Tissue Engineering in Regenerative Medicine (Stem Cell Biology and Regenerative Medicine)

Stem Cell Biology and Regenerative Medicine

Series Editor Kursad Turksen, Ph.D. [email protected]

For further volumes: http://www.springer.com/series/7896

Harold S. Bernstein Editor

Tissue Engineering in Regenerative Medicine

Editor Harold S. Bernstein Cardiovascular Research Institute Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Pediatrics University of California San Francisco San Francisco, CA, USA [email protected]

ISBN 978-1-61779-321-9 e-ISBN 978-1-61779-322-6 DOI 10.1007/978-1-61779-322-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011934681 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)

This book is dedicated to my father, Wallace Carl Bernstein (1923–2010), who taught me to ask questions.

Preface

Over the past decade, significant advances in the fields of stem cell biology, bioengineering, and animal models have converged on the discipline of regenerative medicine. Significant progress has been made leading from preclinical studies through phase 3 clinical trials for some therapies. This volume provides a state-of-the-art report on tissue engineering toward the goals of tissue and organ restoration and regeneration. Examples from different organ systems illustrate progress with growth factors to assist in tissue remodeling; the capacity of stem cells for restoring damaged tissues; novel synthetic biomaterials to facilitate cell therapy; transplantable tissue patches that preserve three-dimensional structure; synthetic organs generated in culture; aspects of the immune response to transplanted cells and materials; and suitable animal models for nonhuman clinical trials. Tissue regeneration, and even stem cell therapy, is not a new concept. As discussed in the cautionary first chapter, efforts toward bone and marrow transplantation have been underway for almost half a century. Steady progress has been made in understanding the criteria for successful cell transplantation, and developing a robust structure for clinical oversight. More recently, pluripotent stem cells, with their capacity for self-renewal and tissue-specific differentiation, have become a prime candidate for tissue engineering and regenerative therapies. More than 100 clinical trials have examined the use of mesenchymal stem/stromal cells. Biochemical and mechanical interactions between the extracellular matrix and cell surface receptors, as well as physical interactions between cells, are now recognized as essential for stem cell self-renewal and differentiation. New technologies for scaffold engineering and fabrication have taken advantage of these observations, and hold promise for repairing tissues requiring a highly specialized niche, such as skeletal muscle. These discoveries have led to clinical trials with bioengineered vascular conduits in children with congenital heart disease, complete hollow organs, and complex organs such as bioartificial livers. An evolving understanding of innate and adaptive immune responses, including the foreign body response, has led to novel approaches to modulating the immune system that facilitate tissue repair. Finally, the development of small animal models for discovery, and large animal models for studies of safety and efficacy, has propelled the field of tissue engineering toward the clinic. vii

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The chapters of this book are organized into six sections: Stem Cells, Biomaterials and the Extracellular Environment, Engineered Tissue, Synthetic Organs, Immune Response, and Animal Models. Each section is intended to build upon information presented in the previous chapters, and set the stage for subsequent sections. Throughout the chapters, the reader will observe a common theme of basic discovery informing clinical translation, and clinical studies in animals and humans guiding subsequent experiments at the bench. I thank the members of my laboratory for their helpful discussion, and my colleagues in Pediatric Cardiology for their support – we all strive to improve the lives of our patients. I appreciate always the encouragement I receive from Tricia Foster, Nathaniel Bernstein, and Katharine Bernstein. I am grateful to the 54 colleagues who have contributed their expertise to this project. We hope that this first edition of Tissue Engineering and Regenerative Medicine will serve as an introduction and guide for students of the field at all levels. San Francisco, CA

Harold S. Bernstein

Contents

Part I  Stem Cells   1 Hematopoietic Stem Cell Transplantation: Reflections on Yesterday and Thoughts for Tomorrow....................... Andrew D. Leavitt

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  2 Human Embryonic Stem Cells in Regenerative Medicine.................. Odessa Yabut and Harold S. Bernstein

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  3 Current Status of Induced Pluripotent Stem Cells.............................. Thach-Vu Ho, Grace Asuelime, Wendong Li, and Yanhong Shi

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  4 Mesenchymal Stromal Cells: Latest Advances.................................... Sowmya Viswanathan and Armand Keating

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Part II  Biomaterials and the Extracellular Environment   5 The Role of Mechanical Forces in Guiding Tissue Differentiation............................................................................. Sean P. Sheehy and Kevin Kit Parker   6 Synthetic Multi-level Matrices for Bone Regeneration....................... Nicholas R. Boyd, Richard L. Boyd, George P. Simon, and David R. Nisbet

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  7 Directing Cell Fate Through Biomaterial Microenvironments........... 123 Kelly Clause, Jonathan Lam, Tatiana Segura, and Thomas H. Barker Part III  Engineered Tissue   8 Basic Considerations with Cell Sheets.................................................. 143 Masayuki Yamato and Sebastian Sjöqvist

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  9 Myocardial Repair and Restoration...................................................... 161 Sharon S.Y. Wong and Harold S. Bernstein 10 Skeletal Muscle Engineering: The Need for a Suitable Niche............ 197 Frédéric Trensz, Anthony Scimè, and Guillaume Grenier 11 Restoring Blood Vessels.......................................................................... 211 Narutoshi Hibino, Christopher Breuer, and Toshiharu Shinoka 12 Engineering Functional Bone Grafts.................................................... 221 Sarindr Bhumiratana and Gordana Vunjak-Novakovic 13 Engineering Functional Cartilage Grafts............................................. 237 Andrea R. Tan and Clark T. Hung 14 Adult Stem Cells and Regeneration of Adipose Tissue....................... 251 Daniel A. Hägg, Bhranti Shah, and Jeremy J. Mao Part IV  Synthetic Organs 15 Hollow Organ Engineering.................................................................... 273 Anthony Atala 16 Engineering Complex Synthetic Organs............................................... 297 Joan E. Nichols, Jean A. Niles, and Joaquin Cortiella 17 Liver Regeneration and Tissue Engineering........................................ 315 Ji Bao, James Fisher, and Scott L. Nyberg Part V  Immune Response 18 Immune Modulation for Stem Cell Therapy........................................ 335 Gaetano Faleo and Qizhi Tang 19 Regenerative Medicine and the Foreign Body Response..................... 353 Kerry A. Daly, Bryan N. Brown, and Stephen F. Badylak Part VI  Animal Models 20 Small Animal Models of Tissue Regeneration...................................... 379 Fernando A. Fierro, J. Tomas Egana, Chrisoula A. Toupadakis, Claire Yellowley, Hans-Günther Machens, and Jan A. Nolta 21 Use of Large Animal and Nonhuman Primate Models for Cell Therapy and Tissue Engineering............................................. 393 Alice F. Tarantal and Karina H. Nakayama About the Editor............................................................................................. 415 Index................................................................................................................. 417

Contributors

Grace Asuelime  Department of Neurosciences, Center for Gene Expression and Drug Discovery, Beckman Research Institute of City of Hope, Duarte, CA, USA Anthony Atala  Wake Forest Institute for Regenerative Medicine, Wake Forest University School of Medicine, 5th Floor, Watlington Hall, Medical Center Boulevard, Winston-Salem, NC 27157, USA Stephen F. Badylak  McGowan Institute for Regenerative Medicine, University of Pittsburgh, 450 Technology Drive, Suite 300, Pittsburgh, PA, USA Ji Bao  Mayo Clinic, Rochester, MN, USA Thomas H. Barker  The Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech, Emory University, 313 Ferst Drive, Atlanta, GA 30332, USA Harold S. Bernstein  Cardiovascular Research Institute, University of California San Francisco, Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Department of Pediatrics, University of California San Francisco, San Francisco, CA 94143-1346, USA Sarindr Bhumiratana  Department of Biomedical Engineering, Columbia University, New York, NY, USA Nicholas R. Boyd  Department of Materials Engineering, Monash University, Clayton, VIC, Australia Richard L. Boyd  Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, VIC, Australia Christopher Breuer  Department of Cardiac Surgery, Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, New Haven, CT, USA

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Bryan N. Brown  Department of Clinical Sciences, Cornell University, Ithaca, NY, USA Kelly Clause  The Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech, Emory University, Atlanta, GA, USA Joaquin Cortiella  Laboratory of Regenerative and Nano-Medicine, Department of Anesthesiology, University of Texas Medical Branch, Galveston, TX, USA Kerry A. Daly  McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA J. Tomas Egana  Department of Plastic and Hand Surgery, University Hospital Rechts der Isar, Technical University of Munich, Munich, Germany Gaetano Faleo  Department of Surgery, University of California, San Francisco, CA, USA Fernando A. Fierro  Department of Internal Medicine, Stem Cell Program and Institute for Regenerative Cures, University of California, Davis, CA, USA James Fisher  Mayo Clinic, Rochester, MN, USA Guillaume Grenier  Étienne-Lebel Clinical Research Center, Department of Orthopedic Surgery, Université de Sherbrooke, 3001–12th Avenue North, J1H 5N4, Sherbrooke, QC, Canada Daniel A. Hägg  Tissue Engineering and Regenerative Medicine Laboratory, Columbia University Medical Center, New York, NY, USA Narutoshi Hibino  Department of Cardiac Surgery, Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, New Haven, CT, USA Thach-Vu Ho  Department of Neurosciences, Center for Gene Expression and Drug Discovery, Beckman Research Institute of City of Hope, Duarte, CA, USA Clark T. Hung  Department of Biomedical Engineering, Columbia University, 1210 Amsterdam Avenue, Engineering Terrace 351, New York, NY 10027, USA Armand Keating  Cell Therapy Program, Princess Margaret Hospital, University Health Network, 610 University Avenue, Suite 5-303, Toronto, ON M5G 2M9, Canada Department of Medicine, University of Toronto, Toronto, ON, Canada Jonathan Lam  Department of Biomedical Engineering, University of California, Los Angeles, CA, USA Andrew D. Leavitt  Departments of Laboratory Medicine and Medicine, UCSF Adult Blood and Marrow Transplant Laboratory, University of California, 513 Parnassus Avenue, Box 0100, San Francisco, CA 94143-0100, USA

Contributors

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Wendong Li  Department of Neurosciences, Center for Gene Expression and Drug Discovery, Beckman Research Institute of City of Hope, Duarte, CA, USA Hans-Günther Machens  Department of Plastic and Hand Surgery, University Hospital Rechts der Isar, Technical University of Munich, Munich, Germany Jeremy J. Mao  Tissue Engineering and Regenerative Medicine Laboratory, Columbia University Medical Center, 630 W. 168 St. – PH7 East, New York, NY 10032, USA Karina H. Nakayama  Departments of Pediatrics, Cell Biology and Human Anatomy, School of Medicine, UC Davis Clinical and Translational Science Center, California ­National Primate Research Center, University of California, Davis, CA, USA Joan E. Nichols  Laboratory of Regenerative and Nano-Medicine, Departments of Internal Medicine and Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA Jean A. Niles  Laboratory of Regenerative and Nano-Medicine, University of Texas Medical Branch, Galveston, TX, USA David R. Nisbet  Research School of Engineering, ANU College of Engineering and Computer Science, The Australian National University, Ian Ross Building 31, North Road, Acton, Canberra, ACT 0200, Australia Jan A. Nolta  Department of Internal Medicine, Stem Cell Program and Institute for Regenerative Cures, University of California, Davis, 2921 Stockton Blvd., Room 1300 , Sacramento, CA 95817, USA Scott L. Nyberg  Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA Kevin Kit Parker  Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, School of Engineering and Applied Sciences, Harvard University, Pierce Hall, Room 321, 29 Oxford St., Cambridge, MA 02138, USA Anthony Scimè  Muscle Health Research Centre, York University, Toronto, ON, Canada Tatiana Segura  Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA, USA Bhranti Shah  Tissue Engineering and Regenerative Medicine Laboratory, Columbia University Medical Center, New York, NY, USA Sean P. Sheehy  Disease Biophysics Group, Wyss Institute for Biologically Inspired Engineering, School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA

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Contributors

Yanhong Shi  Department of Neurosciences, Center for Gene Expression and Drug Discovery, Beckman Research Institute of City of Hope, 1500 E. Duarte Rd, Duarte, CA 91010, USA Toshiharu Shinoka  Department of Cardiac Surgery, Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, 333 Cedar Street, Boardman 204, PO Box 208039, New Haven, CT 06520-8039, USA George P. Simon  Department of Materials Engineering, Monash University, Clayton, VIC, Australia Sebastian Sjöqvist  Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan Andrea R. Tan  Department of Biomedical Engineering, Columbia University, New York, NY, USA Qizhi Tang  Department of Surgery, University of California, Box 0780, 513 Parnassus Avenue, San Francisco, CA 94143-0780, USA Alice F. Tarantal  Departments of Pediatrics, Cell Biology and Human Anatomy, School of Medicine, UC Davis Clinical and Translational Science Center, California National Primate Research Center, University of California, Pedrick and Hutchison Roads, Davis, CA 95616-8542, USA Chrisoula A. Toupadakis  Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA, USA Frédéric Trensz  Étienne-Lebel Clinical Research Center, Université de Sherbrooke, Sherbrooke, QC, Canada Sowmya Viswanathan  Cell Therapy Program, Princess Margaret Hospital, University Health Network, Toronto, ON, Canada Gordana Vunjak-Novakovic  Department of Biomedical Engineering, Columbia University, 1210 Amsterdam Avenue, Engineering Terrace 351, New York, NY 10027, USA Sharon S.Y. Wong  Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA Odessa Yabut  Cardiovascular Research Institute, University of California, San Francisco, CA, USA Masayuki Yamato  Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan Claire Yellowley  Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA, USA

Part I

Stem Cells

Chapter 1

Hematopoietic Stem Cell Transplantation: Reflections on Yesterday and Thoughts for Tomorrow Andrew D. Leavitt

Abstract  Biomedical science is entering a new era with exciting prospects for using cellular therapy to treat a wide spectrum of human diseases from nerve injury to diabetes, myocardial infarction, and more. Hematopoietic stem cell (HSC) transplantation has been used to treat patients for nearly half a century. The experiences and lessons learned over those 50 years are both informative and encouraging. This chapter distills the history of HSC transplantation to provide an orientation to the past that can be used to more wisely navigate the future of cell therapy. The details presented help the reader appreciate that developing novel cell therapy can be a struggle and that chance will likely continue to play a role in future success. However, it also becomes apparent that attention to fundamental details, such as choice of cell type or types, where to obtain the cells, how to handle and process the cells, how to prepare and select patients, how to evaluate success and failure, and how to organize the biomedical community to serve the good of patients, are all critical for new cell therapy to become a reality.

Abbreviations BMT GVHD GVL HLA HSCs

Bone marrow transplantation Graft-versus-host disease Graft-versus-leukemia Human lymphocyte antigen Hematopoietic stem cells

A.D. Leavitt (*) Departments of Laboratory Medicine and Medicine, UCSF Adult Blood and Marrow Transplant Laboratory, University of California, 513 Parnassus Avenue, Box 0100, San Francisco, CA 94143-0100, USA e-mail: [email protected] H.S. Bernstein (ed.), Tissue Engineering in Regenerative Medicine, Stem Cell Biology and Regenerative Medicine, DOI 10.1007/978-1-61779-322-6_1, © Springer Science+Business Media, LLC 2011

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PBSCs Peripheral blood stem cells UCB Umbilical cord blood

1.1 Introduction HSCs are the most studied and well-understood of all adult stem cells, and they provide a model system and paradigm for the more global understanding of stem cell biology [1]. HSCs have also been used clinically for nearly 50  years, with over 55,000 HSC transplants performed around the world in 2009 alone [2]. HSCs and their clinical application, therefore, provide an excellent reference point for discussing the future of stem cell therapy, be it the use of embryonic stem cells and their derivatives or the direct use of tissue-specific adult stem cells. This chapter presents a brief history of HSC transplantation to give perspective and to help inform and orient the reader to issues that will likely be faced as biomedical scientists begin developing tomorrow’s stem cell therapies. Accounts of the history of HSC transplantation have been summarized by others, including a personal account by E. Donnell Thomas who shared the 1990 Nobel Prize in Medicine for his pioneering role in the development of BMT [3, 4].

1.2 Radiation: A Double-Edged Sword Marie Curie (born Maria Sklodowska) shared the 1903 Nobel Prize in physics with Pierre Curie and Henri Becquerel “in recognition of the extraordinary services they have rendered by their joint researches on the radiation phenomena discovered by Professor Henri Becquerel.” She also won the 1911 Nobel Prize in Chemistry “in recognition of her services to the advancement of chemistry by the discovery of the elements radium and polonium, by the isolation of radium and the study of the nature and compounds of this remarkable element.” Tragically, she died on July 4, 1934, from marrow toxicity, reported in various sources as aplastic anemia and/or leukemia, but almost certainly secondary to the chronic radiation exposure she received during her early pioneering studies related to naturally radioactive substances. The bone marrow toxicity of ionizing radiation was appreciated only after much of her initial exposure, and interestingly the field of clinical marrow transplantation relied for decades on the use of ionizing radiation as a preparative regimen to both eradicate underlying malignant disease and to immunosuppress the recipient to facilitate marrow engraftment and HSC repopulation. The highly deleterious effects of radiation on bone marrow were appreciated well before World War II [5], but development and use of the atomic bomb in the 1940s highlighted the marrow toxicity of radium, uranium, and other sources of ionizing radiation. Classified government research to develop treatments for bone marrow toxicity due to atomic bomb radiation exposure was performed in the 1940s under the auspices of the Atomic Energy Commission, but it was not published until

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1950 [6]. Those studies sought “to determine what benefits, if any, may be derived from the transplantation of normal bone marrow in animals that have suffered damage to their bone marrow as a result of single dose roentgen irradiation.” The studies failed to achieve their goal of allogeneic engraftment or to demonstrate any clinically useful effect of marrow transplantation. However, failure was most likely secondary to inadequate radioablation of the recipient animal’s immune system required to achieve engraftment. So, even though the studies failed to achieve their goal [6], they highlighted one of the critical aspects of HSC transplantation – the host is not naturally receptive to foreign cells and the host’s immune system needs to be suppressed to overcome this barrier to cellular therapy. This critical fact is important to consider when developing any future form of allogeneic stem cell therapy.

1.3 Bone Marrow Transplantation: It Is the Cells In 1949, independent investigators reported that lead shielding of the spleen protected mice from the mortality of total body irradiation [7]. Interestingly, it was thought that the beneficial effect was humorally mediated. Even after a 1951 report demonstrated that intravenous or intraperitoneal injections of bone marrow cells protect mice and guinea pigs from the mortality of total body radiation [8], the humoral theory remained the prevailing theory to explain radioprotection. It required an innovative experiment reported in 1955 to begin to convince the research community that radioprotection stemmed from the bone marrow cells themselves engrafting into the recipient [9]. In brief, the investigators knew that skin grafts would not survive if performed between H2-incompatible mice, but the authors showed that skin grafts could survive across H2-incompatible strains if the recipient was first transplanted with marrow from the skin donor [9]. Moreover, skin graft survival required that the irradiated recipient mouse receive marrow from the same mouse strain that provided the skin graft. These findings, as the authors concluded, “are consistent with the cellular repopulation theory of radiation protection.” In 1956, using the then novel technique of genetically traceable donor marrow cells, it was convincingly shown that the radioprotective effect of BMT correlated with engraftment of donor marrow cells in the recipient [10]. The essential role of the marrow cells in radioprotection had finally been established, as had the fact that allogeneic marrow transplantation could work.

1.4 A Rough Clinical Start: Patients Are Always a Bigger Challenge than Mice With the animal transplant data in hand and knowing that radiation could kill leukemic cells, it was only natural for investigators to try to connect these two observations for therapeutic benefit. A 1956 report demonstrated that radiation could be used to

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eradicate leukemia in mice and that bone marrow transplant could rescue the host from the marrow-damaging effects of the radiation treatment [11]. One year later, in 1957, Thomas et al. published the first report of infusing allogeneic marrow into humans when he described his experience with six patients – three with hematologic malignancies (chronic myelogenous leukemia, multiple myeloma, and chronic lymphocytic leukemia), one with ovarian carcinoma, one with metastatic cancer of uncertain origin, and one who had suffered a massive central nervous system bleed [12]. The five patients with malignancies had each received chemotherapy and/or radiation therapy shortly before the marrow infusions. This initial report clearly focused on evaluating the safety and toxicity of the marrow cell infusions and not their therapeutic benefit [12]. There were no deaths attributed to the infused cells, and great effort was taken to assess for pulmonary emboli, which were not found to be a problem clinically or when evaluated at postmortem exam. One case suggested transient engraftment based on circulating blood cell analysis, but no long-term engraftment was demonstrated. The major conclusion was that anticoagulated suspensions of allogeneic marrow cells, strained through fine mesh to remove particulate matter, can be safely given to human recipients, as had been previously demonstrated in animals [13]. In addition to demonstrating relative safety (i.e., no major obvious untoward effects) in a very small number of patients, the authors raised important fundamental issues that are important to consider when developing any type of cell therapy in the future. They discussed the need to establish a clinically relevant cell dose, to develop a preparative regimen to treat the recipient so that their immune system does not reject the allograft, and to define a detailed monitoring system that allows for accurate assessment of toxicity and benefit. The same group reported in 1959 the successful, albeit temporary, eradication of acute lymphocytic leukemia in a patient treated with total body irradiation (Co60) followed by allogeneic BMT from an identical twin [14]. While the patient relapsed 12 weeks later, the case demonstrated that lethal radiation followed by BMT could achieve a remission, even in advanced disease, and it highlighted the importance of immunologically matched donors for efficient engraftment [14]. The authors concluded that transplants of syngeneic marrow are readily achieved in humans, that 1,000 rad of whole body radiation administered properly does not produce troublesome acute radiation sickness in humans, and that whole body irradiation at the 1,000 rad level produces a remission but not a cure of leukemia when followed by infusion of syngeneic marrow. Chemotherapy (cyclophosphamide) was soon added to total body irradiation to help eradicate the underlying disease when employing allogeneic BMT to treat patients with acute leukemia, a preparative regimen that remained in use for several decades. Reports of allogeneic BMT rose steadily over the next few years, with over 60 such transplants reported in 1962. However, enthusiasm rapidly declined as toxicity was clear and success was hard to find; only a few transplants were reported annually through the late 1960s [15]. A 1970 review of all 203 reported allogeneic transplants through 1968 highlighted the dismal state of the field, with few if any true successes. In fact, 125 of the 203 recipients did not even demonstrate evidence of

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engraftment, including 66 of 73 patients with aplastic anemia [15]. Interestingly, the other seven aplastic anemia patients received allogeneic marrow from a syngeneic twin, five of whom had clinical recovery from their disease. This subset of patients provided hope for BMT as a clinical intervention, and the outcome with the identical twins reemphasized the critical importance of immunologic match for a successful engraftment of donor bone marrow. It also highlighted the difference between treating a disease that has a dominant phenotype that is likely to recur, such as leukemia, versus one with a recessive phenotype, such as aplastic anemia. While the late 1950s through the early 1970s was not a good time for clinical success within the BMT field, significant headway was made in critical areas of transplant immunology through the use of animal studies. The advances grew out of studies in the early 1950s that actively developed immune tolerance in young mice [16]. By the mid-1960s, runt disease in mice [17, 18], which is essentially what we call GVHD in the human transplant setting, was becoming well-understood, at least from the perspective of factors related to its development [19, 20]. For example, it was not associated with the injection of syngeneic cells but required antigenic differences between donor and host, and the more pronounced the differences, the more severe the disease. Moreover, persistence of the allogeneic cells was required for persistent disease, and injection of presensitized cells could worsen the problem. Also, one could tolerize the animal prior to transplant and avoid runt disease. These findings continue to influence the field of HSC transplantation today as investigators seek to control GVHD while maintaining therapeutic success, in particular when treating malignant disease. However, as discussed below, the relationship between GVHD and therapeutic success differs with the disease being treated. In parallel with the work in mice, others were using dogs to better understand issues of engraftment, rejection, and GVHD [21, 22]. Dogs, while having a clear disadvantage due to their size and cost of housing, had a distinct advantage in being outbred and large enough for the types of surgical procedures needed to be performed at the time. Dog models demonstrated graft rejection and GVHD, but some became long-term engrafters, true HSC transplant successes, and the search was on to understand why. Ultimately, dog models were used to develop immune serum to allow for the identification of matched allogeneic donors, and it was in this setting that the use of methotrexate to reduce GVHD was developed. By the end of the 1960s, the dog model system had been used to develop a nearly 90% success rate from immunomatched allogeneic outbred donors identified using the serum reagents developed by the investigators [23–25]. They had shown quite clearly in a large animal model that lymphocyte immunophenotyping was critical for the success of allogeneic transplants, something that was proven to be true in human transplants and that continues to be of central clinical importance to this day. GVHD remains a great cause of morbidity and mortality following allogeneic HSC transplantation. Improved antileukemic preparative regimens have made disease recurrence less problematic. However, it is now appreciated that GVHD is a double-edged sword when treating leukemia with allogeneic HSC transplantation. GVHD is itself deleterious, but allogeneic HSC transplant also provides a GVL

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effect that is beneficial and contributes to overall survival. Attempts to separate these two immunologic phenomena are under intense study.

1.5 Finally Some Encouraging Results The disappointing results summarized in 1970 [15] saw many investigators leave the field, but some persevered. They believed that success was possible if they could answer a few key questions – cell dose, patient preparation that can both treat disease and prevent graft rejection, and how to reduce the problem with GVHD. A 1972 publication described four patients with aplastic anemia treated with HLA-A matched sibling donors, giving BMT a much-needed boost. All were opposite sex transplants, so standard karyotyping could determine if blood count return posttreatment was due to endogenous marrow recovery or allogeneic marrow engraftment. One patient died from GVHD with a cellular marrow at 45 days posttransplant, another rejected the transplant and died 67 days after transplant, but two were alive with a robust functioning allogeneic marrow at the time of the report, 138 and 215 days out from transplant. In 1975, the BMT team in Seattle published a two-part review [26, 27] that extensively outlined the scientific rationale for performing BMT and the requirements for successful BMT, including details on the care of the patient, the importance of immunosuppression to allow for engraftment and prevent rejection, the need to eradicate underlying malignancy, and the need for HLA matching. It also established a marrow-nucleated cell count dose that should be met for successful transplant and defined many clinical aspects of GVHD. The review also presented the authors’ results treating 37 patients with aplastic anemia and 73 with end-stage leukemia. While the survivorship was low for the patients with end-stage leukemia, the fact that any were alive 2  years posttreatment was a remarkable success that energized the BMT field. Patients were alive that would otherwise have died if it were not for their BMT. However, the field really took off following a 1977 report describing the outcomes of 100 consecutive patients treated with chemotherapy, total body radiation, and sibling-matched allogeneic transplants for end-stage recurrent leukemia. Thirteen of the patients were apparent “cures” as defined by no recurrence of disease at 2 or more years (some over 4 years) posttransplant [28]. The authors and others realized that success might be much higher if leukemia patients were treated before they relapsed and reached end-stage status of their disease. In 1979, two groups reported on matched, related, allogeneic transplantation for leukemia, demonstrating a nearly 50% survival at 2 years [29, 30]. Bone marrow transplant had worked. Patients were benefiting, and over the next 15 years such transplants became part of mainstream medical care. It is estimated that roughly 60,000 transplants were performed around the world in 2010. The Center for International Blood and Marrow Transplant Research maintains a worldwide database of HSC transplants, including source of cells, underlying disease, and outcome (http://www.cibmtr.org).

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1.6 Not All GVHD Is Bad GVHD was rapidly appreciated to be a major complication of allogeneic transplants, and detailed clinical information on how to define this disorder was included in the 1975 two-part report [26, 27]. However, even as far back as the 1950s, it was speculated that the allogeneic donor cells might also provide a beneficial effect when treating malignant diseases such as leukemia [11]. That is, maybe the same immunologic attack of the normal host tissue could also play a role in destroying the diseased cells. This has turned out to be true, with higher cure rates associated with moderate GVHD. This idea was further supported by findings from identical twin (syngeneic) transplants [31]. It was originally thought that an identical twin would be the ideal donor because of the lack of or minimal GVHD. However, patients with acute myelogenous leukemia who received an allogeneic donation from an identical twin had a significantly higher relapse rate than those who received marrow from an HLA-matched sibling [31]. The twin data highlighted that HLA (-A, -B, -DR, and DQ) matching does not match all immunologic differences, and the ones that remain are sufficient to allow for clinically important GVL effect. This immunological therapeutic value of the allogeneic HSC transplant, GVL, remains a critically important contributor to the cure rate for allogeneic transplants for malignant hematologic diseases. However, it is important to remember that there is no beneficial role for graft-versus-disease when using allogeneic transplantation to treat nonmalignant diseases, such as sickle cell anemia [32] and thalassemia [33]. Innovative approaches to reduce GVHD will be essential if we are to bring this valuable treatment to more patients with nonmalignant hematologic disorders [34]. Congenital immunodeficiencies represent yet another group of disease that can be treated with allogeneic transplantation. As with other nonmalignant diseases, GVHD needs to be minimized at all costs. However, these patients allow for greater HLA mismatch in “the other” direction because the recipient immune system is often unable to mount a host-versus-graft response to reject the marrow. Consequently, more gentle conditioning regimens can often be employed, which translates to less therapy-related toxicity. The immunocompetence of the recipient could have a large impact on trial design and clinical outcomes when identifying initial candidates for novel cell therapies developed in the future.

1.7 Source of Hematopoietic Stem Cells While increasing numbers of people now use the name “hematopoietic stem cell transplantation,” from the start and for many years it was called BMT for obvious reasons. In the original 1957 report entitled “Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy,” the cells infused into the six patients were obtained from fetal (n = 1) or adult (n = 1) cadavers, ribs removed at surgery (n = 1), or the anterior or posterior iliac crest aspiration of a living donor (n = 3).

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By the 1970s, iliac crest marrow aspiration was the standard method for obtaining bone marrow cells for transplantation, a procedure that requires general anesthesia. While the HSCs are required for long-term, sustained engraftment, it is wellappreciated that the transplanted marrow includes many more hematopoietic cell types than just HSCs. The importance of the non-HSC cells for assisting with engraftment remains uncertain, but it is quite clear that the non-HSC progenitor cells play a critical role in providing a more rapid production of circulating allogeneic blood cells following infusion. This aspect of progenitor cells helps protect the patient from infection and bleeding, complications of neutropenia and thrombocytopenia, respectively [35]. Given that transplant morbidity and mortality are directly related to the duration of posttransplant cytopenia, the non-HSC cells in the transplanted material clearly play an important and favorable clinical role. Consequently, as cellular therapy moves to other tissues, it is important to consider the value of cells beyond the stem cells proper. It could be that an overly reductionist or “pure” cell population has less benefit than one that contains critical accessory cells. It became clear in the late 1980s that adequate numbers of HSCs could be obtained from the peripheral blood of patients following administration of newly available human cytokines, such as G-CSF or GM-CSF [36]. Interest grew rapidly in the clinical use of such PBSCs as source material for HSC transplantation, and reports of their use became common in the mid-1990s [35, 37–42]. Clinical trials confirmed their safety and efficacy, and PBSCs rapidly expanded as an HSC source for allogeneic and autologous transplants. G-CSF rapidly became the mobilizing agent of choice [43, 44]. More recently, a CXCR4 inhibitor has been approved as an alternate method for mobilizing PBSCs in a subset of patients. Curiously, the use of PBSCs posed a nomenclature problem for the field. How could PBSC transplants be called bone marrow transplants when the cells were not collected from the bone marrow? Fortunately, BMT is also the acronym for “blood and marrow transplantation,” which is how it is commonly used today. While there is not a simple clinical method to quantify the true HSC content of a PBSC product, standard of care is to use CD34 surface expression as a surrogate marker for HSCs and to dose PBSC transplants based on a desired number of CD34+ cells/kg that ensures engraftment. This contrasts with marrow samples, where the clinical adequacy of the collection is based simply on a nucleated cell count/kg. In either case, it is important to realize that no clear enumeration of HSCs is applied to determine the adequacy of an HSC collection, yet the use of surrogate markers has proven productive and safe for many decades. The limited availability of related, matched allogeneic donors became a problem as the sophistication of HLA matching and the use of transplants grew. While in principle one has a one-in-four chance of finding a sibling match, success is even less in real life. Therefore, the majority of patients who can benefit from a BMT do not have an acceptable sibling donor. The first report of a successful, unrelated HLA-matched (HLA-A, -B, -C, and -DR; four loci, which means eight total alleles) allogeneic transplant for leukemia was reported in 1980 [45]. Finding a match was made possible through the advent of more sophisticated HLA phenotyping, but the

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success in finding this particular donor was the result of pure luck and circumstance. The matched donor was a technician at the Seattle transplant center, where everyone had been HLA typed as part of the center’s studies in HLA typing. The first matched, unrelated allogeneic transplant highlighted the potential value of developing a robust mechanism for identifying unrelated HLA-matched donors. As a direct outgrowth of this particular experience and productive lobbying of the US government by concerned and involved individuals, federal funding was eventually allocated for the development of the National Marrow Donor Program (http://www. marrow.org) in the USA. The program has grown dramatically over the ensuing 25 years, is now linked to other similar programs in Europe and elsewhere, and unrelated donors are identified for thousands of patients each year through the sophisticated international systems. It is a great example of how national boundaries and differences can become invisible when health care and humanity are placed above politics. As a testament to the importance and the success of these programs, more unrelated than related allogeneic transplants were performed in the USA in 2009. UCB HSCs [46] were first demonstrated as a clinically useful option for HSC transplants in 1989 [47] when they were used to treat a patient with Fanconi’s anemia, a nonmalignant, congenital blood disorder. UCB has a number of advantages over other HSC sources, including the lack of risk or discomfort to the donor and the ability to store the product in large banks. The latter point means that one can avoid the need to isolate the HSC product from a donor in a timed fashion relative to the patient’s treatments. It also means that intercurrent health issues do not delay or prevent a donation as they can with a living donor. There is also an apparent advantage related to greater tolerance of HLA mismatching [48]. On the other hand, the limited number of cells in most UCB units precludes their use in older adolescents and adults, a fact that has led to the use of multiple UCB units to treat an adult [49]. Regardless, UCB now occupies a legitimate seat at the table of HSC sources for patients of all ages in need of allogeneic HSC transplantation, and the future establishment of organized public UCB banks will be a big step forward in making UCB cells available to more patients in need [50]. While many efforts have been undertaken, human HSCs have not yet been convincingly generated from human embryonic stem cells, so the clinical application of hESC-derived HSCs remains theoretical.

1.8 Autologous HSC Transplantation Allogeneic HSC transplantation was for many years the primary focus for HSC transplantation, and the most common application was to treat hematologic malignancies. However, it was clear from the beginning that autologous transplants may prove useful if antileukemia regimens could eradicate the disease, thereby making unnecessary the GVL effect achieved with allogeneic transplants. The use of combined chemotherapy and total body irradiation preparative regimens provided such an opportunity, as did subsequent use of all chemotherapy preparative regimens,

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and autologous HSC transplant was found to be curative in a number of patients with acute leukemia [51–53]. While autologous transplants are performed in the setting of clinical remission, there was great concern that relapse could be due to reinfusion of leukemia clones with the transplanted cells. This question was addressed with some of the very first gene therapy trials in which viral vectors were used to mark harvested cells prior to their reinfusion. If the viral vector marked relapsed disease, investigators would know that it came from the harvested and reinfused cell product. Such studies showed that a fraction of relapsed disease does in fact come from reinfusion of malignant cells [54–56]. The risks of autologous and allogeneic transplants differ, with the former having a much higher risk of relapse and no risk of GVHD-related morbidity and mortality. In contrast, allogeneic transplants have a much lower risk of relapse but a significant risk of GVHD-related morbidity and mortality. As risk stratification has evolved, different subsets of patients are preferentially treated with one or the other approach.

1.9 Regulatory Agencies BMT grew up in an era quite different from today when it comes to regulation and oversight. In fact, one might wonder if HSC transplantation could have ever gotten off the ground in today’s regulatory environment. For many decades, procedural decisions and standards were established by individual transplant programs without outside scrutiny. However, as programs grew and more centers opened, it became important for professional organizations to establish rules to guide the field. From this appreciation was born the Federation for Accreditation for Cellular Therapy, the major professional organization that now accredits BMT programs, and accreditation has become an important goal for all centers in the USA. The Federation for Accreditation for Cellular Therapy, originally called the Foundation for the Accreditation of Hematopoietic Cell Therapy, was established in 1996 to develop and implement the inspection and accreditation program of the parent organizations, the International Society for Hematotherapy and Graft Engineering and the American Society of Blood and Marrow Transplant. Training of inspectors began in September 1996 and the first on-site inspections began in September 1997. The Foundation for the Accreditation of Hematopoietic Cell Therapy changed its name to the Federation for Accreditation for Cellular Therapy in December 2001 when it became clear that cellular therapy was growing beyond traditional hematopoietic stem and progenitor cells. The Federation for Accreditation for Cellular Therapy inspects an entire program, including collection, laboratory, and clinical care. The Joint Accreditation Committee of the International Society for Cellular Therapy and the European Group for Blood and Marrow Transplantation launched their first official inspection programs in January 2004, providing Europe a similar accreditation program. The American Association of Blood Banks also inspects and accredits BMT laboratories.

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The Federation for Accreditation for Cellular Therapy and like organizations have done much to make BMT programs safer and more responsive to patient needs. In addition to professional accreditation agencies, such as the Federation for Accreditation for Cellular Therapy and the American Association of Blood Banks, BMT programs in the USA must have all or part of the program licensed with state health care agencies and be registered with the Food and Drug Administration. The governmental organizations work to ensure good practices and to provide an avenue to disseminate information relevant to maintaining a safe operation. They make on-site inspections on a regular basis to ensure that procedures are in place and followed and that clinical outcomes and support are consistent with high-quality care. It behooves the cellular therapy community to put energy into professional organizations that provide oversight of any new cellular therapies that develop. Selfpolicing by informed and interested professionals is the best way to ensure safety and reproducibility and to avoid unwanted and unproductive regulations from outside agencies. For BMT programs in the USA, the Federation for Accreditation for Cellular Therapy and the American Association of Blood Banks provide excellent avenues for working with the states and with the Food and Drug Administration to ensure rational and productive systems.

1.10 Conclusions The history of HSC transplantation offers an informative glimpse into the past, providing a number of experiences that can help guide the future of stem cell therapy. First and foremost is the appreciation that HSC transplantation did not “work” right away. In fact, it took decades before people could speak of meaningful clinical success. However, unlike today’s stem cell activities, the field of HSC transplantation grew up in relative anonymity, a truth that made its initial struggles less likely to derail its efforts. Therefore, the first issue for the stem cell field is to not oversell its product or its timeline for success and to articulate clear and simple goals. While the field of HSC transplantation took a while to gather momentum, there were observations even in the early years that proved informative. For example, the relatively early successful transplant of patients with immunodeficiency syndromes highlighted the fact that some patients provide a more receptive environment for transplant engraftment than do others. Such experiences demonstrate the significant impact of highly selected patient populations on successful outcomes. People developing new cellular therapies need to keep this in mind because nothing breeds success and maintains public support like success. Unlike the development and application of HSC transplantation, most novel cellular therapies being considered today are for nonmalignant diseases. This is an advantage because it typically means not having to eradicate a phenotypically dominant disease and replace it with a normal (phenotypically recessive) new stem cell population. For example, replacing injured nerves or destroyed pancreatic islet cells does not require therapy to remove the diseased cells. However, it could be that the

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environment, i.e., cellular niche where the new cells need to engraft, is damaged or altered in the diseased state leaving it less receptive to new cells, such as in myocardial infarction or diabetes. Consequently, understanding the health and makeup of the engraftment location might be critical for success. The field of cellular therapy, both stem cell and other, also needs to keep in mind that the fundamentals are the key. Just like for HSC transplant, one needs to determine the (minimum) number of cells needed to achieve one’s goal and how to best prepare the patient to receive and accept the transplanted cells. It is envisioned that some cellular therapies will ultimately be developed through modification of autologous cells, but that will not happen tomorrow, so selective immunomodulation will be just as important as it is for current day tissue and organ transplantation. Moreover, consideration should be given to the possible use and benefit of accessory cells, much as the non-HSC progenitor cells help with the clinical success of HSC transplants. Of course, well-designed systems to monitor for toxicity and efficacy are essential to keep the field developing productively. Modern stem cell therapy is growing up under an intense public spotlight. The better the cell therapy community polices itself, the more care it takes to learn from the accreditation and inspection organizations that have developed within the HSC transplant community, the more trust it will be given by the public. Involved members of the scientific community must actively engage regulatory agencies and develop professional oversight groups, much like the HSC transplant community has done. This has resulted in better and safer HSC transplant programs, better data monitoring, and it affords the involved community an efficient mechanism for communication and engagement with government organizations. The future for cellular therapy is promising and exciting, and lessons learned along the way must be carefully and actively used to everyone’s advantage.

References 1. Orkin SH, Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132(4):631–644 2. Pasquini MC, Wang Z (2010) Current use and outcome of hematopoietic stem cell transplantation: CIBMTR Summary Slides. http://www.cibmtr.org. Accessed 9 Jul 2011 3. Perry AR, Linch DC (1996) The history of bone-marrow transplantation. Blood Rev 10(4): 215–219 4. Thomas ED (2005) Bone marrow transplantation from the personal viewpoint. Int J Hematol 81(2):89–93 5. Shouse SS, Warren SL, Whipple GH (1931) II. Aplasia of marrow and fatal intoxication in dogs produced by roentgen radiation of all bones. J Exp Med 53(3):421–435 6. Rekers PE, Coulter MP, Warren SL (1950) Effect of transplantation of bone marrow into irradiated animals. Arch Surg 60(4):635–667 7. Jacobson LO, Marks EK, Robson MJ, Gaston E, Zirkle RE (1949) The effect of spleen protetction on mortality following x-irradiation. J Lab Clin Med 34:1538–1543 8. Lorenz E, Uphoff D, Reid TR, Shelton E (1951) Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Natl Cancer Inst 12(1):197–201 9. Main JM, Prehn RT (1955) Successful skin homografts after the administration of high dosage X radiation and homologous bone marrow. J Natl Cancer Inst 15(4):1023–1029

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10. Ford CE, Hamerton JL, Barnes DW, Loutit JF (1956) Cytological identification of radiationchimaeras. Nature 177(4506):452–454 11. Barnes DW, Corp MJ, Loutit JF, Neal FE (1956) Treatment of murine leukaemia with X rays and homologous bone marrow: preliminary communication. Br Med J 2(4993):626–627 12. Thomas ED, Lochte HL Jr, Lu WC, Ferrebee JW (1957) Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med 257(11):491–496 13. Congdon CC, Uphoff D, Lorenz E (1952) Modification of acute irradiation injury in mice and guinea pigs by injection of bone marrow: a histopathologic study. J Natl Cancer Inst 13(1):73–107 14. Thomas ED, Lochte HL Jr, Cannon JH, Sahler OD, Ferrebee JW (1959) Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 38:1709–1716 15. Bortin MM (1970) A compendium of reported human bone marrow transplants. Transplantation 9(6):571–587 16. Billingham RE, Brent L, Medawar PB (1953) Actively acquired tolerance of foreign cells. Nature 172(4379):603–606 17. Nisbet NW, Heslop BF (1962) Runt disease-II. Br Med J 1(5273):206–213 18. Nisbet NW, Heslop BF (1962) Runt disease. Br Med J 1(5272):129–135,contd 19. Billingham RE (1966) The biology of graft-versus-host reactions. Harvey Lect 62:21–78 20. Billingham RE, Silvers WK (1959) The induction of tolerance of skin homografts in rats with pooled cells from multiple donors. J Immunol 83:667–679 21. Cavins JA, Kasakura S, Thomas ED, Ferrebee JW (1962) Recovery of lethally irradiated dogs following infusion of autologous marrow stored at low temperature in dimethylsulphoxide. Blood 20:730–734 22. Thomas ED, Collins JA, Herman EC Jr, Ferrebee JW (1962) Marrow transplants in lethally irradiated dogs given methotrexate. Blood 19:217–228 23. Epstein RB, Storb R, Ragde H, Thomas ED (1968) Cytotoxic typing antisera for marrow grafting in littermate dogs. Transplantation 6(1):45–58 24. Storb R, Epstein RB, Bryant J, Ragde H, Thomas ED (1968) Marrow grafts by combined marrow and leukocyte infusions in unrelated dogs selected by histocompatibility typing. Transplantation 6(4):587–593 25. Storb R, Rudolph RH, Thomas ED (1971) Marrow grafts between canine siblings matched by serotyping and mixed leukocyte culture. J Clin Invest 50(6):1272–1275 26. Thomas E et  al (1975) Bone-marrow transplantation (first of two parts). N Engl J Med 292(16):832–843 27. Thomas ED et al (1975) Bone-marrow transplantation (second of two parts). N Engl J Med 292(17):895–902 28. Thomas ED et al (1977) One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation. Blood 49(4):511–533 29. Blume KG, Beutler E (1979) Allogeneic bone marrow transplantation for acute leukemia. JAMA 241(16):1686 30. Thomas ED et al (1979) Marrow transplantation for acute nonlymphoblastic leukemia in first remission. N Engl J Med 301(11):597–599 31. Gale RP et al (1994) Identical-twin bone marrow transplants for leukemia. Ann Intern Med 120(8):646–652 32. Johnson FL, Look AT, Gockerman J, Ruggiero MR, Dalla-Pozza L, Billings FT III (1984) Bone-marrow transplantation in a patient with sickle-cell anemia. N Engl J Med 311(12): 780–783 33. Thomas ED et al (1982) Marrow transplantation for thalassaemia. Lancet 2(8292):227–229 34. Hsieh MM et al (2009) Allogeneic hematopoietic stem-cell transplantation for sickle cell disease. N Engl J Med 361(24):2309–2317 35. Korbling M et al (1995) Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. Blood 85(6):1659–1665 36. Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD (1988) Granulocytemacrophage colony stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1(8596):1194–1198

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37. Korbling M et al (1995) Allogeneic blood stem cell transplantation: peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34+ Thy-1dim) and lymphoid subsets, and possible predictors of engraftment and graft-versus-host disease. Blood 86(7):2842–2848 38. Schmitz N et al (1995) Primary transplantation of allogeneic peripheral blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor). Blood 85(6):1666–1672 39. Azevedo WM et al (1995) Allogeneic transplantation with blood stem cells mobilized by rhGCSF for hematological malignancies. Bone Marrow Transplant 16(5):647–653 40. Russell JA et  al (1995) Collection of progenitor cells for allogeneic transplantation from peripheral blood of normal donors. Bone Marrow Transplant 15(1):111–115 41. Bensinger WI et al (1995) Transplantation of allogeneic peripheral blood stem cells mobilized by recombinant human granulocyte colony-stimulating factor. Blood 85(6):1655–1658 42. Dreger P, Suttorp M, Haferlach T, Loffler H, Schmitz N, Schroyens W (1993) Allogeneic granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells for tretment of engrftment failure after bone marrow transplantaion. Blood 81:1404–1407 43. Bensinger WI et  al (1993) The effects of daily recombinant human granulocyte colonystimulating factor administration on normal granulocyte donors undergoing leukapheresis. Blood 81(7):1883–1888 44. Caspar CB, Seger RA, Burger J, Gmur J (1993) Effective stimulation of donors for granulocyte transfusions with recombinant methionyl granulocyte colony-stimulating factor. Blood 81(11):2866–2871 45. Hansen JA, Clift RA, Thomas ED, Buckner CD, Storb R, Giblett ER (1980) Transplantation of marrow from an unrelated donor to a patient with acute leukemia. N Engl J Med 303(10):565–567 46. Broxmeyer HE et al (1989) Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 86(10):3828–3832 47. Gluckman E et al (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 321(17):1174–1178 48. Wagner JE, Gluckman E (2010) Umbilical cord blood transplantation: the first 20 years. Semin Hematol 47(1):3–12 49. Brunstein CG, Laughlin MJ (2010) Extending cord blood transplant to adults: dealing with problems and results overall. Semin Hematol 47(1):86–96 50. Anonymous (2005) Cord blood: establishing a national hematopoietic stem cell bank program, a 2005 report from The Institue of Medicine of The National Academy of Sciences. http://iom. edu/Reports/2005/Cord-Blood-Establishing-a-National-Hematopoietic-Stem-Cell-BankProgram.aspx. Accessed 9 Jul 2011 51. Linker CA (2003) Autologous stem cell transplantation for acute myeloid leukemia. Bone Marrow Transplant 31(9):731–738 52. Linker CA, Damon LE, Ries CA, Navarro WA, Case D, Wolf JL (2002) Autologous stem cell transplantation for advanced acute myeloid leukemia. Bone Marrow Transplant 29(4):297–301 53. Linker CA, Ries CA, Damon LE, Rugo HS, Wolf JL (1993) Autologous bone marrow transplantation for acute myeloid leukemia using busulfan plus etoposide as a preparative regimen. Blood 81(2):311–318 54. Brenner MK et  al (1993) Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients. Lancet 342(8880):1134–1137 55. Brenner MK et  al (1993) Gene-marking to trace origin of relapse after autologous bonemarrow transplantation. Lancet 341(8837):85–86 56. Deisseroth AB et al (1994) Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML. Blood 83(10):3068–3076

Chapter 2

Human Embryonic Stem Cells in Regenerative Medicine Odessa Yabut and Harold S. Bernstein

Abstract  Human embryonic stem cells have the capacity for self-renewal and pluripotency, making them a primary candidate for tissue engineering and regenerative therapies. To date, numerous human embryonic stem cell (hESC) lines have been developed and characterized. In this chapter, we discuss how hESC lines are derived, the means by which pluripotency is monitored, and how their ability to differentiate into all three embryonic germ layers is determined. We also outline the methods currently employed to direct their differentiation into populations of tissuespecific, functional cells. Finally, we highlight the general challenges that must be overcome and the strategies being developed in order to generate highly purified hESC-derived cell populations that can safely be used for clinical applications.

Abbreviations bFGF DKK1 hEB hESC HLA miR RPE

Basic fibroblast growth factor Dickkopf homolog-1 Human embryoid body Human embryonic stem cells Human lymphocyte antigen MicroRNA Retinal pigment epithelium

H.S. Bernstein (*) Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, USA Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA e-mail: [email protected] H.S. Bernstein (ed.), Tissue Engineering in Regenerative Medicine, Stem Cell Biology and Regenerative Medicine, DOI 10.1007/978-1-61779-322-6_2, © Springer Science+Business Media, LLC 2011

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TGFb Transforming growth factor-b VEGF Vascular endothelial growth factor

2.1 Introduction Stem cells have the ability to maintain long-term proliferation and self-renewal. Under specific conditions, stem cells can differentiate into a diverse population of mature and functionally specialized cell types. There are two main types of human stem cells classified according to their source and developmental potential: embryonic and adult, or tissue-specific, stem cells. Human embryonic stem cells are pluripotent cells that can differentiate into all types of somatic and in some cases, extraembryonic tissues. Human adult stem cells are derived from nonembryonic tissues and are capable of generating specific cells from its organ or tissue of origin. Because of the unrestricted potential of human embryonic stem cells (hESCs), these cells have become a highly desirable experimental tool for understanding human development, and are especially attractive for therapeutic applications.

2.2 Sources and Derivation of Human Embryonic Stem Cells hESCs were first derived from the inner cell mass of the blastocyst-stage preimplantation embryo (Fig. 2.1). The inner cell mass is composed of pluripotent cells that are capable of differentiating into the extraembryonic endoderm and the three germ layers that will eventually generate all tissues of the embryo: ectoderm, mesoderm, and endoderm. To generate a hESC line, the cells encompassing the inner cell mass are microsurgically removed and cultured in vitro under specific conditions designed to select cell populations with the capacity to proliferate in the undifferentiated state. Thomson and colleagues reported the first derivation of pluripotent hESCs using this method [1] and were quickly followed by a number of other groups [2–4]. To date, there are 82 hESC lines that adhere to US federal guidelines, many of which are widely used in basic and clinical research. A current list may be found at http://www.grants.nih.gov/stem_cells/registry/current.htm. hESC lines have also been derived from earlier stages of embryonic development, including single blastomeres of 4- or 8-cell stage embryos [5–8] and 16-cell morulae [9, 10] (Fig. 2.1). A single blastomere is considered totipotent and can produce an entire embryo. Thus, blastomere-derived hESCs could circumvent ethical issues surrounding the use of hESCs in biomedical research, since the removal of a single blastomere from an early-stage embryo will, theoretically, not impede the ability of the remaining blastomeres to develop into a normal embryo. hESC lines can also be obtained from parthenogenetic embryos, which are generated when a single egg is fertilized in the absence of male sperm (Fig. 2.1). hESC lines derived using this method can circumvent ethical concerns about the use of embryonic cells since viable embryos are neither created nor destroyed. Parthenote-derived hESC lines have been generated through artificial fertilization of

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Fig. 2.1  Generation of hESC lines from various embryonic sources. Generation of hESC lines undergo three stages. First, donor embryos are obtained after in vitro fertilization or by egg activation (parthenogenetic embryos) and allowed to develop in vitro. Second, pluripotent cells are isolated either from the inner cell mass (ICM) of pre-implantation blastocysts or from 4, 8, or 16-cell stage morulae. Finally, isolated cells are plated in defined hESC medium with or without feeder cell layers to propagate and select for pluripotent cell populations

donor oocytes [11–14]. The ability to derive hESC lines from parthenote-blastocysts is especially attractive not only because of their normal karyotype and their pluripotent properties, but also because these lines contain homozygous major HLA alleles, which could circumvent immunological rejection involved in transplantation therapies (discussed below).

2.3 Characteristics of Pluripotent Human Embryonic Stem Cells hESC lines have been derived from different sources using different methods which can introduce variability between lines. Thus, defining the specific properties and identifying the features of hESCs are critical to their use. In this section, we discuss the guidelines currently used when characterizing new hESC lines.

2.3.1 Cell Morphology and Density Pluripotent hESCs maintain a specific cell morphology and density. hESCs have a high cell nucleus-to-cytoplasm ratio due to an enlarged nucleus and distinct nucleoli.

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Fig. 2.2  Phase contrast images of undifferentiated and differentiating hESCs in culture. (a) A compact colony of proliferating pluripotent hESCs can be seen when cultured in defined medium on mouse embryonic fibroblasts. (b) Floating hEBs are observed at 2 days after induction of differentiation. (c) Differentiating cardiomyocytes appear within adherent cultures at 48 h after plating hEBs onto a gelatin-coated culture dish. Bar, 25 mm

Proliferating pluripotent hESCs form compact and spherical cell colonies when grown on mouse embryonic fibroblast cell layers (Fig. 2.2a). Differentiating hESCs are easily distinguished by the loss of compact morphology and the appearance of flattened cells that form at the edges of the colony. This can be controlled with regular supplementation of fresh growth medium [15].

2.3.2 Expression Profiling A systematic study has been conducted by the International Stem Cell Initiative, a consortium of stem cell researchers from more than 15 countries, on 59 independently derived and commonly used hESC lines in order to identify a panel of molecular markers that are consistently and strongly expressed in pluripotent hESCs [16]. These included developmentally regulated genes such as NANOG, POU domain class 5 homeobox 1 protein (POU5F/OCT4), teratocarcinoma-derived growth factor 1, DNA (cytosine-5-)-methyltransferase 3b, g-aminobutyric acid A receptor b3, and growth differentiation factor 4. The study also established that the collective expression of Stage-Specific Embryonic Antigens 3 and 4, along with keratin sulfate (TRA-1-60, TRA-1-81, GDTM2, and GCT343) and protein (CD9 and Thy1) antigens, are reliable cell surface markers of pluripotent hESCs. Other characteristics of hESCs include the expression of the enzyme alkaline phosphatase, Stem cell factor (or c-Kit ligand), and class 1 HLA. The expression profile of small, noncoding RNAs known as microRNAs, which regulate translational efficiency of target mRNAs [17], has been evaluated in hESCs by several groups [18–22]. These studies identified a number of miR family clusters specifically expressed in pluripotent hESCs. Among these are miR-92b, the miR302 cluster, miR-200c, the miR-368 and miR-154* clusters, miR-371, miR-372, miR-373*, miR-373, and the miR-515 cluster [18, 22]. Functional studies of some

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of these miRs, such as miR-302 and miR-92b, have established roles in pathways that control self-renewal and maintenance of the pluripotent stem cell state [23, 24]. To date, studies that compare the miR expression profiles of all available hESC lines are still lacking. A comprehensive analysis of miR expression profiles is warranted, as this will identify miRs that are expressed across hESC lines, and could be used to select for pluripotent populations, evaluate newly derived hESC lines, and understand mechanisms that regulate basic hESC biology.

2.3.3 Epigenetic Properties Epigenetic mechanisms influence gene expression through heritable modifications in chromosomal or DNA structure, such as DNA methylation, histone modification, and X-chromosome inactivation. Similar to expression patterns of coding genes discussed above, the epigenetic properties of pluripotent hESCs can be used as molecular signatures to distinguish them from other cell types. The chromatin structure of hESCs is generally in an open conformation, making it readily accessible to the transcriptional machinery necessary for the maintenance of pluripotency [25]. It has also been observed that hESC lines display DNA methylation profiles distinct from most other cell types [26]. One study of 14 different lines revealed markedly reduced methylation patterns of CpG dinucleotides when compared to somatic cells. Further analysis revealed that the observed differential methylation of hESCs was specific to promoter regions of pluripotency genes such as OCT4 and Nanog [27]. Thus, the unique epigenetic properties of hESCs likely promote maintenance of the pluripotent state and can be used as a hallmark of undifferentiated hESCs. To date, almost all established female hESC lines analyzed exhibit partial or complete X-chromosome inactivation, a process that occurs as early as the blastocyst stage and leads to methylation of promoter regions. The states and levels of X-inactivation appear to differ between hESC lines, and also between subcultures of each hESC line that are propagated by different laboratories [28–31]. These observations point out that in addition to genetic heterogeneity, the environment and culture conditions can lead to variable and unstable epigenetic states. The variability in X-chromosome inactivation could result in inconsistencies as hESCs are developed for therapeutic applications. Thus, generating an epigenetically naïve hESC line, in which X-chromosome inactivation or other epigenetic modifications have not yet occurred, is an important goal.

2.3.4 Pluripotency of hESCs hESCs are defined in part by their capacity to differentiate, which can be tested using in vivo and in vitro methods. A test of pluripotency in vitro involves determining

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Fig. 2.3  In vivo differentiation of hESCs by teratoma formation. Proliferating cultures of hESCs were used to form teratomas by renal capsule grafting using established methods [35]. (a) An explanted teratoma is shown. (b–f) Teratomas were sectioned and stained with hematoxylin and eosin to identify embryonic tissues. Representative tissues from all three embryonic germ layers can be seen, including endoderm (b), mesoderm (c, d), and ectoderm (e, f). (b) Glandular intestinal structure. (c) Nascent renal tubules and glomeruli within a bed of primitive renal epithelium. (d) Cartilage surrounded by capsule of condensed mesenchyme. (e) Nascent neural tube. (f) Primitive squamous epithelium. Bar, 100 mm

the ability of hESCs to form hEBs when cultured in a nonadherent cell suspension in the absence of feeder cell layers (Fig. 2.2b). hEBs are spherical colonies of differentiating hESCs that contain cell types representative of all three embryonic germ layers [32]. hEBs can be differentiated into specific tissues under suitable culture conditions (Fig. 2.2c). The most commonly used in vivo method to test pluripotency involves the transplantation of undifferentiated hESCs into immunodeficient mice to induce the formation of teratomas [33–36]. Teratomas are benign tumors comprised of disorganized tissue structures characteristic of the three embryonic germ layers. Analysis of embryonic tissues found in teratomas from engrafted hESCs can be used to test their differentiation potential (Fig. 2.3). The ability of hESCs and hEBs to mimic in vitro and in vivo the events occurring during human embryonic development makes them valuable tools for understanding the mechanisms involved in developmental processes, and steppingstones toward the generation of desired cell types suitable for cell therapies.

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2.4 Stem Cell Derivatives and Their Uses for Cell-Based Therapies Cellular insufficiency or deficiency, due to dysfunction or degeneration, respectively, is the root of diseases such as heart failure, neurodegenerative disorders, diabetes, bone marrow failure, and spinal cord injury. For centuries, therapeutic approaches have been limited to the surgical removal of damaged tissues or treatment with pharmacological therapies to ameliorate symptoms and fight infection. Thus, the prospect of replacing damaged or missing cells with new functional cells has shifted the therapeutic paradigm toward restoring tissue function. Deriving specific cell populations from hESCs that could either replace damaged cells or coax neighboring cells to function normally provides a promising strategy for cell-based therapy. With hESCs, it is possible to generate lineage-restricted progenitors that are capable of differentiating into specialized postmitotic cell types such as cardiomyocytes, pancreatic islet cells, chondrocytes, hematopoietic cells, endothelial cells, or neurons. Furthermore, the ability of hESCs to divide indefinitely makes these cells an inexhaustible large-scale source of specific progenitors. Current research studies are focused on identifying and refining ways for directing the differentiation of hESCs that will enrich for pure, homogenous populations of specific cell types. In the following sections, we provide some examples of how differentiation of hESCs can be directed toward specific cell/tissue types, and the potential use of these cell types for clinical applications (Table 2.1).

2.4.1 Endodermal Cell Derivatives of hESCs Endodermal derivatives include cells that populate the lung, liver, and pancreas. Directing the differentiation of hESCs toward definitive endoderm would help generate specific cell types, such as islet cells or hepatocytes, which could be used toward treatment of diseases such as diabetes or liver disease, respectively. D’Amour et al. [37] showed that selective induction of endoderm could be achieved through the addition of high concentrations of activin A, under low serum conditions, and in a stage-specific manner. Activin A mimics the action of Nodal, a ligand that activates TGFb signaling, which in turn leads to the induction of endoderm differentiation. The effect of activin A in inducing definitive endoderm is enhanced when additional factors such as Wnt3a [38] and Noggin [39] are present, or when coupled with the suppression of the phosphoinositide 3-kinase pathway [40]. Induction of definitive endoderm can lead to the generation of specific progenitor populations following the addition of other factors. Among the most successful examples to date is the generation of pancreatic islet progenitors devised by Kroon et al. [41], accomplished through the sequential exposure of hESCs to activin A and Wnt3A, followed by the addition of keratinocyte growth factor or fibroblast growth factor-7 to induce the formation of the primitive gut tube. Subsequently, retinoic

Table 2.1  Examples of methods used to differentiate hESCs into specific cell types Methods to induce differentiation Differentiation factors and/or culture conditions FGF, BMP4, hepatocyte growth factor, Derivation of endodermal Differentiation of hESC into oncostatin M, dexamethasone cells from hESCs definitive endoderm, followed by sequential exposure to Activin A, Wnt3A, keratinocyte growth differentiation factors factor/FGF7, retinoic acid, cyclopamine, noggin Recombinant keratinocyte growth factor Genetic modification of hESCs followed by spontaneous differentiation Human embryoid body formation Serum-free conditions; BMP4 Derivation of mesodermal cells Micromass of dissociated embryoid bodies; BMP2 from hESCs High-density culture of dissociated embryoid bodies; ascorbic acid, dexamethasone Serum-free conditions; bFGF Spin embryoid body formation Serum-free conditions Co-culture with stromal cells Co-culture with stromal cell line M210-B4 for enhanced proliferation of CD34+/CD45+ hematopoietic progenitor cells Dense monolayer of hESCs; activin A, BMP4 Directed differentiation from hESCs by sequential exposure BMP4, BMP4/bFGF/activin A, VEGF/DKK1, to differentiation factors VEGF/DKK1/bFGF Co-culture with primary chondrocytes; poly-d, Directed differentiation from l-lactide scaffold hESCs by the addition of differentiation factors on 3D polymeric scaffolds Cardiac-specific reporter Genetic modification of hESCs followed by spontaneous differentiation Cardiomyocytes [53]

Chondrocytes [72]

Cardiomyocytes [55] Cardiomyocytes [56]

Cardiomyocytes [50] Blood cells [45] T and NK cells [47]

Dendritic cells [46] Chondrocytes [70] Chondrocytes [73]

Lung alveolar cells [68, 69]

Pancreatic islet progenitors [41]

Example of differentiated cells Hepatocytes [42, 43]

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Derivation of ectodermal cells from hESCs

Directed differentiation from hESCs with sequential exposure to differentiation factors Direct differentiation with sequential exposure to differentiation factors on 3D culture with extracellular matrix components

Co-culture with stromal cells and addition of differentiation factors Formation of neural rosettes and addition of differentiation factors

Methods to induce differentiation

Dopaminergic neurons [62] Schwann cells [64]

FGF8, SHh Ciliary neurotrophic factor, neuregulin 1b, dbcAMP Retinoic acid, SHh Withdrawal of FGF2, BDNF; addition of GDNF, NGF, dibutyryl cyclic AMP Serum-free conditions; activin A, nicotinamide B27, thyroid hormone, retinoic acid, FGF2, EGF, insulin BMP4, ascorbic acid

Dopaminergic neurons [59]

FGF8, SHh

Basal keratinocytes [67]

Motor neurons [63] Peripheral sympathetic and sensory neurons [64] Retinal pigment epithelium [66] Oligodendrocytes [65]

Example of differentiated cells

Differentiation factors and/or culture conditions

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acid, cyclopamine, and Noggin are added to inhibit hedgehog and TGFb signaling, and thus induce the differentiation of posterior foregut cells, the source of pancreatic cell progenitors. These are cultured further to generate pancreatic endoderm cells. When these cells are engrafted in immunodeficient mice, they display the histological and structural characteristics of pancreatic islet cells, and are able to sustain insulin production for at least 100 days [41]. In a similar manner, hepatocytes can be obtained after differentiation of hESCs into definitive endoderm [42, 43]. A robust population of functional hepatocytes was generated with the sequential addition of low serum medium, collagen I matrix, and hepatic differentiation factors that include FGF, BMP4, hepatocyte growth factor, oncostatin M, and dexamethasone [43]. These cells expressed known markers of mature hepatic cells, exhibited appropriate function, and were able to integrate and differentiate into mature liver cells when injected into mice with liver injury [43].

2.4.2 Mesodermal Derivatives of hESCs Directing the differentiation of hESCs into mesoderm requires the activation of the TGFb signaling pathway and can be accomplished through the stepwise and dosage-dependent addition of activin A, BMP4, and growth factors, VEGF and bFGF [44]. Mesodermal derivatives have also been successfully obtained by spontaneous differentiation of hESCs through hEB formation without first directing them toward mesoderm. Robust differentiation of hESCs into hematopoietic lineage cells, which give rise to all blood cell types and components of the immune system, has been achieved under serum-free conditions through spin hEB formation [45]. Specific hematopoietic cells, such as functional dendritic cells, have been successfully differentiated from hESCs through spontaneous hEB formation under serum-free conditions with the addition of BMP4 at specific time points [46]. Hematopoietic progenitor cells that give rise to functional T and natural killer cells capable of targeting human tumor cells both in vitro and in vivo have also been derived from hESCs co-cultured with stromal cells [47]. Thus, the ability to differentiate hESCs into hematopoietic lineage cells promises to be useful in improving existing therapies that require blood cell transplantation, and in immune therapies that require induction of the immune response in an antigenspecific manner [48]. Cardiomyocytes, which represent another therapeutically important derivative of mesoderm, have been successfully generated from hESCs using several methods [49]. Through hEB formation, hESCs can spontaneously differentiate into cardiomyocytes under appropriate culture conditions. These cardiomyocytes exhibit morphological, molecular, and electrophysiological properties similar to adult cardiomyocytes [50], and display quantifiable responses to physiological stimuli reminiscent of atrial, ventricular, and pacemaker/conduction tissue [51–54]. Cardiomyocytes have also been generated by directed differentiation with activin A and BMP4 on a dense monolayer of hESCs; these cells successfully form specific

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cardiac lineages when transplanted in  vivo [55]. Another study used additional medium supplements that included VEGF, and the Wnt inhibitor, DKK1, followed by the addition of bFGF to promote cardiomyocyte differentiation from hEBs [56]. Success of these studies was measured by the expression of proteins specific for mature cardiac cells such as cardiac troponin T, atrial myosin light chain 2, and the cardiac transcription factors, Tbx5 and Tbx20.

2.4.3 Ectodermal Derivatives of hESCs The dominant differentiation pathway in hESC cultures leads to the formation of ectoderm, which makes up cells of the nervous system and the epidermis. hESCderived neural progenitor cells are characterized by rosette-like neural structures that form in the presence of growth factors, FGF2 or EGF, through either spontaneous differentiation from an overgrowth of hESCs or after hEBs are plated onto adherent substrates [57, 58]. These neural rosettes have become the signature of hESCderived neural progenitors, capable of differentiation into a broad range of neural cells in response to appropriate developmental signals. Thus, many studies are exploring ways to enhance the formation of neural rosettes in order to generate an enriched population of specific neural cell types. One example is the use of specific stromal cell lines [59]. With this method, stromal cells provide ectodermal signaling factors required for neural induction, as determined in animal model studies, and therefore promote the formation of neural rosettes [60, 61]. The withdrawal of FGF2 and EGF, and the addition of specific compounds can lead to the differentiation of neural rosettes into specific neural subtypes. For example, hESC-derived neural progenitors treated with FGF8 and sonic hedgehog give rise to dopaminergic neurons [62], while treatment with sonic hedgehog and retinoic acid induce motor neuron differentiation [63]. Neural crest stem cells derived from neural rosettes can differentiate into peripheral sympathetic and sensory neurons by withdrawing FGF2/EGF and adding BDNF, GDNF, NGF, and dbcAMP, or into Schwann cells in the presence of CNTF, neuregulin 1b, and dbcAMP [64]. Neuroglial cells, such as oligodendrocytes, are generated with B27, thyroid hormone, retinoic acid, FGF2, epidermal growth factor, and insulin [65]. In 2010, the biotechnology firm, Geron, initiated the first clinical trial with hESCs in the USA using hESC-derived oligodendrocytes to treat acute spinal cord injuries (http://www.clinicaltrials.gov/ct2/archive/NCT01217008). Oligodendrocytes are rapidly lost following acute spinal cord injury leading to demyelination and neuronal loss. In these trials, purified oligodendrocyte progenitor cells derived from hESCs will be injected into the spinal cord of paralyzed patients within 2 weeks of the acute injury. While this first trial is a safety study, the expectation is that these progenitor cells will terminally differentiate into oligodendrocytes and produce myelin, which insulates neuronal cell membranes and is critical for efficient conduction of nerve impulses. Thus, the transplantation of newly differentiated oligodendrocytes is expected to restore myelination of damaged neurons preventing further neuronal death and restoring function.

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Retinal pigment epithelium (RPE) cells are another specific cell type derived from neuroectoderm. These support the neural retina by phagocytosing and renewing the photoreceptor outer segments of rhodopsin. Recent reports have shown that RPE can be induced from hESCs in the presence of nicotinamide and activin A under serum-free conditions [66]. hESC-derived pigmented cells exhibit the morphological and functional properties of RPE cells after transplantation in an animal model of macular degeneration, a disease caused by dysfunction and loss of RPE. These data have led to the second and third clinical trials using hESCs by the biotechnology company, Advanced Cell Technology. For these trials, hESC-derived RPEs will be transplanted directly into the degenerating retinae of patients with Stargardt’s Macular Dystrophy, a juvenile form of macular degeneration, or Dry Age-Related Macular Degeneration, to rescue visual acuity. The launch of these clinical trials heralds the translation of hESC research into therapy for neurodegenerative disease.

2.5 The Promise of hESCs in Tissue Engineering Tissue engineering and regeneration utilize biological substitutes to restore or maintain tissue function. As with cell transplantation, a successfully engineered tissue depends on the generation of the appropriate cell type that is able to provide normal cellular function. Thus, cells suitable for tissue engineering should have the ability to enter a desired differentiation program to produce a specific cell type, and be expandable in vitro to meet the needs of cell transplantation. hESCs provide much promise in tissue engineering and regeneration since hESCs can act as an inexhaustible in  vitro source of differentiated cell types. The potential use of hESCs in tissue engineering include, but are not limited to, organ substitutes, vascularization, and ex vivo cartilage/bone construction. While these applications are discussed in detail in subsequent chapters, brief examples are provided below. Basal keratinocytes, the cells that make up the pluristratified epidermal layer of the skin, have been successfully differentiated from hESCs. Guenou et al. [67] have shown that long-term culture of hESCs in defined medium supplemented with BMP4 and ascorbic acid leads to the directed differentiation of hESCs into basal keratinocytes. These cells express keratins 14 and 5, a6- and b4-integrins, collagen VII, and laminin 5 at levels comparable to postnatal keratinocytes. More importantly, these hESC-derived keratinocytes form a cohesive pluristratified epidermis when placed in 3D culture or when engrafted into immunodeficient mice. These findings prove the feasibility of using hESC-derived keratinocytes as a source of allograft for patients requiring skin restoration. The use of hESCs to treat lung injury has also been an area of active investigation. A significant step toward directed differentiation of lung-specific cells was reported by Wang et al. [68, 69], in which genetically modified hESCs carrying lung-specific reporters under the control of promoters from tissue-specific genes such as surfactant

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protein C, aquaporin 5, and T1a, resulted in the purification of type I and type II alveolar epithelial cells. When engrafted into mice suffering from acute lung injury, these cells terminally differentiated in vivo into type I and type II alveolar epithelial cells and exhibited functional properties that include the capacity for gas exchange and histological amelioration of lung injury. hESCs readily form connective tissue, such as bone or cartilage, as can be appreciated from teratoma formation assays (Fig. 2.3). Thus, hESCs are a valuable source of cells suitable for connective tissue replacement therapy for a number of bone and joint diseases, such as osteoarthritis, which is characterized by the breakdown of cartilage within joints. Most successful and efficient protocols for directing chondrocyte differentiation from hESCs utilize 3D culture systems created by seeding hESCs at high density leading to the formation of a pellet, or by introducing the cells into a synthetic 3D scaffold. Such systems enable cell–cell signaling between the undifferentiated hESCs and mature chondrocytes to stimulate homogeneous and sustained chondrogenic differentiation. For example, single-cell suspension of dissociated hEBs cultured as high-density micromass with BMP2 leads to efficient chondrocyte formation [70]. hESCs co-cultured with primary chondrocytes, or in the presence of osteogenic supplements and polymeric scaffolds, yield cartilaginous- or osteogenic-like cells [71, 72]. More recently, feeder-free 3D culture systems have successfully derived multipotent connective tissue progenitors from hESCs yielding tendon-like structures [73]. The engraftment of these in vitro differentiated tendon structures in injured immunosuppressed mice restored ankle joint movements that rely on an intact Achilles tendon [73]. Furthermore, there is evidence that transplanted chondrogenic cells may exert a stimulatory effect through paracrine mechanisms that promote growth and repair of endogenous cells [74].

2.6 Current Challenges As discussed above, cell therapy with hESCs has begun to enter clinical trials. The International Stem Cell Banking Initiative has been created by the International Stem Cell Forum, a group of national and international stem cell research funding bodies, to develop a set of best practices and principles when banking, testing, and distributing hESCs for clinical application [75]. In the USA, the Food and Drug Administration also monitors these guidelines and has issued recommendations for reviewers of proposals for clinical trials of stem cell therapy (http://www.fda.gov/ BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/ Xenotransplantation/ucm074131.htm). It is important to note that these recommendations do not ensure the quality or efficacy of hESC-derived cells used for clinical application. Rather, these guidelines warrant that the cells used for therapy are reproducible and meet specific criteria to ensure patient safety (Table  2.2). The major safety concerns for the use of hESCs are discussed in the following sections.

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Table 2.2  Requirements for standardization and optimization of hESCs for clinical use Important factors Examples of test methods Cell line identity: must match Short tandem repeat (STR) testing or human leukocyte all alleles of parent cell line antigen (HLA) testing Sterility and pathogen screening Bacteria/fungi/mycoplasma testing by microbiological culture; qPCR analysis for murine viral short interspersed elements (SINE) Genetic/chromosomal stability Analysis of multiple single nucleotide polymorphisms (SNP); karyotype by G-band analysis of 20 metaphase spreads or fluorescent in situ hybridization Epigenetic stability MicroRNA profiling, methylation analysis, X-inactivation Pluripotency Formation of teratomas in immunodeficient mice; flow cytometry to determine hESC-specific antigens such as SSEA-3/4, TRA-1-60, TRA-1-81 Quality and differentiation Gene expression profiling by DNA microarray or qPCR ability analysis to analyze expression of markers of pluripotency or differentiated cell types; ability to form embryoid bodies Functional assays Report on potency, efficacy, and lot-to-lot variability

2.6.1 Xenobiotic-Free Conditions Many of the hESC lines currently in use have been exposed to animal products during isolation of the inner cell mass and propagation of hESCs in vitro. Under these conditions, hESCs could possess animal viruses and other unknown substances capable of eliciting a detrimental immune response in transplanted hosts. Currently, hESC lines under development for clinical use undergo extensive microbiological testing as strictly recommended by the International Stem Cell Banking Initiative. In the USA, the Food and Drug Administration legally requires documentation of the source, potential genetically modified components, and pathogenic agents in any hESCderived cell intended for therapeutic use. Thus, avoiding exposure to xenobiotics is emphasized by law. Recently, replacement media have been developed that would allow maintenance of hESCs in xenobiotic-free conditions. These include xenobioticfree serum replacements such as knockout serum replacer (KSR; Invitrogen) or xenobiotic-free culture media such as HESGRO (Millipore) or TeSR (STEMCELL). Feeder-free culture systems are now being developed to reduce the risk of contamination with foreign agents when hESCs are cultured on animal feeder cell layers. Feeder-free and xenobiotic-free, defined culture media that consist of a combination of recombinant growth factors known to inhibit differentiation and maintain hESCs in the pluripotent state are now commercially available. However, some reports have associated feeder-free culture conditions with greater chromosomal instability and an increased risk of propagating genetically altered hESCs [76]. For this reason, most hESC laboratories practice a surveillance program for genomic instability in cultured lines [36, 53].

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hESC lines derived using human feeder cells have been reported. For example, hESC lines have been successfully derived on human fibroblasts generated from neonatal foreskin [77, 78] and adult skin fibroblasts [79]. Some laboratories deriving new lines have moved exclusively to xenobiotic-free conditions [80]. The ability to derive and maintain new hESC lines using human fibroblast feeder cells represents a significant step toward generating clinical-grade hESCs.

2.6.2 Genetic Abnormalities in hESC Lines The best characterized hESC lines to date are among the earliest lines derived. However, they may not be the best lines for therapeutic applications as many of these lines were derived using animal products. Chromosomal and genomic instability has been detected among several hESC lines, including loss of heterozygosity or copy-number variation in cancer-related genes [81, 82]. Many of these mutations appeared to be induced by prolonged culture, since these changes were not observed in low passage cells. It has been proposed that such karyotypic aberrations occurred with adaptation to the original culture conditions used when the first few lines were being derived and expanded [83]. These observations emphasize the need for complete characterization of hESC lines, particularly the effects of long-term culture, and the design of guidelines for designating therapeutic-grade hESCs.

2.6.3 Enrichment, Directed Differentiation, and Purification Protocols for hESCs A primary safety concern when using pluripotent hESCs is their potential to form germ layer tumors. As discussed above, in vivo transplantation of undifferentiated hESCs in immunodeficient mice results in teratoma formation. Evidence of tumorlike growths has also been observed in differentiated hESC derivatives transplanted in vivo [84, 85]. Thus, it is essential that candidate hESC derivatives intended for use in cell transplantation are free of tumorigenic cells. Another concern is the differentiation of hESC-derived cells into unwanted cell types. For example, the engraftment of inappropriate muscle cells into the myocardium could alter the electrical activity of recipient tissue, provoking arrhythmias [86]. Thus, developing and further optimizing differentiation and purification protocols are necessary to minimize the generation of unwanted cell types for preclinical transplantation experiments and clinical therapy. As discussed earlier in this chapter, enrichment of specific cell types can be achieved using molecules introduced at specific time points during culture. However, many of these methods yield only moderate enrichment that is not yet scalable for clinical application. It may be desirable to enrich first for partially differentiated, proliferative hESC intermediates with specific cell fates. These could then be

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expanded before further differentiation into cells for therapy. For example, the expression of the cell surface antigen, CD133, on proliferating hESCs identifies cells predestined toward a neuroectodermal fate [34]. CD133-positive cells have been selected from cultures of undifferentiated hESCs, and have been observed to differentiate primarily into neuroectodermal cells in vitro and in vivo [34]. In the absence of specific cell surface antigens such as CD133 to identify tissuespecific precursors, molecular beacons have been used to select for specific subpopulations of hESCs. King et al. [33] first demonstrated the utility of this system in isolating live Oct4-expressing pluripotent hESCs in a specific and high-throughput manner. Molecular beacons are single-stranded oligonucleotides that generate fluorescent signals when bound to their target mRNAs, making these cells detectable and selectable by fluorescence-activated cell sorting. More importantly, molecular beacons have a short lifespan within cells and do not alter the function or genomic structure of hESCs. Thus, this method can be used to enrich for desired hESCderived cell populations or used to select against unwanted cell types, such as undifferentiated hESCs that could form tumors.

2.6.4 Circumventing Immune Rejection Using Transplanted hESC-Derived Cells Transplanted hESCs encounter immune rejection [87] because proliferating and differentiated hESCs express class I and II HLA as well as minor histocompatibility antigens at levels sufficient to activate the immune system [87, 88]. Another potential barrier to hESC engraftment can occur through mismatch between donor hESC and recipient ABO blood group antigens. While studies to determine the effects of ABO incompatibility on hESC transplantation are still lacking, this has long been a criterion for successful organ transplantation and thus, it is likely that ABO incompatibility between hESC-donor cells and the recipient would also trigger immune rejection. Ideally, having genetically identical donor and patient cells is the best way to circumvent immune rejection. Thus, there is high interest in developing and using somatic cell nuclear transfer to generate patient-specific hESC lines. Using this technique, the DNA obtained from either a patient’s skin or muscle cell would be transferred into an unfertilized egg that has had its DNA removed. Subsequently, the egg is artificially fertilized and allowed to develop until it reaches the blastocyst stage to derive hESCs. The resultant hESC line would have an immunologic profile matching the patient and could be used for cell therapy. This technique has been conducted successfully in animals using species-specific ESCs, but the bona fide derivation of hESCs through somatic cell nuclear transfer has not yet been reported. Another strategy is to generate hESC lines with the closest match to potential transplant patients. Suggestions have included engineering “universal donor hESCs,” a blood antigen O cell in which the expression of HLA is suppressed, or chimeric hematopoietic cells derived from hESCs capable of inhibiting the immune response when co-transplanted with the desired hESC-derived cells [89]. Alternatively, creating

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hESC banks that store lines representing HLA/ABO combinations that match the majority of potential patients has been proposed. Studies have provided estimates on how many hESC lines would be needed in order to support the needs of a specific population. Taylor et al. [90] estimated that approximately 150 hESC lines could provide an HLA match for most of the population in the United Kingdom. Alternatively, approximately ten parthenote-derived hESC lines that are homozygous for HLA types could be sufficient for a majority of the population. Studies by Nakajima et al. [91] estimated that approximately 170 hESC lines, or 55 hESC lines with homozygous HLA types, would be sufficient for 80% of patients in the Japanese population. These findings demonstrate the feasibility of creating and maintaining a hESC bank with sufficient representation to support a large number of patients. However, in countries such as the USA, many more hESC lines would need to be established to serve its ethnically and genetically diverse population. Given the ethical issues and restrictions on hESC research, and the small number of approved hESC lines currently available, the creation of a hESC bank with a highly diverse collection of cell lines will undoubtedly face enormous challenges.

2.7 Conclusions Research on hESCs has progressed significantly since their first derivation in 1998. The international scientific community has discovered the enormous potential of hESCs as newly derived lines continue to be developed, and differentiation methods into various types of cells are optimized for scientific investigation and clinical use. It is clear that there are still major scientific challenges as well as ethical and legislative issues that must be addressed, especially in the USA. Certainly more questions will emerge as more is understood in the coming years. However, it is encouraging to see that clinical trials involving the use of hESCs in spinal cord injury and macular degeneration have begun. These studies will pave the way toward determining the therapeutic benefit of hESCs in regenerative medicine. Acknowledgments  The authors thank members of the Bernstein Laboratory for helpful discussion. H.S.B. is supported by grants from the National Institutes of Health, the California Institute for Regenerative Medicine, and the Muscular Dystrophy Association. O.Y. is supported by a fellowship from the National Institutes of Health.

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57. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T (2001) Neural progenitors from human embryonic stem cells. Nat Biotechnol 19(12):1134–1140 58. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19(12):1129–1133 59. Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci USA 101(34):12543–12548 60. Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa SI, Sasai Y (2000) Induction of midbrain dopaminergic neurons from ES cells by stromal cellderived inducing activity. Neuron 28(1):31–40 61. Kawasaki H, Suemori H, Mizuseki K, Watanabe K, Urano F, Ichinose H, Haruta M, Takahashi M, Yoshikawa K, Nishikawa S, Nakatsuji N, Sasai Y (2002) Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity. Proc Natl Acad Sci USA 99(3):1580–1585 62. Yan Y, Yang D, Zarnowska ED, Du Z, Werbel B, Valliere C, Pearce RA, Thomson JA, Zhang SC (2005) Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23(6):781–790 63. Li XJ, Du ZW, Zarnowska ED, Pankratz M, Hansen LO, Pearce RA, Zhang SC (2005) Specification of motoneurons from human embryonic stem cells. Nat Biotechnol 23(2):215–221 64. Lee G, Kim H, Elkabetz Y, Al Shamy G, Panagiotakos G, Barberi T, Tabar V, Studer L (2007) Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat Biotechnol 25(12):1468–1475 65. Nistor GI, Totoiu MO, Haque N, Carpenter MK, Keirstead HS (2005) Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. Glia 49(3):385–396 66. Idelson M, Alper R, Obolensky A, Ben-Shushan E, Hemo I, Yachimovich-Cohen N, Khaner H, Smith Y, Wiser O, Gropp M, Cohen MA, Even-Ram S, Berman-Zaken Y, Matzrafi L, Rechavi G, Banin E, Reubinoff B (2009) Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells. Cell Stem Cell 5(4):396–408 67. Guenou H, Nissan X, Larcher F, Feteira J, Lemaitre G, Saidani M, Del Rio M, Barrault CC, Bernard FX, Peschanski M, Baldeschi C, Waksman G (2009) Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study. Lancet 374(9703):1745–1753 68. Wang D, Haviland DL, Burns AR, Zsigmond E, Wetsel RA (2007) A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells. Proc Natl Acad Sci USA 104(11):4449–4454 69. Wang D, Morales JE, Calame DG, Alcorn JL, Wetsel RA (2010) Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice. Mol Ther 18(3):625–634 70. Toh WS, Yang Z, Liu H, Heng BC, Lee EH, Cao T (2007) Effects of culture conditions and bone morphogenetic protein 2 on extent of chondrogenesis from human embryonic stem cells. Stem Cells 25(4):950–960 71. Bielby RC, Boccaccini AR, Polak JM, Buttery LD (2004) In vitro differentiation and in vivo mineralization of osteogenic cells derived from human embryonic stem cells. Tissue Eng 10(9–10):1518–1525 72. Vats A, Bielby RC, Tolley N, Dickinson SC, Boccaccini AR, Hollander AP, Bishop AE, Polak JM (2006) Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment. Tissue Eng 12(6):1687–1697 73. Cohen S, Leshansky L, Zussman E, Burman M, Srouji S, Livne E, Abramov N, Itskovitz-Eldor J (2010) Repair of full-thickness tendon injury using connective tissue progenitors efficiently derived from human embryonic stem cells and fetal tissues. Tissue Eng Part A 16(10):3119–3137 74. Toh WS, Lee EH, Guo XM, Chan JK, Yeow CH, Choo AB, Cao T (2010) Cartilage repair using hyaluronan hydrogel-encapsulated human embryonic stem cell-derived chondrogenic cells. Biomaterials 31(27):6968–6980

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Chapter 3

Current Status of Induced Pluripotent Stem Cells Thach-Vu Ho, Grace Asuelime, Wendong Li, and Yanhong Shi

Abstract  The discovery of induced pluripotent stem cells (iPSCs) has “spiced up” the stem cell research field in the last few years. It has made tremendous progress in a very short time by demonstrating that adult fibroblasts could be reprogrammed into iPSCs using pluripotency factors. This suggested that cell fates are not as permanent as initially thought, but rather possess a degree of plasticity. Unsurprisingly, induced pluripotent stem cell technology still faces many technical obstacles before safe and high-quality human iPSCs can be generated for therapeutic applications. This chapter examines the current status of iPSC technology and new methods for ­inducing pluripotency and its use in modeling human disease.

Abbreviations iPSCs ESCs ICM SCNT MEFs OSKM

Induced pluripotent stem cells Embryonic stem cells Inner cell mass Somatic cell nuclear transfer Mouse embryonic fibroblasts Oct4, Sox2, Klf4, and c-Myc

Y. Shi (*) Department of Neurosciences, Center for Gene Expression and Drug Discovery, Beckman Research Institute of City of Hope, Duarte, CA, USA e-mail: [email protected] H.S. Bernstein (ed.), Tissue Engineering in Regenerative Medicine, Stem Cell Biology and Regenerative Medicine, DOI 10.1007/978-1-61779-322-6_3, © Springer Science+Business Media, LLC 2011

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3.1 Introduction Stem cells are a unique population of cells that possess the ability to self-renew and differentiate into multiple lineages. ESCs are derived from the ICM of the blastocyst [1]. Cells isolated from ICM have the ability to differentiate into any cell type derived from the three germ layers (Fig. 3.1a), but are unable to generate extraembryonic tissues and thus lack the capacity to become a complete organism [2]. This restriction in cell potency suggests that ESCs are pluripotent but not totipotent. Nonetheless, ESCs can be differentiated into many tissue-specific cells, which hold promise for tissue and stem cell replacement therapies. Derivation of stem cells via SCNT or therapeutic cloning presents a potential advantage over ESCs for clinical use, since donor-derived stem cells could theoretically be used to treat human disease without fear of immune rejection. SCNT is the introduction of a nucleus from a somatic cell into an enucleated oocyte [3]. After inoculation, the oocyte is coaxed into becoming a fertilized embryo (Fig.  3.1b). ICM is removed at the blastocyst stage and is allowed to differentiate into tissue-specific cells in  vitro. Evidence indicates that SCNT stem cells are indistinguishable from ESCs, supporting the therapeutic potential of SCNT stem cells [4]. Unfortunately, ESCs and SCNT stem cells also carry limitations in therapeutic application. Evidence shows that ESC transplantation can evoke an immune response due to the lack of patient specificity [5]. While SCNT stem cells may be donor-specific, and therefore bypass immune rejection, SCNT stem cells have not yet been achieved in humans despite significant effort. It is also important to point out that the efficiency of SCNT is low due to epigenetic changes during the cloning process [4, 6]. Moreover, the use of SCNT to make ESCs requires the generation of possibly viable embryos that raises ethical concerns for many people. For these reasons, stem cell researchers have been exploring alternative approaches that would eliminate the use of human embryos. A major breakthrough in the stem cell field came when Takahashi and Yamanaka demonstrated the conversion of somatic cells to pluripotent stem cells using four transcription factors, OSKM [7] (Fig.  3.1c). Thereafter, several studies have ­verified that iPSCs could be derived using various combinations of transcription factors and have demonstrated the use of only one or two factors to induce ­pluripotency [8–13]. The ability of iPSCs to differentiate into multiple lineages and give rise to chimeric mice suggested that iPSCs are functionally similar to ESCs [14]. Moreover, iPSCs could lead to patient-specific cell lines, which would overcome concerns of immune rejection. The aim of this chapter is to review the current status of iPSC technology and discuss the challenges that iPSCs face before their application in the clinical setting.

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Fig. 3.1  A schematic overview of the generation of pluripotent stem cells. (a) Embryonic stem cells are derived from the inner cell mass of a fertilized egg in  vitro. (b) In somatic cell nuclear transfer, the nucleus of the somatic cell is inserted into an enucleated egg. The nucleus is stimulated to develop into a blastocyst. (c) Adult somatic cells are reprogrammed by overexpression of defined factors into induced pluripotent stem cells. Pluripotent stem cells can differentiate into tissue-specific cells of the three germ layers

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3.2 Molecular Profiling of iPSCs Since the advent of iPSCs, many studies have examined the properties of iPSCs, hoping to identify the key factors that regulate pluripotency and improve the reprogramming process [8, 15, 16]. Several studies have compared iPSCs and ESCs at the genomic level to determine whether iPSCs and ESCs are distinct at the molecular level [17–20]. While no study has yet proven that iPSCs are functionally equivalent to ESCs, many studies have shown that iPSCs may be very similar to ESCs [14]. To be ESC-like, iPSCs have to demonstrate self-renewal and several pluripotency criteria. The most stringent criterion of pluripotency is the yield of live organisms through tetraploid complementation [21]. Recent work demonstrated that iPSCs have passed the pluripotency criteria by generating live mice through tetraploid complementation assay [22–24]. While iPSCs exhibit ESC-like characteristics (Fig. 3.2), ESCs and iPSCs have subtle differences in gene expression profiles, epigenetic modification, and mitochondrial regulation [16–20]. Recent gene expression data demonstrated that iPSCs and ESCs may be distinguishable at the gene expression level [17, 20]. Data from Chin et al. [17] suggested that the gene expression profile of late-passage human iPSCs (beyond 37 passages) was highly correlated to human ESCs while the gene expression profile of earlypassage human iPSCs (less than 5 passages) was significantly different from that of human ESCs [17]. This observation suggests that reprogramming is a gradual process and that passage numbers must be taken into consideration when evaluating iPSCs.

Fig. 3.2  A general comparison between ESCs and iPSCs. Many studies have demonstrated that ESCs and iPSCs have common as well as distinct characteristics. The significance of these similarities and differences to tissue regeneration remain to be elucidated

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Human iPSCs are also distinguished from human ESCs at the epigenetic level [25, 26]. Experimental data suggested that the epigenetic variations observed between iPSCs and ESCs may have been caused by incomplete reprogramming of human somatic cells to human iPSCs [17, 19, 25]. In fact, the methylation pattern of CpG islands in human iPSCs can be significantly different from the parent somatic cells and ESCs [25]. This suggests that certain loci are incompletely reprogrammed. Ghosh et al. [19] proposed that human iPSCs have the ability to retain “transcriptional memory” of donor cells. Based on evidence from mice, Kim et al. [16] similarly suggested that iPSCs retain “epigenetic memory” of their tissue donors. DNA methylation is an important criterion for comparison of iPSCs and ESCs, and the state of histone H3 lysine 27 trimethylation and histone H3 lysine 4 trimethylation is commonly used to analyze chromatin modification in pluripotent stem cells. Through pairwise comparison of genes occupied by trimethylated histone H3 lysine 27 and lysine 4 in human iPSCs and human ESCs, Guenther et al. [20] found that human iPSCs were not significantly different from human ESCs for genes studied by this method. Data from Chin et al. [17] provided similar observation on histone H3 lysine 27 trimethylation within promoter regions and concluded that human ESCs and human iPSCs were nearly identical in their histone methylation pattern. Thus far, the question as to whether iPSCs and ESCs are equivalent is clear. They are not equivalent, but rather quite similar.

3.3 Progress in Reprogramming the Pluripotent State iPSC technology has exciting potential for disease modeling, drug development, and toxicity screening as previously mentioned. Nonetheless, the molecular mechanism of reprogramming remains elusive and it is only with further understanding of the cellular pathways involved in reprogramming that iPSC technology can possibly move from the bench to the clinic. In recent years, much effort has been focused on refining reprogramming methodologies. For example, iPSC technology needs to overcome the low reprogramming efficiency observed using Yamanaka’s original method, along with finding suitable viral-free methods for iPSCs’ derivation before moving toward clinical application. Viral-mediated transduction is the most used method so far for delivering the reprogramming transcription factors into somatic cells. One concern regarding the use of viral vector-mediated transduction is the integration of viral DNA into the host genome. Permanent integration of viral DNA can result in the development of cancer and also holds the possibility of being passed through the germ line [27]. As such, the expression of retroviral transgenes for OSKM may hold serious consequences as recently reported [13, 28]. Several groups, however, have successfully generated iPSCs using plasmids, piggyBac transposons, adenoviruses, recombinant proteins, and synthetic mRNAs [29–33]. Despite the fact that virus-free iPSCs can be generated, the efficiency remains extremely low (0.0001–0.1%). Reprogramming efficiency is influenced by many variables, some of which may be unknown. Several groups have attempted to remove or substitute the four transcription

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factors with small molecules [8, 10, 13]. However, it is important to note that certain factors such as Oct4 cannot be excluded, confirming that Oct4 is a key regulator of pluripotency. For example, Shi et al. [10] demonstrated that mouse embryonic fibroblasts could be induced to iPSCs by Oct4, Klf4, and small molecules, BIX and BayK. BIX and BayK are specific inhibitors of histone methyltransferase G9a and L-type calcium channel, respectively [10]. The authors concluded that BIX/BayK improved the reprogramming efficiency. Similarly, Huangfu et  al. [8] increased reprogramming efficiency (~1%) of human fibroblasts with a combination of Oct4, Sox2, and valproic acid, a histone deacetylase inhibitor. Recently, iPSCs were generated from MEFs with only Oct4 and small molecules (tranylcypromine, valproic acid, CHIR99021, and 616452) [13]. The Oct4-iPSCs were capable of germ line transmission in chimera formation, which was indicative of pluripotency. The authors argued that tranylcypromine (H3K4 demethylation inhibitor), valproic acid, CHIR99021 (GSK3-b inhibitor), and 616452 (TGF-b inhibitor) improved reprogramming efficiency by reducing epigenetic barriers [13]. Meanwhile, reprogramming efficiency was reported to improve as much as 50-fold with sodium butyrate treatment leading to a reprogramming efficiency of 15–20% [34]. In these studies, Mali and colleagues virally transduced IMR90 fibroblasts with OSKM followed by small-molecule treatment (RG108, BIX01294, valproic acid, sodium butyrate) 2 days after viral transduction. They observed that sodium butyrate facilitated epigenetic changes and stimulated pluripotency-associated genes in iPSCs. Sodium butyrate also efficiently stimulated reprogramming using a PiggyBac transposon delivery system [34]. More recently, human iPSCs were generated using Oct4 and chemical compounds, including sodium butyrate [35]. Small molecules may provide an important new path to reprogramming since these molecules can sufficiently replace the expression of one or more of the originally described reprogramming transcription factors.

3.4 iPSCs as Models of Disease The fundamental goal of regenerative medicine is to replace or restore normal function to damaged or aged and/or congenitally defective human cells, tissues, or organs. Animal models have been extensively used despite the fundamental differences and limitations compared to humans [36–38]. This raises concerns as to whether animal models accurately predict the effectiveness of the proposed therapies [37]. iPSCs have several advantages, including an abundant source of cells (self-renewal), ability to generate tissue-specific cell types (differentiation), and ability to generate autologous cell lines. Nonetheless, Saha and Jaenisch [39] reiterated the difficulty of using iPSCs to study human diseases in culture. The disease progression in the patient is much more dynamic than any model can possibly mimic. Therefore, Saha and Jaenisch suggested that the progression of the disease can be accelerated by exposing human iPSCs to environmental stimulus, such as oxidative stress. Moreover, modulating culture conditions to mimic the microenvironment similar in the patient may be essential when using iPSCs to model human diseases.

3  Current Status of Induced Pluripotent Stem Cells Table 3.1  Disease-specific iPSCs Disease Sickle cell anemia Amyotrophic lateral sclerosis Adenosine deaminase deficiency-related severe combined immunodeficiency Shwachman-Bodian-Diamond syndrome Gaucher disease type III Duchenne and Becker muscular dystrophy Parkinson’s disease Huntington disease Juvenile-onset, type 1 diabetes mellitus Down syndrome (trisomy 21) Lesch-Nyhan syndrome Acute myocardial infarction Spinal muscular atrophy Fanconi anemia Myeloproliferative disorder LEOPARD syndrome Angelman and Prader–Willi syndromes Rett syndrome Fabry disease Globoid cell leukodystrophy Mucopolysaccharidosis VII Long QT syndrome Inherited metabolic disorders of the liver and other liver diseases Lung diseases Fragile X syndrome Bombay blood group Vascular disease

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Stem cell origin Mouse Human Human

Reference [59] [60] [61]

Human Human Human Human, rats Human, monkey Human Human Human Mouse Human Human Human Human Human Human Mouse Mouse Mouse Human Human

[61] [61] [61] [48, 61–63] [61, 64] [61, 65] [61] [61] [66] [67] [68] [69] [41] [47] [70] [71] [71] [71] [72] [73, 74]

Human Human Human Human

[75] [76] [77] [78]

To date, iPSC technology has already been used to generate disease-specific iPSC lines (Table 3.1). For example, LEOPARD syndrome, also known as multiple lentigines syndrome, is an autosomal dominant condition [40] caused by a missense mutation in the nonreceptor protein tyrosine phosphatase type 11 gene [41]. The clinical manifestations in LEOPARD syndrome patients may include hypertrophic cardiomyopathy, multiple lentigines, and deafness [42]. Two common mutations associated with LEOPARD syndrome are Y279C and T468M. Clinical testing for LEOPARD syndrome involves sequence analysis of the nonreceptor protein tyrosine phosphatase type 11 gene after clinical manifestations have been established. Currently, there is no cure for LEOPARD syndrome. Recent studies have established human iPSC lines with a heterozygous T468M mutation from two patients [41]. Carvajal-Vergara and colleagues derived human iPSC lines from the fibroblasts of two LEOPARD syndrome patients using OSKM. They demonstrated that these human iPSC lines were able to differentiate into three germ layers. Hypertrophic cardiomyopathy is manifested in a majority of LEOPARD

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syndrome patients. Data from iPSC studies revealed that human iPSCs were able to differentiate into hematopoietic and cardiac lineages and demonstrated that LEOPARD syndrome iPSC-derived cardiomyocytes showed similar hypertrophic features when compared to wild-type iPSC-derived and human ESC-derived cardiomyocytes [41]. The generation of functional cardiomyocytes from human iPSCs also supports their potential for cardiomyocyte replacement therapy for myocardial infarction, which is characterized by the loss of cardiomyocytes due to the imbalance of blood supply [43]. However, the current inefficient processes for reprogramming patient-specific cells have to be improved before this becomes a therapeutic reality. Neurodegenerative diseases represent another area of research that requires disease-specific models to study the mechanism underlying the disease. Most neurodegenerative diseases in humans, including Angelman and Prader–Willi syndromes, are caused by genetic mutations. Angelman syndrome is characterized by a mutation in the UBE3A gene that is expressed only from maternal chromosomes. In contrast, Prader–Willi syndrome is distinguished by the loss of paternal expression of SNORD116 snoRNAs [44]. Studies demonstrated that both Angelman and Prader– Willi syndromes are caused by the deletion or lack of expression of seven genes on chromosome 15q11–15q13 [45]. Clinical manifestations of Angelman syndrome include developmental delay, movement disorder, speech disorder, and behavior problems [46]. Children with Prader–Willi syndrome, on the other hand, experience obesity, hypogonadism, short stature, and mental retardation [45]. Currently, there is no specific treatment for patients with Angelman and Prader–Willi syndromes and no autologous disease model with which to test potential therapies. Recently, Chamberlain et  al. [47] generated human iPSCs from children with Angelman and Prader–Willi syndromes using retroviral vectors expressing OSKM and LIN28. Additionally, they assessed DNA methylation to screen for epigenetic changes in the resulting iPSC lines. Both Angelman and Prader–Willi syndrome human iPSC lines showed normal DNA methylation patterns compared to control human iPSCs. The group also demonstrated that Angelman syndrome iPSCs differentiated into functional neurons in  vitro. This is an important step toward designing an Angelman syndrome human iPSC model. They also explored the regulatory mechanism of UBE3A in Angelman syndrome human iPSCs. Within the last few years, several iPSC models have been derived from patient fibroblasts, including ones for Parkinson’s disease [48], familial dysautonomia [49], and several others, to study the mechanisms and explore novel compounds for treatment. However, many more human diseases await mechanistic elucidation using iPSCs. Another application for iPSC technology is in drug efficacy and toxicity screening. Current technologies allow screening and evaluating thousands of compounds to target human diseases [50]. Increased knowledge of biological networks allows pharmaceutical companies to narrow the number of targets and relevant compounds for specific human diseases [51, 52]. However, nonhuman models used for current screening provide inefficient reaction mimicry of the compounds in humans [36, 50]. Disease-specific iPSC lines would alleviate this problem by allowing direct analysis of compound efficacy in a human system. For example, Lee et  al. [49]

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derived patient-specific familial dysautonomia iPSCs to study the disease mechanism. Familial dysautonomia is caused by gene mutations in the IkB kinase complexassociated protein, which results in the degeneration of sensory neurons [53]. Lee and colleagues used three parameters, mutant IkB kinase complex-associated protein splicing, neurogenesis, and diseased iPSC-derived neural crest precursor function, to monitor the effects of drug treatment. This study suggested that the potential of disease-specific iPSC model for drug screening is feasible.

3.5 Bypassing the Pluripotency State In recent years, several groups have explored the idea of direct conversion (or induced transdifferentiation) to generate tissue-specific cells. For example, rat exocrine pancreatic cells were treated with leukemia inhibitory factor and epidermal growth factor in vitro to generate insulin-producing beta cells [54]. Shortly after, Zhou and colleagues found that the expression of transcription factors Ngn3, Pdx1, and Mafa could reprogram pancreatic exocrine cells to b-cells in adult mice in  vivo [55]. Recently, mouse tail fibroblasts were converted to neurons capable of generating action potentials and forming functional synapses using the transcription factors Ascl1, Brn2 (also known as Pou3f2), and Myt1l [56]. Most recently, Ieda et al. [57] examined whether the key regulators of cardiac development could directly convert cardiac fibroblasts into cardiomyocytes. They found that three transcription factors, Gata4, Mef2c, and Tbx5, were able to convert mouse cardiac fibroblasts and dermal fibroblasts into cardiomyocytes in vitro and in vivo while maintaining a global gene expression pattern intermediate between the ICM and endogenous cardiomyocytes. They also demonstrated that the transdifferentiation of functional beating cardiomyocytes was more rapid and up to 20% more efficient then iPSC reprogramming [57]. While these groups successfully provided evidence for the potential of transdifferentiated cells in animal models, Szabo et al. [58] were the first to illustrate this concept in human cells. Through the transduction of human fibroblasts with lentivirus expressing Oct4 and cultured with cytokine supplements known to support hematopoietic progenitor development, they were able to derive multipotent hematopoietic progenitors that give rise to the myeloid, erythroid, and megakaryocytic lineages [58]. Concurrently, they verified that the conversion does not require passage through a pluripotent stem cell state [58]. To date, the advantages of using directly converted cells for therapy include, but are not limited to, higher induction efficiency and lower risk of tumorigenesis. It is important to consider that further verification of the similarities between transdifferentiated cells and primary tissue-specific cells is required, as well as an appreciation that unlike stem cells, adult cells cannot easily be expanded and may require larger numbers of primary cells.

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3.6 Conclusion iPSCs hold much promise for therapy, as well as for drug screening and disease modeling, especially when one considers the ethical and immunological complications associated with ESC and SCNT use. Currently, iPSC technology is at an early stage of development, but is making rapid progress. The future of iPSC technology has enormous potential, but as with many approaches using stem cells, more work is needed to bring iPSC technology to therapeutic application. Acknowledgments  We apologize to colleagues whose work could not be cited due to space limitations. T.H. is supported by a stem cell research internship program of the California Institute for Regenerative Medicine and California State University at Long Beach. W.L. is supported by a postdoctoral fellowship from the California Institute for Regenerative Medicine. Y.S. is supported by the National Institutes of Health/NINDS (R01 NS059546 and RC1 NS068370) and the California Institute for Regenerative Medicine (TR2-01832).

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71. Meng XL, Shen JS, Kawagoe S, Ohashi T, Brady RO, Eto Y (2010) Induced pluripotent stem cells derived from mouse models of lysosomal storage disorders. Proc Natl Acad Sci USA 107:7886–7891 72. Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flugel L, Dorn T, Goedel A, Hohnke C, Hofmann F, Seyfarth M, Sinnecker D, Schomig A, Laugwitz KL (2010) Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N Engl J Med 363:1397–1409 73. Rashid ST, Corbineau S, Hannan N, Marciniak SJ, Miranda E, Alexander G, Huang-Doran I, Griffin J, Ahrlund-Richter L, Skepper J, Semple R, Weber A, Lomas DA, Vallier L (2010) Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest 120:3127–3136 74. Ghodsizadeh A, Taei A, Totonchi M, Seifinejad A, Gourabi H, Pournasr B, Aghdami N, Malekzadeh R, Almadani N, Salekdeh GH, Baharvand H (2010) Generation of liver disease-specific induced pluripotent stem cells along with efficient differentiation to functional hepatocyte-like cells. Stem Cell Rev 6:622–632 75. Somers A, Jean JC, Sommer CA, Omari A, Ford CC, Mills JA, Ying L, Sommer AG, Jean JM, Smith BW, Lafyatis RA, Demierre MF, Weiss DJ, French DL, Gadue P, Murphy GJ, Mostoslavsky G, Kotton DN (2010) Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem cells 28:1728–1740 76. Urbach A, Bar-Nur O, Daley GQ, Benvenisty N (2010) Differential modeling of fragile X syndrome by human embryonic stem cells and induced pluripotent stem cells. Cell Stem Cell 6:407–411 77. Seifinejad A, Taei A, Totonchi M, Vazirinasab H, Hassani SN, Aghdami N, Shahbazi E, Yazdi RS, Salekdeh GH, Baharvand H (2010) Generation of human induced pluripotent stem cells from a Bombay individual: moving towards “universal-donor” red blood cells. Biochem Biophys Res Commun 391:329–334 78. Freund C, Davis RP, Gkatzis K, Ward-van Oostwaard D, Mummery CL (2010) The first reported generation of human induced pluripotent stem cells (iPS cells) and iPS cell-derived cardiomyocytes in the Netherlands. Neth Heart J 18:51–54

Chapter 4

Mesenchymal Stromal Cells: Latest Advances Sowmya Viswanathan and Armand Keating

Abstract  Over the past decade, the study of mesenchymal stromal cells (MSCs) has moved rapidly from in vitro and animal models to randomized clinical trials. Despite the challenges of defining MSCs, a consensus has emerged on culture methodology and their characterization, including the requirement for a minimum immunophenotype. Mechanisms of action in tissue regeneration have matured from the simple notion of transdifferentiation to effects on endogenous progenitors and promotion of an anti-inflammatory environment. Clinical investigation with MSCs now covers a wide variety of diseases, and sources of MSCs other than the bone marrow continue to be identified. Genetically engineered MSCs may provide more effective agents of tissue regeneration but will require careful preclinical study. Nonetheless, challenges remain: the need for appropriate preclinical models, informative clinical trials, good manufacturing practice cell production, and long-term trials follow-up.

Abbreviations ALS AMI AT AT-MSC

Amyotrophic lateral sclerosis Acute myocardial infarction Adipose tissue Adipose tissue-derived mesenchymal stromal cell

A. Keating (*) Cell Therapy Program, Princess Margaret Hospital, University Health Network, ON, Canada Department of Medicine, University of Toronto, Toronto, ON, Canada e-mail: [email protected] H.S. Bernstein (ed.), Tissue Engineering in Regenerative Medicine, Stem Cell Biology and Regenerative Medicine, DOI 10.1007/978-1-61779-322-6_4, © Springer Science+Business Media, LLC 2011

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bFGF BDNF BM BM-MSC CD CDAI DC Dkk-1 DMSO EAE FBS FGF GvHD GM-CSF GMP HGF HLADR HLA-G5 HUCPVCs IBD IDO IGF-1 LIF MI MHC MSCs NGF NK NO NT-3 PD PDGF PGE2 SDF-1 SSEA-3/4 TGF-b T-reg TSG-6 UC UCB VCAM-1 VEGF

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Basic fibroblast growth factor Brain-derived neurotrophic factor Bone marrow Bone marrow-derived mesenchymal stromal cell Crohn’s disease Crohn’s disease activity index Dendritic cell Dickkopf-1 Dimethyl sulfoxide Experimental autoimmune encephalomyelitis Fetal bovine serum Fibroblast growth factor Graft-versus-host disease Granulocyte macrophage colony-stimulating factor Good manufacturing practice Hepatocyte growth factor Human leukocyte antigen DR Human leukocyte antigen G5 Human umbilical cord perivascular cells Inflammatory bowel disease Indoleamine-pyrrole 2,3-dioxygenase Insulin-like growth factor-1 Leukemia inhibitory factor Myocardial infarction Major histocompatibility complex Mesenchymal stromal cells Nerve growth factor Natural killer Nitric oxide Neurotrophin-3 Parkinson’s disease Platelet-derived growth factor Prostaglandin E2 Stromal cell-derived factor-1 Stage specific embryonic antigen-3/4 Transforming growth factor-beta Regulatory T cell Tumor necrosis factor inducible gene-6 protein Umbilical cord Umbilical cord blood Vascular cell adhesion molecule-1 Vascular endothelial growth factor

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4.1 MSC Definition Since the first published report by Friedenstein and colleagues [1] describing the expansion of an adherent, spindle-shaped population of cells from whole human BM, MSCs have been expanded from numerous sources including skeletal muscle, AT, UC, synovium, dental pulp, amniotic fluid, and other tissues [2]. There has been a tendency in the field to identify this heterogeneous population with different terminology, including multipotent stromal cells, mesenchymal stem cells, marrow stromal cells or MSCs, without rigorous discrimination of their “stemness” properties [3]. For this reason, we prefer the term “mesenchymal stromal cells” (the acronym MSC still applies) as recommended in a white paper from the International Society for Cell Therapy [4]. Here, we define MSCs according to the minimum criteria established by the International Society for Cellular Therapy [5], i.e., greater than 95% cells must express CD105, CD73, and CD90, as measured by flow cytometry, and less than 2% should be positive for CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA Class II, and must be able to differentiate into osteoblasts, adipocytes, and chondroblasts under standard in  vitro differentiating conditions. It is reassuring that McGonagle et  al. [6] have demonstrated that MSCs are not merely an in  vitro manifestation of an unknown cell in vivo by detecting primary cells in the BM with an immunophenotype indistinguishable from that of cultured MSCs: CD45lo, CD271+, CD105+, CD90+, and CD10+. The origin of MSCs remains unproven. There are several hypotheses, some suggesting that MSCs may be skeletal stem cells [7, 8], others suggesting an embryonic remnant of pluripotent cells [9, 10], and still others suggesting a neural crest origin [11]. It is especially intriguing that MSCs bear a resemblance in immunophenotype, location and function to pericytes [12–14] and thus would be expected to be present in all vascularized tissues, especially during inflammation or injury. This change in paradigm of MSCs from self-renewing multilineage precursors to pleiotropic pericytes is the subject of ongoing investigation.

4.2 Sources of MSCs 4.2.1 Adipose Tissue In the adult, MSCs have been isolated outside the BM in AT [15]. This highly complex tissue contains adipocytes, pre-adipocytes, fibroblasts, vascular smooth cells, endothelial cells, monocytes, macrophages, and lymphocytes [16], stores and provides energy, and also serves as a dynamic hormone-producing organ involved in a number of physiological and pathological processes. AT-MSCs are

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readily available in large amounts (100 mL to 1 L) with relatively little morbidity through liposuction procedures [17]. MSCs are found at a higher frequency in AT than in BM, ranging from 1–10 per 1,000 cells [18] to 25–50 per 1,000 cells [19]. AT-MSCs also exhibit higher proliferation rates than BM-MSCs [20]. Comparative analyses of MSCs from BM and AT show that there is little difference in terms of morphology or immunophenotype [21]. Similar to their BM counterparts, AT-MSCs differentiate into multiple cell types including adipocytes, myocytes, osteocytes, chondrocytes, hepatocytes, neurons, pancreatic cells, endothelial cells, and cardiomyocytes (reviewed in [22]). Similar to BM-MSCs, AT-MSCs are immunosuppressive [23], lack HLA-DR expression and can therefore be used therapeutically in allogeneic transplantation with low risk of immune-mediated rejection. AT-MSCs exhibit similar cell surface antigens to BM-MSCs [20, 24] and secrete growth factors such as VEGF, HGF, and IGF-1 [25].

4.2.2 Umbilical Cord Blood The UC contains two arteries and one vein, which are surrounded by mucoid connective tissue called Wharton’s Jelly. Nonhematopoietic cells have been isolated from the connective tissue of the cord using different isolation and enzymatic processing techniques by many groups [26–28]. UCB-MSCs are a multipotent stromal cell population that are plastic-adherent and share BM-MSC surface markers such as CD73, CD90, and CD105 (reviewed in [29]), but have lower expression of CD106 and HLA-DR. UCB-MSCs also exhibit pluripotent transcription factors such as Oct-4, Nanog, and Sox-2 but several magnitudes lower than that expressed in embryonic stem cells [30]. UCB-MSCs also express Rex-1, SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81 [31]. Additionally, UCBMSCs, like myofibroblasts, express vimentin, desmin, and/or alpha-smooth muscle actin [29, 32, 33]. Isolation of this rare cell (1:200 million) is found in only an average of 29% of cords although this can be improved to 60% by optimizing the isolation process [21]. UCB-MSCs proliferate more rapidly compared with BM-MSCs [34] and maintain expansion and differentiation properties longer in culture [35].

4.2.3 Human Umbilical Cord Perivascular Cells UCB-MSCs are a heterogeneous group, coming from different zones of the connective tissue (subamniotic, intervascular, and perivascular) and exhibiting overlapping but distinctive features [36]. A distinct population of MSCs are obtained by enzymatic digestion of UC perivascular tissue [37]. These cells, HUCPVCs, can be isolated with high efficiency (100%) compared with UCB and have high clonogenic potential with a CFU-F frequency of 1:300 [37]. HUCPVCs exhibit multilineage differentiation potential in vitro and in vivo as recently confirmed at the clonal level [38]. They express cell surface markers similar to BM-MSCs but have higher levels

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of CD146 [39]. HUCPVCs, like UCB-MSCs, exhibit a higher proliferation rate and can be propagated in culture for longer periods than BM-MSCs [37].

4.3 Immune Modulation MSCs have inherently low immunogenicity, lacking MHC Class I and co-­ stimulatory molecules for T-cell recruitment [40], rendering them safe for mismatched allogeneic transplantation. Conflicting evidence [41], however, suggests that allogeneic, but not syngeneic, gene-modified murine MSCs may elicit an immune response in immunocompetent mice [42]. MSCs are generally considered immunosuppressive and numerous mechanisms have been proposed. MSCs secrete soluble factors such as IDO [43], NO [44], TGF-b1, HGF [45], PGE2 [46], HLA-G5 [47], LIF, and IL-10 (reviewed in [48]) to exert their immunosuppressive effects in a systemic manner. Best studied is the suppression of T-cell proliferation, although the exact mechanism of inhibiting this proliferation is not fully understood with some groups suggesting MSC-mediated cell-cycle arrest in G1 phase while others argue for MSC-mediated apoptosis [49–52]. MSCs have also been shown to recruit and support proliferation of regulatory T-cells [53], which in turn inhibit T-cell proliferation and cytokine production. MSCs also mediate the ­suppression of other cell types such as NK cells, although they can also be targets of NK cell killing [54]. MSCs further inhibit DC proliferation [55] and maturation [56] and can decrease production of pro-inflammatory cytokines [46]. MSCs also inhibit monocyte differentiation and modulate macrophage activity [57]. High doses of MSCs appear to inhibit B-cell proliferation and differentiation through paracrine action, while cell–cell contact increases antibody production [58]. MSC-mediated immunosuppression has been demonstrated in vivo in disease animal models, including experimental autoimmune encephalomyelitis [59, 60] and allogeneic skin grafts [61]. In vivo administration leads to inhibition of pathogenic antibodies, due to metalloproteinase processing of CCL2 produced by MSCs [60, 62]. Xenogeneic infusions of human MSCs are not immunologically recognized by immunocompetent rodents and can account for dramatic improvements in models of MS, stroke, colitis, IBD, and MI. The ability of MSCs to inhibit T-cell responses in a non-MHC-restricted manner [45] has led to the successful treatment of steroid-resistant acute GvHD [63]. However, the mechanisms of immune suppression by MSCs in patients are not well understood, in part, because the in vivo fate of MSCs is poorly documented.

4.4 MSC Therapy for Autoimmune Diseases Because of their immunosuppressive properties and demonstration of safety and feasibility in GvHD, there is increased interest in MSCs for treating autoimmune and inflammatory disorders, including Crohn’s disease, diabetes, and MS.

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4.4.1 Crohn’s Disease The immunosuppressive properties of MSCs make them well suited for inflammatory diseases such as IBD and CD. In a murine model of IBD, AT-MSCs reduced systemic and mucosal levels of pro-inflammatory cytokines, increased IL-10 secretion, induced T-regs in mesenteric lymph nodes, and reduced microscopic signs of colitis [64]. In a pilot study, MSCs isolated from patients with refractory CD had intact immunosuppressive properties [65]. MSCs were expanded and infused into nine patients; three showed a clinical response as measured by CDAI but in three others disease progressed requiring surgery. This pilot study showed no apparent benefit for patients with severe refractory luminal disease. AT-derived cells encapsulated in fibrin glue were used to treat complex perianal fistula associated with CD (n = 14) and those of cryptoglandular origin (n = 35) in a Phase II trial [66]. There was a 71% response rate (fistula closure) in the treatment group compared with a 16% response in the group receiving fibrin glue alone. In a Phase II trial sponsored by Osiris Therapeutics, two infusions of allogeneic MSCs were administered at 2 or 8 × 106 cells/kg in nine moderate-to-severe CD patients. This led to a reduction in CDAI in all nine by day 28; three of nine patients had a clinical response determined by a reduction in CDAI by 150 [67]. There was no correlation of dose with outcome. Based on these results, a Phase III trial has been initiated with 207 patients with a CDAI between 250 and 450, and who have previously failed therapy with at least one steroid, an immunosuppressant, and a biological agent. The primary endpoint for this trial is disease remission, defined as a CDAI at or below 150 by day 28.

4.4.2 Diabetes Mellitus Human MSCs can improve diabetes although the mechanism is not well understood [68]. For example, paracrine secretion is implicated in a study of type 1 diabetic mice in which infusion of MSCs resulted in a significant reduction of blood glucose within 1 week that reached near euglycemic values a month later. These animals showed an increase in morphologically normal beta-pancreatic islets and normal glomeruli, suggesting therapeutic potential of these cells [69]. In contrast, other studies report the derivation of insulin-secreting cells by differentiating MSCs isolated from UCB. Blood sugar levels were reduced upon xenotransplantation into NOD mice and the histological presence of insulin-secreting cells with human nuclei and C-peptide was detected in the liver [70]. Another strategy for insulin treatment involves electroporating MSCs with the endogenously active glucoseresponsive promoter, EGR1 [71]. Mice receiving modified human MSCs exhibited dose-responsive corrections of hyperglycemia, improved glucose tolerance, and reduced body weight. In a clinical trial, 25 patients with type II diabetes received

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concomitant intrapancreatic autologous BM stem cell infusions and hyperbaric oxygen treatment and showed improved metabolic control and reduced insulin requirements 1 year later [72].

4.4.3 Multiple Sclerosis Preclinical evaluation in EAE models shows that MSCs can suppress clinical manifestations [59], although the effect was apparent only when cells were injected at disease onset or peak and not during the chronic phase. Rodents exhibiting EAE receiving intravenous and intraventricular infusions of MSCs had almost twice the number of axons as control animals [73]. A case report using intrathecal and intravenous infusions of UCB-MSCs to treat a patient with MS showed improvements in sensory impairment, an expanded disability status scale score, and a reduced T2 lesion load by MRI, with no side effects [74]. A pilot study of autologous MSCs in ten patients similarly demonstrated safety and feasibility [75].

4.5 MSC Therapy for Neurodegenerative Diseases MSCs are highly interactive with their microenvironment, and share protein, RNA, and mitochondria with damaged tissue, which may be particularly relevant in treating neurodegenerative diseases [76]. Increasing numbers of preclinical and clinical studies are being reported investigating the effects of MSCs on diseases such as ALS, PD and on acute and chronic ischemic stroke, and spinal cord injuries. MSCs have been shown to stimulate the proliferation, migration, and differentiation of endogenous neural stem cells [77], promote neuronal survival [78] and neurite outgrowth [79], and protect neurons against oxidative stress [80] through the secretion of soluble factors such as BDNF [81], Wnt antagonist Dkk-1 [82], and NGF. Trophic factors secreted by MSCs promote neuronal growth while modifying the tissue microenvironment [79]. MSCs also promote oligodendrogenesis [83]. In animal studies, transplantation of BM-MSCs into the brain of immunodeficient mice markedly increased the proliferation of endogenous neural stem cells [77]. The common theme of these studies is that the effect of MSCs lies in stimulating endogenous cells to enhance the repair of neural tissue.

4.5.1 Amyotrophic Lateral Sclerosis Several groups have demonstrated that intraparenchymal delivery of human MSCs is safe and can delay loss of motor neurons in rodents [84]. Human MSCs

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t­ ransplanted directly into the spinal cords of transgenic SOD1 mice [85] migrated throughout the spinal cord and delayed loss of motor neurons, prolonging motor performance. A pilot study in ALS was conducted by transplanting autologous, cultureexpanded MSCs into a surgically exposed spinal cord (T7–T9 level). No significant side effects were reported [86]. Magnetic resonance imaging performed 3 and 6 months after transplantation did not show structural changes of the spinal cord or abnormal cell proliferation when compared with baseline scans. Three months after cell implantation, four patients exhibited a significant slowing of muscle strength decline in the proximal muscle groups of lower limbs. A Phase II clinical trial using MSCs is underway in Europe, and the FDA has recently approved a Phase I trial in the USA.

4.5.2 Parkinson’s Disease The goal of cellular therapy of PD is to replace lost neurons in the substantia nigra with healthy dopaminergic neurons or to prevent further neuronal loss. In a rat model of PD, MSC transplantation resulted in behavioral changes that correlated with partial restoration of dopaminergic markers and vesicular striatal pool of dopamine [87]. MSCs engineered to express neurotrophic factors may be superior, since BDNF-modified MSCs transplanted into a 6-hydroxydopamine-induced lesion model of PD showed behavioral improvements and reduced dopamine depletion compared to unmodified MSCs [88]. Recently, Venkataramana et  al. [89] reported the first open-label clinical pilot study with a single dose of autologous MSCs transplanted by stereotaxic surgery into the striatum of seven patients with advanced PD. At 10–36 months follow-up, there were no safety concerns or serious adverse events reported, but no efficacy outcomes could be concluded.

4.5.3 Stroke MSCs have been used in the treatment of experimental stroke [90–93] and exert an effect in rats even when administered 1 month after the stroke [92]. However, very few transplanted cells were detected during the 1-year tracking experiment, suggesting that MSC differentiation and replacement of neurons is an unlikely primary mechanism [92]. Secretion of factors from MSCs including BDNF, NT-3, VEGF, NGF, bFGF, and IGF-1 may play a role and promote functional repair [90, 91]. This was confirmed in a recent study that showed MSC-treated grafts had higher levels of BDNF, NT-3, and VEGF compared with saline control grafts, which accelerated proliferation of neuronal progenitors in the subventricular zone [94].

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A single randomized study of culture-expanded MSCs was conducted in 30 patients with cerebral infarcts within the middle cerebral arterial territory, and severe neurological deficits [95]. Only five received autologous 108 culture-expanded MSCs intravenously while 25 other patients served as controls. Outcomes improved in the MSC-treated patients compared with control patients as measured by the Barthel Index and Rankin Score at 3 and 6 months, but differences were not statistically significant at 12 months. No significant toxicities were identified. Clinical trials of a MSC-like multipotent cellular product, MultiStem®, were recently approved by the FDA to treat stroke in the USA.

4.5.4 Spinal Cord Injuries Many studies have documented successful engraftment of MSCs into the injured spinal cord [96]. Some evidence suggests that MSCs may reduce the acute inflammatory response to spinal cord injury and decrease astrocyte, microglia, and macrophage reactivity [96]. In 2005, Park et al. [97] reported the first trial of MSCs in six patients with spinal cord injury and showed that autologous whole BM injected directly into the site of spinal cord injury, along with intravenous infusions of GM-CSF slightly improved neurological function in five patients. The same researchers later treated 18 patients with MSCs and 13 patients in a control group (decompression and spinal fusion surgery) [98]. Thirty percent of patients who had received cells during the acute injury stage, 33% during the chronic injury stage, and 8% of the control group demonstrated an increase in neurological function. In another study, MSC injection into the cerebrospinal fluid produced improvement in the quality of life score only for patients with acute but not chronic injuries [99].

4.6 MSC Therapy for Cardiovascular Disease MSCs have been shown to mediate functional improvement in a number of ­animal models of cardiac injury, including a porcine AMI model [100], a canine chronic myocardial ischemia model [100, 101], a pig heart failure model [102], and a rodent model of dilated cardiomyopathy [103]. Mechanisms mediating this repair, however, remain unclear; early reports claimed that MSCs transdifferentiate into a cardiomyocyte phenotype [104–107] although differentiation to a mature functioning cardiomyocyte has not been demonstrated. Other groups citing evidence of low engraftment have proposed paracrine mechanisms [108, 109] that include recruitment of resident cardiac stem cells [110], inhibition of fibrosis [111], cardioprotection (likely through secretion of HGF, TGF-b, VEGF, IGF-1, stanniocalcin 1, and GM-CSF), promotion of neoangiogenesis [112], and improvement in cardiac contractility [113]. Another hypothesis is

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that MSCs, which only appear to be transiently present in the infarcted myocardium, modulate the inflammatory microenvironment of ischemic tissue by up-regulation of a number of genes including TSG-6 [114], an anti-inflammatory protein produced by monocytes, macrophages, and DCs [115]. This was confirmed when the beneficial effect of MSCs was abrogated in siRNA knock-down of TSG-6 and rescued by infusion of recombinant TSG-6. Clinical studies involving interventional delivery [intramyocardial (PROMETHEUS trial) and transendocardial (TAC-HFT)] of autologous MSCs are currently ongoing. The only trial involving systemic delivery of allogeneic MSCs is the Provacel trial by Osiris Therapeutics. They reported a randomized Phase I trial of allogeneic MSCs administered intravenously in 53 patients after AMI [116]. There were comparable adverse events between the MSC-treated and placebo groups and no serious events related to cell treatment at 2 years. Unlike preclinical studies [117], the MSC-treated group reported improved pulmonary function. At 6 months however, there was no difference in ejection fraction between MSC-treated versus placebo groups by echocardiography, but analysis of a subgroup using MRI revealed significant persistent improvement at 12 months. A larger Phase II study of 220 patients is underway. MSCs have been evaluated in small numbers of patients with administration via the intracoronary route [118]. In all trials, preliminary data showed improvement in ejection fraction and perfusion defects in MSC-treated groups, although controls were not included in early studies.

4.7 Manufacturing Considerations Culture expansion of MSCs from BM, AT, UC, fetal tissue, and other sources is required for clinical use because the cells are present in very low frequencies in these tissues. GMP is required to produce clinical-grade MSCs to meet multiple morphological, immunophenotypic, functional, karyotypic, safety, and sterility criteria prior to infusion into patients. A common protocol for the expansion of MSCs from the European Group for Blood and Marrow Transplantation has since been adopted by many groups participating in multicenter trials [119]. A tenet of GMP is the use of certified, pathogen-free reagents. Despite remarkable clinical advancements in this field, MSCs are still expanded in traditional culture media containing FBS [119]. This can be concerning as MSCs have been shown to retain FBS proteins and increase the risk of sensitization [120]. Platelet lysates represent an efficient alternative to FBS as demonstrated by many groups [121, 122] although there may be some limiting effects on the functional behavior of differentiated MSCs [123]. MSCs expanded in platelet lysate were administered to GvHD patients; although there were no safety concerns, a lower overall response of 54% was reported at a 28-day time point [124]. It is not clear whether this reduced efficacy is related to the design of the clinical protocol or due to the FBS substitution.

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Human MSCs can also be expanded in serum-free medium supplemented with a cocktail of factors, including recombinant human PDGF-BB, bFGF, and TGF-b1 and appear to retain their immunophenotypic, colony-forming, and differentiation potential [125]. However, exposure to growth factors may induce MHC Class II expression and cause aneuploidy [126]; consequently, follow-up is needed to assess the long-term safety of these defined culture conditions. Production of xenogeneic contaminant-free adhesion proteins (e.g., fibronectin, lamenin, vitronectin) to facilitate attachment of MSCs to culture surfaces also poses a challenge. GMP-grade MSCs used for clinical trials are typically early passage, although there are no significant differences between MSCs passaged up to P7 in terms of immunosuppressive properties [127]. Early-passage MSCs are still preferred for increased safety and efficacy as continuous culture of MSCs over several passages may result in the accumulation of karyotypic abnormalities [128]. A confounding factor is how passage numbers are really measured in different systems with different seeding densities and definitions of confluency. Population doublings are a more instructive term, as we have proposed [129] and MSCs expanded for fewer population doublings (90%) hepatocyte replacement in livers of mice mutant for Fah, Rag2, and the common g-chain of the interleukin receptor in the absence of the protective drug, 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione [24]. The human liver chimeric mice have provided a model for hepatitis B and C virus infection and treatment research [25]. The unique capacity of normal human hepatocytes to expand in the liver of a bioengineered mouse is based on a strong selective advantage for the transplanted cells to survive compared to the host cells. However, a limitation in the repopulated FAH deficient mouse is related to the absolute number of primary human hepatocytes that can be obtained. Thus, the generation of bioengineered Fah-null homozygous pigs is underway for much larger scale expansion of human hepatocytes.

17.6 Sources of Hepatocytes 17.6.1 Human Donor Livers Primary human hepatocytes would be a desirable option for hepatocyte transplantation as well as internal and external liver assist devices; however, human hepatocytes from a human source are not a good option because human livers are preferentially allocated for transplant. Alternatively, at least one group has reported isolating human hepatocytes from livers unsuitable for organ donation [26].

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17.6.2 Immortalized Human Hepatocyte Lines In an attempt to bypass the limitations associated with terminally differentiated hepatocytes, several groups have attempted to immortalize hepatocytes via spontaneous transformation, telomerase introduction, and retroviral transfection of the simian virus 40 large T antigen. To date, the hepatoblastoma C3A line, a subclone of the HepG2 cell line, is the only human-based cell line to be tested clinically in BAL device trials [27]. Limitations of C3A cells are their reduced levels of cytochrome P450 activity, ammonia removal, and amino acid metabolism compared to primary hepatocytes. Reduced ammonia removal by C3A cells is due to reduced expression of urea cycle genes [28].

17.6.3 Primary Pig Hepatocytes In place of primary human hepatocytes, xenogeneic cells could be a potential cell source for bioartificial systems. Because of differentiated metabolic functions, unlimited supply, and a high yield of cells, primary porcine hepatocytes have been most commonly used in several liver support devices undergoing preclinical and clinical evaluation [29]. Concerns regarding the use of porcine hepatocytes in human treatment include the risk of humoral and cellular immunologic response, transmission of porcine endogenous retrovirus, and potential function mismatch between porcine proteins and their human counterparts.

17.6.4 Stem Cells In recent years, great advances have been made in the production of stem cellderived hepatocytes. A large number of studies have utilized liver-derived stem cells including fetal liver stem cells (hepatoblasts) and adult liver stem cells (oval cells) to generate primary hepatocytes. But these hepatic progenitor cells are rare within liver tissue, with hepatoblasts comprising only 0.1% of fetal liver mass, and oval cells comprising 0.3–0.7% of adult liver mass. Other studies have induced ESCs to differentiate into hepatocyte-like cells. Induction involves exposure of ESCs to Wnt3a signaling to mimic events within the developing embryo, however, this results in limited functionality and incomplete maturity. In addition, ethical concerns may limit clinical application of ESCs. Hepatocytes can also be generated from hematopoietic stem cells and either bone marrow or adipose tissue mesenchymal stem cells through both transdifferentiation and fusion [30]. However, stem cell-derived hepatocytes have not yet been shown to fully function as primary hepatocytes.

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Reprogramming of adult somatic cells to iPSCs with the introduction of a defined set of transcription factors addresses the concerns of embryo destruction to produce ESC cells [31, 32]. Recent progress in iPSC technology may provide an individualized approach to the treatment of genetic disorders by providing cells that are theoretically identical to the patient for treatment, thereby avoiding immune rejection without immunosuppressive drug therapy [33, 34].

17.7 Application of Synthetic Liver 17.7.1 Diagnostic: Drug Discovery and Toxicity Testing The pharmaceutical industry uses high-throughput assessment of drug metabolism, toxicology, distribution, and pharmacokinetics from in  vitro hepatocyte culture assays. The microtiter plate (96-, 384- and 1,536-well), which is a miniaturized and parallel version of a conventional tissue culture dish, is most commonly used as the industry standard for high-throughput liver cell assays for drug discovery and toxicology. To provide better, faster, and more efficient prediction of in vivo toxicity and clinical drug performance, microfabricated cell arrays and microfluidics were recently developed to optimize liver-specific function of primary hepatocytes [13]. Microfabricated hepatocyte cultures exhibit characteristic patterns of gene expression phase I/II metabolism, canalicular transport, secretion of liver-specific products, and susceptibility to hepatotoxins [35]. Advances in microfluidics include the development of in vitro models of physiologically based pharmacokinetics. These models are designed to mimic physiological architecture and dynamics to allow for extrapolation of key in vivo drug parameters from in vitro cell culture assays and animal studies.

17.7.2 Therapeutic Liver transplantation is the only available treatment for severe end-stage liver disease. But the organ shortage and the need for life-long immunosuppression still limit its application. Thus, there are demands for alternative treatments for liver failure, including hepatocyte transplantation, hepatic tissue engineering, and extracorporeal BALs, which utilize isolated hepatocytes based on the belief that these cells should express full functionality in substituting for the normal liver. 17.7.2.1 Extracorporeal Devices BAL devices are extracorporeal temporary liver support systems that are analogous in concept to kidney dialysis machines, but use living hepatocytes within a bioreactor. These living hepatocytes can provide synthetic functions, regulation, and selective

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detoxification of water-soluble and protein-bound waste substances. BALs are expected to stimulate regeneration of injured liver, increase spontaneous recovery, and/or bridge to liver transplantation with the goal of improving patient survival [27]. To fulfill those goals, the BAL should contain high cell densities, maintain longterm well-differentiated hepatocyte function, and reduce mass transfer limitations. 17.7.2.2 Oxygenation Oxygen is an important component in the hepatic microenvironment since primary hepatocyte energy production is highly dependent on oxidative phosphorylation. Thus, BALs must overcome the issue of the high oxygen uptake rate of hepatocytes and the relatively low solubility of oxygen in aqueous media [4]. To ensure a sufficient supply of oxygen, some BAL designs use an in-line oxygenator in the extracorporeal perfusion circuit, while other designs incorporate an oxygenator into the bioreactor. To improve oxygen delivery, some BAL designs have employed oxygen carriers such as emulsified fluorocarbon and hemoglobin or additional fibers to carry gaseous oxygen directly into the bioreactor [36]. 17.7.2.3 Immunologic and Membrane Considerations To protect hepatocytes from the host immune system, most BAL designs employ selective membranes to prevent direct contact between patient blood and hepatocytes. In these designs, mass transfer is determined by the molecular weight cutoff of the membrane. Mass transfer refers to the transport of immunoglobulins and toxins out of the patient’s circulation and transport of hepatic proteins from the BAL into the patient’s circulation. Hollow fiber membranes with a molecular weight cutoff between 100 and 200 kDa appear optimal in providing immunoprotection of allogeneic/xenogeneic hepatocytes and serving as a barrier against zoonoses, without impairing removal of toxins [37]. 17.7.2.4 Cell Mass On the basis of data from human liver surgical resection, the maintenance of normal human liver function occurs with 10–30% of the liver mass. With an average liver mass of 1,500 g and hepatocytes comprising 90% of liver cell mass, this corresponds to 100–400 g of hepatocytes. This mass serves as the adequate mass for BALs to treat patients with liver failure. Hepatocyte mass of BALs that have undergone clinical testing has ranged from 75 g of cryopreserved porcine hepatocytes [29] up to 400 g of immortalized human C3A cells [27]. On the horizon is the SRBAL, a novel extracorporeal device equivalent to 40% of the hepatocyte mass of a normal human liver (Fig. 17.3). Anchorage-independent spherical aggregates of hepatocytes (i.e., spheroids) engineered by a novel rocked mixing technique serve as the source of detoxification activity in the SRBAL [21].

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Fig. 17.3  Features of the Mayo SRBAL. (a) Schematic of generic modular extracorporeal cellbased liver support device. (b) Components of SRBAL set-up. (c, d) Photograph and schematic of spheroid reservoir demonstrating fenestrated funnel configuration used to keep hepatocyte spheroids in suspension during continuous perfusion of the reservoir

17.7.2.5 Transplantation of Hepatocytes Hepatocyte transplantation is also a potential therapy for the treatment of numerous liver disorders. Hepatocytes are infused directly into the portal vein or indirectly into the spleen and then undergo blood flow-mediated translocation into the hepatic sinusoids. Intraportal injection of hepatocytes can cause transient portal hypertension; thus, intrasplenic delivery is often preferred for hepatocyte transplantation. On the basis of animal and human data, transplantation of hepatocytes corresponding to