-SCs) were used in the treatments of human IBD and induced colitis in mice (Quante and Wang, 2009). Indeed, BM-SC transplantation can effectively treat both ulcerative colitis and Crohn’s disease (Brittan et al., 2007). These stem cells can ameliorate induced colitis in murine model by homing to the inflamed colon and enhancing microcirculation and tissue regeneration in mice (Khalil et al., 2007). Alleviation of experimental colitis in mice was also reported using adipose tissue derived MSCs that

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induce T-regulatory cells and increase interleukin 10 levels in treated mice (Gonzalez et al., 2009). A recent study has determined the potential therapeutic effects of multiple sources of MSCs of fetal, adult, and embryonic origin and compared their roles in treating acute IBD using murine model (Kagia et al., 2019). Interestingly, more histopathological and clinical improvements were shown only in acute IBD murine model that received MSCs derived from the bone marrow or umbilical cord (Kagia et al., 2019). The MSC-based therapy of IBD has been recently reviewed (Gre´goire et al., 2017).

8.2.1.2 Roles of ESCs and other stem cells in the therapy of accessory digestive organs The application of different types of stem cells in the regenerative therapy of gastrointestinal and liver diseases, such as intestinal failure and known liver diseases, has attracted the attention of researchers and clinicians worldwide. For example, human ESC-derived hepatocytes (or derived from other human stem cell types) represent a potential liver disease treatment. Using specific protocol and culture medium, Basma et al. (2009) have derived cells with a hepatocyte phenotype from human ESCs in culture. These cells can secret significant human albumin and α1-antitrypsin levels when growing in culture and secrete moderate human albumin and α1-antitrypsin levels into the serum after transplantation in rodent models for more than 60 days, suggesting a promising therapeutic approach using human ESCs (Basma et al., 2009). However, this approach has several challenges, including the potential immune rejection of ESCs that is also not patient specific. Alternative strategies include the generation of patient-specific ESCs, which can be used for autologous transplantation (Quante and Wang, 2009). Interestingly, a recent study has developed an efficient protocol for marmoset ESC differentiation into functional hepatocyte-like cells (Aravalli et al., 2020). These ESC-derived hepatocyte-like cells can express many hepatocyte-specific markers, including α-fetoprotein, albumin, α-1 antitrypsin, and asialoglycoprotein receptor 1 and are functionally competent. These cells may, therefore, valuable for both drug metabolism studies and cell transplantation therapy for liver diseases (Aravalli et al., 2020). MSCs have antiinflammatory properties that can facilitate their roles in tissue repair and regeneration. Indeed, MSCs can be differentiated to form hepatocytes and contribute to liver repair and regeneration when infused into hepatic failure mouse models (Kuo et al., 2008). Granulocyte colony-stimulating factor increases MSC mobilization and promotes the activation of endogenous liver stem cells (Oval cells), which are partially derived from the bone marrow, suggesting that this factor may facilitate liver tissue repair and regeneration (Piscaglia et al., 2007). Furthermore, BM-SCs are probably involved in human liver repair (Gehling et al., 2005), and their therapeutic potential was investigated using intraportal autologous transplantation that leads to clinical improvements of some cases (am Esch et al., 2005; Terai et al., 2006). Remarkably, evidences of the

8.3 Conclusions

therapeutic potentials of human ESC-MSC-derived molecules that provide trophic supports to hepatocytes have been reported (Lotfinia et al., 2016). In addition, a contribution of transplanted human induced mesenchymal stem cells (iMSCs) to the in vivo regeneration of liver was reported, suggesting a promising tool for treating inherited diseases in the liver (Spitzhorn et al., 2018).

8.2.1.3 Role of ESCs and other stem cells in the gastrointestinal and related diseases ESCs may play a role in some neurodegenerative disorders of the gastrointestinal tract. Deriving neural precursor cells from ESCs is well established (Pera et al., 2004; Gerrard et al., 2005; Zhang, 2006; Elkabetz et al., 2008; Koch et al., 2009), and ESCs were successfully induced toward a neural crest lineage that forms enteric nervous system (ENS; Lee et al., 2007; Kawaguchi et al., 2010). The ESC-derived neural crest stem cells could colonize explanted gut tissues and differentiate to form the enteric glial and neuronal cells (Lee et al., 2007), and several studies on their development of functional neuromuscular connections are currently underway. Furthermore, the incomplete migration of the enteric ganglion cell precursors during the development of the intestine causes the relax failure of the aganglionic segment of the colon, leading to the development of Hirschsprung disease as a motor disorder of the gut (aganglionic gut). Regenerative stem cell based therapy can play an important role in gastrointestinal motility disorders, including those related to the Hirschsprung disease. For example, Metzger et al. (2009) utilized postnatal human gut mucosal tissues in the ex vivo generation of ENS stem cells. This study used endoscopy to get cells from human postnatal gut mucosal tissues, before growing in culture to form neurosphere-like bodies that were transplanted into an animal model of aganglionic gut (Metzger et al., 2009). Interestingly, the transplanted neurosphere-like bodies colonized the mucosal tissues before differentiation into the neurones and glial cells of the ENS (Metzger et al., 2009). The roles of other stem cell types such as induced pluripotent stem cells, bone marrow stem cells, and MSCs in intestinal diseases will be described in detail in three chapters of this book.

8.3 Conclusions The ESC pluripotent potential enables their differentiation into intestinal epithelial and immune cells, allowing these cells to restore the intestinal epithelium and the immune balance in the murine model of colitis. Indeed, recent achievements in stem cell research and therapy have provided possibilities for using these cells in treating several gastrointestinal and related diseases. For example, stem cells could be used in the treatment of IBD due to their homing properties to the injury sites, and differentiation capacity into both lymphocyte and epithelial cells that

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modulate both immune response and damages of tissues. Other types of stem cells are also important for the treatment of IBD and/or colitis, including MSCs, HSCs, and BM-SCs. These stem cells bring hope for transferring stem cell based therapy/approach from in vivo animal models of IBD to human patients. In addition, human ESC-derived hepatocytes represent a potential liver disease treatment and can be used in drug metabolism studies and cell transplantation therapy for liver diseases. Moreover, ESCs may play a role in some neurodegenerative disorders of the gastrointestinal tract. Notably, the use of adult or tissue-specific stem cells in the gut, liver, and pancreas related diseases is a promising approach that has significant clinical applications. However, these stem cells also represent cancer stem cell source and, therefore, more studies are still needed to evaluate the risks versus benefits of these promising therapies.

References am Esch 2nd, J.S., et al., 2005. Portal application of autologous CD133 1 bone marrow cells to the liver: a novel concept to support hepatic regeneration. Stem Cell 23, 463 470. Aravalli, R.N., Collins, D.P., Hapke, J.H., Crane, A.T., Steer, C.J., 2020. Hepatic differentiation of marmoset embryonic stem cells and functional characterization of ESC-derived hepatocyte-like cells. Hepat. Med. 12, 15 27. Baharvand, H., Hassani, S.N., 2013. A new chemical approach to the efficient generation of mouse embryonic stem cells. Methods Mol. Biol. 997, 13 22. Barker, N., van de Wetering, M., Clevers, H., 2008. The intestinal stem cell. Genes Dev. 22, 1856 1864. Basma, H., et al., 2009. Differentiation and transplantation of human embryonic stem cellderived hepatocytes. Gastroenterology 136, 990 999. Brittan, M., Alison, M.R., Schier, S., Wright, N.A., 2007. Bone marrow stem cell-mediated regeneration in IBD: where do we go from here? Gastroenterology 132, 1171 1173. Chagastelles, P.C., Nardi, N.B., 2011. Biology of stem cells: an overview. Kidney Intl. Suppl. 1 (3), 63 67. Coraux, C., Nawrocki-Raby, B., Hinnrasky, J., et al., 2005. Embryonic stem cells generate airway epithelial tissue. Am. J. Respir. Cell Mol. Biol. 32 (2), 87 92. Elkabetz, Y., Panagiotakos, G., Al Shamy, G., et al., 2008. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 22, 152 165. Gehling, U.M., et al., 2005. Partial hepatectomy induces mobilization of a unique population of haematopoietic progenitor cells in human healthy liver donors. J. Hepatol. 43, 845 853. Gerrard, L., Rodgers, L., Cui, W., 2005. Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cell 23, 1234 1241. Gonzalez, M.A., Gonzalez-Rey, E., Rico, L., Buscher, D., Delgado, M., 2009. Adiposederived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology 136, 978 989.

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Gre´goire, C., Lechanteur, C., Briquet, A., Baudoux, E´., Baron, F., Louis, E., et al., 2017. Review article: mesenchymal stromal cell therapy for inflammatory bowel diseases. Aliment. Pharmacol. Ther. 45 (2), 205 221. Ilic, D., Ogilvie, C., 2017. Concise review: human embryonic stem cells—what have we done? What are we doing? Where are we going? Stem Cell 35 (1), 17 25. Kagia, A., Tzetis, M., Kanavakis, E., Perrea, D., Sfougataki, I., Mertzanian, A., et al., 2019. Therapeutic effects of mesenchymal stem cells derived from bone marrow, umbilical cord blood, and pluripotent stem cells in a mouse model of chemically induced inflammatory bowel disease. Inflammation 42 (5), 1730 1740. Kawaguchi, J., Nichols, J., Gierl, M.S., et al., 2010. Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos. Development 137, 693 704. Khalil, P.N., et al., 2007. Nonmyeloablative stem cell therapy enhances microcirculation and tissue regeneration in murine inflammatory bowel disease. Gastroenterology 132, 944 954. Koch P., Opitz T., Steinbeck J.A., et al. A rosette-type, self-renewing human ES cellderived neural stem cell with potential for in vitro instruction and synaptic integration. Proc. Natl. Acad. Sci. USA. 2009;106:3225 3230. Kuo, T.K., et al., 2008. Stem cell therapy for liver disease: parameters governing the success of using bone marrow mesenchymal stem cells. Gastroenterology. 134 (2111 2121), e1 e3. Lee, G., Kim, H., Elkabetz, Y., et al., 2007. Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat. Biotechnol. 25, 1468 1475. Lotfinia, M., Kadivar, M., Piryaei, A., Pournasr, B., Sardari, S., Sodeifi, N., et al., 2016. Effect of secreted molecules of human embryonic stem cell-derived mesenchymal stem cells on acute hepatic failure model. Stem Cell Dev. 25 (24), 1898 1908. McIntyre, B.A., Alev, C., Mechael, R., et al., 2014. Expansive generation of functional airway epithelium from human embryonic stem cells. Stem Cell Transl. Med. 3, 7 17. Metzger, M., Caldwell, C., Barlow, A.J., Burns, A.J., Thapar, N., 2009. Enteric nervous system stem cells derived from human gut mucosa for the treatment of aganglionic gut disorders. Gastroenterology. 136 (2214 2225), e1 e3. Murry, C.E., Keller, G., 2008. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661 680. Pera, M.F., Andrade, J., Houssami, S., et al., 2004. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J. Cell Sci. 117, 1269 1280. Piscaglia, A.C., Shupe, T.D., Oh, S.H., Gasbarrini, A., Petersen, B.E., 2007. Granulocytecolony stimulating factor promotes liver repair and induces oval cell migration and proliferation in rats. Gastroenterology 133, 619 631. Quante, M., Wang, T.C., 2009. Stem cells in gastroenterology and hepatology. Nat. Rev. Gastroenterol. Hepatol. 6 (12), 724 737. Sadeghian Chaleshtori, S., Dezfouli, M.R., Dehghan, M.M., Tavanaeimanesh, H., 2016. Generation of lung and airway epithelial cells from embryonic stem cells in vitro. Crit. Rev. Eukaryot. Gene Expr. 26 (1), 1 9. Sa´nchez, L., Gutierrez-Aranda, I., Ligero, G., Rubio, R., Mun˜oz-Lo´pez, M., Garcı´a-Pe´rez, J.L., et al., 2011. Enrichment of human ESC-derived multipotent mesenchymal stem cells with immunosuppressive and anti-inflammatory properties capable to protect against experimental inflammatory bowel disease. Stem Cell 29 (2), 251 262.

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Singh, S.R., Hou, S.X., 2008. Lessons learned about adult kidney stem cells from the malpighian tubules of Drosophila. J. Am. Soc. Nephrol. 19, 660 666. Singh, U.P., Singh, N.P., Singh, B., et al., 2010. Stem cells as potential therapeutic targets for inflammatory bowel disease. Front. Biosci. (Sch. Ed.) 2, 993 1008. Spitzhorn, L.S., Kordes, C., Megges, M., Sawitza, I., Go¨tze, S., Reichert, D., et al., 2018. Transplanted human pluripotent stem cell-derived mesenchymal stem cells support liver regeneration in Gunn rats. Stem Cell Dev. 27 (24), 1702 1714. Srivastava, A.S., Feng, Z., Mishra, R., Malhotra, R., Kim, H.S., Carrier, E., 2007. Embryonic stem cells ameliorate piroxicam-induced colitis in IL10-/- KO mice. Biochem. Biophys. Res. Commun. 361, 953 959. Terai, S., et al., 2006. Improved liver function in liver cirrhosis patients after autologous bone marrow cell infusion therapy. Stem Cell 24, 2292 2298. van der Flier, L.G., Clevers, H., 2009. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu. Rev. Physiol. 71, 241 260. Wang, P., Hou, S.X., 2010. Regulation of intestinal stem cells in mammals and Drosophila. J. Cell Physiol. 222, 33 37. Wright, N.A., 2000. Epithelial stem cell repertoire in the gut: clues to the origin of cell lineages, proliferative units and cancer. Int. J. Exp. Pathol. 81, 117 143. Zhang, S.C., 2006. Neural subtype specification from embryonic stem cells. Brain Pathol. 16, 132 142. Zhang, Q., Shi, S., Liu, Y., Uyanne, J., Shi, Y., Shi, S., et al., 2009. Mesenchymal stem cells derived from human gingiva are capable of immunomodulatory functions and ameliorate inflammation-related tissue destruction in experimental colitis. J. Immunol. 183, 7787 7798.

CHAPTER

Stem-cell therapy with bone marrow (hematopoietic) stem cells for intestinal diseases

9

Mahmoud Shaaban Mohamed1, Mahmoud I. Elbadry2 and Chao-Ling Yao3 1

Zoology Department, Faculty of Science, Assiut University, Assiut, Egypt Internal Medicine Department, Division of Hematology, Faculty of Medicine, Sohag University, Sohag, Egypt 3 Department of Chemical Engineering, National Cheng Kung University, No. 1, University Road, Tainan, Taiwan

2

9.1 Introduction The hematopoietic blood machinery made up of many cell sorts with certain functions: red blood cells (erythrocytes), which bring oxygen into the tissue; platelets (derived from megakaryocytes) help prevent bleeding; and granulocytes (neutrophils, basophils, and eosinophils) and macrophages (known as myeloid cells) fight foreign infections caused by bacteria, fungi, and other parasites. Many of these cells are also involved in the transformation of tissues and bones and the elimination of dead cells. B lymphocytes produce antibodies, while T lymphocytes can directly kill or isolate inflammatory cells that are diagnosed as foreign to the body, including many virus-infected cells and most cancerous cells. Many blood cells are short-lived and need a continuous replenishment; a person needs about one hundred billion new hematopoietic cells daily. The further production of these cells depends directly on hematopoietic stem cells (HSCs), the ultimate and only source of all these cells (Baum et al., 1992). A HSC is a cell isolated from the blood or bone marrow that renew itself, can differentiate various specialized cells (Fig. 9.1), can mobilize out of the bone marrow into circulating blood, and is subject to programed cell death, apoptosis, a process in which cells that are harmful or unnecessary destroy themselves (Baum et al., 1992; Wilson and Trumpp, 2006). A major focus of HSC basic research since the 1960s has been the identification and characterization of these stem cells. Since HSCs look and behave like normal white blood cells in culture, this was a tough challenge and makes it difficult to identify them based on morphology (size and shape). Even today, scientists

The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00005-9 © 2021 Elsevier Inc. All rights reserved.

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FIGURE 9.1 Stem cell differentiation from bone marrow. Schematic diagram showing hematopoietic stem cells with the potential to differentiate into various cell types. Source: Created with BioRender.com.

have to rely on proteins on the cell surface that serve only roughly as markers for white blood cells (Wilson and Trumpp, 2006; Szade et al., 2018). The identification and characterization of HSCs began with studies on mice, which laid the foundation for studies in humans. The challenge is daunting, as about one in 10,000 to 15,000 bone marrow cells are believed to be stem cells. In the blood, the proportion drops to 1 in 100,000. To this extent, scientists began to develop tests for the detection of self-renewal and plasticity of HSCs (Patt and Maloney, 1972). The “major standard” for proving that a cell derived from the bone marrow of the mouse is actually an HSC is still based on the same evidence described above and used in mice many years ago. This means that the cells are injected into a mouse that has received a dose of radiation sufficient to kill its own bloodproducing cells. When the mouse recovers and all kinds of blood cells (which carry a genetic marker from the donor animal) reappear, the transplanted cells are assumed to contain stem cells (Patt and Maloney, 1972). These investigations have shown that there seem to be two families of HSCs. When bone marrow cells from the transplanted mouse can be transplanted into another lethal mouse and recover its hematopoietic system over a few months, they are considered long-term stem cells capable of self-renewal. Besides, other cells

9.2 Bone-marrow HSC niche

from the bone marrow can instantly reproduce all different types of blood cells, but under normal circumstances they cannot renew themselves in the long term and are termed short-term progenitor cells. In addition, progenitor cells are comparatively immature cells that are precursors to an entirely differentiated cell of the same tissue pattern. Moreover, they are able to multiply, but they have only a limited potential to differentiate into more than one cell type, as HSCs do. For instance, a blood progenitor cell can produce only one red blood cell. Harrison and colleagues reported that short-term blood progenitor cells in a mouse can restore hematopoiesis for 3 4 months (Patt and Maloney, 1972; Maloney and Patt, 1975). The longevity of short-term human stem cells is not well-established. A proper stem cell capable of self-renewal must renew itself throughout the life span of an organism. It is these long-term replicating HSCs that are most essential for the establishment of HSCbased cell therapies. Lamentably, until now, researchers have been unable to distinguish between long-term and short-term cells when they are dismissed from the bloodstream or bone marrow. The key obstacle with tests to verify long-term stem cells and short-term progenitor cells is they are opaque, costly, time-consuming, and cannot be handled in humans. There are now some tests that examine cells in culture for their potential to form primitive and long-lived cell colonies, but these tests are not admitted as evidence that a cell is a long-term stem cell. Some genetically modified mice may receive transplanted human HSCs to test the self-renewal and hematopoietic capacities of cells during the life of a mouse, but the appropriateness of this test for cells in humans, who may live for decades, is controversial (Maloney and Patt, 1975). The complication of the HSC tests has contributed to two mutually contradictory research obstacles: definitively demonstrating the HSC and making it expand in a culture dish or increase its number. Faster research progress in characterizing and using HSCs would be possible if they could be grown in the laboratory without problems. Conversely, advances in establishing growth conditions appropriate for HSCs and reproducing cells would be faster if researchers could reliably and easily identify true HSCs (Schofield, 1978).

9.2 Bone-marrow HSC niche The relationship between bone-marrow production (hematopoiesis) and bone development (osteogenesis) was first identified in the 1970s in research showing that first bone and then vascularized bone marrow developed after subcutaneous transfer of whole, unmanipulated bone marrow (Maloney and Patt, 1975). The term “niche” for the specific HSC bone-marrow microenvironment was first minted by Schofield, who proposed that HSCs are in immediate contact with bone, and that cell cell communication was responsible for the ostensibly unlimited proliferative efficiency and inhibition of maturation of HSCs (Schofield, 1978).

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Various mutant mice, in which hematopoiesis is insufficient, due to primary defects in bone development or remodeling, have involved osteoblasts and osteoclasts in the formation and function of the bone-marrow HSC surroundings or niche. For illustration, mice missing core binding factor α1 (CBFα1; also known as RUNX2), one of the earliest osteoblast-specific transcription factors, have deficient bone-marrow hematopoiesis and considerable extramedullary hematopoiesis, owing to deficiencies in osteoblast differentiation and the subsequent failure to develop bone (Deguchi et al., 1999; Ducy et al., 2000). Nevertheless, whether the hematopoietic deficiency is an indirect effect caused by the lack of an appropriate bone-marrow cavity or whether the deficiency in CBFα1 immediately affects hematopoiesis remains unknown. Therefore different other mouse mutants with defects in bone development and remodeling have been reported (Karsenty and Wagner, 2002; Asada et al., 2017), but promising effects on hematopoiesis have not been determined.

9.3 Clinical applications of HSCs The clinical utilization of stem cells possesses immense promise, although the application of most types of adult stem cells is either currently untested or is in the initial stages of clinical investigation (Koc¸ et al., 2000). The sole exception is HSCs, which have been used clinically since 1959 and are used progressively routinely for transplantation, albeit almost entirely in a nonpure type. By 1995, more than 40,000 transplants were performed annually world-wide (Rizzo, 2005). Currently the main signs for bone marrow transplantation are either hematopoietic cancers (leukemias and lymphomas), or using high-dose chemotherapy for nonhematopoietic malignancies (cancers in other organs). Other signs include disorders that include genetic or gained bone marrow failure, including aplastic anemia, thalassemia, sickle cell anemia, and increasingly, autoimmune disorders.

9.3.1 Leukemia Strict regulation of HSC division is required to ensure a sustained pool of regenerating cells without overgrowth of immature cell types. The uncontrolled growth of immature hematopoietic cells is considered a paradigm for malignant growth and is in fact the most likely scenario for at least some cancers, including acute myeloid leukemia and chronic myeloid leukemia (CML) (Hope et al., 2004). Whether this is a fundamental mechanism for all cancers remains to be seen, but the identification of subgroups of breast cancer cells that are transmitted to recipient animals and can cause tumors has increased the possibility that stem cells could exist for many cancers. The study of normal stem cell regulation could therefore shed light on pathways that are important for disrupting self-renewal in malignant tumors.

9.3 Clinical applications of HSCs

One of the first clinical applications of HSCs was the therapy of blood cancer, leukemia, and lymphoma arising from the uncontrolled spread of white blood cells. In these applications, the cancer cells of the patient were destroyed by radiation or chemotherapy and then replaced by a bone marrow transplant or, as today, by a transplantation of HSCs from the peripheral circulation of a matched donor. A matching donor is usually a sister or brother of the patient who has inherited similar human leukocyte antigens (HLAs) on the surface of their cells. Blood cancers involve acute lymphoblastic leukemia, acute myeloblastic leukemia, CML, Hodgkin’s disease, multiple myeloma, and non-Hodgkin’s lymphoma (Attar and Scadden, 2004). Chemotherapy for acute leukemia acts on both malignant and proliferating transit-amplifying cells and causes a considerable reduction in white cells and platelets in 1 2 weeks. Resting cells, such as bone marrow stem cells and memory T-cells, are comparatively unchanged. When chemotherapy is stopped, red cells, white cells, and platelets recover in about 10 14 days. The most vital cell at this time is the neutrophil. Thus, the failure of these cells to recover may result in lethal infections. Neutrophil recovery can be stimulated by application of granulocyte colony-stimulating factor. Recovery is mitigated by the production of a new population of white and red cell precursors from chemotherapy-resistant stem cells. The phenotype of the cell that could achieve this is CD341 , Thy110, and descent negative. An even more primitive stem cell may indeed be CD34-. The characteristic features of human bone marrow stem cells are that they can produce all hematopoietic colonies in vitro, repopulate the bone marrow, or cause hematopoiesis in SCID-Hu mice. Cluster of differentiation (CD) marker is a single or group of molecules on the surface of a cell that is highly specific to that cell, allowing one to identify it among others. CD markers representative of various phases of white cell poiesis can be utilized to characterize different leukemias and lymphomas, the response to chemotherapy, and the differentiating populations during recovery. Furthermore, cyclic administration of chemotherapy may be therapeutic of some leukemias, indicating that the most primary bone marrow stem cell is not influenced. The expression of gene resistance in multiple drugs may partly illustrate this resistance. In some cases of leukemia, the malignant genetic event occurs in the most primitive stem cells, so ablative irradiation or chemotherapy are required to obliterate all malignant stem cells (Attar and Scadden, 2004). Stem cells are then substituted with bone marrow or circulating blood stem cells. Although cytokine-liberated autologous blood stem cells are frequently utilized to prevent graft versus host reactions owing to T-cells in the blood or marrow of allogeneic donors, a mild graft-versus-host disease (GVHD) response after transplantation has been proven to be effective in withdrawing residual host tumor cells. The evaluations of bone marrow transplantation clearly show this is an effective therapy for acute and some chronic leukemias and that malignant transformation accordingly occurs either in the most primitive bone marrow stem cell or in an initial transit-amplifying cell. Improvements in therapy in younger patients over the past 35 years have mainly been due to dose

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intensification and better supportive care. Allogeneic hematopoietic stem cell transplantation (HSCT) may be utilized to secure release in those patients who are considered at high risk of relapse. A plethora of new agents—involving those selected at specific biochemical pathways and immunotherapeutic procedures—are now in trial based on improved understanding of disease pathophysiology. Thus these developments provide adequate grounds for optimism, although fatality remains high, particularly in older patients (Khwaja et al., 2016).

9.3.2 Cancer One of the most dramatic new applications of HSC transplantation is activating cells that attack contrastingly untreatable tumors. A group of researchers in NIH’s intramural research program lately reported this proposal to cure metastatic kidney cancer. Just under half of the 38 patients treated so far have had their tumors depressed. The research protocol is now expanding to therapy of other solid tumors that resist standard therapy, including cancer of the lung, prostate, ovary, colon, esophagus, and pancreas (Childs et al., 2000). This experimental treatment is based on an allogeneic stem cell transplant of an HLA-adapted sibling whose HSCs are collected peripherally. The patient’s own immune system is suppressed, but not totally overwhelmed. The donor’s cells are transfused into the patient, and for the following 3 months, doctors closely observe the patient’s immune cells, using DNA fingerprinting to follow the engraftment of the donor’s cells and regrowth of the patient’s own blood cells. They should also judiciously inhibit the patient’s immune system as required to prevent his/her T cells from attacking the graft and to decrease GVHD (Childs et al., 2000). A study by Joshi et al. shows that umbilical cord blood (UCB) and peripherally collected human HSCs in a test tube are antitumor active against leukemia and breast cancer cells (Joshi et al., 2000). Implanted in a mouse model that tolerates human cells, HSCs attack human leukemia and breast cancer cells. Although untreated UCB lacks natural killer lymphocytes that can kill tumor cells, researchers have discovered they can greatly increase the activity and number of these cells with the cytokines IL-15, at least in test tubes and mice (Swart et al., 2017).

9.4 Autologous hematopoietic stem cell transplantation for autoimmune diseases Autologous HSCT is the sole treatment that can induce long-term, drug-free, and symptom-free remission in various refractory autoimmune rheumatic diseases (Fig. 9.2). The purpose of HSCT in autoimmune disease is the elimination of autoreactive immune cells and the regeneration of a naive, self-tolerant immune system. Clinical remission in autoimmune disease after HSCT is the outcome of a true reconfiguration of the immune system rather than long-term

9.4 Hematopoietic stem cell transplantation

FIGURE 9.2 Autologous hematopoietic stem cell transplantation. Schematic illustration showing autologous hematopoietic stem cell (HSC) transplantation sequential steps. The procedure is started after collecting HSCs from the patient’s bone marrow. Then, the collected HSC grafts are stored in liquid nitrogen until they are required for transplantation. The patient’s immune cells will be defeated after high-dose chemotherapy along with immune ablative preparation regimens. The cryopreserved HSCs will be injected into the patient intravenously, and then the reconstitution of the hematopoietic systematization will develop 10 14 days after transplantation, with full restoration from chemotherapy existing between 3 and 6 months. Source: Created with BioRender.com.

immunosuppression (Sureda et al., 2015; Alexander et al., 2015). The first HSCT for autoimmune diseases was conducted in 1995 and the Autoimmune Diseases Working Group of the EBMT was established in 1996 (Tyndall and Gratwohl, 1997). EULAR has gradually cooperated with the EBMT. Outside Europe, the Center for International Bone Marrow Transplant Registry (CIBMTR) and the NIH in the United States have interacted with major HSCT programs in Australia, Brazil, China, and the United States (Snowden et al., 2012). In 1997 a consensus report on stem cell transplants in autoimmune diseases was drafted on behalf of EULAR and the EBMT, and an internationally coordinated clinical program was launched (Tyndall and Gratwohl, 1997). Retrospective analyses from the EBMT autoimmune disease registry, the largest registry gathering HSCT information on autoimmune diseases, were accompanied by CIMBTR investigations. These investigations, together with small, potential phase I and II trials, supported the expediency, safety, and effectiveness of HSCT in several severe, therapy-resistant autoimmune diseases (Snowden et al., 2012). These studies also led to large-scale Phase II and Phase III HSCT studies in several autoimmune diseases. Besides, in

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2012 it was judged that around 3000 patients with autoimmune diseases had been cured with HSCT worldwide. The sequential steps for autologous HSCT include careful patient selection, chemotherapy-based stem cell mobilization, stem cell harvesting, conditioning, stem cell infusion, supportive care for neutrophils and lymphocytes recovery, and posttransplant follow-up. HSCT resets the immune system by renewing the CD41 T cell compartment and the Treg cell population, which is accompanied by an increment in the number of Treg cells and the reestablishment of TCR heterogeneity and function (Swart et al., 2017).

9.5 HSCs for the treatment of genetic blood cell diseases Most inherited blood cell diseases such as primary immunodeficiencies, hemoglobinopathies, memory and metabolic diseases, congenital cytopenia, and stem cell deficiencies can be cured by transplantation of allogeneic HSCs. Moreover, the transplanted genetically normal HSCs can serve as a constant source of blood cells of all lineages and eliminate these disorders from a single treatment with a lifetime benefit (Walters, 2015). While there are typically high success rates when an HLA-identical sibling donor is applicable, HSCT results are generally less successful in less wellmatched allogeneic donors (either haplo-identical family members or unrelated donors) (Boelens et al., 2013). Lowered HLA matching between recipient and donor raises the risk of transplant rejection and transplant versus host disease (GVHD). The rejection of an HSC graft usually causes the patient in a perilous position, with a critical need to reconstitute hematopoiesis to prevent complications from prolonged pancytopenia (anemia, infection, bleeding). The first donor may not be attainable (e.g., UCB units are not connected to their origin) and a matching second donor cannot be recognized. Thus GVHD is a significant cause of transplant morbidity and even mortality and can deceive a chronic rheumatologic-like inflammatory/fibrotic disease with demand for continuous immune suppression and the consequent risks of infection and toxicity (Cooke et al., 2017). Immediately before and after the allogeneic transplant, high levels of immune suppression are necessary to reduce immunological risks, but these treatments also add to morbidity. Continuous progress has been made in reducing GVHD in allogeneic HSCT, including improved transplantation technology by removing selective T cell populations (TCR-α/ß-depletion, naive T cell depletion) and by using cyclophosphamide after transplantation (Muccio et al., 2016). Nevertheless, immune obstacles and insufficient suitable donors present significant clinical obstacles to the successful use of allogeneic HSCT in a wider variety of diseases. Autologous HSCT, in which the patient’s HSCs are gene-modified, should completely avoid the most important immunological complications of allogeneic HSCT, which could provide progressing results for patients with genetic blood cell disorders. Particular diseases demand the expression of the gene

9.6 HSCs for the treatment of intestinal diseases

inserted into HSCs in cells of one or more hematopoietic lineages (e.g., red blood cells, neutrophils, lymphocytes). Insufficient immunogenicity with autologous cells permits using reduced intensity of the pretransplant preparation to create space in the marrow niche to promote HSC engraftment, weighing to what is demanded for efficient allogeneic HSCT. Therefore HSCs are long-lived and multipotent, so gene alteration in HSCs should contribute to continuous gene alteration among the diverse lineages (Laurenti and Gottgens, 2018). The hematopoietic system is a perfect aim for gene therapy due to the ease with which HSCs can be obtained for ex vivo gene manipulation, efficient gene-alternation, and readministration as an intravenous infusion. HSCs are traditionally obtained from bone marrow extracted from the iliac crests under general anesthesia. Multiple aspirations are operated with the aim of collecting 10 20 mL of bone marrow per kilogram of body weight of the recipient. Alternatively, HSCs can be obtained as cytokine (e.g., G-CSF)-mobilized peripheral blood stem cells (PBSC) collected by leukopheresis. Hematopoietic growth factors, involving GM-CSF and G-CSF, or CXCR4 inhibitors, have increased quantities of circulating hematopoietic stem and progenitor cells (HSPC) by 30 1000 times (Brave et al., 2010). PBSCs are now the dominant clinical HSC source for allogeneic and autologous transplants for the routine and successful treatment of multiple blood cell disease using cutting-edge techniques. Over three decades, gene therapy with HSCs has evolved from ineffectiveness to the ability to essentially cure several disorders. The pathway was not linear but demanded multiple iterative bench-to-bedside cycles. It is probable that treatments using other stem cells will also have progression and setbacks. However, the underlying assumptions for cellular treatments are so impressive, it is proper that multiple innovative stem cell-based therapies will be established. The lessons from the field of HSC gene therapy may stipulate some help for researchers conducting the translational procedure (Morgan et al., 2017).

9.6 HSCs for the treatment of intestinal diseases 9.6.1 Inflammatory bowel diseases At present, many gastrointestinal (GI) diseases are a major cause of the increasing mortality of children and adults each year. Furthermore, these patients may survive with the high expense of the parenteral nutrition (PN), which sustains in the long-term survival of the patients (Mohamed et al., 2015). HSC have been used to treat various intestinal diseases, including Crohn’s disease. Crohn’s disease is a category of inflammatory bowel disease (IBD). It produces inflammation of the digestive tract, which can cause abdominal pain, severe diarrhea, fatigue, weight loss, and malnourishment. Despite major improvements accomplished by biological treatments in Crohn’s disease, some patients have treatment-resistant disorder and require complementary therapies. The autologous stem cell transplantation in

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Crohn’s disease (ASTIC) trial was the first randomized controlled assessment in Crohn’s disease and compared early versus deferred autologous HSCT in patients with resistant Crohn’s disease (Qiu et al., 2017). Early case sequence and singlecenter cohort studies suggested that a HSCT could produce sustained clinical advantage. A recent investigation analyzed the HSC transplantation results of 40 patients, there were considerable advances in clinical disease efficacy, quality of life, and endoscopic disease effect after 1 year; 50% of this group had fulfilled mucosal healing, a critical endpoint in Crohn’s disease, and correlated with enhanced long-term results (Lindsay et al., 2017). Ditschkowski et al. found that 10 out of 11 patients [seven with Crohn’s disease and four with ulcerative colitis (UC)] were free of IBD after allogeneic stem cell transplantation for hematological malignancy with a median follow-up of 34 months (Ditschkowski et al., 2003). Another study presented the advancement of all patients undergoing HSCT (including the post-HSCT direction, following the lagged HSCT of the control group). Compared to baseline, there was tremendously notable progress at 1 year for Crohn’s Disease Activity Index AI, patient documented results, quality of life (based on IBDQ and EQ-5D forms), and ileocolonoscopic findings, based on the simple endoscopic score for Crohn’s disease (SES-Crohn’s disease). Remarkably, 26% of patients had no ileocolonoscopic evidence of CD (SES-Crohn’s disease score of 0 in all segments examined) and there was perfect healing of active ulceration in 50% of patients. There was a modest relation in outcomes for Crohn’s disease AI and on ileocolonoscopic assessment. On univariate analysis, baseline factors associated with steroid free clinical remission (Crohn’s disease AI , 150) at 1 year include an inflammatory phenotype with colonic localization and a high SES-Crohn’s disease score, demonstrating equitably active disease. Therefore this result suggests that some patients may have failed to respond because their symptoms more exhibited secondary structural or functional notabilities of prolonged severe disease. There was no proposal that HSCT had a favorable influence on fistula healing (Hawkey and Hommes, 2017). In an experimental study on nonmyeloablative allogeneic HSCT, data from nine Crohn’s disease patients were published after 5 years of follow-up (LopezGarcia et al., 2017). In three patients, unselected matched sibling PBSCs were utilized and UCB was used in six patients. The incorporation criteria for allogeneic HSCT were related to the strictness and refractoriness to common medications comparable to those reported in other studies. The nonmyeloablative conditioning regimen used was cyclophosphamide, alemtuzumab, and fludarabine. Moreover, a Calcineurin inhibitor for 6 9 months was used to inhibit GVHD. No patient died during transplantation. No patient suffered acute GVHD, and one had hindered chronic GVHD, one patient died 3 months after allogeneic UCB HSCT from dispersed adenovirus, and the other eight patients were alive without CD therapy or disease indications. Curiously, of five allogeneic UCB receivers, none had GVHD, and none had CD31 and CD331 donor engraftment after 6 months (Lopez-Garcia et al., 2017; Ruiz et al., 2020). Reduced microcirculation is involved in the pathogenesis of IBD. It is believed that stem cells or endothelial progenitor cells contribute to tissue

9.6 HSCs for the treatment of intestinal diseases

regeneration through neoangiogenesis or vasculogenesis in ischemia or inflammation-related diseases. An earlier study demonstrated that transplantation of immortalized CD342 stem cells isolated from mouse bone marrow and peripheral blood can expedite mucosal repair in a pattern of DSS-induced colitis in a nonmyeloablative context. They established the transplanted cells, promoted new vessel development and accordingly overwhelmed the defective vasculogenesis associated with the pathogenesis of IBD (Khalil et al., 2007). Preceding investigations using animal models of IBD have demonstrated that transplanted bone marrow cells contribute to tissue repair by producing epithelial cells, activated myofibroblasts, and can also contribute to neovasculogenesis in the inflamed colon (Brittan et al., 2007). Moreover, prior examinations induced experimental colitis in wild-type rats, and then utilized BM-HSCT for medication. These results illustrated that an enormous mass of bone marrow-derived cells were engaged in regeneration of the colon after experimental colitis in rats (Komori et al., 2005). Mendelian disorders in glucose-6-phosphate metabolism can be related with IBD. A recent study utilized whole genome sequencing which successfully demonstrated a homozygous variant in the glucose-6-phosphatase G6PC3 gene [c.911dupC; p.Q305fs 82] in an adult patient with congenital neutropenia, lymphopenia and childhood-onset, therapy-refractory Crohn’s disease. Because G6PC3 is expressed in various hematopoietic and nonhematopoietic cells, it was doubtful whether allogeneic HSCT would aid this patient with intestinal inflammation. Furthermore, the study indicated that HSCT resolves G6PC3-associated immunodeficiency and the Crohn’s disease phenotype (Bolton et al., 2020). Crohn’s disease can be complicated with other diseases including Myelodysplastic syndrome (MDS). This case has already been reported in the past; however, HSCT is seldom operated. Recently, researchers reported a case of Crohn’s disease with MDS, then performed an allogeneic HSCT, which appeared promising treatment method (Zhang et al., 2020).

9.6.2 HSCs for mucosal healing Recently, mucosal healing has become a required part of clinical response and a treatment goal based on treat-to-target therapeutic strategies in IBD. Stem cells are important to regulate congenital inflammation and to heal the mucosal injury. Since stem cells find their way to the injury positions and differentiate into the cell types needed at this site to heal the injured tissue, they can be a promising tool for IBD patients. Accordingly, stem cell therapy has been considered for IBD patients. Thus HSCs showed promising results for improving disease control, especially in IBD patients who have turned to current anti-IBD therapy without resistance. Cell-based therapies can not only improve tissue regeneration by inhibiting disease activity, but also reduce the risk of developing colitis-related carcinomas. Consequently, these cells differentiate into epithelial or immunomodulatory cells and therefore can restore normal mucosal tissue and barrier integrity.

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Furthermore, stem cell therapy is an extension of immunomodulatory therapies, rather than inhibiting a particular pathway, many antiinflammatory pathways could be inhibited by stem cell transplantation, and, hence, stem cell based therapies themselves may ascertain more successful than other therapies that focus either on a specific inflammation pathway or inflammation in general (Salem and Selby, 2017).

9.7 Conclusion, challenges, and future directions Allogeneic HSCT is a treatment for numerous diseases, including intestinal diseases. Although allogeneic HSCTs are becoming increasingly safe, use of allogeneic stem cells is hindered by many serious complications, involving the absence of an appropriate donor, GVHDs, and infectious complications due to immunosuppression (Zeiser and Vago, 2019). The primary drawback is the insufficient search for a suitable HLA-suitable donor for transplantation. Worldwide, only 30% of patients requiring allogeneic HSCT will have a suitable sibling donor. Although alternative transplant sources are convenient, it is reported that the probability of finding a suitable donor with HLA agreement and donation readiness is complicated due to ethnic background (Ballen et al., 2012). Acute GVHD remains a clinical defiance and a significant source of morbidity and mortality following allogeneic HSCT. Traditionally, GVHD has been dispensed into acute and chronic GVHD depending on the time of onset of the GVHD symptoms. GVHD appears on or before the 100th day after transplantation, defined as acute GVHD, and the onset of chronic GVHD occurs after the 100th day. Nevertheless, this transitory variation is relatively inconsistent, as patients may exhibit classic symptoms of acute GVHD even after day 100, and chronic symptoms may occur before 100 days posttransplantation. Acute GVHD recognition should be approved by biopsy of an affected organ if feasible; in addition, other non-GVHD complications involving the skin, liver, and GI tract should be ruled out, including cytomegalovirus enteritis or drug eruption from medications (Kanda et al., 2020). However, ultimate diagnosis of acute GVHD needs amalgamation of all available clinical data, because the sensitivity of these biopsies is only relatively 60%. Thus the development of diagnostic tests for acute GVHD is needed. HSCs have been demonstrated to be a potential therapy for disease control in refractory Crohn’s disease. Continuing in-depth analysis is guaranteed to fully understand the complicated cellular processes. Moreover, information from ongoing Phase III clinical trials will afford a valuable roadmap for the future of stem cell therapy. Cellular therapy should not be confined to HSCs, other cellular therapies should continue to be investigated from a preclinical perspective. Future research should concentrate on improving safety and feasibility, with the purpose of enhancing quality of life for the patient.

References

References Alexander, T., Bondanza, A., Muraro, P.A., Greco, R., Saccardi, R., Daikeler, T., et al., 2015. SCT for severe autoimmune diseases: consensus guidelines of the European Society for Blood and Marrow Transplantation for immune monitoring and biobanking. Bone Marrow Transpl. 50 (2), 173 180. Asada, N., Takeishi, S., Frenette, P.S., 2017. Complexity of bone marrow hematopoietic stem cell niche. Int. J. Hematol. 106 (1), 45 54. Attar, E.C., Scadden, D.T., 2004. Regulation of hematopoietic stem cell growth. Leukemia 18, 1760 1768. Ballen, K.K., Koreth, J., Chen, Y.B., Dey, B.R., Spitzer, T.R., 2012. Selection of optimal alternative graft source: mismatched unrelated donor, umbilical cord blood, or haploidentical transplant. Blood 119 (9), 1972 1980. Baum, C.M., Weissman, I.L., Tsukamoto, A.S., Buckle, A.M., Peault, B., 1992. Isolation of a candidate human hematopoietic stem-cell population. Proc. Natl. Acad. Sci. U. S. A. 89 (7), 2804 2808. Boelens, J.J., Aldenhoven, M., Purtill, D., Ruggeri, A., Defor, T., Wynn, R., et al., 2013. Outcomes of transplantation using various hematopoietic cell sources in children with Hurler syndrome after myeloablative conditioning. Blood 121 (19), 3981 3987. Bolton, C., Burch, N., Morgan, J., Harrison, B., Pandey, S., Pagnamenta, A.T., et al., 2020. Remission of inflammatory bowel disease in glucose-6-phosphatase 3 deficiency by allogeneic haematopoietic stem cell transplantation. J. Crohns Colitis 14 (1), 142 147. Brave, M., Farrell, A., Ching Lin, S., Ocheltree, T., Pope Miksinski, S., Lee, S.L., et al., 2010. FDA review summary: Mozobil in combination with granulocyte colonystimulating factor to mobilize hematopoietic stem cells to the peripheral blood for collection and subsequent autologous transplantation. Oncology 78 (3 4), 282 288. Brittan, M., Alison, M.R., Schier, S., Wright, N.A., 2007. Bone marrow stem cell-mediated regeneration in IBD: where do we go from here? Gastroenterology 132 (3), 1171 1173. Childs, R., Chernoff, A., Contentin, N., Bahceci, E., Schrump, D., Leitman, S., et al., 2000. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. N. Engl. J. Med. 343, 750 758. Cooke, K.R., Luznik, L., Sarantopoulos, S., Hakim, F.T., Jagasia, M., Fowler, D.H., et al., 2017. The biology of chronic graft-versus-host disease: a task force report from the National Institutes of Health Consensus Development Project on criteria for clinical trials in chronic graft-versus-host disease. Biol. Blood Marrow Transpl. 23 (2), 211 234. Deguchi, K., Yagi, H., Inada, M., et al., 1999. Excessive extramedullary hematopoiesis in Cbfa1-deficient mice with a congenital lack of bone marrow. Biochem. Biophys. Res. Commun. 255 (2), 352 359. Ditschkowski, M., Einsele, H., Schwerdtfeger, R., Bunjes, D., Trenschel, R., Beelen, D.W., et al., 2003. Improvement of inflammatory bowel disease after allogeneic stem-cell transplantation. Transplantation 75 (10), 1745 1747. Ducy, P., Schinke, T., Karsenty, G., 2000. The osteoblast: a sophisticated fibroblast under central surveillance. Science 289 (5484), 1501 1504. Hawkey, C.J., Hommes, D.W., 2017. Is stem cell therapy ready for prime time in treatment of inflammatory bowel diseases? Gastroenterology 152 (2), 389 397. e382.

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Hope, K.J., Jin, L., Dick, J.E., 2004. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat. Immunol. 5 (7), 738 743. Joshi, S.S., Tarantolo, S.R., Kuszynski, C.A., Kessinger, A., 2000. Antitumor Therapeutic Potential of Activated Human Umbilical Cord Blood Cells against Leukemia and Breast Cancer. Clin. Cancer Res. 6 (11), 4351 4358. Kanda, J., Umeda, K., Kato, K., Murata, M., Sugita, J., Adachi, S., et al., 2020. Effect of graft-versus-host disease on outcomes after pediatric single cord blood transplantation. Bone Marrow Transpl. 55 (7), 1430 1437. Karsenty, G., Wagner, E.F., 2002. Reaching a genetic and molecular understanding of skeletal development. Dev. Cell 2 (4), 389 406. Khalil, P.N., Weiler, V., Nelson, P.J., Khalil, M.N., Moosmann, S., Mutschler, W.E., et al., 2007. Nonmyeloablative stem cell therapy enhances microcirculation and tissue regeneration in murine inflammatory bowel disease. Gastroenterology 132 (3), 944 954. Khwaja, A., Bjorkholm, M., Gale, R.E., Levine, R.L., Jordan, C.T., Ehninger, G., et al., 2016. Acute myeloid leukaemia. Nat. Rev. Dis. Prim. 2, 16010. Koc¸, O.N., Gerson, S.L., Cooper, B.W., et al., 2000. Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J. Clin. Oncol. 18 (2), 307 316. Komori, M., Tsuji, S., Tsujii, M., Murata, H., Iijima, H., Yasumaru, M., et al., 2005. Involvement of bone marrow-derived cells in healing of experimental colitis in rats. Wound Repair Regen. 13 (1), 109 118. Laurenti, E., Gottgens, B., 2018. From hematopoietic stem cells to complex differentiation landscapes. Nature 553 (7689), 418 426. Lindsay, J.O., Allez, M., Clark, M., Labopin, M., Ricart, E., Rogler, G., et al., 2017. Autologous stem-cell transplantation in treatment-refractory Crohn’s disease: an analysis of pooled data from the ASTIC trial. Lancet Gastroenterol. Hepatol. 2 (6), 399 406. Lopez-Garcia, A., Rovira, M., Jauregui-Amezaga, A., Marin, P., Barastegui, R., Salas, A., et al., 2017. Autologous hematopoietic stem cell transplantation for refractory Crohn’s disease: efficacy in a single-centre cohort. J. Crohns Colitis 11 (10), 1161 1168. Maloney, M.A., Patt, H.M., 1975. On the origin of hematopoietic stem cells after local marrow extirpation. Proc. Soc. Exp. Biol. Med. 149 (1), 94 97. Mohamed, M.S., Chen, Y., Yao, C.L., 2015. Intestinal stem cells and stem cell-based therapy for intestinal diseases. Cytotechnology 67 (2), 177 189. Morgan, R.A., Gray, D., Lomova, A., Kohn, D.B., 2017. Hematopoietic stem cell gene therapy: progress and lessons learned. Cell Stem Cell 21 (5), 574 590. Muccio, L., Bertaina, A., Falco, M., Pende, D., Meazza, R., Lopez-Botet, M., et al., 2016. Analysis of memory-like natural killer cells in human cytomegalovirus-infected children undergoing alphabeta 1 T and B cell-depleted hematopoietic stem cell transplantation for hematological malignancies. Haematologica 101 (3), 371 381. Patt, H.M., Maloney, M.A., 1972. Bone formation and resorption as a requirement for marrow development. Proc. Soc. Exp. Biol. Med. 140 (1), 205 207. Qiu, X., Feng, J.R., Chen, L.P., Liu, S., Zhang, M., Zhou, Z., et al., 2017. Efficacy and safety of autologous hematopoietic stem cell therapy for refractory Crohn’s disease: a systematic review and meta-analysis. Medicine (Baltim.) 96 (26), e7381.

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Rizzo, J.D., 2005. Clinical bone marrow and blood stem cell transplantation. Bone Marrow Transpl. 35, 319. Ruiz, M.A., Junior, R.L.K., Piron-Ruiz, L., Saran, P.S., Castiglioni, L., de Quadros, L.G., et al., 2020. Medical, ethical, and legal aspects of hematopoietic stem cell transplantation for Crohn’s disease in Brazil. World J. Stem Cell 12 (10), 1113 1123. Salem, G.A., Selby, G.B., 2017. Stem cell transplant in inflammatory bowel disease: a promising modality of treatment for a complicated disease course. Stem Cell Investig. 4, 95. Schofield, R., 1978. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cell 4 (1 2), 7 25. Snowden, J.A., Saccardi, R., Allez, M., Ardizzone, S., Arnold, R., Cervera, R., et al., 2012. Hematopoietic SCT in severe autoimmune diseases: updated guidelines of the European Group for Blood and Marrow Transplantation. Bone Marrow Transpl. 47 (6), 770 790. Sureda, A., Bader, P., Cesaro, S., Dreger, P., Duarte, R.F., Dufour, C., et al., 2015. Indications for allo- and auto-SCT for haematological diseases, solid tumours and immune disorders: current practice in Europe, 2015. Bone Marrow Transpl. 50 (8), 1037 1056. Swart, J.F., Delemarre, E.M., van Wijk, F., Boelens, J.J., Kuball, J., van Laar, J.M., et al., 2017. Hematopoietic stem cell transplantation for autoimmune diseases. Nat. Rev. Rheumatol. 13, 244 256. Szade, K., Gulati, G.S., Chan, C.K.F., Kao, K.S., Miyanishi, M., Marjon, K.D., et al., 2018. Where hematopoietic stem cells live: the bone marrow niche. Antioxid. Redox Signal. 29 (2), 191 204. Tyndall, A., Gratwohl, A., 1997. Blood and marrow stem cell transplants in auto-immune disease: a consensus report written on behalf of the European League against Rheumatism (EULAR) and the European Group for Blood and Marrow Transplantation (EBMT). Bone Marrow Transpl. 19 (7), 643 645. Walters, M.C., 2015. Update of hematopoietic cell transplantation for sickle cell disease. Curr. Opin. Hematol. 22 (3), 227 233. Wilson, A., Trumpp, A., 2006. Bone-marrow hematopoietic-stem-cell niches. Nat. Rev. Immunol. 6, 93 106. Zeiser, R., Vago, L., 2019. Mechanisms of immune escape after allogeneic hematopoietic cell transplantation. Blood 133 (12), 1290 1297. Zhang, Y., Lou, L.L., Shi, X.D., Lu, S.S., Zhang, L.X., Huang, X., et al., 2020. Allogeneic hematopoietic stem cell transplantation for Crohn disease complicated with myelodysplastic syndrome: a case report. Medicine (Baltim.) 99 (10), e19450.

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Role of mesenchymal and other stem cell therapy in intestinal diseases

10

Jingwen Liu1 and Deming Jiang2 1

Laboratory of Gastroenterology Department, Zhejiang University School of Medicine, Hangzhou, P.R. China 2 Biosensor National Special Laboratory, Key Laboratory for Biomedical Engineering of Education Ministry, Department of Biomedical Engineering, Zhejiang University, Hangzhou, P.R. China

10.1 Introduction In recent years, the multidifferentiation potential of stem cells has made it possible to repair the damaged intestinal mucosa of patients and implement the functions of immune regulation and reconstruction. There are many basic and clinical studies that are advancing stem cell therapy. Both undifferentiated embryonic stem cells and induced pluripotent stem cells can cause embryo tumors after transplantation. Therefore, when embryonic stem cells and induced pluripotent stem cells are used to generate differentiated cells, the complete elimination of undifferentiated cells is a critical step in the clinical application of cell therapy. On the contrary, adult stem cells are isolated from mature organs, and their differentiation potential is more limited than embryonic stem cells and induced pluripotent stem cells. Therefore, adult stem cells are considered to be multipotent progenitor cells. The most recognized adult stem cells are hematopoietic stem cells (HSCs), which can be concentrated from bone marrow or collected from peripheral blood. After transplantation into the appropriate recipients, HSCs will reorganize and repair all mature blood lines. Bone marrow stem cell (BSC) transplantation has been accepted for the treatment of many kinds of blood diseases and intestinal diseases (Swenson and Theise, 2010). For other refractory and fatal diseases, the risk of bone marrow transplantation (BMT) is superior to infection, bleeding, and graft-versus-host disease. Mesenchymal stem cells (MSCs) are a completely different type of adult stem cells. MSCs can be derived from bone marrow, cartilage, and fat. The differentiation potential of MSCs in mesoderm tissues such as fat, cartilage, and bone may be more limited. Transplanting MSCs does not recombine hematopoietic cell lines, but it changes the host’s immune response.

The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00008-4 © 2021 Elsevier Inc. All rights reserved.

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Multiple adult stem cells, especially MSCs, have strong proliferative capacity, multidirectional differentiation potential, and the function of positioning and repairing to damaged tissues. Under certain conditions, they can be differentiated into a certain type of adult stem cells or laterally into a variety of tissue cells. Therefore, it has attracted people’s attention and become a new hot spot for cell transplantation treatment of intestinal diseases, including inflammatory bowel disease (IBD), radiation enteritis, colorectal cancer, and so forth. This chapter reviews the application of MSCs and other stem cells in the treatment of intestinal diseases.

10.2 Mesenchymal stem cells As early as 1867, the famous pathologist Cohnhein discovered that non-HSCs in bone marrow could differentiate into fibroblasts that heal wounds. Since Friedenstein et al. (1987) isolated and cultured MSCs from guinea pig bone marrow for the first time in 1966, research on its properties and potential applications has never ceased. Caplan (1991) proposed that these MSCs could differentiate into all mesenchymal cells and had the hypothesis of promoting mesenchymal tissue regeneration. This hypothesis was not confirmed by Pittenger et al. (1999) until 1999. As one of the most widely used stem cells for the treatment of intestinal diseases, MSCs are present in almost all tissues. In addition to bone marrow, they can also be derived from fat, umbilical cord blood, placenta, amniotic fluid, milk, nerves, skin, and dental pulp (Bellavia et al., 2014; Takebe et al., 2017; Shih et al., 2005). MSCs from different sources are similar in function and screening method, but there are certain differences. Among them, bone marrow mesenchymal stem cells (BMSCs) are widely used. Comparing with BMSCs, umbilical cord-derived MSCs are higher in content, weaker in immunogenicity, more primitive in state, and stronger in self-proliferation and multidirectional differentiation, but it needs to do a good job of mobilizing mothers and their families. MSCs derived from adipose tissue do not require bone marrow puncture and other operations. They are easy to obtain and have abundant sources but relatively few clinical studies and incomplete technology.

10.2.1 Mechanism of MSC treatment of intestinal diseases MSCs cultured in vitro are small in size and have a large nucleus-to-plasma ratio. They grow in a fusiform or irregular triangle shape on the surface of the support. There is an oval nucleus in the center of the cell, which extends two to three antennae with different lengths (Le Blanc and Mougiakakos, 2012). In order to standardize MSCs from different sources, in 2006, the International Cell Therapy Association reached three minimum standard consensus (Horwitz et al., 2005): (1) adherence; (2)

10.2 Mesenchymal stem cells

surface antigens including CD731 , CD901 , CD1051 , CD11b2 , CD142 , CD342 , CD452 , CD192 , CD79a2 , and HLA-DR2 ; and (3) the ability to differentiate into osteoblasts, chondrocytes, and adipocytes in vitro. MSCs can participate in immune regulation and have the ability of selfrenewal and multidirectional differentiation. Thus, its application in the treatment of intestinal diseases is not a single process, but the result of multiple factors and mechanisms (Fig. 10.1).

10.2.1.1 Promoting intestinal repair MSCs have the potential for self-renewal and multidirectional differentiation and can promote the repair of damaged tissue in the intestine. MSCs have chemoattract activity towards damage and inflammation in the intestine, which is called “homing” (Le Blanc and Ringden, 2007). MSCs have been reported to be isolated from intestinal mucosa and submucosa (Lanzoni et al., 2009). BMSCs expressing green fluorescent protein were transplanted into a colitis rat model, and after 28 days, the epithelial cells expressing green fluorescent in the rat colon reached 37.6%, proving that MSCs have the ability to differentiate into colon epithelial cells to promote intestinal repair (Komori et al., 2005). MSCs can specifically migrate and home to damaged tissues, where they can differentiate into functional cells to replace damaged cells. In the experimental rat colitis model, some MSCs can develop into myofibroblasts after reaching the site of inflammation and upregulate the expression of α-actin and desmin (Hayashi et al., 2008).

FIGURE 10.1 Multiple factors and mechanisms of MSCs in the treatment of intestinal diseases.

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Some studies also believe that although MSCs can replace damaged cells by their own differentiation, their tissue repair characteristics are mainly due to their ability to secrete a variety of cytokines to promote regeneration of intestinal epithelial cells and vascular cells (Chen et al., 2013). Meanwhile, MSCs inhibit tissue apoptosis and fibrosis by secreting proteolytic enzymes and angiogenic factors (Chen et al., 2013). The above results show that MSCs can be directed to migrate, colonize the intestinal injury and inflammation sites, be divided into intestinal epithelial cells and secrete various cytokines, and then play a role in promoting intestinal repair.

10.2.1.2 Immune regulation Activated macrophages extracted from the intestines of patients were co-cultured with MSCs and found that the secretion levels of tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) decreased significantly (Gonzalez et al., 2009). MSCs can inhibit M1-type macrophages and promote their differentiation into M2 (Volarevic et al., 2010). MSCs can also play a role in immune regulation by inhibiting the proliferation and toxicity of natural killer cells, reducing the proliferation and activation of B cells, and inhibiting the maturation of T cells and dendritic cells (Sun et al., 2012). MSCs can also regulate Th17/Treg cell balance by inhibiting STAT3 activation, so that experimental enteritis can be relieved. In addition, many researches show that interferon-γ (IFN-γ) alone or in combination with TNF-α, IL-1α, etc. can induce MSCs to secrete IL-10, cyclooxygenase-2, prostaglandin E2, and other soluble factors or enzymes that involved in MSCs-mediated immunosuppression (DelaRosa et al., 2009).

10.2.1.3 Paracrine mechanism MSCs-derived exosomes (MSCs-EXOs) are membrane vesicles containing active substances such as functional proteins, mRNA, and microRNA secreted by MSCs, with a diameter of 30 100 nm. MSCs-EXOs play an important role in the transmission of information between MSCs and damaged cells of the intestine and can participate in the regulation of intestinal inflammation and injury repair (Phinney and Pittenger, 2017). Study has found that MSCs-EXO treatment can effectively relieve intestinal inflammation in mice with colitis, which reduce the mRNA and protein levels of nuclear transcription factors kappaBp65 and TNF-α while decreasing pro-inflammatory factor IL-1β level and increasing antiinflammatory factor IL-10 level (Yang et al., 2015). In addition, MSCs-EXOs can suppress the expression of macrophage chemokines CX3CL1 and TNF-α and upregulate the expression of IL-10 and reduce the local inflammatory response (Zou et al., 2014).

10.2.2 Application of MSCs in the treatment of IBD IBD refers to a group of intestinal diseases of unknown etiology, including ulcerative colitis (UD) and Crohn’s disease (CD). Due to environmental factors acting

10.2 Mesenchymal stem cells

on genetically susceptible patients, with the participation of intestinal flora, it leads to the body’s immune and inflammatory reactions, thereby causing chronic damage to the intestinal mucosa. At present, MSCs are widely used in both IBD preclinical basic research and clinical trials. Most preclinical basic studies have confirmed that MSCs have a good therapeutic effect on IBD animal models, but clinical trials are still needed to further evaluate the effectiveness and safety of MSCs in human IBD treatment.

10.2.2.1 Preclinical basic research A large number of preclinical basic studies have been carried out at the cellular level and animal models to verify the therapeutic effect of MSCs in IBD. The animals used in the experiment were mostly colitis rat, mouse, or guinea pig models induced by dextran sodium sulfate and trinitrobenzenesulfonic acid. So far, most studies have shown that MSCs can effectively improve the symptoms and histopathological scores of IBD animal models. It has been found that MSCs may suppress intestinal inflammation through immunomodulation and relieve mouse colitis after MSCs acting on a mouse model of colitis and the concentration of inflammatory factors such as TNF-α and IL-1 in the colon tissue determined (He et al., 2012). A study transferred green fluorescent protein to allogeneic MSCs and found that the therapeutic effect of MSCs may be related to the redistribution of Treg cells (Zuo et al., 2013). Some studies applied MSCs derived from human umbilical cord blood, bone marrow, and adipose tissue to animal models, respectively, which can also achieve the purpose of promoting symptom relief and pathological tissue repair (Banerjee et al., 2015; Robinson et al., 2015; Xie et al., 2017). In a mouse model of CD fibrosis, it was found that MSCs could play an anti-intestinal fibrosis role by regulating the inflammatory environment, inhibiting the TGF-β/Smad pathway, and improving epithelial-mesenchymal transition (Lian et al., 2018). Although the relevant basic research data is rich, it cannot fully represent the feasibility of clinical application. The main reasons include (Chinnadurai et al., 2015): (1) the etiology and pathogenesis of human IBD are complex, and animal models are often induced by drugs, which cannot accurately simulate the characteristics of human IBD. (2) The functions of MSCs from different species are different.

10.2.2.2 Clinical trials Nearly 500 clinical trials of MSCs registered with the National Institutes of Health (NIH) are currently underway, involving a variety of diseases from IBD and multiple sclerosis to cartilage repair (Squillaro et al., 2016).

10.2.2.2.1 Application status of MSCs in the treatment of IBD Judging from the results of the currently completed phase I III clinical trials of MSCs in the treatment of CD with anal fistula, more than half of the patients can be completely relieved, at least two-third of the patients respond to the treatment,

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and there is no serious MSCs-related adverse reactions reported. This fully demonstrates the effectiveness and safety of MSCs in the treatment of CD with anal fistula. The currently completed clinical trials of MSCs in the treatment of IBD have shown that both allogeneic and autologous MSCs have a certain clinical response rate and remission rate for the treatment of IBD. Garcia-Olmo et al. (2005) used autologous fat-derived MSCs to locally treat five patients with CD. Except for one patient who withdrew from the experiment due to cell culture contamination, of the remaining four patients, a total of eight fistulas, 75% of the fistula healed after 8 weeks of treatment, and the other 25% of the fistula did not heal completely, but its leakage was also significantly reduced, and no obvious side effects occurred during the treatment. In the clinical trial of Ciccocioppo et al. (2011), of the 10 patients who completed the experiment, 7 patients achieved complete healing of the fistula, and 3 patients had incomplete healing of the fistula. Panes et al. (2016) used allogenic fat-derived MSCs (Cx601) in clinical trials. Of the 107 patients with complex CD, 53 patients had fistula healing and the time required for healing was shortened. Molendijk et al. (2015) used allogeneic BMSCs to treat 21 patients with refractory CD combined with anal fistula. The results showed that local administration of MSCs was not related to the patient’s serious adverse reactions. Local injection of low-dose MSCs was better than high-dose MSCs. These studies have confirmed the effectiveness and safety of local injection of MSCs for refractory CD with anal fistula. In addition, many trials used allogenic MSCs to treat refractory IBD without rejection and serious side effects related to MSC infusion during treatment (Zhang et al., 2018a; Liang et al., 2012; Forbes et al., 2014). The patient’s intestinal inflammation improved significantly. Dhere et al. (2016) used autologous BMSCs to treat 12 patients with refractory CD and found that all patients were well tolerated by MSC infusion, and no dose-limiting toxicity was seen.

10.2.2.2.2 Sources of MSCs for clinical applications The sources of MSCs for clinical treatment include autologous and allogeneic. Autologous MSCs have no immune rejection because they are from the host. Studies have shown that MSCs extracted from patients with CD are similar in phenotype to those of healthy people and have the same function (Dietz et al., 2017). The low immunogenicity of MSCs makes it possible for allogeneic MSCs to be used clinically. It has been reported that the treatment of refractory CD with allogenic MSCs has a considerable effect, and of the 16 cases involved in the experiment, only 1 case had side effects, but the side effects cannot be determined whether they are related to MSC infusion (Bernardo et al., 2009). The MSCs used in many clinical trials today are allogenic, but there are no reports of serious side effects or complications. The advantage of allogeneic MSCs compared with autologous MSCs is that patients do not need to suffer from the pain of obtaining materials, and they have a wide range of sources. However, the genetic properties of allogeneic MSCs are different from those of the host. Compared with

10.2 Mesenchymal stem cells

autologous MSCs, their efficacy, safety, and long-term effects need to be further verified.

10.2.2.2.3 Mode of administration of MSCs The mode of administration includes systemic administration and local administration. Systemic administration is mainly by intravenous infusion, while local administration includes intraperitoneal injection and intra-fistula injection. Local injection of MSCs into the fistula has proved to be one of the effective ways to treat CD combined with anal fistula. Dietz et al. (2017) conducted a 6-month phase I clinical trial using autologous MSCs attached to a bioabsorbable matrix and placed into a fistula. 10 of 12 patients (83%) achieved complete clinical healing. Intravenous injection of MSCs has the characteristics of easy and minimally invasive, but the injected MSCs mainly stay in the lungs and rarely reach the intestinal inflammatory site and can really play a role (Castelo-Branco et al., 2012). Although interventional treatment of MSCs injected through the superior mesenteric artery can increase the number of MSCs that reach the intestinal inflammation site, but because of its invasive operation and the risk of complications, it is rarely used clinically. In summary, which method of administration is more conducive to the relief of clinical symptoms depends on the specific circumstances, and more experimental verification is needed.

10.2.3 Application of MSCs in the treatment of radiation enteritis Clinical radiotherapy is a major treatment for malignant tumors such as the abdomen and pelvis during the perioperative period. However, because the intestinal epithelium is highly sensitive to radiation, it leads to the destruction of the mucosal barrier and the increased permeability of the intestine, causing intestinal dysfunction and radiation intestinal damage. It has been reported that MSCs can repair skin, lung, and gastrointestinal tissues damaged by radiation (Wang et al., 2007). MSCs will not be rejected by the transplant and can effectively repair the tissues, which has a good effect on radiation enteritis. Sun et al. (2009) treated baboons that had been irradiated with MSCs. The results showed that baboons had the most implanted MSCs in the gastrointestinal tract, suggesting that MSCs can act on radiation-damaged intestinal tissue. Some study has found that the amount of insulin-like growth factor-1 (IGF-1) secreted by BMSCs can inhibit p53 upregulated modulator of apoptosis (PUMA) through the PI3K/AKT/P53 signaling, thereby preventing the apoptosis of intestinal stem cells (ISCs) due to radiation damage (Qiu et al., 2010). Through experimental research, Zhang et al. (2008) found that the expression of SDF-1 factor in the radioactive intestine increased. Modification of the mCXCR4 gene (receptor of SDF-1) can significantly improve the ability of BMSCs to colonize and differentiate into the intestine and promote the repair of radiation intestinal injury.

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Implantation of exogenous BMSCs can accelerate the self-renewal of intestinal epithelial cells and at the same time help restore the intestinal secretory function. Semont et al. (2006) found that after injecting BMSCs into rats with radiation enteritis, the length of small intestinal villi increased, which further confirmed the repairing effect of BMSCs in intestinal injury. Some studies have found that injecting BMSCs into rats with radioactive intestinal injury can regulate the homeostasis of small intestinal epithelial cells, promote the proliferation of radioactive injured epithelial cells, and inhibit their necrosis (Semont et al., 2010). Some scholars believe that MSCs of syngeneic mice can promote the recovery of hematopoietic function in mice with severe radiation injury. After the intestinal blood flow state is restored, good blood perfusion helps to absorb intestinal nutrients, strengthen the intestinal barrier function, resist the invasion of foreign microorganisms, and restore radioactive intestinal damage early.

10.2.4 Application of MSCs in the treatment of liver transplantation-related bowel disease At present, liver transplantation has become the most effective treatment for patients with various end-stage liver diseases, but complicated gastrointestinal dysfunction after liver transplantation becomes the key to threatening the success of the operation. Studies have found that injecting MSCs into a rat model with autoimmune enteropathy can reduce the number of mesenteric lymph nodes during the active phase of the disease and can inhibit the aggregation of active T cells in the mesenteric lymph nodes. The acute graft-versus-host disease mechanism is that donor mature T cells encounter activation and proliferation of host antigen-presenting cells. MSCs can inhibit the activation and proliferation of T cells, change the level and composition of cytokines, and suppress immune responses. In addition, MSCs can affect the migration of T cells and DCs, induce the expansion of Treg cells, inhibit the secretion of antibodies by B lymphocytes, and regulate the secretion of soluble factors such as NO and DO. These immune regulatory mechanisms can reduce intestinal dysfunction caused by rejection after transplantation. Studies have shown that MSCs can reduce the level of malondialdehyde in rats with ischemia-reperfusion liver injury and reduce the release of large amounts of oxygen-free radicals caused by lipid peroxidation, so it can be speculated that MSCs can also reduce intestinal damage caused by a large number of free radicals. Under the condition of ischemia and hypoxia caused by blocked portal blood flow, BMSCs can secrete various cytokines to improve the survival and proliferation ability of intestinal epithelial cells and participate in tissue repair (Weil et al., 2009). VEGF and other angiogenic proteins secreted by MSCs participate in the vascularization of the injured area and promote local blood perfusion (Schuleri et al., 2007). At the same time, VEGF can also prevent the adhesion of leukocytes

10.3 Hematopoietic stem cells

and chronic inflammation and reduce the ischemic reaction caused by inflammation (Scalia et al., 1999). HGF secreted by MSCs can promote the proliferation of vascular endothelial cells, increase angiogenesis, and reduce apoptosis (Guo et al., 2008); FGF promotes the proliferation and vascularization of endothelial cells and can also make BMSCs have a higher survival rate under hypoxia (Song et al., 2005).

10.2.5 Application of MSCs in the treatment of intestinal tumor Current research believes that MSCs are an excellent anticancer drug “porter,” which can effectively deliver drugs to the damaged site. Recent studies have found that MSCs play an important role in the development of tumors. It can promote the proliferation, invasion, and migration of tumors, as well as the formation of tube, and even can enhance the resistance of tumors. Studies have shown that MSCs isolated from human colorectal cancer tissue can help tumor cells escape aging through the P53/P21 pathway and promote the growth of colorectal cancer cells in vivo (Li et al., 2020). Another study suggests that IL-6 secreted by human colorectal cancer-derived MSCs enhances the migration and invasion of colorectal cancer cells through JAK2/STAT3 signaling and may provide new therapeutic or preventive targets (Zhang et al., 2018b). MSCs can alleviate cancer progression by modulating immune components in the tumor microenvironment. The latest research by Sabine et al. (Francois et al., 2019) has determined the effect of MSC treatment on the progress of solid tumors and found that MSCs slow down the occurrence and development of colon cancer by regulating the immune components of the tumor microenvironment. CD681 mononuclear macrophage infiltration was reduced by 50% in colon adenocarcinoma of rats treated with MSCs, while the number of CD31 lymphocytes increased twice. MSCs also reprogram macrophages to become regulatory cells involved in phagocytosis, thereby inhibiting the production of pro-inflammatory cytokines. In addition, MSCs restored Th17 cell activity while reducing NK and rTh17 cell activity and Treg recruitment. Importantly, the use of MSCs after radiotherapy not only reduces the damage to healthy tissues but also slows the tumor growth. Scientists believe that after cancer radiotherapy, MSCs are given a possible safe and innovative treatment option for healing tissues.

10.3 Hematopoietic stem cells HSCs are a type of adult stem cells that are found in the bone marrow, blood, and umbilical cord. HSCs have the potential for high value-added and multidirectional differentiation and can still maintain complete hematopoietic function after bone marrow removal. HSCs can not only differentiate into blood cell lines but also transform into nonhematopoietic cells under certain conditions (Gazouli et al., 2014).

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This is called plasticity of HSCs. HSC transplantation can be divided into bone marrow HSC transplantation, peripheral blood HSC transplantation, umbilical cord HSC transplantation, and autologous and allogeneic transplantation according to different sources. HSCs are rarely used to treat UC alone. They are usually used to treat severe blood diseases with IBD. This is mainly because the treatment of most HSCs involves myeloablative and matching problems and complications such as transplantation failure, graft resistance to host disease, infection, hepatic vein occlusion, and intestinal ulcer aggravation, which limit its application. Moreover, for patients with autologous transplantation, the immune system is likely to revert to the previously pathological inflammatory system within a short time after “restart,” thus losing the significance of transplantation. Rabian et al. (2016) retrospectively analyzed cases of allogeneic HSC transplantation in patients with hematological malignancies combined with IBD nationwide and found that the 48-month overall survival rate was not significantly different from that of the control group. Mehta et al. (2018) even considered that the transplantation of HSCs would increase the mortality of UC in the analysis of a large national sample, and if patients with UC are complicated by primary sclerosing cholangitis or tuberculosis and cytomegalovirus infection, the mortality after transplantation will further increase. This is quite inconsistent with the results reported in previous small samples. Because the surface antigenicity of umbilical cord HSCs is weak, umbilical cord transplantation avoids the high cost of bone marrow removal and immune matching and various complications and is relatively safe. Karaca et al. (2016) reported an early allogeneic bone marrow HSC transplantation in an 11-monthold IBD female patient with familial IL-10 and its receptor deficiency. The clinical symptoms of chronic diarrhea, perianal abscess, and recurrent infection completely disappeared. Some scholars have adjusted the direction to apply HSC transplantation to specific genetically pathogenic special IBD populations, such as the successful application of very early onset IBD and X-linked primary immunodeficiency disease. Although studies have shown that HSC transplantation can successfully treat people with specific genetically specific IBD, it is still not widely used clinically. The adverse reactions caused by the large dose of chemically toxic drugs used before transplantation are not optimistic, especially in the infection caused by immunosuppression.

10.4 Intestinal stem cells ISCs are a type of adult stem cells located at the base of the intestinal mucosal crypts. Normally, they continue to proliferate, differentiate, and migrate to the top of the crypts. Therefore, the intestinal mucosa is updated every 3 5 days. ISCs

10.4 Intestinal stem cells

can divide into a daughter cell that is the same as the original cell and a daughter cell that has the ability to differentiate. The latter differentiates into different types of intestinal cells: goblet cells, Paneth cells, endocrine cells, M cells, and intestinal absorption cells. Although the function of ISCs is quite different from other cells in the intestine, there are still great difficulties in directly distinguishing them from other intestinal cells. In the past 40 years, two theories have been perfected. One is the “stem cell region” model proposed by LEBLOND et al., which believes that the crypt base columnar cells (CBCs) are ISCs. The proteins Lgr5, Rnf43, and Troy are considered to be several highly expressed markers in CBCs (Nishioka et al., 2018; Min et al., 2016; Carroll et al., 2018). The other is the “ 1 4 model” proposed by POTTEN. It is believed that ISCs are located on Paneth cells and surrounded by 16 cells in a circle. The genes HOPX and LRIG1 are considered to be distinguishing markers (Munoz et al., 2012). Many signal pathways have important regulatory effects on ISCs. This includes Wnt (Wingless) and Hh (Hedgehog) signaling pathways, which are closely related to the proliferation of ISCs. Wnt pathway is the main signal that promotes the self-renewal and regeneration of ISCs in mammals. Tian et al. (2016) believe that it may inhibit the expression of JAK-STAT signal of ISCs through an involuntary pathway, that is, activating the Wnt signaling pathway of intestinal epithelial absorption cells, thereby stopping the proliferation of ISCs. ISCs maintain homeostasis and deal with past damage through the Bmp and Notch pathways. Studies have observed that the loss of Notch leads to the decrease of Lgr5 1 stem cells accompanied by transient secretory cell proliferation (Carulli et al., 2015). The microenvironment is also very important for the regulation of ISCs. Athiyyah established a rat model exposed to pathogenic Escherichia coli (serotype O55: B5) and found that Lactobacillus plantarum IS-10506 can activate ISCs to produce obvious anti-inflammatory effects (Athiyyah et al., 2018), which shows that microenvironments such as tract flora can also affect the activities of stem cells. ISCs are scarce in number, not concentrated in distribution, and lack of highly specific markers. Its specific protein Lgr5 has been shown to only exist on the surface of some ISCs and it is difficult to bind to specific antibodies or identify. Other markers are located in the cell, lacking a large-scale identification method, so their transplantation cases are relatively rare, and they are limited to animal experiments and lack large-scale clinical practice. Yui et al. (2012) used Lgr5 as a marker to screen out ISCs and induced them to proliferate in vitro to become intestinal “microorgans” and transplanted them into the intestines of UC mice. It was found that the graft closely adhered to the intestinal wall injury and produced the same normal tissue rich in multiple cells as the intestinal epithelium. Okamoto et al. (Okamoto and Watanabe, 2015, 2016; Okamoto et al., 2020) currently develops autologous ISC-transplantation therapy for IBD patients. This strategy is divided into two stages: first, the ISCs obtained from endoscopic biopsy samples of IBD patients are expanded ex vivo, and second, those ex vivo

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FIGURE 10.2 Workflow for the organoid transplantation process of ISCs for intestinal diseases.

cultured ISCs are transplanted and delivered through targeted endoscopy (Fig. 10.2). However, there are several points that must be verified or confirmed for such a therapy. First, the feasibility of effective ex vivo expansion of IBD patient-derived ISCs is still uncertain and needs to be verified. Second, from the perspective of clinical safety, the transplantable donor cells need to be further confirmed whether the cultured organoids can be expanded and maintained. For example, the tumorigenic or potential pathogens of these organoids may expand, so they should be carefully excluded. In addition, an efficient endoscopic method for delivering live organoids to targeted lesions needs to be established. Finally, the benefits of ISC-transplantation should be confirmed by a prospective study in IBD patients. Also, the optimization of the application of ISC-transplantation in combination with existing treatments should be carefully verified. This shows that ISCs have a good prospect in the treatment of IBD, but there are still many problems to be overcome.

10.5 Conclusions and prospects Mesenchymal and other stem cells can treat intestinal diseases through various mechanisms such as immune regulation and promotion of intestinal repair. These stem cells have shown a certain therapeutic effect for the treatment of intestinal diseases, especially for patients whose traditional methods are ineffective or cannot accept side effects. Stem cell therapy is a new hope, indicating that the application of mesenchymal and other stem cells has a good prospect in the years to come. It deserves the attention of more scientific researchers and clinicians. However, the current experimental data mostly come from preclinical basic

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research, with relatively few clinical trial results and insufficient treatment experience. For this reason, more research is needed to confirm the safety and effectiveness of stem cell therapy and to explore more methods to improve its therapeutic effect on intestinal diseases. There are still many problems in the application of mesenchymal and other stem cells. First, the selection of stem cells is restricted by various objective conditions such as donor sources, culture methods, and lack of clinical application experience, and there is no clear type selection criterion. Next, regarding various measurement calculations for stem cell transplantation, there is no international uniform standard for transplantation methods. Moreover, a large number of longterm follow-up works of clinical trials are not perfect. Not all clinical trials have proved the effectiveness of stem cells for intestinal diseases, and there are still many patients with ineffective stem cell transplantation therapy. Stem cells have a low homing rate in the body, and more stem cells are retained in the liver and lungs, and their effects are unknown. Finally, the safety of stem cell transplantation needs to be further strengthened. For example, it is reported that stem cells are related to certain malignant tumors. The side effects of stem cell transplantation, especially HSC transplantation, such as cell embolism, infection, and other issues mentioned above, need to be paid attention to. Since more and more experiments are devoted to stem cell research, positive and negative results are frequently reported. If the mechanism of action can be clarified and the common problems in the transplantation process can be solved, then the road to cure a variety of intestinal diseases with mesenchymal and other stem cells is at hand. At the same time, stem cell therapy will be applied to a broader field, so as to better serve the clinic.

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Munoz, J., Stange, D.E., Schepers, A.G., van de Wetering, M., Koo, B.K., Itzkovitz, S., et al., 2012. The Lgr5 intestinal stem cell signature: Robust expression of proposed quiescent ’ 1 4’ cell markers. EMBO J. 31, 3079 3091. Nishioka, M., Suehiro, Y., Sakai, K., Matsumoto, T., Okayama, N., Mizuno, H., et al., 2018. TROY is a promising prognostic biomarker in patients with colorectal cancer. Oncol. Lett. 15, 5989 5994. Okamoto, R., Watanabe, M., 2015. Perspectives for regenerative medicine in the treatment of inflammatory bowel diseases. Digestion 92, 73 77. Okamoto, R., Watanabe, M., 2016. Investigating cell therapy for inflammatory bowel disease. Expert. Opin. Biol. Ther. 16, 1015 1023. Okamoto, R., Shimizu, H., Suzuki, K., Kawamoto, A., Takahashi, J., Kawai, M., et al., 2020. Organoid-based regenerative medicine for inflammatory bowel disease. Regen. Ther. 13, 1 6. Panes, J., Garcia-Olmo, D., Van Assche, G., Colombel, J.F., Reinisch, W., Baumgart, D.C., et al., 2016. Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn’s disease: A phase 3 randomised, double-blind controlled trial. Lancet 388, 1281 1290. Phinney, D.G., Pittenger, M.F., 2017. Concise review: MSC-derived exosomes for cell-free therapy. Stem Cell 35, 851 858. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., et al., 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143 147. Qiu, W., Leibowitz, B., Zhang, L., Yu, J., 2010. Growth factors protect intestinal stem cells from radiation-induced apoptosis by suppressing PUMA through the PI3K/AKT/p53 axis. Oncogene 29, 1622 1632. Rabian, F., Porcher, R., Sicre de Fontbrune, F., Lioure, B., Laplace, A., Nguyen, S., et al., 2016. Influence of previous inflammatory bowel disease on the outcome of allogeneic hematopoietic stem cell transplantation: A matched-pair analysis. Biol. Blood Marrow Transpl. 22, 1721 1724. Robinson, A.M., Miller, S., Payne, N., Boyd, R., Sakkal, S., Nurgali, K., 2015. Neuroprotective potential of mesenchymal stem cell-based therapy in acute stages of TNBS-induced colitis in guinea-pigs. PLoS One 10, e0139023. Scalia, R., Booth, G., Lefer, D.J., 1999. Vascular endothelial growth factor attenuates leukocyte-endothelium interaction during acute endothelial dysfunction: essential role of endothelium-derived nitric oxide. FASEB J. 13, 1039 1046. Schuleri, K.H., Boyle, A.J., Hare, J.M., 2007. Mesenchymal stem cells for cardiac regenerative therapy. Handb. Exp. Pharmacol. 195 218. Semont, A., Francois, S., Mouiseddine, M., Francois, A., Sache, A., Frick, J., et al., 2006. Mesenchymal stem cells increase self-renewal of small intestinal epithelium and accelerate structural recovery after radiation injury. Adv. Exp. Med. Biol. 585, 19 30. Semont, A., Mouiseddine, M., Francois, A., Demarquay, C., Mathieu, N., Chapel, A., et al., 2010. Mesenchymal stem cells improve small intestinal integrity through regulation of endogenous epithelial cell homeostasis. Cell Death Differ. 17, 952 961. Shih, D.T., Lee, D.C., Chen, S.C., Tsai, R.Y., Huang, C.T., Tsai, C.C., et al., 2005. Isolation and characterization of neurogenic mesenchymal stem cells in human scalp tissue. Stem Cell 23, 1012 1020. Song, H., Kwon, K., Lim, S., Kang, S.M., Ko, Y.G., Xu, Z., et al., 2005. Transfection of mesenchymal stem cells with the FGF-2 gene improves their survival under hypoxic conditions. Mol. Cell 19, 402 407.

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Squillaro, T., Peluso, G., Galderisi, U., 2016. Clinical trials with mesenchymal stem cells: an update. Cell Transpl. 25, 829 848. Sun, Q., Ming, L., Thomas, S.M., Wang, Y., Chen, Z.G., Ferris, R.L., et al., 2009. PUMA mediates EGFR tyrosine kinase inhibitor-induced apoptosis in head and neck cancer cells. Oncogene 28, 2348 2357. Sun, Y.Q., Deng, M.X., He, J., Zeng, Q.X., Wen, W., Wong, D.S., et al., 2012. Human pluripotent stem cell-derived mesenchymal stem cells prevent allergic airway inflammation in mice. Stem Cell 30, 2692 2699. Swenson, E., Theise, N., 2010. Stem cell therapeutics: potential in the treatment of inflammatory bowel disease. Clin. Exp. Gastroenterol. 3, 1 10. Takebe, Y., Tatehara, S., Fukushima, T., Tokuyama-Toda, R., Yasuhara, R., Mishima, K., et al., 2017. Cryopreservation method for the effective collection of dental pulp stem cells. Tissue Eng. Part. C. Methods 23, 251 261. Tian, A., Benchabane, H., Wang, Z., Ahmed, Y., 2016. Regulation of stem cell proliferation and cell fate specification by Wingless/Wnt signaling gradients enriched at adult intestinal compartment boundaries. PLoS Genet. 12, e1005822. Volarevic, V., Al-Qahtani, A., Arsenijevic, N., Pajovic, S., Lukic, M.L., 2010. Interleukin1 receptor antagonist (IL-1Ra) and IL-1Ra producing mesenchymal stem cells as modulators of diabetogenesis. Autoimmunity 43, 255 263. Wang, J., Boerma, M., Fu, Q., Hauer-Jensen, M., 2007. Significance of endothelial dysfunction in the pathogenesis of early and delayed radiation enteropathy. World J. Gastroenterol. 13, 3047 3055. Weil, B.R., Markel, T.A., Herrmann, J.L., Abarbanell, A.M., Meldrum, D.R., 2009. Mesenchymal stem cells enhance the viability and proliferation of human fetal intestinal epithelial cells following hypoxic injury via paracrine mechanisms. Surgery 146, 190 197. Xie, M., Qin, H., Luo, Q., He, X., He, X., Lan, P., et al., 2017. Comparison of adiposederived and bone marrow mesenchymal stromal cells in a murine model of Crohn’s disease. Dig. Dis. Sci. 62, 115 123. Yang, J., Liu, X.X., Fan, H., Tang, Q., Shou, Z.X., Zuo, D.M., et al., 2015. Extracellular vesicles derived from bone marrow mesenchymal stem cells protect against experimental colitis via attenuating colon inflammation, oxidative stress and apoptosis. PLoS One 10, e0140551. Yui, S., Nakamura, T., Sato, T., Nemoto, Y., Mizutani, T., Zheng, X., et al., 2012. Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5 (1) stem cell. Nat. Med. 18, 618 623. Zhang, J., Gong, J.F., Zhang, W., Zhu, W.M., Li, J.S., 2008. Effects of transplanted bone marrow mesenchymal stem cells on the irradiated intestine of mice. J. Biomed. Sci. 15, 585 594. Zhang, J., Lv, S., Liu, X., Song, B., Shi, L., 2018a. Umbilical cord mesenchymal stem cell treatment for Crohn’s disease: a randomized controlled clinical trial. Gut Liver 12, 73 78. Zhang, X., Hu, F., Li, G., Li, G., Yang, X., Liu, L., et al., 2018b. Human colorectal cancer-derived mesenchymal stem cells promote colorectal cancer progression through IL-6/JAK2/STAT3 signaling. Cell Death Dis. 9, 25. Zou, X., Zhang, G., Cheng, Z., Yin, D., Du, T., Ju, G., et al., 2014. Microvesicles derived from human Wharton’s Jelly mesenchymal stromal cells ameliorate renal ischemiareperfusion injury in rats by suppressing CX3CL1. Stem Cell Res. Ther. 5, 40. Zuo, D., Liu, X., Shou, Z., Fan, H., Tang, Q., Duan, X., et al., 2013. Study on the interactions between transplanted bone marrow-derived mesenchymal stem cells and regulatory T cells for the treatment of experimental colitis. Int. J. Mol. Med. 32, 1337 1344.

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CHAPTER

General discussion, conclusion remarks, and future directions

11 Ahmed El-Hashash1,2

1

The University of Edinburgh-Zhejiang International Campus (UoE-ZJU Institute), Haining, P.R. China 2 Centre of Stem Cell and Regenerative Medicine, Schools of Medicine & Basic Medicine, Hangzhou, P.R. China

Intestinal diseases have major health and economic impacts and have a high mortality in different countries in the world. Because of their complexity, identifying and developing more effective therapeutic approaches for intestinal diseases are still challenging and, therefore, need more research. Intestinal stem cells are well reported and used to investigate stem cell biology since they are easily accessible and renew rapidly. They are well maintained throughout the human life. Structurally, the intestinal epithelium in humans consists of crypts containing both intestinal stem cells and other cell types (the proliferative compartment) and villi that represent intestinal mucosa folds and contain the differentiation cell compartment. Multipotent intestinal stem cells can generate all cell types of the intestinal lineage that exist in the intestinal surface, including goblet cells, enterocytes, Paneth cells, and endocrine cells (Arrighi, 2018). Recently developed cutting-edge in vivo and in vitro research approaches and tools have facilitated the investigation of the role of stem cells in the intestine and their potential applications in intestinal repair, regeneration, and diseases. Indeed, the intestine is among the leading organs, in which several cutting-edge in vitro and in vivo research tools and approaches are recently developed and used to investigate stem cell biology/function and the potential applications of stem cells in the treatment of intestinal diseases. Intestinal stem cells are easily accessible and well maintained throughout the human life and renew rapidly. The recently developed cutting-edge approaches/tools include genetic editing in vivo and in vitro, murine and human organoid cultures, human-induced pluripotent cell models of diseases, genetically engineered mice, haploid cells for genetic as well as compound screening paradigms, and stem cell transplantation for treatments of human diseases.

The Intestine. DOI: https://doi.org/10.1016/B978-0-12-821269-1.00002-3 © 2021 Elsevier Inc. All rights reserved.

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11.1 Advances of organotypic intestinal cell culture and gene editing/engineering in intestinal repair, regeneration, and diseases: current challenges and future prospectives The term “organotypic model” originally referred to explanted tissue that retains the same or highly similar functions with its in vivo counterpart (Randall et al., 2011; Dedhia et al., 2016). In the last decade, this definition continued to evolve to include various ex vivo and in vitro cell culture systems emerging in the field of organ development and tissue engineering. The organotypic models of human intestine can be divided into two categories based on the type of the stem cells they originate from, including human adult stem cells (hASCs) and human pluripotent stem cells (hPSCs)-derived models. These organotypic intestinal cell culture models share common features, including their stem cell (multipotent or pluripotent) origin and their requirement for the presence of minimum stem cell niche components for their long-term survival in vitro. In addition, they share the capability to self-renew and self-organize into 3D structures composed of intestinal epithelium by undergoing multilineage differentiation (Spence et al., 2011; Sato et al., 2011; Zachos et al., 2016; Munera et al., 2017; Wallach and Bayrer, 2017; Fair et al., 2018; Singh et al., 2020). The organotypic intestinal cell cultures currently represent significant advancements in modeling human intestinal development, composition, and function. However, they still suffer from several shortcomings, despite their numerous strengths. These organotypic cell cultures were used in modeling intestinal functions. Indeed, remarkable progress has been made towards emulation of human intestinal cell and tissue-level functions using human organotypic intestinal cell cultures. However, these models do not fully recapitulate the complexity and organ-level physiology of the native human intestine, as they lack cellular components (i.e., enteric neurons, immune cells, and vasculature), mechanical forces (i.e., shear and stretch), and appropriate geometry of extracellular microenvironment. Thus, a number of recent studies have focused on improving the complexity of human organotypic intestinal cell cultures models through the use of codevelopment and co-culture methods as well as various engineering-based approaches This was discussed in detail in Chapter 2. Furthermore, various biology and engineering-based approaches have been exploited to develop organotypic culture systems that more holistically recapitulate the cellular complexity and dynamic nature of native human intestine and therefore more accurately model complex organ-level functions. Moving forward, in parallel to the continued advancement of these models, research efforts should focus on improving their fidelity, reproducibility and scalability. Adoption and democratization of these improved in vitro tools across the research community will undoubtedly enhance our understanding of human biology in the contexts of

9.4 Organotypic intestinal cell culture and gene editing

intestinal development, homeostasis, and disease. This was described in detail in Chapter 2. Over the past several years, precision medicine has been moving from a mere idea to a tangible reality of the medical practice. Personalized medicine or personalized care refers to a treatment designed only for one patient and considers the influence of individuals’ genes, environment and lifestyle to tailor specific interventions. Examples of diseases that would benefit from personalized care strategies include cystic fibrosis, advanced and metastatic cancer (Dekkers et al., 2013; van de Wetering et al., 2015; Pauli et al., 2017; Howell et al., 2018) and inflammatory bowel disease (IBD; McGovern, 2014). Remarkably, the gastrointestinal tract (GI)-derived organoid cultures allow designing tailored personalized treatments since they carry both the genetic and epigenetic make-up of the tissue of origin. Notably, induced pluripotent stem cells can be used to generate organoids to study unique genetic mutations without the need for invasive intestinal biopsies. Research conducted on human organoids provides a novel perspective on what we have previously learned on the pathophysiology of diseases, including enteropathogens and protozoa infections, autoimmune diseases, and cancer. Furthermore, they allow the development of physiologically relevant organotypic co-culture models that incorporate different cell lineages, including epithelium, immune, mesenchymal, vascular cells, and microbiota to reproduce the complexity of the GI micro-milieu. Finally, considerations are offered on the ethical implications regarding the growing number of living biobanks of human organoids and the need for worldwide harmonized regulations that protect the individuals’ privacy without hindering scientific progress in using this new powerful tool. These aspects on the role of human gastrointestinal organoids in discovery and translational medicine have been discussed in detail in Chapter 3. Gene therapy is currently highly promising strategy for treating different human diseases and is based on the replacement of a faulty gene(s) or adding a new gene to the intestine. Increased potential therapeutic targets in the intestine has been reported for clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based gene editing technology. The current application of CRISPR/Cas9 system in prenatal gene editing is a promising therapeutic approach for intestinal disease and disorders. However, a major challenge facing gene editing technology is preventing off-target mutagenesis, which could be solved by developing modified Cas9 or other enzymes. Tissue engineering is a rapidly growing filed that applies the principals of both bioscience and bioengineering sciences for developing new biological substitutes that can improve or restore the functions of damaged of failed tissues. As discussed in Chapter 6, despite recent progresses in the study of intestinal stem cells, organoid culture and tissue-engineered small intestine, there are many challenges that face the regeneration of small intestine probably due to its complex architecture and function. Therefore, more research and development of novel methodology and technology are still needed to enhance small intestinal repair and regeneration, particularly using large animals with gastrointestinal system

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that is similar to humans to verify murine model study results. In addition, more studies are needed on the functional regeneration of tissue-engineered small intestines, including secretory, absorptive, and immune functions. Since there is currently donor shortage and life-long immunosuppression that limit intestinal transplantation, much attention has been paid to tissue-engineered small intestines since they could offer large constructs of small intestine for therapeutic applications. However, some reseeded tissue-engineered small intestines do not function well in vivo, despite having a very similar structure to native adult human small intestine (Finkbeiner et al., 2015). This is may be because these matrices could prevent the infiltration of important cells, including vascular cells and the imitated vascularization of intestinal tissues can badly affect intestinal cell function, integration and survival (Schneeberger et al., 2017). Generation of microchannels in these matrices using 3D bioprinting or similar techniques before repopulation with induced pluripotent stem cell (iPSC)-derived endothelial cells and other vessel supporting cells could be one approach to solve this problem (Gao et al., 2015; Vecchione et al., 2016; Qi et al., 2020). Interestingly, the current availability of both iPSC-derived endothelial cells and vessel supporting cells such as smooth muscle cells can facilitate the generation of patient-specific human intestinal organoids using iPSCs from the same individual (Qi et al., 2020). Furthermore, the functions of tissue-engineered intestines, including secretion, contractility, and motility, could also be improved by incorporating enteric neurons that improve the innervation of these engineered intestine. Indeed, proper innervation is a key in small intestinal functions and innervation-related defects can lead to several intestinal diseases (Brookes et al., 2016; Mourad et al., 2017).

11.1.1 Role of pluripotent stem cells in intestinal repair, regeneration, and diseases Recent progress has been achieved in investigation of the roles of pluripotent stem cells in intestinal repair, regeneration, and diseases. For instance, iPSCs hold great potential in the generation of patient-specific pluripotent stem cells for human disease modeling, drug development, and individualized cell-based therapy for various diseases. iPSCs offer a novel method of research into intestinal diseases especially with their ability to generate intestinal organoids. iPSCs are largely involved in both basic science and clinical research related to some important intestinal diseases, including colorectal cancer (CRC), Hirschsprung disease (HSCR), and IBD, which are discussed in Chapter 7. Other types of stem cells also play important roles in intestinal disease, repair, and regeneration. For example, the pluripotent potential of embryonic stem cells (ESCs) enable their differentiation into intestinal epithelial and immune cells, allowing these cells to restore the intestinal epithelium and the immune balance in the murine model of colitis. Indeed, recent achievements in stem cell research and therapy have provided possibilities for using these cells in treating several

9.4 Organotypic intestinal cell culture and gene editing

gastrointestinal and related diseases. For instance, stem cells could be used in the treatment of IBD due to their homing properties to the injury sites and differentiation capacity into both lymphocyte and epithelial cells that modulate both immune response and damages of tissues. In addition, mesenchymal stem cells (MSCs), hematopoietic stem cells (HSCs), and bone marrow-derived stem cells (BM-SCs) play important roles in the treatment of IBD and/or colitis. These stem cells bring hope for transferring stem cell-based therapy/approach from in vivo animal models of IBD to human patients. In addition, human ESC-derived hepatocytes represent a potential liver disease treatment and can be used in drug metabolism studies and cell transplantation therapy for liver diseases. Moreover, ESCs may play a role in some neurodegenerative disorders of the GI. Notably, the use of adult or tissue-specific stem cells in the gut, liver, and pancreas-related diseases is a promising approach that have significant clinical applications. However, these stem cells also represent cancer stem cell source, and, therefore, more studies are still needed to evaluate the risks versus benefits of these promising therapies. More attention has been recently devoted to the role of MSCs and HSCs in intestinal repair, regeneration, and diseases. As discussed in Chapter 10, MSCs and other stem cells can treat intestinal diseases through various mechanisms such as immune regulation and promotion of intestinal repair. These stem cells have shown a certain therapeutic effect for the treatment of intestinal diseases, especially for patients whose traditional methods are ineffective or cannot accept side effects. Stem cell therapy is a new hope, indicating that the application of mesenchymal and other stem cells has a good prospect in the years to come. It deserves the attention of more scientific researchers and clinicians. However, the current experimental data mostly come from preclinical basic research, with relatively few clinical trial results and insufficient treatment experience. For this reason, more research is needed to confirm the safety and effectiveness of stem cell therapy and to explore more methods to improve its therapeutic effect on intestinal diseases. Since more experiments are devoted to stem cell research, positive and negative results are frequently reported. If the mechanism of action can be clarified and the common problems in the transplantation process can be solved, then the road to cure a variety of intestinal diseases with MSCs and/or other stem cells is at hand. At the same time, stem cell therapy will be applied to a broader field to better serve the clinic. Finally, there are still many problems in the application of MSCs and other stem cells in intestinal repair, regeneration, and diseases. For example, the selection of stem cells is restricted by various objective conditions such as donor sources, culture methods, and lack of clinical application experience, and there is no clear type selection criterion. In addition, regarding various measurement calculations for stem cell transplantation, there is no international uniform standard for transplantation methods. Moreover, many long-term follow-up works of clinical trials are not perfect. Indeed, not all clinical trials have proved the effectiveness of stem cells for intestinal diseases, and there are still many patients with

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ineffective stem cell transplantation therapy. Stem cells also have a low homing rate in the body, and more stem cells are retained in the liver and lungs, and their effects are unknown. Furthermore, the safety of stem cell transplantation needs to be further strengthened. For example, it is reported that stem cells are related to certain malignant tumors. The side effects of stem cell transplantation, especially HSC transplantation, such as cell embolism, infection, and other issues mentioned above, need to be paid attention to.

References Arrighi, N., 2018. Definition and classification of stem cells. In: Arrighi, N. (Ed.), Stem Cells: Therapeutic Innovations Under Control Elsiver, pp. 1 45. Brookes, S., Chen, N., Humenick, A., Spencer, N.J., Costa, M., 2016. Extrinsic sensory innervation of the gut: Structure and function. Adv. Exp. Med. Biol. 891, 63 69. Dedhia, P.H., et al., 2016. Organoid models of human gastrointestinal development and disease. Gastroenterology 150 (5), 1098 1112. Dekkers, J.F., Wiegerinck, C.L., de Jonge, H.R., Bronsveld, I., Janssens, H.M., de Winterde Groot, K.M., et al., 2013. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat. Med. 19 (7), 939 945. Fair, K.L., Colquhoun, J., Hannan, N.R.F., 2018. Intestinal organoids for modelling intestinal development and disease. Philos. Trans. R. Soc. Lond. B Biol. Sci. 373 (1750). Finkbeiner, S.R., Freeman, J.J., Wieck, M.M., El-Nachef, W., Altheim, C.H., Tsai, Y.H., et al., 2015. Generation of tissue-engineered small intestine using embryonic stem cellderived human intestinal organoids. Biol. Open. 4 (11), 1462 1472. Gao, Q., He, Y., Fu, J.Z., Liu, A., Ma, L., 2015. Coaxial nozzle-assisted 3D bioprinting with built-in microchannels for nutrients delivery. Biomaterials 61, 203 215. Howell, K.J., Kraiczy, J., Nayak, K.M., Gasparetto, M., Ross, A., Lee, C., et al., 2018. DNA methylation and transcription patterns in intestinal epithelial cells from pediatric patients with inflammatory bowel diseases differentiate disease subtypes and associate with outcome. Gastroenterology. 154 (3), 585 598. McGovern, D., 2014. Personalized medicine in inflammatory bowel disease. Gastroenterol. Hepatol. (N. Y.) 10 (10), 662 664. Mourad, G.H., Barada, K.A., Saade, N.E., 2017. Impairment of small intestinal function in ulcerative colitis: Role of enteric innervation. J. Crohn’s Colitis 11 (3), 369 377. Munera, J.O., et al., 2017. Differentiation of human pluripotent stem cells into colonic organoids via transient activation of BMP signaling. Cell Stem Cell 21 (1), 51 64. e6. Pauli, C., Hopkins, B.D., Prandi, D., Shaw, R., Fedrizzi, T., Sboner, A., et al., 2017. Personalized In vitro and in vivo cancer models to guide precision medicine. Cancer Discov. 7 (5), 462 477. Qi, D., Shi, W., Black, A.R., et al., 2020. Repair and regeneration of small intestine: A review of current engineering approaches. Biomaterials 240, 1 18. Randall, K.J., Turton, J., Foster, J.R., 2011. Explant culture of gastrointestinal tissue: A review of methods and applications. Cell Biol. Toxicol. 27 (4), 267 284. Sato, T., et al., 2011. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141 (5), 1762 1772.

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Schneeberger, K., Spee, B., Costa, P., Sachs, N., Clevers, H., Malda, J., 2017. Converging biofabrication and organoid technologies: The next frontier in hepatic and intestinal tissue engineering? Biofabrication 9 (1), 013001. Singh, A., et al., 2020. Gastrointestinal organoids: A next-generation tool for modeling human development. Am. J. Physiol. Gastrointest. Liver Physiol 319 (3), G375 G381. Spence, J.R., et al., 2011. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470 (7332), 105 109. van de Wetering, M., Francies, H.E., Francis, J.M., Bounova, G., Iorio, F., Pronk, A., et al., 2015. Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell. 161 (4), 933 945. Vecchione, R., Pitingolo, G., Guarnieri, D., Falanga, A.P., Netti, P.A., 2016. From square to circular polymeric microchannels by spin coating technology: A low cost platform for endothelial cell culture. Biofabrication 8 (2), 025005. Wallach, T.E., Bayrer, J.R., 2017. Intestinal organoids: New frontiers in the study of intestinal disease and physiology. J. Pediatr. Gastroenterol. Nutr. 64 (2), 180 185. Zachos, N.C., et al., 2016. Human enteroids/colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology. J. Biol. Chem. 291 (8), 3759 3766.

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Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Acute myeloid leukemia, 134 Adenomatous polyposis coli (APC), 68 69, 77 78, 106 Adult intestinal stem cells (ISCs), 89 90, 102 103 Adult stem cells (ASCs), 29, 31f, 61 62 Alzheimer’s disease, 102 Angiotensin-converting enzyme 2 (ACE2), 41 Antimicrobial peptides (AMPs), 10 11 Autoimmune diseases, 136 138 Autologous Stem Cell Transplantation International Crohn’s Disease (ASTEC), 47

B β-catenin, 105 Bioengineering, 20 22 B lymphoma Mo-MLV insertion region 1 (Bmi1), 89 Bone marrow-derived stem cells (BM-SCs), 125 126, 168 169 Bone marrow transplantation (BMT), 147 Budding organoids (BOs), 65 66

C Cadherin-17, 106 Cancer, 136 Cancer stem cells (CSCs), 104 Celiac disease (CeD), 35 36 Center for International Bone Marrow Transplant Registry (CIBMTR), 136 138 Chromosomal instability (CIN), 68 69 Chronic myeloid leukemia (CML), 134 Clustered regularly interspaced short palindrome repeats (CRISPR), 60 Co-culture, 19 20 Co-development, 18 19 Colorectal cancer (CRC), 36 37, 77 79, 104 106, 168 advantages for, 78 79 drug screening for, 106 engineered organoids for, 68 69 hallmark of progression of, 78 79 invasion and metastasis of, 77 78 COVID-19, 41 42. See also Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Coxsackievirus B, 111

CRISPR/Cas 9 brief introduction of, 75 77 in colorectal cancers, 77 79 gene editing tools, 81 82 in gut microbiota, 81 in inflammatory bowel disease, 79 81 limitations and perspectives, 82 83 Crohn’s disease (CD), 3, 36 37, 79 80, 103 104, 108, 139 140, 150 151 Crohn’s Disease Activity Index (CDAI), 140 Crypt base columnar cells (CBCs), 157 Cyclooxygenase-2 (COX-2), 81 82 Cyclospora cayetanensis, 110 Cystic fibrosis (CF), 32 35 Cystic fibrosis transmembrane conductance regulator (CFTR) gene, 34, 81 82 Cystic fibrosis transmembrane receptor (CFTR), 12 13

D Disruptor of telomeric silencing 1-like methyltransferase (DOT1L), 82

E Embryonic stem cells (ESCs), 61, 63, 88, 101, 123 application of, 88 differentiation of, 88 for inflammatory bowel disease, 124 127 gastrointestinal and related diseases, 127 stem cell types, 125 126 therapy of accessory digestive organs, 126 127 pluripotent potential of, 168 169 treatments of, 123 124 Enteric nervous system (ENS), 11 12, 127 Enteric neural crest cell (ENCC), 19 Enterocyte, 2, 10 11 Enteroendocrine cells (EECs), 10 11 Epidermal growth factor (EGF), 65 66 Ethylnitrosourea (Enu), 79 Extracellular matrix (ECM), 87 88

F Familial adenomatous polyposis (FAP), 106 Fetal enterospheres (FEnS), 38 Fetal intestine-derived progenitor cells (FIPCs), 65 66 Fibroblast growth factor 4 (FGF4), 103

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G Gastric cancer, 69 70 Gastric inhibitory polypeptide (GIP), 14 15 Gastrointestinal (GI) diseases, 139 140 celiac disease, 35 36 cystic fibrosis, 34 35 ethical perspective on organoids biobanks, 48 49 Helicobacter pylori, 39 40 human, 30 32 inflammatory bowel diseases, 36 37 necrotizing enterocolitis, 38 39 nomenclature and distinction, 29 30 Salmonella enterica serovar Typhi, 40 41 SARS-CoV-2, 41 42 Toxoplasma gondii, 42 43 transplant application, 47 48 treatment of, 33t tumoroids in immuno-oncology, 46 47 in precision medicine, 43 46 Gastrointestinal (GI) tract, 79 80, 167 Gene therapy, 59 60, 167 Genetically engineered mouse models (GEMMs), 79 Genome/gene editing, 60 61, 166 170 engineered organoids for colorectal cancer, 68 69 gastric cancer, 69 70 intestinal diseases, 64 65 intestinal stem cell therapy for, 65 66 in intestinal enteroids, 66 68 of stem cells, 61 therapeutic applications, 61 64 adult stem cells, 61 62 embryonic stem cells, 63 induced pluripotent stem cells, 63 64 mesenchymal stem cells, 62 63 tools, 81 82 Giardia duodenalis, 110 Gluten-free diet (GFD), 35 36 Glycine amidinotransferase (GATM), 80 Graft-versus-host disease (GVHD), 135 136 Gut microbiota, 81

H Head and neck squamous cell carcinoma (HNSCC), 29 30 Helicobacter pylori, 39 40 Hematopoietic stem cells (HSCs), 59 60, 125, 131, 147, 155 156, 168 169 autologous hematopoietic stem cell transplantation for autoimmune diseases, 136 138 bone-marrow, 133 134

challenges, 142 clinical applications of, 134 136 cancer, 136 leukemia, 134 136 future directions, 142 genetic blood cell diseases, 138 139 identification and characterization of, 132 inflammatory bowel diseases, 139 141 for mucosal healing, 141 142 plasticity of, 155 156 transplantation of, 156 Hematopoietic stem cell transplantation (HSCT), 47, 135 136 allogeneic, 142 autologous, 136 138 Hepatocellular carcinoma (HCC), 45 Hirschsprung disease (HSCR), 107 108, 127, 168 potential engrafting model for, 107 108 therapeutic strategies for, 107 Homing, 149 Human adult stem cells (hASCs), 5 6, 166 reductionist nature of, 7 Human colonic organoids (HCOs), 5 6 Human embryonic stem cells (hESCs), 6 7 Human gastric organoid (hGO), 39 40 Human intestinal organoids (HIOs), 5 6, 38, 103 static culture of, 16 in vivo maturation of, 20 Human leukocyte antigens (HLAs), 112, 135 Human pluripotent stem cells (hPSCs), 5 6, 166

I Immune dysfunction, polyendocrinopathy, enteropathy, X-linked (IPEX), 64 65 Immunoglobulin (Ig), 42 43 Induced pluripotent stem cells (iPSCs), 1, 6 7, 29, 31f, 60, 63 64, 88 89, 101, 168 clinical trials, 111 112 intestinal organoids, 103 104 iPSC-based disease modeling, 102 103 limitations, 112 113 organoids in intestinal diseases colorectal cancer, 104 106 hirschsprung disease, 107 108 inflammatory bowel disease, 108 110 parasite, 110 viruses, 110 111 potential application of, 3 role of, 168 170 Inflammatory bowel diseases (IBD), 32 34, 36 37, 79 81, 89 90, 108 110, 124 127, 139 141, 147 148, 150 153, 167 animal models of, 140 141

Index

clinical applications on, 125 clinical syndrome of, 79 80 clinical trials, 151 153 exact pathogen of, 80 genetic predisposition, 80 mucosal damage, 109 pathogenesis and progression, 108 pathogenesis of, 140 141 preclinical basic research, 151 therapeutic method for, 81 tissue fibrosis characteristic of, 109 treatment of, 125 in vivo animal models of, 127 128, 168 169 Infliximab (IFX), 109 Interferon-γ (IFN-γ), 80 International Society for Stem Cell Research (ISSCR), 48 49 Intestinal epithelium, 10, 13 14 Intestinal stem cells (ISCs), 47, 65, 102 103, 153, 156 158 dosing and optimal expansion of, 70 gene editing of, 66 self-renewing cell divisions of, 65 66 Intrahepatic cholangiocarcinoma (ICC), 45

K Kirsten rat sarcoma 2 viral oncogene homolog (KRAS), 77 78

L Lactobacillus rhamnosus, 13 14 Lactococcus lactis, 81 Large intergenic noncoding RNA ribonucleic acids-regulator of reprogramming (lincRNA-ROR), 105 Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), 89 Leukemia, 134 136 Listeria monocytogenes, 81

M Mesenchymal stem cells (MSCs), 61 63, 90, 125, 147 155, 168 169 application of, 147 148 hematopoietic stem cells, 155 156 IBD, 150 153 clinical trials, 151 153 preclinical basic research, 151 immune regulation, 150 intestinal stem cells, 156 158 intestinal tumor, 155

liver transplantation-related bowel disease, 154 155 paracrine mechanism, 150 proliferation of, 91 92 promoting intestinal repair, 149 150 radiation enteritis, 153 154 Microfabrication techniques, 20 21 Microfold (M) cell, 15 16 Molecular biology, 1 Multiple intestinal neoplasia (Min), 79 MutL homolog 1 (MLH1), 78 79 Myelodysplastic syndrome (MDS), 141 Myeloid cell, 131

N National Institutes of Health (NIH), 151 Necrotizing enterocolitis (NEC), 38 39 Neural crest stem cells (NCSCs), 107 Noggin, 65 66 Non-Hodgkin’s lymphoma, 135

O Organotypic intestinal cell culture models definition, 5 7 generation methods and characteristics of, 8f modeling of intestinal functions using, 12 16 organ-level functions, toward emulation of, 16 22 bioengineering, 20 22 co-culture, 19 20 co-development, 18 19 structure and functions, 7 12

P Pancreatic ductal adenocarcinoma (PDAC), 45 Parasite, 110 Parenteral nutrition (PN), 64 65, 70, 139 140 Patient-derived organoids (PDO), 44 Peptide transporter 1 (PEPT1), 12 13 Peripheral blood mononuclear cells (PBMC), 46 47 Peripheral blood stem cells (PBSC), 139 P-glycoprotein (P-gp), 20 Primary liver carcinoma (PLC), 45 Protospacer adjacent motif (PAM), 77

R Regenerative medicine, 1 Retrovirus, 60 Rotavirus, 111 RUNX2, 134

175

176

Index

S

T

Salivary gland, 48 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), 41 42. See also COVID-19 Short bowel syndrome (SBS), 64 65 Short-chain fatty acids (SCFAs), 9 10 Short guide RNA (sgRNA), 75, 77 Sickle cell disease (SCD), 62 Single nucleotide polymorphisms (SNPs), 80 Small intestine biological tissue-engineered grafts, 90 92 decellularization of tissues/organs, 91 92 composition and structure of, 9f stem cells, 88 90 adult intestinal stem cells, 89 90 embryonic stem cells, 88 induced pluripotent stem cells, 88 89 mesenchymal stem cells, 90 structure and cellular components of, 87 88 tissue-engineered, 92 93 transplantation in intestinal regeneration, 93 Sodium-glucose cotransporter 1 (SGLT1), 12 13 Sodium-hydrogen exchanger 3 (NHE3), 12 13 Staphylococcus aureus, 81 Stem cell genetic engineering (SCGE), 60 Streptococcus thermophilus, 75 76

Three-dimensional (3D) bioprinting, 94, 168 Tissue engineering, 1, 167 168 Toll-like receptor 4 (TLR4), 80 Toxoplasma gondii, 42 43, 110 Transcription activator-like effector (TALEN) proteins, 75, 82 Transit-amplifying (TA), 10 Trichinella spiralis, 16 Tumor microenvironment (TME), 29 30

U Ulcerative colitis (UC), 36 37, 79 80, 108, 139 140 Umbilical cord blood (UCB), 136

V Virus, 110 111

X Xenotransplantation, 69

Z Zinc finger nucleases (ZFNs), 60, 75