Stem Cells in Ophthalmology [1 ed.] 9789386056511, 9789962678762

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Stem Cells in Ophthalmology EDITORS Daniel H. Scorsetti, MD, PhD Victor L. Perez, MD Jose Alvaro Pereira Gomes, MD, PhD

An Editorial Branch of Jaypee Brothers Medical Publishers (P) Ltd.

PRODUCTION Editor-in-Chief: Samuel Boyd, MD Production Director: Kayra Mejia Digital Composition: Laura Duran International Communications: Sheyla Marengo MARKETING Director Sales & Marketing Latin America: Srinivas Chaubey Customer Service: Miroslava Bonilla Sales Manager: Tomas Martinez © Copyright, English Edition, 2015 for Jaypee - Highlights Medical Publishers, Inc. All rights reserved and protected by Copyright. No part of this publication may be reproduced, stored in retrieval system or transmitted in any form by any means, photocopying, mechanical, recording or otherwise, nor the illustrations copied, modified or utilized for projection without the prior, written permission of the copyright owner. Due to the fact that this book will reach ophthalmologists from different countries with different training, cultures and backgrounds, the procedures and practices described in this book should be implemented in a manner consistent with the professional standards set for the circumstances that apply in each specific situation. Every effort has been made to confirm the accuracy of the information presented and to correctly relate generally accepted practices. The authors, editors, and publisher cannot accept responsibility for errors or exclusions or for the outcome of the application of the material presented herein. There is no expressed or implied warranty for this book or information imparted by it. Any review or mention of specific companies or products is not intended as an endorsement by the authors or the publisher. Daniel H. Scorsetti, MD, PhD; Victor L. Perez, MD; Jose Alvaro Pereira Gomes, MD, PhD “STEM CELLS IN OPHTHALMOLOGY” ISBN: 978-9962-678-76-2 Published for:

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Jaypee - Highlights Medical Publishers, Inc. City of Knowledge International Technopark, Bldg. 237 Gaillard Highway, Clayton Panama Rep. of Panama Phone: (507) 301-0496 / 97 - Fax: (507) 301-0499 E-mail: [email protected] Worldwide Web: www.jphmedical.com

EDITORS

Daniel H. Scorsetti, MD, PhD Chairman and Professor of Ophthalmology Director, Board of Ophthalmology School of Medicine, USAL Chief of Cornea, CEOS Buenos Aires, Argentina

Victor L. Perez, MD Director, Ocular Surface Center Walter G. Ross Distinguished Chair in Ophthalmic Research Professor of Ophthalmology, Microbiology and Immunology University of Miami Miller School of Medicine Bascom Palmer Eye Institute Miami, FL (USA)

Jose Alvaro Pereira Gomes, MD, PhD Professor of Ophthalmology/Universidade Federal de S. Paulo – UNIFESP/EPM Director, Anterior Segment Area and Advanced Ocular Surface Center (CASO) UNFESP/EPM President, Sociedad Panamericana de Cornea (PanCornea) Sao Paulo, Brazil

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STEM CELLS IN OPHTHALMOLOGY STEM CELLS IN OPHTHALMOLOGY

ACKNOWLEDGEMENTS Medicine is constantly advancing and ophthalmology is one of the fields that best demonstrates this. Stem cells were discovered over 50-years ago, however a boom in publications as regenerative therapy only began in the year 2000, and has enjoyed steady growth since that time. In 2012, two Nobel Prizes were awarded to researchers, Sir John Gurdon and Shinya Yamanaka for their contributions to the field of stem cells, discovering that mature cells can be reprogrammed to become pluripotent. Their findings have revolutionized our understanding of how cells and organisms develop. The ocular surface has had the privilege of being one of the pioneers in the field of stem cell use because of the ease of access and the clinical and scientific evidence of the presence of these cells in the limbus permitting reconstructed therapies using limbal stem cell transplantation. Other areas of ophthalmology, such as the retina, orbit and trabecular meshwork, etc., have evolved significantly, and now have very promising treatments. My first book of Stem Cells in Ophthalmology was published in 2011, and its important repercussions coupled with the latest technological advances in the field convinced me to develop this new textbook with two long-time friends and world-renowned physician-scientists as co-editors. Without the intensive, unrivaled commitment, and remarkable meticulousness of Jose AP Gomes and Victor L. Perez, this book would not have been possible. The value of this book has been greatly enhanced by the chapters written by our international expert colleagues. Their contribution is greatly appreciated and the main reason for the much anticipated success of this valuable resource to ophthalmologists and trainees worldwide. Last but not least, I would like to thank Karen A. Hodous, who spared neither time nor effort coordinating the materials and liaising with each author. And to Felipe Valenzuela, who in the final hour of preparation and review, and with “fresh eyes”, provided review support and constructed the book index. I would also like to thank the PanAmerican Cornea Society (PanCornea), the Bascom Palmer Eye Institute at the University of Miami, the School of Medicine at Salvador University in Buenos Aires (USAL) and the Universidade Federal de São Paulo (UNIFESP/EPM) for their support of this work. Finally, a special thanks to our wives and children who have allowed us the time to work on this project. Daniel H. Scorsetti, MD, PhD

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PREFACE The use of stem cell therapy in medicine has evolved from general ideas to structured concepts that are now on trial in different fields including ophthalmology. This book is certainly a testimony to what has been achieved, and most importantly, where we are and what still needs to be done in order to discover and develop new therapeutic approaches that will contribute to the control of a variety of ocular diseases. Since the beginning, the cornea was seen as an important and easy target for conducting research because of its anatomy and other peculiar aspects, including the facility of observation and access. Therefore, it is not surprising that the studies on the cornea and the ocular surface of the eye have become one of the more advanced areas in cell biology. Nevertheless, in the last decade, investigators working on other parts of the eye, such as the retina, have also achieved promising results to be explored and confirmed in the near future. This amplification of the stem cell field in ophthalmology has also seen the realization of fantastic new discoveries in other fields of basic research showing just how important long-term investments in stem cell treatment and therapies are. Drs. Victor L. Perez from Miami, Daniel H. Scorsetti from Buenos Aires and José AP Gomes from São Paulo, are excellent clinical and surgical ophthalmologists, who lead research teams with international recognition. This book represents part of the pioneering work done by them and by some of the most distinguished investigators that work in this still mostly experimental field. We expect (and we need) that it will continue to be developed, helping medicine to advance for the benefit of our patients. I am certain that this book will be very useful to many who are interested in, or currently working in ophthalmology. It will help with the understanding of the present situation of research in the different subfields of ophthalmology. And most certainly, will be a “go to” resource in the planning of future research endeavors in stem cells, specifically.

Prof. Rubens Belfort Jr. Head Professor Esciola Paulista de Medicina São Paulo Hospital, Brazil Member Academia Ophtahlmologica Internationalis

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STEM CELLS IN OPHTHALMOLOGY

PROLOGUE

The use of stem cell therapy to treat and potentially cure human diseases has become a realistic concept in the last decade. The field of regenerative medicine using cell-based therapy is here to stay and grow. Many fields in medicine are adopting this approach to treat patients with degenerative and acquired disorders where organ regeneration is necessary. This “vision” of hope has also become a very important part in the future of vision science research and translational ophthalmic medicine. Interestingly, ophthalmology has been a pioneer specialty in the use of cell-based therapy, specifically, in the successful treatment of ocular surface disorders using limbal stem cell transplantation. In contrast to other organs, the eye provides a unique scenario where direct visualization and direct access to the target affected organ are readily accessible. This provides a perfect setting where end-points of success are clear and local delivery of cells can be efficiently performed. In fact, the eye may provide an excellent platform to test efficacy and safety of regenerative medicine. Understanding, that the field of stem cell biology and therapy is rapidly evolving, we have tried to capture in present time where in ophthalmology we stand with respect to the use of stem cell therapy to regenerate all the structures of the visual system and ocular adnexa. From lacrimal gland to the optic nerve, the use of cell-based therapy has been applied to most of the structures of the eye. In some, major advances have been achieved, while in others, early steps have been initiated. In this book, basic stem cell biology and immunity will be reviewed and unique advances or present state-of-the-art use of these cells in ocular tissue will also be covered. As we move into the practice of translational medicine, the final chapter will cover the description of the facilities and governmental requirements needed to establish an ocular stem cell program. With the participation of recognized experts from all over the world, we hope that this comprehensive collection becomes a stepping stone to scientific interaction in the exciting field of cell therapy! Victor L. Perez, MD Daniel H. Scorsetti, MD, PhD Jose Alvaro P. Gomes, MD, PhD

.

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STEM CELLS IN OPHTHALMOLOGY

CONTRIBUTING AUTHORS

Alex Barash, MD

Priscilla C. Cristovam, PhD

Department of Ophthalmology

Post-Doctoral Research Associate

Black Family Stem Cell Institute

Centro Avancado de Superficie Ocular (CASO)

Ichan School of Medicine at Mount Sinai,

Department of Ophthalmology

New York, NY

Universidade Federal de S. Paulo-UNIFESP/EPM

Rubens Belfort Jr., MD, PhD

São Paulo, Brazil

Full Professor Department of Ophthalmology

Juan O. Croxatto, MD

Universidade Federal de

Department of Ophthalmic Pathology

S. Paulo-UNIFESP/EPM

Fundacion Oftalmologica Argentina

São Paulo, Brazil

“Jorge Malbran”, Buenos Aires, Argentina

Sanjoy Bhattachary, M. Tech., PhD Associate Professor

Fernando Cruz-Guilloty, PhD

Neurodegenerative Disease & Vision Research

Howard Hughes Medical Institute Fellow of the

Bascom Palmer Eye Institute

Life Sciences Research Foundation

University of Miami Miller School of Medicine

Bascom Palmer Eye Institute

Miami, FL

University of Miami Miller School of Medicine Miami, FL

So-Hyang Chung, MD Department of Ophthalmology

Bruno Diniz, MD

Catholic University of Seoul

Doheny Eye Institute, Los Angeles, CA

Seoul, South Korea

Department of Ophthalmology, Universidade Federal

Roberto A. Cohen, MD

de S. Paulo-UNIFESP/EPM

Associate Professor of Ophthalmology

São Paulo, Brazil

School of Medicine, USAL. Buenos Aires Private Practice,

Ali R. Djalilian, MD

Cornea and Anterior Segment

Department of Ophthalmology and

Tucuman, Argentina

Visual Sciences University of Illinois at Chicago Chicago, IL

vii

STEM CELLS IN OPHTHALMOLOGY STEM CELLS IN OPHTHALMOLOGY

Harminder S. Dua, MD, PhD

Yuntao Hu, MD, PhD

Chair and Professor of Ophthalmology

Peking University Eye Center

Academic Ophthalmology, Division of Clinical

Peking, China

Neuroscience, University of Nottingham Honorary Consultant, University Hospital,

Mark S. Humayun, MD, PhD

Queens Medical Centre

Professor of Ophthalmology

Nottingham, England

Doheny Eye Institute, Keck School of Medicine

Jose J. Echegaray, MD, PhD

at the University of Southern California

Post-Doctoral Research Fellow,

Los Angeles, CA

Opthalmic Pathology Bascom Palmer Eye Institute

Irina Kerkis, PhD

University of Miami Miller School of Medicine

Director of the Genetics Laboratory

Miami, FL

Instituto Butantan São Paulo, Brazil

Medi Eslani, MD Department of Ophthalmology

Shigeru Kinoshita, MD

and Visual Sciences

Chair and Professor

University of Illinois at Chicago

Department of Ophthalmology

Chicago, IL

Kyoto Prefectural University of Medicine Kyoto City, Japan

Rodigo A. Brant Fernandes, MD Doheny Eye Institute,

Noriko Koizumi, MD

Keck School of Medicine at the University of

Professor, Department of Biomedical Engineering

Southern California, Los Angeles, CA

Doshisha University

Universidade Federal de

Kyoto City, Japan

S. Paulo-UNIFESP/EPM São Paulo, Brazil

Friedrich E. Kruse, MD Professor and Chairman,

Jose Alvaro Pereira Gomes, MD, PhD

Department of Ophthalmology

Associate Professor of Ophthalmology

Friedrich-Alexander University of

Universidade Federal de

Erlanger-Nümberg

S. Paulo-UNFESP/EPM

Erlangan, Germany

Director, Anterior Segment Area and Advanced Ocular Surface Center (CASO) UNFESP/EPM Universidade Federal de S. Paulo-UNIFESP/EPM São Paulo, Brazil

viii

Contributing Authors

Praful Kumar, MD

Helen P. Makarenka, PhD

Senior Resident,

Assistant Professor

Cornea and Refractive Services

Department of Cell and Molecular Biology

Rajendra Prasad Centre for Ophthalmic Sciences

The Scripps Research Institute

All India Institute of Medical Sciences

La Jolla, CA

New Delhi, India

Ikeda Lal, MS, FICO

Jodhbir Singh Mehta, MD, BSc, MBBS, FRCS

Research Fellow, Cornea and

Head, Corneal and External Eye Disease

Anterior Segment Services

Senior Consultant, Refractive Service

LV Prasad Eye Institute,

Singapore National Eye Center

Kallam Anji Reddy Campus

Head, Tissue Engineering and Stem Cells Group

LV Prasad Marg, Banjara Hills

Associate Professor,

Hyderabad, Andhra Prades, India

Duke-NUS Medical School Adjunct Associate Professor, School of Material

Richard K. Lee, MD, PhD

Science & Engineering and School of Mechanical

Associate Professor of Ophthalmology

& Aerospace Engineering,

Department of Cell Biology and Anatomy

Nanyang Technological University

Neuroscience Graduate Program

Singapore, Japan

Bascom Palmer Eye Institute University of Miami Miller School of Medicine

Johannes Menzel-Severing, PhD, MSc

Miami, FL

Department of Ophthalmology Friedrich-Alexander

Matthew Lovatt, PhD

University of Erlanger-Nümberg

Institute of Medical Biology

Erlangan, Germany

A*STAR (Agency for Science, Technology and Research)

Babyla Geraldes Monteiro, BHS

Singapore, Japan

Secretaria da Saúde Genetics Laboratory, Butantan Institute

Mauricio Maia, MD, PhD

São Paulo, Brazil

Universidade Federal de S. Paulo-UNIFESP/EPM São Paulo, Brazil

ix

STEM CELLS IN OPHTHALMOLOGY

Asadolah Movahedan, MD

Victor L. Perez, MD

Department of Ophthalmology and

Professor of Ophthalmology

Visual Sciences

Microbiology and Immunology

University of Illinois at Chicago

Walter G. Ross Chair Ophthalmic Research

Chicago, IL

Director, Ocular Surface Center Bascom Palmer Eye Institute

Naoki Okumura, MD

University of Miami Miller School of Medicine

Assistant Professor

Miami, FL

Department of Biomedical Engineering Doshisha University, Kyoto City, Japan

Cameron Pole, BS

Department of Ophthalmology

Bascom Palmer Eye Institute

Kyoto Prefectural University of Medicine

University of Miami Miller School of Medicine

Kyoto City, Japan

Miami, FL

Gary Swee-Lim Peh, MD

Naresh Polisetti, PhD

Singapore Eye Research Institute, Singapore

Integrative Regenerative Medicine (IGEN)

Duke-Nus Graduate Medical School

Centre and Department of Physics,

Singapore, Japan

Chemistry and Biology, Linköping University, Linköping, Sweden

Daniel Pelaez, PhD Research Assistant Professor

Caio Regatieri, MD, PhD

Ophthalmology Department

Adjunct Professor of Ophthalmology,

Bascom Palmer Eye Institute

Federal University of Sao Paulo

University of Miami Miller School of Medicine

Adjunct Assistant of Ophthalmology,

Miami, FL

New England Eye Center Tufts Medical School, Boston, MA

Fernando M. Penha, MD, PhD

Department of Ophthalmology and Black Family

Department of Ophthalmology

Stem Cell Institute

Bascom Palmer Eye Institute

Ichan School of Medicine at Mount Sinai

University of Miami Miller School of Medicine

New York, NY

Miami, FL Universidade Federal de S. Paulo-UNIFESP/EPM São Paulo, Brazil

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Contributing Authors

Peter S. Reinach, PhD

Virender S. Sangwan, MD

Professor

Director, Clinical Research

Wenzhou Medical University

Cornea and Anterior Segment Services

School of Ophthalmology and Optometry

Ocular Immunology and Uveitis Services

Wenzhou, P. R. China

LV Prasad Eye Institute, Kallam Anji Reddy Campus

Ramiro Ribeiro, MD

LB Prasad Marg, Banjara Hills

Doheny Eye Institute, Los Angeles, CA

Hyderabad, Andhra Pradesh, India

Department of Ophthalmology, Hospital Universitário de Curitiba – FEMPAR,

Ursula Schlötzer-Schrehardt, PhD

Curitiba, Brazil

Department of Ophthalmology Friedrich-Alexander University of

Jose Reinaldo S. Ricardo, MD, PhD

Erlanger-Nümberg

Research Associate, Centro Avancado de

Erlangan, Germany

Superficie Ocular (CASO) Department of Ophthalmology, Universidade

Daniel H. Scorsetti, MD, PhD

Federal de S. Paulo-UNIFESP/EPM

Chairman and Professor of Ophthalmology

São Paulo, Brazil

Director, Board of Ophthalmology School of Medicine, USAL

Enrique Salero, PhD

Chief of Cornea, CEOS

Research Assistant Professor

Buenos Aires, Argentina

Ophthalmology Department Bascom Palmer Eye Institute

Ozlem Barut Selver, MD

University of Miami Miller School of Medicine

Department of Ophthalmology

Miami, FL

Dokuz Elul University Izmir, Turkey

Dalia G. Said, MD, FRCS Consultant Ophthalmologist, University Hospital

Namrata Sharma, MD

Queens Medical Centre, Nottingham, England

Additional Professor

Assistant Professor, Research Institute of

Rajendra Prasad Centre for Ophthalmic Sciences

Ophthalmology, Cairo, Egypt

All India Institute of Medical Sciences New Delhi, India

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STEM CELLS IN OPHTHALMOLOGY

Ashton Shirazi, MD

J. Mario Wolosin, PhD

Department of Ophthalmology

Professor, Department of Ophthalmology

Black Family Stem Cell Institute

Black Family Stem Cell Institute

Ichan School of Medicine at Mount Sinai

Carl Ichan School of Medicine at Mount Sinai

New York, NY

New York, NY

Donald TH Tan, MD, MBBS, FRCSG, FRCSE, FRCOphth, FAMS

Gary Hin-Fai Yam, PhD

Medical Director,

and Visual Sciences

Singapore National Eye Center

The Chinese University of Hong Kong

Professor, Department of Ophthalmology

Shatin, Hong Kong, China

Assistant Professor of Ophthalmology

Yong Loo Lin School of Medicine, National University of Singapore

Aaron M. Yeung, MD, PhD

Academic Chair, Ophthalmology Academic

Clinical Lecturer, Academic Ophthalmology,

Clinical Program, Duke-NUS Graduate Medical

Division of Clinical Neuroscience

School

University of Nottingham

Adjunct Professor,

Nottingham, England

Nanyang Technological University Singapore, Japan

Driss Zoukhri, PhD Department of Diagnosis and Health Promotion

Zheng Wang, MD

Tufts University School of Dental Medicine

Department of Ophthalmology

Department of Neurosciences

Black Family Stem Cell Institute

Tufts University School of Medicine

Ichan School of Medicine at Mount Sinai

Boston, MA

New York, NY

Sara Tullis Wester, MD Assistant Professor of Clinical Ophthalmology Oculofacial plastic and Reconstructive Surgery Orbital Surgery and Oncology Bascom Palmer Eye Institute University of Miami Miller School of Medicine Miami, FL

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CONTENTS Chapter 1: Stem Cells......................................................................................

Enrique Salero, PhD



Daniel Pelaez, PhD

Chapter 2: Ocular Surface Epithelia Side Population Stem Cells: Isolation, Molecular Signatures and Approaches to Activation and Self Renewal for Tissue Regeneration........... ....

Alex Barash, MD



Ozlem Barut Selver, MD



So-Hyang Chung, MD



Zhen Wang, MD



Ashton Shiraz, MD



Peter S. Reinach, PhD



J. Mario Wolosin, PhD

1

15

Chapter 3: Cultivation In Vitro and Ex Vivo................................................... 37

Virender S. Sangwan, MD



Praful Kumar, MD



Ikeda Lal, MD, FICO



Namrata Sharma, MD

Chapter 4: Avoiding Rejection: Immunological Aspects for Successful Stem Cell Therapy......................................................

Fernando Cruz-Guilloty, PhD



Victor L. Perez, MD

Chapter 5: Ocular Surface Stem Cells: Science and Surgery........................

61

Harminder S. Dua, MD, PhD Aaron Yeung, MD, PhD Dalia G. Said, MD, FRCS

Chapter 6: Corneal Stromal Keratocytes and Endothelial Cells...................

51

81

Gary Hin-Fai Yam, MD Matthew Jason Lovatt, MD Gary Swee-Lim Peh, MD Donald Tan, MD Jodhbir Singh Mehta, MD xiii

STEM CELLS IN OPHTHALMOLOGY

Chapter 7:

Restricted Pluripotent Cells and Their Integration

into the Trabecular Meshwork.............................................................. Cameron Pole, BS Richard K. Lee, MD, PhD Sanjoy K. Bhattacharya, PhD

93

Chapter 8: Crystalline Lens Stem Cells, Reparation and Regeneration.......

101



Roberto A. Cohen, MD Juan O. Croxatto, MD Daniel H. Scorsetti, MD, PhD

Chapter 9:

Stem Cells in the Orbit and Ocular Adnexal Tissue...................

Sara T. Wester, MD

Chapter 10: Cell Therapy for Total Limbal Stem Cell Deficiency...................

xiv

135

Noriko Koizumi, MD Naoki Okumura, MD Shigeru Kinoshita, MD

Chapter 12: Cell Therapy for Retinal, Optic Nerve and Visual Tract Diseases....................................................................

125

Jose Reinaldo da Silva Ricardo, MD, PhD Priscila Cardoso Cristovam, PhD Babyla Geraldes Monteiro, BHS Rubens Belfort Jr., MD, PhD Irina Kerkis, PhD Jose Alvaro Pereira Gomes, MD, PhD

Chapter 11: Cell Therapy for Corneal Endothelial Dysfunction....................

109

Rodrigo A. Brant Fernandes, MD Bruno Diniz, MD Ramiro Ribeiro, MD Fernando M. Penha, MD, PhD Yuntao Hu, MD, PhD Mauricio Maia, MD, PhD Mark Humayun, MD, PhD Caio V. Regatieri, MD, PhD

149

Contents

Chapter 13: Role of Stem Cells in Lacrimal and Meibomian Gland Development and Regeneration..................................................





Driss Zoukhri, PhD Helen P. Makarenkova, PhD

Chapter 14: Towards the Use of Non-Ocular Cell Lineages in Ocular Surface Disease.................................................................

195

Medi Eslani, MD Asadolah Movahedan, MD Ali R. Djalilian, MD

Chapter 17: Protocols, Facilities, Equipment, Materials and Regulations for Establishing a Human Stem Cell Program.......

187

Fernando Cruz-Guilloty, PhD Victor L. Perez, MD

Chapter 16: Gene Therapy................................................................................

179

Johannes Menzel-Severing, MD, MSc Naresh Polisetti, PhD Ursula Schlötzer-Schrehardt, PhD Friedrich E. Kruse, MD

Chapter 15: Induced Pluripotent Stem Cells and Their Potential to Treat Eye Diseases...................................................................

165

217

Jose J. Echegaray, MD, PhD Fernando Cruz-Guilloty, PhD Priscila Cardoso Cristovam, PhD José Alvaro P. Gomes, MD, PhD Victor L. Perez, MD

Index....................................................................................................................... 227

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STEM CELLS IN OPHTHALMOLOGY

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CHAPTER

1

Stem Cells Enrique Salero, PhD Daniel Pelaez, PhD

INTRODUCTION Stem cells are undifferentiated cells that are present in the embryonic, fetal and adult stages of life and give rise to the differentiated cells that constitute tissue and organs. In the post-natal and adult stages of life, tissuespecific stem cells are found in differentiated organs and are of crucial importance for maintaining tissue homeostasis and for tissue repair after injury. The major characteristics of stem cells are: (a) self-renewal, which is the ability to extensively proliferate while maintaining an undifferentiated phenotype, (b) clonality, which the capacity to form clonal colonies arising from a single cell, and (c) potency, the ability to differentiate into different cell types and across germ layers (Figure 1). The ability to differentiate, one of the three main characteristics of stem cells, varies between stem cells depending on their origin and their derivation. For example, embryonic stem cells derived from the blastocyst have a greater ability for self-renewal and potency, while stem cells found in adult tissue have limited self-renewal since they do not proliferate as extensively and can only differentiate into germ layer-specific cells.

Classification Based on Differentiation Potential Stem cells can be categorized according to their differentiation potential into: totipotent, pluripotent, multipotent, oligopotent, and unipotent. i) Totipotent cells are the most undifferentiated cells and are found in early development. A fertilized oocyte (zygote) and the cells (bastomeres) of the first two divisions are totipotent cells. At the 16-cell stage, the outer cells of the embryo are allocated to two lineages: the trophoblast lineage, which will form part of the placenta; and the bipotential inner cell mass (ICM), which generates the epiblast and the hypoblast. The epiblast and hypoblast will form the embryo and the yolk sac, respectively.[1] ii) Pluripotent Embryonic Cells are derived from epiblast cells in the ICM of the blastocyst and are termed pluripotent because they are able to differentiate into cells that arise from the 3 germ layers ectoderm, endoderm, and mesoderm from which all tissues and organs develop.[2] This property of pluripotency is confined to the epiblast cells that persist only a few days, however, a pluripotent state can be maintained indefinitely in vitro by placing epiblast cells in culture through derivation of embryonic stem cells lines. iii) Multipotent Cells are found in most

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STEM CELLS IN OPHTHALMOLOGY

Figure 1. Diagram depicting stem cell characteristics, division, and differentiation. 1. Stem Cell symmetric division with 2. Self-renewal of stem cell niche. 3. Clonogenicity of stem cells. 4. Asymmetric division and lineage commitment to progenitor cell stage. 5. Terminal differentiation. 6. Potency: differentiated cells of different germ layers. tissues and differentiate into cells from a single germ layer.[3] Mesenchymal stem cells (MSCs) are the most recognized and widely studied multipotent cell. MSC can be derived from a variety of tissue including bone marrow, adipose tissue, bone, cartilage and muscle, Wharton’s jelly, umbilical cord blood, and peripheral blood.[4] iv) Oligopotent Cells are able to self-renew and form 2 or more lineages within a specific tissue; for example, the ocular surface of the pig, including the cornea, has been reported to contain oligopotent stem cells that generate individual colonies of corneal and conjunctival cells.[5] Hematopoietic stem cells (HSCs) are a typical example of oligopotent stem cells, as they can differentiate into both myeloid and lymphoid lineages.[6] v) Unipotent stem cells can self-renew and differentiate into only one specific cell type and form a single lineage. Examples of this are spermatogonial stem cells that can only form sperm and muscle stem cells, which give rise to mature muscle cells and not any other cells.[7,8]

2

Classification Based on Origin and Source Stem cells can also be classified according to their origin. i) Embryonic Stem Cells (ESCs) are pluripotent, derived from the ICM of the blastocyst, a stage of the preimplantation embryo, 5 to 6 days post-fertilization.[9] ESCs can self-renew and generate all cell types in the body in-vivo, as well as in culture and induce teratoma formation after being injected into immunosuppressed mice. However, ESCs are not able to generate the extra embryonic trophoblast lineage.[10] Cells from the inner cell mass are separated from trophoblasts and transferred to a culture dish under very specific conditions in order to develop ESC lines.[11] ESCs are identified by the presence of transcription factors such as Nanog and Oct4, which maintain the stem cells in an undifferentiated state, and endow them with the capability of selfrenewal.[12,13] ii) iii) Epiblast Stem Cells (EpiSC) are pluripotent cells derived from the epiblast of the

CHAPTER 1: Stem Cells implanted embryo or derived from cells of the germ-line lineage. The epiblast is a single layer of epithelial cells that originates from the ICM after implantation of the embryo at day 5.5 to 7.5, but prior to gastrulation. EpiSC display flat colony morphology, grow poorly as singlecell clones, have more limited developmental potential than ESCs, and are highly inefficient at generating chimeras.[14] iv) Embryonic and Adult Germ Cells are pluripotent cells derived from the germline lineage. When cultivated in adequate growth conditions, primordial germ cells isolated from embryonic day 8.5 embryos generate ES-like cells, termed embryonic germ cells (EGCs). These cells are pluripotent, capable of generating teratomas and chimeras. Similarly, in the newborn and adult male gonads, spermatogonial stem cells (SSCs) are located on the basement membrane of seminiferous tubules. While these cells show very low proliferative efficiency when they are explanted in-vitro, they can induce teratomas and chimeras.[15] v) Fetal Stem Cells (FSCs) have emerged as an intermediate phenotype between ESCs and adult stem cells.[16] FSCs are neither fully pluripotent nor multipotent when compared with their adult counterparts; FSCs appear to be more primitive, with higher growth kinetics, smaller cell size, active telomerase and greater plasticity, while lacking teratoma formation abilities.[16,17] These features may represent an advantage for regenerative medicine because they might be easier to reprogram.[18] FSCs can be isolated from fetal tissues such as blood, liver, bone marrow, pancreas, spleen and kidney, and from the supportive extra-embryonic structures such as placenta, cord blood and Wharton jelly from the umbilical cord’s outer region and amniotic fluid.[19,20] FSC populations are heterogeneous with respect to phenotypic features, properties and cell marker expression, which depend on their tissue of origin and gestational age. FSC subpopulations include stromal/mesenchymal stem cells, hematopoietic stem cells (HSCs) and pro-pluripotent cells. Examples of FSCs derived from extra-embryonic fetal tissues are: a) Amniotic fluid stem cells that represent a heterogeneous population derived from the three germ layers. These cells clonally expand like MSCs and express stem cell markers such as Oct4 (also known POU5F1), Nanog and SSEA-4. b) Umbilical cord blood stem cells, which are becoming a major source of multipotent stem cells for therapeutic applications. These cells are a rich source of hematopoietic and MSC populations and express ESC markers such as Oct4, as well as some MSC markers. c) Wharton’s jelly stem cells are stromal cells from the umbilical cord’s outer region. They express ESC markers such as Oct4, Sox2 and

Nanog, as well as mesenchymal markers. d) Amniotic membrane stem cells constituted by two populations from the inner layer (amniotic epithelial cells) or from the outer layer (amniotic membrane MSC) have been isolated and exhibit a variable of degree of differentiation potential. e) Placenta stem cells, exhibit markers of pluripotency (SSEA-4, Oct4, Stro-1 and Tra1-81), and mesenchymal cell markers. These cells are capable of invivo differentiation into neural, glia, insulin positive cells, and hepatocytes.[21] vi) Postnatal Stem Cells have been successfully isolated from a variety of human and experimental animal tissues, including bone marrow, peripheral blood, neural tissue, skeletal muscle, epithelium, dental pulp, and periodontal ligament. These cells are capable of self-renewal and differentiating into different tissues and many of them have proven useful in stem cell therapies and tissue engineering applications.[22,23] vii) Adult Stem Cells are derived from adult tissues, which has an inherent advantage of possible autologous cell sourcing, thus minimizing the concerns for rejection or ethical controversies.[24,25] Several studies have demonstrated that transplantation of adult stem cells restores damaged organs in-vivo, such as bone tissue repair and revascularization of ischemic cardiac tissues via stem cell differentiation and generation of new specialized cells.[26,27] Other studies have shown that cultured adult stem cells secrete various molecular mediators with anti-apoptotic, immunomodulatory, angiogenic, and chemo-attractant properties that promote repair.[28] viii) Tissue-Resident Stem Cells are found in some tissues and organs in the adult and are meant to renew and repair the tissues following injury. Such endogenous repair mechanisms are critically dependent on the availability and activation of these tissue-resident stem cells that generate tissuespecific, terminally differentiated cells.[29] Studies suggest that these cells originate during ontogenesis and remain in a quiescent state until local stimuli activate their proliferation, differentiation or migration.[30,31] Tissue-resident stem cells reside in a stem cell niche.[32] The majority of tissue-resident stem cells are dormant but are activated by specific signals during injury and repair.[33] This property is critical to maintaining a population of cells that do not perform other functions apart from generating tissue-specific cells during repair and regeneration processes.[34] ix) Cell Fusion is the generation of pluripotent cells, which has been successfully achieved using somatic cells, combined with EGCs or ESCs, to reprogram the nuclei of the somatic cell after fusion with these embryonic cell types.[35-37] This procedure yields tetraploid cells

3

STEM CELLS IN OPHTHALMOLOGY resembling EGCs in which the originally inactive somatic X chromosome in female cells becomes reactivated and the differential DNA methylation of imprinted loci is erased. After introduction into diploid host blastocysts, those tetraploid cells are able to contribute to chimeric embryos. However, any such contribution is modest and is attributable to the tetraploid nature of the cells.[35,38] Piccolo et al. have identified epigenetic factors from the ten-eleven translocation (Tet) family of DNA demethylases, Tet1 and Tet2, as having a critical yet distinct role in reprogramming somatic nuclei during cell fusion.[39] x) Somatic Cell Nuclear Transfer (SCNT) is the process by which the nucleus from an adult cell is transferred into an enucleated oocyte that initiates subsequent development.[40] Like the fused nucleus of the sperm and egg during fertilization, the somatic nucleus is reprogrammed by the oocyte cytoplasm to generate a cloned animal, such as Dolly the sheep[41] resetting any hallmark of aging that the somatic nucleus bore upon transplantation. In the 1960s, Gurdon demonstrated that the full battery of genetic material present at fertilization and necessary to give rise to an adult organism is maintained in the cells throughout development and maturation to adulthood.[42] The nuclei of adult somatic cells, just like the genetic material in the adult sperm or egg, can have pluripotency restored in the context of the oocyte cytoplasm. xi) Induced Pluripotent Stem Cells (iPSCs) are generated pluripotent cells by reprogramming adult somatic cells through the forced expression of transcription factors, and share characteristics of ESCs. Takahashi and Yamanaka produced the first iPSCs by ectopic expression of defined transcription factors Oct4, Sox2, Krüppel-like factor 4 (Klf4) and Myc (also known as c-Myc), collectively known as the OSKM factors on mouse fibroblasts and adult human dermal fibroblast.[43,44] OSKM factors can reprogram the cells to a pluripotent state and make them similar to ESCs in terms of morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent-specific genes, and telomerase activity. Furthermore, the researchers demonstrated that iPSCs could differentiate into cell types of the 3 germ layers in-vitro.[43,44] iPSCs are currently useful tools for drug development, modeling of disease, and regenerative medicine, but although these cells express identical characteristics of pluripotent stem cells,[45] it is not yet known if iPSCs and ESCs would significantly differ in clinical practice. Retroviral vectors, used to introduce OSKM reprogramming factors into adult cells, and expression of oncogenes like Myc limit the use of iPSCs in a clinical setting since the vectors used to introduce transcription factors to adult cells can cause cancers. To

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avoid the use of oncoprotein Myc, somatic cells can be reprogrammed by one single factor, Oct4, or by a substituted combination of other factors.[46] There are recent approaches to achieve this goal including the use of non-retroviral vectors, such as chemical compounds, plasmids, adenovirus, and transposons.[47-50] Despite the safety issues, this innovative discovery has created a powerful tool to reprogram somatic adult cells to earlier undifferentiated stages and generating iPSCs, thereby creating an identical match to the cell donor and thus avoiding immunologic concerns of cell transplantation.

SELF-RENEWAL Self-renewal refers to the capacity of a cell to maintain its identity after division. Self-renewal is the process by which a stem cell divides asymmetrically or symmetrically to generate one or two daughter stem cells that have a developmental potential similar to the mother cell. The choice of a stem cell to undergo self-renewal is carried out by two cell division mechanisms, which fulfill two different requests by the tissue:[51] i) asymmetric self-renewal, in which each stem cell divides into one stem and one differentiated cell, allows maintaining a constant number of stem cells, which is generally sufficient under physiological conditions; ii) symmetric self-renewal, in which each stem cell gives rise to two daughter stem cells, leads to an expansion of the stem cell pool, a condition required after tissue injury or in diseased conditions causing loss of differentiated cells.[52] In asymmetric cell division, the mitotic process leads to polarization and asymmetric segregation of components essential for the cell fate determination so that once cell division is completed, one daughter cell has received RNAs, proteins and other molecules that maintain the undifferentiated program, whereas the other cell receives lineage commitment factors. In symmetric cell division, the two daughter cells receive the same factors and the decision for commitment and differentiation is not linked to mitosis, rather it is a later event that can involve the newly formed cells.[53] Symmetric or asymmetric divisions are not mutually exclusive, and a mixture of these two mechanisms can be used on subsequent divisions. During mid to late gestation, some mammalian progenitor cells are able to make a developmentally regulated transition from largely symmetric to predominantly asymmetric divisions. Similarly, adult stem cells dividing asymmetrically under steady-state conditions retain the capability to divide symmetrically to restore stem cell pools depleted by injury or disease.[52]

CHAPTER 1: Stem Cells To expand, pluripotent stem cells must undergo symmetric self-renewal divisions in which each daughter cell maintains pluripotency. Pluripotent stem cells must fulfill two requirements to be self-renewing: they must maintain the pluripotency state following division and they must continue to divide. For most mammalian stem cells, such as HSCs and neural stem cells, this means that self-renewal is division with maintenance of multipotency. For stem cells that make a single type of daughter cell, e.g., spermatogonial stem cells, selfrenewal is division with maintenance of the unipotent undifferentiated state. Some of the mechanisms involved in stem cell self-renewal broadly regulate the proliferation of many cells, though a surprising number of these mechanisms preferentially regulate stem cell self-renewal.[54,55] As most mature cells have a limited lifespan, the capacity of stem cells to both replenish aging mature cells and perpetuate themselves through self-renewal is crucial to the maintenance of tissue homeostasis throughout the lifetime of an organism. The size of stem cell populations depends on the balance between self-renewal and cell differentiation. When the rate of self-renewal is higher than that of differentiation, the stem cell population expands; whereas when the self-renewal rate is lower than the rate of differentiation, the population declines owing to exhaustion.[56] Cellintrinsic networks cooperate with signals from the microenvironment to fine-tune the self-renewal capacity of stem cells and to maintain homeostasis.[57] Self-renewal is not unique to stem cells, some types of restricted progenitors and differentiated cells, such as restricted glial progenitors and lymphocytes, can also self-renew,[58,59] although their differentiation potential is more restricted. Although stem cells have extensive self-renewal potential, this does not necessarily mean that these cells actually self-renew extensively under physiological conditions. For example, most HSCs are quiescent most of the time and may undergo a limited number of self-renewing divisions in normal adult mice.[60] Neural crest stem cells undergo a limited number of self-renewing divisions in vivo before differentiating [61] despite having the potential for massive self-renewal in culture.[62] Thus stem cells may be fated in-vivo to execute many fewer divisions than they have the potential to undergo. This endows stem cells with the potential to repair tissues after injuries that involve much higher regenerative demands than are encountered under normal physiological conditions. Stem cells reside in a dynamic, specialized micro environment, denoted as a niche, which provides

extracellular cues to allow stem cell survival and identity. Moreover, the niche dynamically regulates stem cell behavior, maintaining a balance between quiescence, self-renewal and differentiation.[63] Despite their high potential to proliferate, the niche maintains stem cells in a quiescent and low metabolic state to prevent stem cell exhaustion.[64] Moreover, the niche is thought to protect stem cells from the accumulation of gene mutations that may lead to their malignant transformation into cancer cells.[65] The deregulation of the stem cell niche plays a key pathogenic role in a number of diseases associated with tissue degeneration, aging and tumorigenesis.[66] Both quiescent and active stem cell subpopulations coexist in several tissues and in separate yet adjoining locations. In these niches, the precise regulation of the balance between symmetric and asymmetric divisions is critical for maintaining proper stem cell number and for fulfilling the needs for differentiated cells within the surrounding tissue.[63] The ability of a stem cell to seed in its niche represents one of the most important features of the niche itself, and the proper binding between stem cells and their niche is essential to maintain the stem cell pool for longterm self-renewal. Thus, the niche establishes a sort of crosstalk between the state and necessity of the tissue and the proper functioning of the stem cell pool. [67] Niches are highly specialized for each type of stem cell, with a defined anatomical localization, and they are composed by stem cells and by supportive stromal cells (which interact with each other through cell surface receptors, gap junctions and soluble factors), together with the extracellular matrix (ECM) in which they are located. Moreover, blood vessels carry systemic signals and provide a conduit for the recruitment of inflammatory and other circulating cells into the niche, whereas neural inputs transmit distant physiological cues to the stem cell microenvironment. The diverse and dynamic composition of the ECM provides controlled biochemical, physical, structural, and mechanical properties to the different niches. In summary, not only the niche components influence stem cell behavior, but also the interactions between stem cells and their niche are reciprocal, since stem cells are able to remodel the niche and secrete ECM components in response to the signals they receive from it.[68]

IMMORTALITY AND SENESCENCE Immortality is established when a cell loses its cell cycle checkpoint pathways (p53/p16/pRb). The concept of immortality differs when considering the germ line and ESCs. For the germ line, immortality is dependent

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STEM CELLS IN OPHTHALMOLOGY on adaptive change and natural selection promoting survival and reproductive success associated with advantageous changes in the genome, and is estimated over evolutionary time. For ESCs, the criteria for immortality implies self-renewal, the maintenance of pluripotency after many, presumably indefinite passages in culture.[69,70] Progeny with changes that would confer a selective growth advantage are deemed unfavorable and discarded because they are usually the result of karyotypic changes.[71] Thus, immortality of ESCs lines is assured by selecting progeny with properties as close to the parental cells as possible, not by the natural selection of competitive reproductive advantage. A feature of germ line immortality that is important for adult stem cells is the ability to ensure that genetic information is passed on with the highest fidelity to successive generations. Cells must have robust mechanisms to resist and/or repair damage to the genome.[72] For ESCs, the maintenance of genome stability is essential to their value as tools for research and potential therapeutic vehicles.[73] For adult stem cells in-vivo, possessing the capacity to resist, detect and repair changes in the genome (such as telomere shortening and mutation accumulation) underlies their ability to participate in tissue homeostasis and repair across an organism’s lifespan. In the 1960s Leonard Hayflick showed that normal human cells could not divide indefinitely in culture and named this phenomenon as the Hay flick limit.[74] The Hayflick limit is the result of a progressive cell divisiondependent shortening of telomeres, a process named senescence.[75] Cellular senescence is a complex cellular response to a variety of stressors that results in irreversible growth arrest, alteration of gene expression profiles, epigenetic modifications and altered secretome. There are extrinsic and intrinsic inducers that promote cell senescence: i) Extrinsic inducers are related with advanced glycation end products, angiotensin II, IGFBP7, interlukin (IL-6), IL-8, GROα and PAI can induce cellular senescence in different cell types.[76] ii) Intrinsic inducers of cellular senescence are either the progressive telomere erosion that is associated with cell proliferation or the formation of irreparable DNA lesions that induce a persistent DNA damage response, which keeps the cells alive but arrests their proliferation. Once senescence is established in the cells, they are locked into a senescent phenotype through a global induction of heterochromatin, which results in the formation of senescence-associated heterochromatin foci, where cyclin-dependent kinase inhibitor p16INK4A and oncogenes seem to play an important role.[76,77]

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Telomeres are ribonucleoprotein heterochromatic structures at the ends of chromosomes that protect them from degradation and from being detected as double-stranded DNA breaks. Telomeres consist of tandem repeats of the (TTAGGG)n sequence, bound by a six-protein complex known as shelterin/telesome complex, which is crucial to maintaining the structure and function of telomeres at the chromosomal ends in mammalian cells. During the process of DNA synthesis and cell division, telomeres are shortened as a result of the incomplete replication of linear chromosomes, which is called “end-replication problem”. Telomere homeostasis and its structural integrity help to protect chromosome ends from attrition, which can cause chromosome recombination, end-to-end fusion, DNA damage, and genome instability. Incomplete DNA replication of telomeres results in progressive telomere shortening, which can eventually lead to telomere uncapping and cell cycle arrest/senescence. Progressive telomere shortening is proposed to be one of the mechanisms underlying organismal aging. Telomere length is maintained by telomerase, a reverse transcriptase encoded by the telomerase reverse transcriptase (TERT) and telomerase RNA component (TERC) genes, which adds telomeric repeats de novo about 40-100pb after each round of cell division.[78,79] This amount is a constant for various types of human cells and provides a kind of mitotic counter.[80] Alternative ways to maintain telomere length have also been described, such as alternative lengthening of telomeres (ALT), which relies on homologous recombination between telomeric sequences. Current evidence suggests that telomere elongation mechanisms are regulated by the epigenetic status of telomeric chromatin and by the telomere-binding proteins. Both telomeric and subtelomeric regions are enriched in histone marks characteristic of repressed heterochromatin domains, such as trimethylation of H3K9 and H4K20 and binding of heterochromatin protein 1 (HP1).[78,79] Cellular senescence is not an inevitable fate of all cells though; pluripotent cells such as ESC and iPSCs in culture can bypass the Hay flick limit. At the embryo stage, telomeres are effectively elongated by a possible ALT pathway from zygote to blastocyst. However, during early expansion, ESCs obtained from ICM of blastocyst in-vitro continuously elongate their telomeres to a relatively stable level in a telomerase dependent way, although the ALT pathway cannot be excluded in this process. Similarly, both telomerasedependent and independent mechanisms may coexist in the iPSCs reprogramming process.

CHAPTER 1: Stem Cells In summary, ESCs and iPSCs, need to elongate their telomeres during the derivation process to stabilize their genomic DNA and keep their self-renewal and pluripotency status. Telomerase activity regulation is necessary for maintaining ESC and iPSCs pluripotency and modulating their differentiation. Interestingly, the longest telomeres appear to exist in adult stem cells where they become shortened as stem cell function declines. Additionally, higher expression of the shelterin/ telosome complex component TRF1 correlates with higher pluripotency of stem cells. Consequently, long telomeres and high TRF1 level have been proposed as stem cell markers.[81] Indeed, telomere status may serve as a benchmark to evaluate the quality of ESCs and iPSCs, which should have important implications in the application of iPSCs in regenerative medicine.

STEM CELLS DERIVED FROM THE EYE Stem cells are also found in the eye of postnatal and adult organisms. Several studies have discovered stem-cell populations in different eye regions (Figure 2). i) Retinal stem cells (RSCs) have been isolated from the marginal periphery of the neurosensory mammalian retina. RSCs

isolated from early developmental stages can develop into all retinal cell types.[82] In postnatal tissues, RSCs can be isolated from the developing mouse neuroretina at embryo stage and postnatal day 1 (P1), and possess self-renewal abilities and can generate several retinal cell types.[83] Similarly, RSCs can be obtained from retinal pigmented ciliary margin located between the iris and the retina[84] of adult mouse, rat[85,86] and humans, which can self-renew, proliferate clonally in-vitro and give rise to retinal-specific cell types, including photoreceptors, bipolar cells and Müller glia cells.[86,87] ii) Limbal stem cells (LSCs) reside within the basal epithelium of the limbus at the bottom of the palisades of Vogt. LSCs are a population of stem/progenitor cells that continuously renew the corneal epithelium. LSCs can self-renew and express stem cell markers such as Oct4, Nanog, Sox2, as well as SSEA4.[88] LSCs produce progenitor cells that proliferate, migrate centripetally, differentiate, and replace lost cornea epithelial cells. Dysfunction or loss of LSCs in combination with the destruction of their niche alters the homeostasis of the corneal epithelium that is important for the structural integrity of ocular surface, and thus for cornea transparency and visual function.[89,90] iii) Müller glia cells (MGCs) are the

Figure 2. Localization of stem cell populations in postnatal and adult eye. Schematic diagram of a section of adult mammalian eye showing the stem cells niches (red) described by several groups. Limbal stem cells (LSCs) localized in basal epithelium of limbus that renewal continuously the cornea epithelium of the ocular surface; retinal stem cells (RSCs) are isolated from the ciliary body (CB) and fetal and postnatal retinas that generate several retinal progenitor cell types and glia; Muller glia (MGCs) isolated from fish and mammals can differentiate into retinal progenitors cells, in fish MGCs can differentiated into photoreceptors neurons; Retinal pigment epithelium (RPE) have a stem cell population (RPESCs) that have regenerative capacity after injury in amphibians, while in mammals they can differentiated into neural progenitors and mesenchymal cell types in vitro. This figure has been adapted from Salero et al. (2012)[96] and Osakada et al. (2009).[125] GCL, Ganglion Cell Layer; INL, inner nuclear layer; ONL, outer nuclear layer.

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STEM CELLS IN OPHTHALMOLOGY Table 1 - Identification of stem cell population in the eye. Type of cell

Location Ciliary margin in rodent and human eyes

Retinal Stem Cell Fetal and Neonatal retinas

Various cell types including photoreceptors, bipolar cells, Müller glia in vitro

Limbal Stem Cell

Basal layer of the limbal epithelium

Cornea epithelium

Retinal Müller glia

Inner nuclear layer of the retina

Lamina-specific retinal neurons

RPE Stem Cell

Retinal pigment epithelium

Neural progenitors, mesenchymal lineages

mayor glial cell type of the retina. Müller glia can regenerate damaged retina in lower vertebrates and can be activated in mice when growth factors are administered.[91] In-vitro, MGC progenitors express stem cell markers such as Sox2, Myc and Klf4; they are multipotent cells and produce neural lineages.[92,93] iv) Retinal pigment epithelium (RPE) is a polarized monolayer that originates from the neuroepithelium of the anterior neural plate in the developing embryo. Although RPE is not a defined part of the retina, it is an essential supporting tissue involved in retinol cycling, nutrient transport, growth factor production and phagocytosis of photoreceptor outer segments. In adult amphibians, the neural retina and RPE have overlapping regenerative capacity after injury. After extensive retinal damage, there is an activation of a progenitor/stem cell population in the RPE layer (RPESCs).[94,95] Salero et al. have shown that adult human RPE can express stem cell factors such as Sox2, Myc and Klf4, they can self-renew, proliferate clonally in-vitro, activate to a multipotent cell and differentiate into neural progenitors and mesenchymal lineages.[96] (Table1)

STEM CELL APPLICATIONS IN OPHTHALMOLOGY As in other medical fields, stem cells have been employed for ophthalmologic purposes in a myriad of ways. The application spectrum of stem cells in ophthalmology ranges from their use as models of disease progression to regenerative medicine and tissue engineering approaches using stem cells and biomaterial scaffolds. Below is a review of the different applications of stem

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Differentiation capacity

cells in ophthalmology and discussion about the current state of development and future projections.

DIFFERENTIATION AND TISSUE REPLACEMENT The traditional paradigm of stem cell use for regenerative medicine relies on the cells capacity for self-renewal and lineage potency. Under this paradigm, stem cells are viewed as a source of replacement cells for lost or damaged tissues and give rise to new fully differentiated, functional tissues. This approach is very widely used in several key aspects of ophthalmology, such as retinal disease[97,98] and corneal grafts.[90] The eye represents a unique organ for regenerative studies, not only due to the fact that it possesses within itself several stem cell populations capable of regenerating its own structures, but it is also one of the few organs for which humans have a duplicate system that contains a viable source of autologous, contralateral, tissue-specific stem cells. Several researchers employ these characteristics to their work and attempt to elucidate the mechanisms by which these ocular stem cells differentiate and activate endogenous repair mechanisms. Researchers have so far identified naturally-occurring stem or progenitor cells for corneal and retinal structures. Corneal stem cells have been identified as residing in the limbus[90] and from there these limbal stem cells (LSC) regulate cell turnover and regeneration of the different corneal layers. Understanding the biology of these cells and how they maintain their undifferentiated state, as well as what stimuli can drive them down different commitment pathways, can allow us to develop strategies to activate

CHAPTER 1: Stem Cells endogenous repair mechanisms and achieve functional regeneration of these tissues following disease or injury. Similarly, this knowledge can someday lead to the creation of undifferentiated eye stem cell banks for use in allografts for people who do not possess a viable source of these cells. In their study, Mei et al.[99] identified Frizzled 7 (Fz7, a member of the Frizzled family of receptors) as a key biomarker for undifferentiated LSC capable of giving rise to mature corneal structures. In their study, Mei et al. demonstrated that Fz7 was highly expressed in the basal limbal epithelium and co-localized with markers for limbal stem cells such asΔNp63α, N-cadherin, and keratin 14 (K14), while the Fz7 marker was decreased in cells expressing the mature corneal marker, keratin 12 (K12). Similarly, Li et al.[100] demonstrated that LSC reside in a complex niche microenvironment consisting of a limbal niche, or stem, cells and limbal stromal cells, which all contribute differently to the activation and maturation of progenitor cells towards mature corneal structures. Hara et al,[101] showed that corneal endothelial progenitors retain some characteristics of the cranial neural crest contribution to the periocular mesenchyme and were capable of generating mature cornea endothelium when transplanted into rabbit corneas. Even though not as well characterized or reported, several stem and progenitor cell populations have also been described for the retina, which can be exploited to promote retinal regeneration. These retinal progenitor and stem cells have been identified at the retinal margin, retinal pigmented epithelium (RPE), and Müller cells within the retina among others.[98] Yet little is still known about how these cells undergo activation from quiescence and what cues, intrinsic or extrinsic, drive their differentiation down the various retinal phenotypes. Retinal stem/progenitor cell biology and characterization is an active field of research. For example, Miyake et al.[97] describe a novel, previously unknown, retinal stem cell (RSC) population in vertebrate organisms residing in the ciliary marginal zone (CMZ) which was capable of regenerating the entire retina 30 days after complete retinal removal in Xenopus Tropicalis.[97] Studies such as these highlight the complexity and limited understanding we still have of these regenerative mechanisms in the retina. And while natural stem and progenitor cell populations represent an ideal source for regeneration, these are sometimes not available for use in therapy due to an inherent lack of these stem cell pools in certain individuals, loss of the stem cell reservoir due to trauma, disease or surgery; and limitations in the isolation,

expansion and phenotypic maintenance of these cells in-vitro. Thus, several researchers explore the possible use of other better characterized stem cell sources for differentiation into ophthalmic tissues. One of the most widely used extraocular sources of stem cells for tissue repair and regeneration are MSC found in various tissues and organs. These stem cells represent a readily accessible, easily expandable and highly versatile source of cells for various tissues. One of the possible applications of MSC that is being explored is the differentiation of these cells down epithelial lineages in order to repair ocular surface and corneal defects.[102] Recently, Yao et al.[103] employed a transplantation of bone marrow-derived MSC into an animal model of corneal wound healing in the acute stage of an alkali burn. The MSC administered subconjuctivally were capable of enhancing corneal regeneration and reducing corneal neovascularization as compared to the media controls. Furthermore, in the animals receiving the MSC treatment, the group was able to demonstrate an attenuation of the inflammatory response following corneal alkali burn.[103] Other stem cells sources, such as adipose-derived stem cells have also been explored for their potential use in corneal regeneration. Ma et al.[104] explored the use of autologous rabbit adipose derived stem cells (rASC) on a poly (lactide-co-glycolide) (PLGA) scaffold to repair corneal defects due to mechanical injury. The group was able to demonstrate that rASC were capable of generating transparent grafts with collagen fibril distribution profiles similar to those found in native corneas. Furthermore, the stem cell scaffolds inhibited the neovascularization of the corneal surface and resulted in functional keratocyte differentiation of the ASCs after 24 weeks of transplantation.[104] The use of extraocular stem cell sources for retinal regeneration is also an active field of research. Similarly to their use in the cornea, adipose derived stem cells,[105] MSC,[106] iPSC[107,108] have all been used by various researchers to generate various retinal phenotypes in vitro. In their study, Rezanejad et al.[105] demonstrated that a single transcription factor, Pax6 transduced using lentiviral vectors, was capable of differentiating ASC into retinal progenitors (RP), RPE cells and photoreceptors in vitro. Similarly, Maeda et al.[108] showed that iPSCs derived from umbilical cord or human fibroblasts were capable of differentiating to functional RPE by growth factor supplementation in vitro. When transplanted into a blind animal model, these cells aided in the recovery of visual function by replacement of native RPE cells with

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STEM CELLS IN OPHTHALMOLOGY iPSC-RPE cells.[108] Finally, Goldernberg-Cohen et al.[106] demonstrated that bone marrow-derived MSC were capable of engrafting into an ischemic retinal model and differentiating down microglial lineages when co-administered with neurotrophic factors BDNF and CNTF.[106] While we are still learning how to differentiate stem cells down the various needed ophthalmic lineages and maintain their correct phenotype when transplanted, there are other challenges to the use of stem cells for tissue repair that can only be addressed by multidisciplinary fields, such as tissue engineering.

TISSUE ENGINEERING APPROACH IN THE EYE The studies on the use of stem cells in conjunction with stimulatory factors and biomaterial scaffolds for delivery and engraftment highlight the need for a more comprehensive tissue engineering approach to stem therapies in the eye. The transplantation of stem cell suspensions alone has limitations in terms of proper stratification, localization and engraftment, as well as phenotypic maintenance of the transplanted cells. Therefore, the field of tissue engineering for the eye has evolved to find solutions to these challenges by incorporating biomaterial scaffolds, cell sources, and biochemical factor incorporation as a multidisciplinary approach to tissue regeneration in the eye. Several studies have been initiated that combine the use of stem/ progenitor cells with natural and synthetic scaffolds to achieve the correct incorporation of newly formed regenerative tissues. This approach becomes crucial for structures in which shape can affect the overall outcome of the regenerative therapy, such as the cornea that possesses a highly-ordered hierarchical cellular structure.[109] Several approaches to deliver stem and progenitor cells to the corneal surface are being studied for the effectiveness in maintaining transplant morphology, clarity and viability. To this end, several researchers have used amniotic membranes as a suitable scaffold material to deliver stem cells to the corneal surface.[110-112] In the study by Lai et al,[110] the group went further to chemically modify the amniotic membrane with a zero-length crosslinker, carbodiimide, and demonstrated that higher levels of crosslinking resulted in increases in water content, light transmittance, and resistance to enzymatic degradation of the membranes. Furthermore, the higher crosslinked membranes exhibited better limbal epithelial cell growth and higher expression of LSC markers ΔNp63α and ABCG2.[110] While a natural membrane material

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may be a more intuitively viable substrate, these results show that synthetic optimization can enhance some of the required characteristics of the scaffold and the cells themselves. Other researchers seek to develop fully synthetic biomaterial constructs to achieve this goal.[109,113-115] Brown et al.[113] used a modified contact lens with a plasma polymer coated surface to culture and deliver the cells to the corneal surface. The group go on to demonstrate that the modified surface promotes cell adhesion, proliferation, stem cell retention, and cell transfer to a corneal wound bed in a rabbit model of limbal stem cell deficiency (LSCD).[113] Whether natural or synthetic, the ideal membrane scaffold for corneal reconstruction would be one that allows for the maintenance of the phenotypic expression of limbal stem cell markers and their niche, as well as one that promotes the correct differentiation and hierarchical ultrastructure found in mature corneas, meanwhile keeping an optically clear and breathable surface for proper regeneration to take place.

PROMOTERS OF ENDOGENOUS REPAIR AND SURVIVAL One of the most unexpected outcomes of the early clinical trials using stem cell therapies in humans has been a complete redrawing of the traditional stem cell paradigm. From the results obtained in preliminary clinical studies, we have found that stem cells can exert most of their regenerative potential as mediators of the host microenvironment and promote endogenous repair mechanism activation, progenitor cell activation and regeneration through a paracrine effect. Furthermore, as discussed above the paracrine effect can have an immunomodulatory role that can be exploited in transplantation. Stem cells as biological pumps for several survival and regenerative factors are now being employed widely in regenerative medical applications, including those in the eye. In retinal disease, for example, several stem cells have been shown to confer some neuroprotective effect against degenerative diseases. Bone marrow-derived mesenchymal stem cells were shown to ameliorate the retinal degeneration in a rat model of retinal dystrophy when delivered as a thin layer of cells into the subretinal space.[116] Similarly, in a rat model of diabetic retinopathy, adipose tissuederived mesenchymal stem cells were actually shown to have a regenerative effect in hyperglycemic-stressed retinas and helped reduce vascular leakage and promote survival.[117] Furthermore, the study by Mead et al.[118] demonstrated that dental pulp stem cells transplanted

CHAPTER 1: Stem Cells intravitreally into a mouse model of optic nerve crush exerted a neuroprotective effect on retinal ganglion cell and promoted their survival through the secretion of neurotrophic factors from the transplanted stem cells.[118] The studies by Machalinska et al.[119] and Jung et al.[120] went even further by transplanting genetically modified MSC and neural stem cells, respectively. These cells were modified to force the expression of neurotrophic factors to help promote retinal repair. Yet other researchers employ the immunomodulatory properties of stem cells to minimize the risk of rejection of transplanted cells and tissues. Lee et al.[121] use intravenous infusion of human MSC into a mouse model of corneal allotransplantation and demonstrated a reduced amount of CCR7+ antigen presenting cells in the animals receiving the stem cell infusion.[121] The natural tendency of stem cells to regulate their own niche, as well as their unique ability to withstand high levels of exogenous stresses by secreting paracrine factors has revolutionized the way we approach stem cell therapies. We now view stem cell regenerative therapies as a milieu of replacement cell administration, microenvironmental modulation and endogenous repair activation and proliferation working in synchrony to achieve the desired results.

STEM CELLS TO MODEL DISEASE PROGRESSION One final application for which stem cells have emerged as a tremendously valuable tool is the modeling of disease progression. With the advent of induced pluripotency, the stem cell field has been revolutionized by the possibility to revert somatic, fully differentiated cells back to an embryonic state. Coupled with the advances in background genetic profiling for specific diseases, this technology has enabled researchers to investigate the molecular mechanisms by which degenerative diseases progress throughout development. Researchers can now take somatic cells from an autistic individual, for example, reprogram them back to their embryonic state and then monitor the progression of neural differentiation of the cells. This technique can help elucidate early biomarkers for different diseases, which can serve diagnostic, prophylactic and therapeutic purposes. Similarly, it can provide researchers with indications as to the divergence point during development at which the cells no longer follow normal patterns of commitment and behavior. This novel approach to understanding the molecular biology of a disease is now being applied to

the different structures in the eye and to understanding different inherited and acquired pathologies in human beings. This approach has been employed to gain better molecular understanding of various pathologies, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP).[122] Phillips et al.[123] employed iPSC from an individual with a genetic mutation in a region of the visual system homeobox 2 (VSX2) transcription factor to investigate its role in the development of the retina and was able to elucidate multiple roles for this VSX2 gene. Similarly, several other researchers are using this technique to explore other ocular pathologies.[124] The usefulness of this technique will undoubtedly rely on our concomitant advancements in bioinformatics and genetic analyses and will require many decades before we can understand the complex interactions at the genetic, epigenetic and proteomic levels that govern the progression of disease.[125] Nevertheless, once figured out, this knowledge will provide us with the necessary knowledge to address the fundamental causal mechanisms behind the various eye diseases. References 1.

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Ocular Surface Epithelia Side Population Stem Cells: Isolation, Molecular Signatures and Approaches to Activation and Self Renewal for Tissue Regeneration Alex Barash, MD, Ozlem Barut Selver, MD So-Hyang Chung, MD, Zheng Wang, MD Ashton Shiraz, MD, Peter A. Reinach, PhD J. Mario Wolosin, PhD

INTRODUCTION Over 6 million people worldwide are afflicted with corneal blindness.1 Due to the immuno-privileged nature of the central corneal epithelium and stroma, transplantation of the central cornea can cure many corneal conditions that impair vision. However, in cases where the tissue defect extends beyond the central cornea into the limbal zone, corneal transplantation may no longer be curative. This can occur in chemical and thermal burns, ocular cicatricial pemphigoid, Stevens-Johnson syndrome, microbial infections, and chronic inflammation. Such conditions result in corneal vascularization, conjunctivalization, scarring, and opacification, even if a corneal transplant is performed. To understand the causes of this ineffectiveness and its source, limbal stem cell deficiency (LSCD),2 we need to understand the special biology features of the epithelial lining protecting the cornea. All stratified epithelia such as those from cornea, skin, buccal, conjunctival or nasal tissue are constantly undergoing self-renewal through proliferation, maturation and desquamation at the exposed surface. Lineage survival depends on a ‘universal‘ stem cell (SC)-based growth and differentiation plan outlined below.

When the tissue is at rest, a rare stem cell, localized in a specialized ‘niche’, remains in a quiescent state for long periods of time.3-5 Infrequently, this stem cell enters the cell cycle to yield one daughter stem cell and on more differentiated cells. The asymmetry in daughter cell yield may reflect an intrinsic feature of stem cells, asymmetric cell division, or become established after a symmetric division based on the relative distance of the cells to the stem cell nurturing ‘niche’, i.e. niche driven cell fate. As progenitors move progressively farther from being stem cells, this subsequent progeny displays a much higher proliferative rate. However, after a number of replications, the cells spontaneously arrest their cell cycling and enter terminal differentiation. Since it is this rapid proliferation that balances for the cell attrition at the surface, the rapidly proliferating cells have been termed transient amplifying (TA) cells. True limbal SCs may be extremely rare. When the corneal epithelium (CoE) of a mouse chimera incorporating similar proportions of LacZ-positive and negative cells is stained, the stain identifies about 50 LacZ positive cell zones alternating with 50 wild type cell zones, suggesting the existence of only about 100 originating clones.6

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STEM CELLS IN OPHTHALMOLOGY The Dual Domain Arrangement of the Corneal Epithelium; an Achiles’ Heel and Limbal Stem Cell Deficiency

cornea, but variations in MUC 16 expression may confer similar differences in cohesiveness.11

A unique feature of the corneal epithelium is the segregation of its stem and precursor cells to the encircling outer limbal (Li) region, where a developed vascular bed arriving from the conjunctiva ends. This conclusion is unavoidable because only within the basal region of the limbus are cells devoid of K3 and K12, the two keratins associated with corneal lineage differentiation.7 All basal and stratified cells in the cornea express these keratins. Given that the tissue specific cytokeratin expression starts sharply at the limbalcorneal interface and that early progeny of bona fide stem cells ought to be in the proximity of the stem cell, it is rational to conclude that most if not all progenitors of value for tissue regeneration are contained within the limbal zone. This dual-domain evolutionary arrangement is likely to reflect the need of serum as a component of the stem cell niche on the face an avascular cornea. This domain segregation coupled to the narrowness of the limbal zone represents the Achilles’ heel of this vision critical tissue. Since the limbal-corneal epithelium (LCE) is surrounded by the much larger conjunctival (CNJE) epithelium, the limbal zone must also act as a physical domain boundary and barrier. Overall the cells of the limbal zone are sufficiently different than the cells over the cornea proper to be distinguishable by average cell size.8 The fact that the limbus is the zone where long term proliferating cells exist would contribute a proliferative pressure component to resist CNJE encroachment. In addition several experimental observations suggest that a high degree of intercellular cohesiveness within the limbus may also contribute to a barrier property. Secondly, when the same corneas are subjected to a controlled devitalization by exposure to heptanol, the entire central corneal epithelium sloughs off within minutes, while the epithelium over the limbal zone remains attached for a much longer period, even as dye staining confirms that all the cells have been devitalized. In rabbits, central corneal stratification (but not intra-limbal stratification) coincides with de novo expression of a α-2,3 sialyltransferase enzyme that places negative charges at the end of o-mucin chains that extend outwardly in perpendicular to the cell surface.9,10 We have speculated that such expression differences between limbus and cornea may underpin the observed differences in suprabasal cell cohesiveness. It is presently not known whether an equivalent absolute limbal to corneal gene expression difference exists in the human

Thus any condition that results in a limbal defect, whether structural due to physicochemical or thermal damage or functional, due to deficiency of functioning stem cells to replenish TA populations, allows for opportunistic domain encroachment by the CNJE (Figure 1). The CNJE is closely related to the LCE in both structure and gene architecture. However, in spite of its close embryological proximity, the conjunctival epithelium does not have the same surface properties of the corneal epithelium. Its ion permeability is higher. This results in a higher rate of fluid imbibition from the anterior surface that places a higher load on the corneal endothelium fluid efflux activity and may overwhelm it, resulting in stromal swelling. Refractoriness to pathogen attachment is also lower due to a less perfect surface. Finally, the CNJE does not possess the anti-angiogenic properties of the corneal epithelium. The vascularization that ensues the encroachment of the CNJE on the corneal domain is likely to allow stem cells from the conjunctival lineage to establish themselves in this zone. The loss of surface protection and barrier functions resulting from the replacement of the LCE by the CNJE leads to a chain

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Figure 1. Distribution of cells in the limbal corneal epithelium (top panel) and the consequences of limbal stem cell deficiency or limbal epithelial loss; the conjunctival epithelium encroach into the central cornea. Putatively, conjunctival stem cell may become establish in the neovascularized ocular surface (bottom panel).

CHAPTER 2: Ocular Surface Epithelia Side Population Stem Cells of events that gradually degrades corneal function and eventually leads to blindness. The role of the segregation of the LCE to the limbal zone was first recognized by Kenyon and Tseng.12 They proposed that multiple states of full or partial corneal blindness could be attributed to limbal stem cell deficiency as the root of all the different corneal defects. Soon after, Huang and Tseng provided a robust experimental demonstration of the essential role of the limbus for central corneal healing in a rabbit model.13 Since then, there has been substantial progress in the clinical application of autologous limbal transplantation for the treatment of monocular limbal deficiencies using limbal epithelial cells harvested from the contralateral eye and expanded in culture.14,15 Allografts using limbal cells from cadaver donor corneas have also been implemented.16 However, whether as a result of required immunosuppressive agents, a persistent sub-clinical inflammatory state in spite of the immunosuppression, or due to other unseen incompatibilities, allogeneic transplants have a much lower rate of success than do autologous transplants. Clearly immunological and phenotypic compatibility are best in autologous transplants from the contralateral eye, but the number of donor cells that can be harvested without risk to the donor eye is very small. Hence, understanding the limbal stem cell is critical to develop the best methodologies to expand this cell ex vivo while maintaining its regenerative properties. The essential features and composition of adult SCs in general, and of the limbal stem cell, in particular, remains poorly understood. A thorough understanding of this nature will be essential to develop optimal methodologies for limbal epithelial expansion in culture maintaining or even increasing the percentile of stem and precursor cells with regenerative potential, as well as optimal conditions for transplantation and protection of regeneration-able precursor cells during the critical engraftment period. Thus multiple efforts are being made to isolate the bonafide stem cell for its full characterization. Isolation of Stem Cells by the ABCG2-dependent Side Population Phenotype The first step for in-depth characterization of a stem cell is its isolation in viable form. However, for many years, except in the case of the hematopoietic system, this feat remained elusive. A breakthrough for a more or less generalized method of somatic stem cell isolation

derived from Goodell et al17 observation that a small cohort of cells (0.05 percent!) that resisted stain by the supravital nuclear dye Hoechst 33342 are a component of the bone marrow stem cell population with excellent in vivo engrafting/repopulating capacity. Exclusion was abolished by verapamil, a known inhibitor of ATP Binding Cassette (ABC) transporters associated with multidrug resistance (MDR) activity indicating that the resistance was due to an efflux transport activity, rather than nuclear features that excluded the dye. Due to its location towards the blue side in dual emission flow cytometry plots, the cell cohort has been termed a side population (SP). Intriguingly, while human hematopoietic SCs for bone marrow transplants are isolated according to the CD34hi phenotype, the SP cells isolated by Goodell were CD34lo.18 However, when CD34lo are engrafted in the irradiated mouse they yield or convert into CD34hi, suggesting that the SP may contain the most primitive bone marrow SC cohort within a complex stem cell hierarchy.19 In fact, the conversion may reflect the transition from the most primitive, fully quiescent bone marrow stem cells, to activated stem cells.20 Subsequent to the initial findings on SP in bone marrow hematopoietic stem cells, Zhou et al21 reported that Hoechst 33342 exclusion, and hence the SP phenotype is specifically due to the transport activity of the G2 subtype of ATPase binding cassette transporters (ABCG2) and, that in fact, this transporter is present in many SCs. This finding opened a new avenue to isolate SCs cells in viable form by flow cytometry as SP cells. Incidentally, ABCG2 is also known as Breast Cancer Resistance Protein (BCRP); the multidrug resistance transporter that confers chemotherapeutic resistance against agents such as doxorubicin to metastatic breast cancer cells. These agents are DNA intercalating planar compounds that are highly genotoxic because they induce strand breaks that activate the p53 apoptosis pathway. Thus, the teleological explanation of the close relationship between ABCG2 and stem cells is that this is the transporter that nature has developed to protect the long-lived tissue founding cells from dangerous DNA damaging compounds. The interest in finding ways to improve the effect of chemotherapeutic agents has generated intense search for ABCG2 specific inhibitors. The acceptance of a stem cell/ABCG2 relationship accelerated stem cell research in many fields including the ocular surface epithelia.22-26 Typically, limbal or conjunctival tissue is incubated with Dispase for the separation of a pure epithelial sheet from underlying stroma. Incubation with trypsin for 15-25 min results in

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STEM CELLS IN OPHTHALMOLOGY single cell suspensions. These cells are incubated with Hoechst 3342 and sorted into SP and non-SP (nSP) cohorts as indicated in Figure 2. Several features of the thus isolated limbal (LiE) and conjunctival (CNJE) epithelial SP cells were consistent with their putative status as stem cells. However, definitive proof of status was attained by tracking the source of the SP cells. BrdU was administered to young rabbits via subcutaneous osmotic pumps until ~3 months of age (for up to 17 days) when their rate of growth slows markedly. At the end of this period, the pumps were removed and the animals were either sacrificed or maintained for up to 2 months before tissue harvesting. After enucleation, the bulk of the limbus and conjunctiva was used to isolate SP and nSP cell cohorts. Using only the center of the G0/G1 cohort as the nSP population is advantageous because,

Figure 2. Generation of side populations by ABCG2mediated Hoechst 33342 efflux. Right panel: The fluorescent dye Hoechst 33342 enters cells passively and intercalates in DNA. At saturation, the degree of stain is proportional to the amount of DNA per cells. Cells in the G2/M phases have twice as much stain as cell in G0/G1. Cells in S phase have intermediate values. The transporter ABCG2 effluxes the dye and thus ABCG2hi cells develop less Hoechst stain. In addition, because Hoechst is a bathochromic dye with blue and red emissions and its red/ blue emission ratio increases when the degree of nuclear stain saturation (due to orbital interactions) under-saturated cells not only have lower blue and red intensities but have a lower blue/red ratio. They have been denominated blue side population (SP) cells. Left panels: The complete elimination of the dye exclusion by the highly specific inhibitor fumitremorgin C (FTC) proves that in the human limbal epithelium the Hoechst exclusion is due to the efflux activity of ABCG2.

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since SP cells are slow cycling, both cohorts exclude cells in the S and G2/M stage, avoiding gene expression differences associated with cycle stages. These cells were deposited on glass slides using a cytospin centrifuge and stained for BrdU. Small sections of tissues were used to generate formalin fixed, paraffin embedded sections and those were also stained for BrdU distribution. The results depicted in Figure 3, showed that; a) at the end of the two weeks Brdu loading all basal cells and basal proximal suprabasal cells within the ocular surface have acquired BrdU, an expected result given the rapid growth rate less than 90 day old rabbit; b) after the two month chase period between 5-10% of the basal cells in the limbal and conjunctival domains stain strongly for BdrU while most other basal cells were stain free or have only residual levels as indicated by faint staining; and c) the SP cohort incorporated a much higher percentile of high BrdU content than the main non-SP cohort. Thus, we concluded that the SP population represents a concentrate of cells that in vivo were identifiable as the label retaining, slow cycling cells, the most trusted characteristic of epithelial stem cells. In spite of this demonstration, a disappointing experimental finding,

Figure 3. Methodology used to relate the cells collected in the SP flow cytometry cohort with the in vivo cycling status of the cells just prior to cell harvesting, staining and sorting. Naote that after 38 days of label chasing there are label-retaining cells in the basal cell layer and that after sorting the SP cohorts from either limbus or conjunctiva are very rich in high BrdU cells. This experiment shows also that after 38 days of BrdU chasing there were still labeled cells in the supra-basal strata, near the epithelial surface (white arrowheads). Hence, in a second experiment the chase was extended to two full months.

CHAPTER 2: Ocular Surface Epithelia Side Population Stem Cells confirmed in two independent studies, was that the SP cells were unable or less able to start clonogenic growth on irradiated 3T3 feeder cells than the non-SP cells. Since the capacity to generate long term proliferation holoclones according to the principles enunciated by Barrandon and Green for mouse keratinocytes27 is commonly used to prove cell stemness, including in the limbal epithelial system.28 This low clonal capacity would seem to negate the stem cells nature of the isolated ocular surface epithelial cells SP. However, more recent studies on keratinocytes provide a more nuanced perspective. A highly clonogenic fraction of mouse keratinocytes can be isolated by their ability to rapidly bind to collagen type IV surfaces. Yet in an in vivo repopulation assay these cells showed only short term repopulating capacity, suggesting a TA provenance.29,30 Hence, clonogenic index may not be directly related to stemness. In our studies we demonstrated that; a) in spite of its low clonal ability only cells within the SP were able to form colonies after exposure to phorbol ester during the first 72 hrs, a feature associated with the unique ability of epidermal stem cells to survive this treatment, and b) after 14 days of clonal growth the SP population contained, in addition to large clones similar to those observed in the non-SP, small clones, which, upon mechanical removal of the large ones continued to grow for the next 10 days to form large classical holoclones, suggesting a growth delay consistent with a slow cycling origin. Finally, as described above, there is sound evidence that the number of ‘core’ stem cells in the limbus is extremely small. Consistent with the very low percentiles of SP cells observed in most tissues, we found that the human limbus of older adults contains at maximum one thousand SP cells (representing 0.3-0.5% of the tested cells). Percentiles in the rabbit or pig were 2-3 times higher. The SP number compares well with the calculation of less than 100 founding cells calculated from the chimerization experiment of Collinson in the small mouse cornea.6 Another difference between the mature adult human and young rabbit (1.5 mM calcium). Under these conditions only live basal cells can be expected to attach. Hoechst loading was then performed in these physiologically stable conditions. Subsequently, cells were subjected to very short trypsin treatment and immediately sorted. Test experiments demonstrated that; a) there were no SP cells in the nonattaching fraction, and b) the central cornea contained only vestiges of SP cells. The conjunctival epithelium yielded slightly higher SP percentiles. The first fundamental problem with trying to obtain a human limbal SP molecular signature (the set of genes that show a significantly different level of expression in

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STEM CELLS IN OPHTHALMOLOGY the SP with respect to the nonSP cohort) in the human limbal side population stem cell is the very low number of cells available. Robust results required a minimum of 30 ng total RNA equivalent to about 15,000 flow cytometry sorted cells or 15 corneal pairs for each 3-4 microarray replicates. Facing this resource obstacle, we performed the study first with human conjunctiva, where the number of cells per individual tissue is about 10 times the amount in the limbus. Thus, each independent microarray run, four of which were required to obtain statistically robust data, required the cellular proceed of two independently gathered and processed pairs of human conjunctival cadaver donor tissue. We reasoned that, considering the common embryological origin of the LCE and CNJE, similar control of biological fate in the adult epithelia by PAX6 and a common growth environment, there maybe a high degree of similarity in the molecular signatures in both cases. This hypothesis was probed in a comparison of the molecular signatures for pig LiE and CNJE SP cells using a microarray that probes about one third of the porcine genome. In all three cellular systems, pig LiE and CNJE and human CNJE, the microarray comparative analysis indicated the unique character of the SP cell cohort. In all three cellular systems, pig LiE, pig CNJE and human CNJE, the microarray comparative analysis indicated the unique character of the SP cell cohort. The microarray identified approximately 9000 distinct transcripts in both pig LiE and pig HCNJ, of which only 40% could be annotated. Of those transcripts, 382 and 296 were either over- or under-expressed in the LiE SP cells using a 2-fold cut off.37 The pig study confirmed our expectation that the molecular signature on LiE and CNJE SP cells will display substantial similarities (Table 1). Eighty percent of the genes over-expressed or under-expressed in the LiE SP cells did so too on the CNJE SP cells. Furthermore, comparison against the results obtained within the human CNJE showed that most genes that were over-expressed in the pig CNJE, if represented in the human microarray, were also over-expressed in the latter tissue (Table 1). The pig limbal SP cells molecular signature was consistent with enrichment of precursor epithelial cell, such as enhanced expression of K15, K19, CXCR4, ALDH1A1 and reduced levels of connexin 43 (Table 2). DAVID’s functional annotation clustering using DAVID’s human gene set as background showed that within the over-expressed gene, the largest annotation clusters were those associated with regulation of cellular and biological developmental processes. Overrepresentation analysis showed that the GO_TERMS

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Table 1 - The most SP over-expressed genes in the pig LiE and correlation with over-expression in the pig and human CNJE. SP/nSP ratios Gene

p-LiE

p-CNJE

h-CNJE

1. CXCR4

14.01

24.31

14.51

2. MXD1

9.02

9.53

4.07

3. GPX2

8.74

4.96

2.17

4. MGST1

6.69

3.51

1.88

5. GRTP1

6.10

3.55

4.22

6. RHCG

5.70

5.07

2.04

7. LNX1

5.54

2.62

8.43

8. VNN2

5.44

2.82

5.42

9. HIST1H2BD

5.34

3.74

4.42

10. GNA14

4.54

2.72

4.04

11. ZMYND8

4.50

4.01

NL

12. FBXO32

4.49

4.53

8.16

13. ABLIM1

4.37

2.30

1.81

14. CEBPD/B

4.29

9.76

5.36

15. BLNK

4.28

2.89

2.47

16. CGN

4.21

2.57

3.60

17. TMM54

4.13

3.72

NL

18. CRYAB

4.10

2.40

2.13

19. MPN

4.06

2.89

NL

20. PIR

4.04

3.17

2.08

21. HSP70

3.99

4.97

NL

22. ELF3

3.86

5.63

7.47

25. EEA1

4.34

A

A

26. ZAR1

5.36

A

A

NL, not listed in the human array; A, not expressed in the CNJE. with the most significant over-representation were related to regulatory inhibition of cellular biological and metabolic activity, i.e., consistent with a slow cycling or quiescent cell origin. Conversely, analysis of the underexpressed gene set essentially showed that the SP cells were underactive in terms of the synthesis of ribosomes and cellular organelles. Other notable features were a substantial armamentarium of proteins that should protect the cells from intrinsic or extrinsic oxidative

CHAPTER 2: Ocular Surface Epithelia Side Population Stem Cells Table 2 Over-expressed genes by categories relevant to stem cells in the pig LiE.

Category

R

Associated with stem cell CXCR4

14.51

ALDH1A1

3.59

KRT19

3.06

KRT15

2.64

GJA1

0.33 Control of hormonal

DUSP5

2.77

DUSP1

2.13

DUSP14

2.14

DUSP16

2.07

P57/Kip2

3.12

P27/Kip1

1.97

Modulation of MYC MXD1

9.02

MXI1

3.45

MYC

0.86 Stem cell replication

HES1

2.58

ID2

2.47

ID1

2.19

Protection against ROS damage SH3BGRL3

12.70

GPX2

8.74

MGST1

6.69

MGST2

2.34

GSR

2.25

HAGH

2.07

SQRDL

2.87

MXI1

3.45

DHRS3

2.68

NQO1

2.56

RRM2

2.46

DHRS8

2.34

Protection against xenobiotic insult CYP1A1

3.52

CYP2C19

6.42

Basal cell markers KRT5

1.07

KRT14

1.12

damage or ectopic xenotoxicity and gene sets that may underpin slow cycling phenotype. In particular every dual specificity phosphatase (DUSP), the set of enzymes that collectively dephosphorylate and neutralize each of the terminal (t) kinases of the mitogen activated kinase cascades ERK1/2, p38 and JNK1/2 (collectively referred henceforth as tMAPKs) is over-expressed in the pig limbal and conjunctival epithelium (Table 2). As argued below, these over expressions may be the most relevant component in a constellation of cellular factors conspiring to establish the slow cycling phenotype. The SP and nSP cohorts showed no difference in the expression of the universal basal epithelial cytokeratins K5 and K14. Many of the features found in the pig cells and additional ones were more patent in the microarrays from the human CNJE.36 Several factors contribute to the quantitative difference. Firstly, genomic representation is much larger in the human microarray used (Affymetrix Human Genome U133 Plus 2.0 Array) and annotation is more extensive. In fact, because many genes are represented by multiple transcripts, in some cases basis for statistical analysis was a paired comparison of 8, 12 or even 16 transcripts, as shown in (Table 3) for the MYLIP gene. The array identified 10,266 unique known genes in the CNJE. Of those, about 10% were overexpressed at >2-fold in the SP cells, suggesting the unique nature of these cells within the whole population of basal cells. For this review we have extracted and categorized genes or sets of genes whose over- or under- expression provide support to the notion that ocular surface SP cells are highly enriched in stem cells (Table 4). Set A includes genes whose proteins are known to be associated with somatic stem cells. B1 through B6 represents genes that can be related to one or more of the slow cycling and other quiescent properties observed in somatic stem cells. C1 through C4 gathers genes that can all be linked to cellular survival mechanisms. D includes two highly under-expressed cytoskeletal proteins associated with smooth muscle actin that may be linked to motile properties of the SP. E represents the under-expression in the SP cell of the anti-angiogenic thrombospondin 1 gene. Section F describes differentially expressed genes that may affect global stem cell behavior, and finally G includes homeobox genes. A. CXCR4 and K15. Cxcr4 is a pleotropic chemokine receptor that controls hematopoietic stem cell homing

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STEM CELLS IN OPHTHALMOLOGY

Table 3 - Analysis of SP/nSP ratios for a gene with multiple transcript representation in the Affymetrix 133 plus microarray.

SP: SI and P/A status Exp.

Ratio

NSP:SI and P/A status

t-test

SP/ nSP

Av. SI

P-A

1

2

3

5

PPPP 1240

11.59

107

PPAP

68

160

68

132 0.160

5556

PPPP 2513

8.53

295

PPPP

181

442

166

391 0.110

812

3722

PPPP 1572

7.50

210

PPPP

106

236

196

302 0.142

2520

7470

PPPP 3733

7.17

520

PPPP

234

756

330

760 0.07

1

2

3

4

1

720

697

457

3088

2

1420

1450

1626

3

952

803

4

2625

2316

P-A

Av. SI

p