Islet Transplantation and Beta Cell Replacement Therapy [1 ed.] 0824728629, 9780824728625, 9781420016512

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ISLET TRANSPLANTATION AND

BETA CELL REPLACEMENT THERAPY

ISLET TRANSPLANTATION AND

BETA CELL REPLACEMENT THERAPY

Edited by

A. M. James Shapiro University of Alberta Edmonton, Alberta, Canada

James A. M. Shaw University of Newcastle Newcastle upon Tyne, UK

Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017  2007 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-2862-9 (Hardcover) International Standard Book Number-13: 978-0-8247-2862-5 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Islet transplantation and beta cell replacement therapy / edited by A. M. James Shapiro, James A. M. Shaw. p. ; cm. Includes bibliographical references. ISBN-13: 978-0-8247-2862-5 (hardcover : alk. paper) ISBN-10: 0-8247-2862-9 (hardcover : alk. paper) 1. Islands of Langerhans–Transplantation. 2. Pancreatic beta cells–Transplantation. 3. Diabetes–Treatment. I. Shapiro, A. M. James. II. Shaw, James A. M. [DNLM: 1. Diabetes Mellitus, Type 1–surgery. 2. Islets of Langerhans Transplantation. 3. Insulin-Secreting Cells–transplantation. WK 815 I82 2007] RD599.5.I84I82 2007 2007022615 617.50 570592–dc22 Visit the Informa web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

Foreword

As James Shapiro points out in the first chapter of this superbly edited book on islet transplantation and beta-cell replacement therapy for diabetes, the concept is old but the realization is recent. Within five years of the late 19th century experimental observations of von Mering and Minkowski, definitively showing that diabetes was inevitable after extirpation, and that the pancreas had to have an “internal secretion” regulating blood glucose levels, attempts began to supply the unidentified and obviously missing substance to diabetic patients by injecting pancreatic extracts or by transplanting pancreatic fragments. Clinical success with a pancreatic extract (the insulin of Banting and Best) was achieved decades before complete reversal of diabetes by islet transplants in animal models. In the interim, immediately vascularized pancreas transplants were clearly shown to reverse diabetes, and the clinical application that began in 1966 continues to this day. However, pancreas transplants are associated with a relatively high incidence of surgical complications, and the entire gland is transplanted solely to supply one cell—the only one missing in type 1 diabetes: the beta cell. Thus, if there ever was a solid-organ transplant that could be replaced by a cellular transplant, pancreas for islet transplantation was it. If there was ever an indication for a wholesale transfer from major to minimally invasive surgery, this is it. The rationale for and progress towards this goal is laid out in this book. As noted by Shapiro, the morbidity associated with pancreas transplants in the early days was an incentive to develop islet transplantation. Indeed, many thought the transition would be rapid. However, as the results of pancreas transplants improved, the immediate need for clinical application of islet transplantation diminished, probably delaying its development. Likewise, the promise of islet transplantation was probably partially responsible for the underuse of pancreas transplants, even when the results did improve. Progress in clinical islet transplants has reached a point where the results approach those of pancreas transplants, with surgical complications truly minimized. This book is not only a practical guide for the current application of islet transplantation, but also a blueprint for the future.

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Foreword

The patients currently selected for beta-cell replacement therapy are those for whom being immunosuppressed is judged less onerous than remaining diabetic, or those already in need of immunosuppression—that is, diabetic recipients of a kidney transplant. Expansion of islet transplantation to the general diabetic population will require reduction of the side effects of immunosuppression or a practical method of inducing immunological tolerance. Expansion to all diabetics will require a source of beta cells other than the limited number of deceased human donors, either from human stem cells or by use of xenografts. Leaders in the field, including Shapiro, have contributed chapters on each of these topics as well as on the current technical aspects and challenges of islet transplantation. A bonus is the inclusion in the first chapter of the final reminiscence of the father of islet transplantation, Paul Lacy. This book is a monument to the hope of every diabetic—to be free of the need for exogenous insulin with the least risk possible. David E. R. Sutherland, MD, PhD Professor and Head, Division of Transplantation Director, Diabetes Institute for Immunology and Transplantation Department of Surgery University of Minnesota Minneapolis, Minnesota, U.S.A.

Preface

Transplantation of pancreatic segments to replace the “internal secretion” missing in diabetes was first attempted in 1893, several years before the discovery of insulin. Despite ongoing refinement of insulin formulations and delivery devices over the last 85 years, including recent progress towards the closed loop, bioartificial pancreas, insulin replacement by conventional injection therapy remains inextricably linked to hypoglycemia. Transplantation of whole pancreas, together with its blood supply, can entirely prevent significant hypoglycemia while normalizing overall glycemic control. This major invasive procedure is necessarily associated with morbidity and mortality and will remain suitable for only a minority. The turn of the millennium heralded a new dawn in the management of Type 1 diabetes, with realization of reproducible insulin independence, and liberation from disabling hypoglycemia following transplantation of isolated islet cells. This book is dedicated to further refinement of all aspects of this procedure, with the aim of achieving the best possible outcomes for future recipients and providing these outcomes to suitable candidates worldwide. A successful clinical program is dependent on a truly multidisciplinary approach. Our goal in editing this first edition of Islet Transplantation and Beta Cell Replacement Therapy is to bring together leading proponents from each discipline critical to current and future success in the field. The contributing authors have excelled in providing a uniformly accessible text for all involved in clinical transplantation and the underpinning basic research. The approach is practical and focused on improving clinical outcomes for those with Type 1 diabetes. A wealth of experience is provided for those setting up new programs. We are confident that the level of detail and insight presented within a unified, structured format will ensure that this volume is equally well-thumbed in established centers of excellence. In the early chapters, a historical perspective is followed by an excellent exposition of the true impact and far-reaching consequences of hypoglycemia for those living with all forms of exogenous insulin replacement therapy. The subsequent chapters outline the key role of the endocrinologist in holistic assessment and selection of islet transplant recipients, in addition to facilitating a truly informed decision agreed to by both the patient and the multidisciplinary team that takes into account risks as well as potential benefits. v

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Clinical success cannot be achieved without the highest quality pancreas resection and preservation in tandem with expert islet isolation, validation, and, ultimately, transplantation. Each of these challenges is addressed in turn in Chapters 4 through 7. This section is completed by a practical guide to safe and effective management of islet immunosuppressant regimens. True understanding of the factors underlying attrition of islet function over time will only be gained by enhanced graft monitoring posttransplantation. The importance and future promise of in vivo islet imaging, in addition to metabolic monitoring of the recipient, are addressed next. This is followed by hard-earned advice on setting up a new clinical islet transplant program, outlining potential models and pitfalls for a costeffective and sustainable integrated approach. The parallel requirement for optimally designed and implemented clinical trials is explored in Chapter 12. Chapter 13, “Clinical Outcomes and Future Directions in Islet Transplantation,” highlights the most recent clinical milestone—reproducible insulin independence following transplantation of islets purified from a single donor pancreas. The importance of maintaining islets in culture enabling pre-operative stabilization and induction, in addition to successful transplantation of islets purified at a distant isolation facility, is discussed next as a prelude to Chapter 15, which provides a vision into the future of islet immunosuppressant protocols. Potential approaches for those already immunosuppressed with a functional renal graft are specifically considered. The last four chapters address the need for new sources of beta cells to meet future clinical needs through xenotransplantation, generation of new insulin-producing cells from adult tissue, and, ultimately, stem cell banks. Finally, the multitude of ways in which gene therapy may impact on clinical practice in islet transplantation and beta cell replacement within the foreseeable future are reviewed. We are extremely grateful to all of the contributing authors, without whom our aspirations to realize a truly state-of-the-art work in these pioneering days would have foundered. We also wish to acknowledge the invaluable support and enthusiasm of our associate editors Camillo Ricordi and Jonathan Lakey, in addition to Dana Bigelow and her tireless team at Informa Healthcare. This book is dedicated to the memory of Paul Lacy, whose prescient words, recorded from his final public lecture and printed in our opening chapter, continue to resonate for all of us lucky enough to be involved in trying to shape future chapters for those with diabetes. A. M. James Shapiro James A. M. Shaw

Contents

Foreword David E. R. Sutherland Preface v Contributors ix

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1. A Historical Perspective on Experimental and Clinical Islet Transplantation 1 A. M. James Shapiro 2. Hypoglycemia in Type 1 Diabetes: The Need for a New Approach Stephanie A. Amiel

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3. Patient Selection and Assessment: An Endocrinologist’s Perspective 57 Peter A. Senior 4. The Surgical Aspects of Pancreas Procurement for Pancreatic Islet Transplantation 81 Neal R. Barshes, Timothy C. Lee, Ian W. Udell, Christine A. O’Mahony, F. Charles Brunicardi, John A. Goss, and A. M. James Shapiro 5. Pancreas Preservation for Islet Isolation 99 Mohammadreza Mirbolooki and Jonathan R. T. Lakey 6. Aspects and Challenges of Islet Isolation 115 Mohammadreza Mirbolooki, Jonathan R. T. Lakey, Tatsuya Kin, Travis Murdoch, and A. M. James Shapiro 7. Percutaneous Portal Vein Access: Radiological Aspects Richard J. Owen

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8. Care of the Islet Transplant Recipient: Immunosuppressive Management and Complications 147 Raquel N. Faradji, Pablo Cure, Camillo Ricordi, and Rodolfo Alejandro 9. Islet Graft Monitoring and Imaging Christian Toso and Thierry Berney

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10. Metabolic Measures of Islet Function and Mass After Islet Transplantation 193 R. Paul Robertson vii

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11. Challenges in Setting Up a New Islet Transplant Program Paul R. V. Johnson

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12. Key Factors to Consider in Setting Up Clinical Trials in Islet Cell Transplantation: A Nursing Coordinator’s Perspective 215 Barbara S. DiMercurio 13. Clinical Outcomes and Future Directions in Islet Transplantation Faisal Al-Saif and A. M. James Shapiro 14. Culture and Transportation of Human Islets Between Centers Hirohito Ichii, Antonello Pileggi, Aisha Khan, Chris Fraker, and Camillo Ricordi

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15. Development and Application of Contemporary Immunosuppression in Human Islet Transplantation 269 Dixon B. Kaufman 16. Pig Islet Xenotransplantation—Update and Context Daniel R. Salomon 17. Approaches to b-cell Regeneration and Neogenesis Susan C. Campbell and Wendy Macfarlane 18. Stem Cell Approaches for b-cell Replacement Enrique Roche and Bernat Soria 19. Diabetes Gene Therapy 327 Peter S. Chapman and James A. M. Shaw Index

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283 293

Contributors

Rodolfo Alejandro Department of Medicine, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Faisal Al-Saif Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Stephanie A. Amiel London, U.K.

King’s College School of Medicine, King’s College,

Neal R. Barshes Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Thierry Berney Department of Surgery, University of Geneva Hospital, Geneva, Switzerland F. Charles Brunicardi Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Susan C. Campbell Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle, U.K. Peter S. Chapman Matthew J. Ryan Veterinary Hospital, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. Pablo Cure University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Barbara S. DiMercurio Adult General Clinical Research Center, University of Colorado Health and Science Center, Denver, Colorado, U.S.A. Raquel N. Faradji Department of Medicine, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A.

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Contributors

Chris Fraker DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. John A. Goss Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Hirohito Ichii DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Paul R. V. Johnson Oxford Islet Transplant Program, Nuffield Department of Surgery, University of Oxford, and Department of Pediatric Surgery, John Radcliffe Hospital, Oxford, U.K. Dixon B. Kaufman Department of Surgery, Division of Transplantation, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, U.S.A. Aisha Khan DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Tatsuya Kin Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Jonathan R. T. Lakey Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Timothy C. Lee Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Wendy Macfarlane School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, U.K. Mohammadreza Mirbolooki Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Travis Murdoch Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada Christine A. O’Mahony Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A. Richard J. Owen Department of Radiology, University of Alberta, Edmonton, Alberta, Canada

Contributors

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Antonello Pileggi DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. Camillo Ricordi DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, and Clinical Islet Transplant Program, Cell Transplant Center, Diabetes Research Institute, Miami, Florida, U.S.A. R. Paul Robertson Pacific Northwest Research Institute and the Departments of Medicine and Pharmacology, University of Washington, Seattle, Washington, U.S.A. Enrique Roche Instituto de Bioingenieria, Universidad Miguel Hernandez, Elche, Alicante, Spain Daniel R. Salomon Department of Molecular and Experimental Medicine, The Scripps Research Institute, Center for Organ and Cell Transplantation, Scripps Health, La Jolla, California, U.S.A. Peter A. Senior Division of Endocrinology, University of Alberta, Edmonton, Alberta, Canada A. M. James Shapiro Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada James A. M. Shaw Diabetes Research Group, Institute of Cellular Medicine, University of Newcastle, Newcastle upon Tyne, U.K. Bernat Soria CABIMER, Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Andalucia, Spain Christian Toso Department of Surgery, University of Alberta Hospital, Edmonton, Alberta, Canada, and Department of Surgery, University of Geneva Hospital, Geneva, Switzerland Ian W. Udell Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, U.S.A.

1 A Historical Perspective on Experimental and Clinical Islet Transplantation A. M. James Shapiro Department of Surgery, Walter C. Mackenzie Health Center, University of Alberta, Edmonton, Alberta, Canada

INTRODUCTION It is always sobering to reflect back on history and appreciate how much was accomplished with seemingly primitive tools, and especially to realize that very few ‘original’ ideas have not been considered, attempted, tried, and perhaps dismissed many years before their time. Such is definitely the case with islet transplantation for diabetes. The first clinical attempt at islet transplantation in the treatment of diabetes occurred on December 20th, 1893, 28 years before the discovery of insulin, and was published in the following year in the British Medical Journal (1) (Fig. 1). Dr WatsonWilliams and his surgical colleague Mr. Harsant, working at the Bristol Royal Infirmary in England, transplanted three pieces of freshly slaughtered sheep’s pancreas, “each the size of a Brazil nut,” into the subcutaneous tissues of a 15-year-old boy dying from uncontrolled ketoacidosis. Their

Figure 1 Original article header in the British Medical Journal, December 1894. Source: From Ref. 1. 1

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operation, performed under chloroform anesthesia, was completed “within twenty minutes of the death of the sheep.” This seemingly simple experiment is remarkable from many aspects— not least the fact that Watson-Williams and Harsant’s first instincts were to use xenograft pancreas tissue and not human tissue as a potentially unlimited source. Secondly, their transplant took place without any immunosuppression, in the hope that tolerance to xenogeneic tissue would naturally occur. It was not until almost 50 years later that the mechanisms underlying allograft rejection, the concepts underlying immunological tolerance, and how the immune system could be in check with powerful non-specific immunosuppression began to emerge. Nonetheless, a major hope and focus of research remains the induction of immunological tolerance to xenogeneic tissues, and this still seems like an almost insurmountable barrier. In Watson-Williams and Harsant’s case, there was temporary improvement in the patient’s clinical condition before the tissue was rapidly rejected and death occurred three days later. Even Watson-Williams and Harsant’s idea was not new, for Oscar Minkowski had already carried out a similar procedure in a pancreatectomized dog in the previous year (1892), and had described a temporary reduction in glycosuria (2). These experiments were published just three years after Joseph von Mering and Oscar Minkowski had discovered that the pancreas was linked to diabetes by surgical removal of a dog’s pancreas with onset of polyuria and glycosuria (3) (Fig. 2). The first clinical attempts to transplant fragmented pancreatic tissue as allografts in patients with diabetes is attributed to the pioneering surgeon Frederick Charles Pybus from Newcastle-upon-Tyne, England, where in July 1916 Pybus procured cadaveric human pancreas fragments and transplanted these subcutaneously in two patients (4). One of the two patients showed temporary reduction in glycosuria before rejection ensued (Fig. 3). Four years later, on October 31st, 1920, the idea occurred to Frederick Banting that ligation of the pancreatic duct in dogs might lead to acinar degeneration and enhanced recovery of the “internal secretions” for treatment of diabetes (5). The effect was dramatic, and ongoing studies by Banting, Best, Collip, and Macleod rapidly led to the introduction of exogenous insulin into clinical practice in 1922. By the following year, Eli Lilly and Company was producing insulin in virtually unlimited quantities for the widespread treatment of diabetics (6). In 1923, it appeared that diabetes had been ‘cured’ by insulin. It only slowly became apparent that while insulin could prevent the devastating and rapidly fatal death sentence from ketoacidosis, it really only provided a very protracted stay of execution, with diabetes becoming an incurable illness with most patients developing one or more end-stage debilitating or life-shortening secondary complication.

A Historical Perspective on Experimental and Clinical Islet Transplantation

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Figure 2 Oscar Minkowski discovered the link between the pancreas and diabetes (1892).

Thus the idea of endocrine replacement therapy lay fallow, and there was little incentive to continue with futile attempts to transplant fragments of pancreatic tissue. It was not until the late 1960’s that Paul E. Lacy

Figure 3 Original article header in The Lancet, 1924, published eight years after the experiments took place. Source: From Ref. 4.

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initiated islet transplantation studies in mice using islets isolated from the pancreas rather than the large pieces of devascularized tissue used previously. Extracting islets from the pancreas was not an easy task. The human pancreas contains 1–14.8 million islets of mean diameter 157 µm constituting only 0.8–3.8% of the total mass of the gland (7,8). In 1911, Bensley stained islets within the guinea pig pancreas using a number of dyes, and was able to pick free the occasional islet for morphological study (9). Armed with watchmaker’s forceps, hypodermic needles, and a binocular microscope, Claus Hellerstro¨m developed methods in 1964 for free-hand micro-dissection of small numbers of islets for biochemical and physiological study (10). These techniques were effective in an obese, hyperglycemic strain of mouse with uniquely large islets, but were impractical in most other species. Prompted by a need for large-scale isolation to further in vitro studies, Moskalewski introduced a mechanical and enzymatic method of dispersion of pancreatic tissue in 1965 using bacterial collagenase derived from Clostridium histolyticum (11). Although the collagenase destroyed many islets, it did permit complete separation of islets from surrounding acinar tissue, with demonstrable viability in culture and appropriate b-cell degranulation in hyperglycemic challenge. Paul E. Lacy and colleague Dr. Kostianovsky introduced two further modifications in 1967 that considerably improved islet yield and recovery (12).a Mechanical disruption of the pancreas by ductal injection of a balanced salt solution greatly increased the subsequent penetration of collagenase, with consequential enhanced islet release. They further discovered that islets could be separated from digested acinar tissue by differential density elutriation on discontinuous sucrose gradients, but these islets failed to release insulin in response to a glucose challenge, which was presumed to be the result of hyperosmolar sucrose injury from cellular dehydration and islet exhaustion. Lindall et al. found that replacement of sucrose gradients with Ficoll (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), a high molecular weight polymer of sucrose, led to more efficient islet separation, and David Scharp and colleagues further showed that dialyzed Ficoll provided islets that responded appropriately in vitro (13,14). These preliminary studies paved the way for transplantation studies in diabetic rodents. Younoszai et al. in 1970 were the first to demonstrate amelioration of the diabetic state in rats by intraperitoneal implantation of allografted islets, with improvement in glycuria but only temporary improvement in glycemia (15). Two years later, Ballinger and Lacy showed

a

Paul E. Lacy gave his final address, three weeks before he passed away, at a historic meeting in Philadelphia. A complete transcript of his account of the progress and challenges of islet isolation and transplantation concludes this chapter.

A Historical Perspective on Experimental and Clinical Islet Transplantation

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sustained improvement (but not complete correction) of chemical diabetes in rats receiving 400–600 islets delivered intraperitoneally or intramuscularly, with graft excision inducing return of diabetes (16). It was not until Rechard and Barker transplanted larger numbers of islets (800–1200) into the peritoneal cavity in 1973 that chemically induced diabetes was effectively cured for the first time (17). Searching for optimal sites for islet implantation, Kemp et al. found that intraportal embolization of only 400–600 rodent islets to an intrahepatic site resulted in complete reversal of diabetes within 24 hours, whereas a similar islet load placed intraperitoneally or subcutaneously was inadequate (18). Portal embolization was thus recognized to be the most efficient site for islet implantation in the rodent, with the benefit of high vascularity, proximity to islet-specific nutrient factors, and physiological first-pass insulin delivery to the liver. It has subsequently become apparent that once embolized to the liver, islets undergo a process of angiogenesis and neovascularization to form a microvascular network and to re-establish nutritional blood supply. In the mouse, capillary sprouts and arterioles arise within 2 to 4 days, interconnect by day 6, and the process is completed by day 10 to 14 (19). These vessels are of host origin, pierce the islet, and branch into capillaries within the centre of the graft (20). Furthermore, it was shown that a physiological “core-to-mantle” perfusion is reinstated for optimal intercellular b-to-alpha/delta sensing and signaling for optimal insulin and glucagon control (21). Similar techniques of islet isolation and purification were not successful when applied to the more dense and fibrous pancreas of larger animals, including the human gland. Mirkovitch et al. were the first to reverse pancreatectomy-induced diabetes by intrasplenic autotransplantation of partially digested pancreatic tissue in dogs (22); intravenous glucose tolerance tests were indistinguishable from normal controls, even if less than half the pancreas was used for tissue digestion. Warnock and others subsequently showed that islet autografts prepared by enzymatic digestion and mechanical dispersion could reliably reverse the diabetic state in dogs (23). Simon Griffin et al. further showed that up to three recipients could be normalized by one donor graft when non-purified pancreatic tissue was infused intrasplenically (24). Unfortunately the human spleen is not distensible as in the dog, where the spleen serves as an important role in autotransfusion in the face of life-threatening hemorrhage. Attempts to transplant impure or partially purified tissue intrasplenically in humans have met with considerable morbidity, including splenic rupture (25), wedge splenic infarction, and portal vein thrombosis in a high proportion of recipients, although insulin independence has been achieved in the autograft setting (26). Investigators resorted to intraportal transplantation of impure pancreatic homogenates in dogs and ultimately in humans, leading to

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disastrous outcomes, including disseminated intravascular coagulation, portal vein thrombosis, and the sequelae of portal hypertension, hepatic infarction, and, in some cases, liver failure (27–29). Mehigan et al. found that the addition of heparin and aprotinin to the tissue preparation at the time of transplantation could ameliorate the risk of disseminated intravascular coagulation (30). Recent progress has occurred in the science of islet isolation, based on evolution of an enzymatic pancreatic dissociation process that provides more consistent high yields of viable human islets for transplantation. The techniques used currently evolved in a strong international collaborative effort with a select number of islet isolation laboratories. The early history and development of the current state-of-the-art isolation methods are reviewed below. Recent methods have increased the efficiency of the process, and have had major impact in enhancing the consistency and quality of highly purified islet preparations for safe transplantation into patients. The evolution towards current techniques is outlined below. Improvement in the isolation and purification of islets from the large animal pancreas became a major focus of intensive study in several laboratories, using the canine pancreas as the pre-clinical model. Intraductal injection of collagenase directly into the pancreatic duct was shown by Horaguchi and Merrell, and subsequently by Noel et al., to be the most effective way to dissociate the pancreas for high yield islet isolation, with up to 57% recovery of the total islet mass (31,32). Trans-ductal collagenase delivery, whether by direct injection (33) or continuous perfusion (34,35), was able to cleave the islet–acinar interface more readily than any method described previously, but still led to significant islet destruction through inadvertent islet enzyme penetration (36). However, the process did permit successful isolation of islets from the pig (37), monkey (38), and human pancreas (33). The approach was further refined to allow precise control of the temperature and perfusion pressure (39). Lakey et al. demonstrated that retrograde intraductal Liberase-HI (Roche Pharmaceuticals, Indianapolis, Indiana, U.S.A.) delivery using a recirculating controlled perfusion system provided superior human islet recovery and survival when compared to syringe loading (40). By providing control over perfusion pressures and Liberase temperature during loading of the enzyme into the pancreas, the recirculating controlled perfusion system more effectively delivers the Liberase to the interface of the islet–acinar interface resulting in a greater separation of islets from the surrounding exocrine tissue (40) A major advance came with the introduction of a semi-automated dissociation chamber and process originally developed by Ricordi et al. in 1988, modifications of which have now become the universal standard for successful high yield large animal and human islet isolation (41). The

A Historical Perspective on Experimental and Clinical Islet Transplantation

Figure 4

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Ricordi continuous digestion chamber and automated shaker.

collagenase-distended pancreas is placed inside a stainless steel chamber containing glass marbles and a 500 µm mesh screen and mechanically dissociated by gentle agitation, with tissue samples evaluated sequentially to determine the end-point before liberated islets become fragmented by overdigestion (Fig. 4). This novel approach minimized trauma to the islets in a continuous digestion process with the collection of free islets as they are liberated from the digestion chamber. A comparison of manual and automated methods of islet isolation clearly demonstrated superiority of the automated method (35,42–44). Since the introduction of this technique, many laboratories around the world have utilized this system for the isolation of islets from canine, pig, and human islets. Large-scale purification of human islets of suitable quality for safe transplantation into the human portal vein was enhanced considerably by the introduction of an automated refrigerated centrifuge system (COBE 2991) by Lake et al., which permitted rapid large volume Ficoll gradient processing in a closed system 600 ml bag (45) (Fig. 5A). When Ficoll is made up in Euro-Collins solution (Euro-FicollÔ), hypertonic exposure of the exocrine component reduces osmotic swelling and enhances differential isletexocrine density improving purification, but results in significant b-cell stress with degranulation and loss of insulin content (46,47). Until recently, a major limitation to successful pancreatic digestion has been the source, quality, and variability in collagenase activity, and contaminants in various enzyme blends. A new class of highly purified enzyme blend (Liberase), containing collagenase I and II, thermolysine, clostripain, and clostridial neutral protease, with low endotoxin activity, has

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Figure 5 (A) COBE 2991Ô cell apheresis system. (B) Final packed cell volume is less than 4 cc Processor (Gambro BCT, Inc., Lakewood, Colorado, U.S.A.) after purification.

consistently provided enhanced islet yield, viability, and function from the human pancreas, and has been an important advance to the field (48–51). Refinements in manufacture have largely eliminated the lot-to-lot variability in crude enzyme effectiveness for islet isolation (50,51). Liberase has proven to be superior to crude collagenase preparations by consistently yielding large numbers of islets without compromising the functional viability (51) (Fig. 6). Despite the key advances in collagenase quality, intraductal enzyme delivery, automated dissociation, and purification outlined above, inconsistency remains in the overall success of the islet isolation procedure, which may reflect variability in donor-related factors (donor inotropic need,

A Historical Perspective on Experimental and Clinical Islet Transplantation

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Figure 6 Low-endotoxin Liberase (Roche Pharmaceuticals, Indianapolis, Indiana, U.S.A.) collagenase—significant improvement in consistency and yield (blend of type I and type II collagenase with thermolysine).

duration of cardiac arrest, hyperglycemia, age, and obesity in the donor, in addition to the skills of the local procurement team) (52). EARLY CLINICAL TRIALS OF ISLET TRANSPLANTATION Prior to the introduction of the Edmonton Protocol in the year 2000, a total of 447 human islet allografts, and 3,185 fetal or neonatal islet allografts and xenografts were carried out in 79 institutions, as reported to the Islet Transplant Registry (53–55). These results were interesting as the excellent success of islet transplantation in small and large animals in the laboratory and of human islet autotransplantation after pancreatectomy stood in contrast to the striking lack of success of islet allotransplantation in the treatment of patients with autoimmune Type 1 diabetes. The first series of clinical islet allograft transplants in Type 1 diabetic patients immunosuppressed with azathioprine and corticosteroids were reported by Najarian et al. in 1977, and followed shortly after reports of successful cure of diabetes by islet transplantation in rats (56). It was anticipated that human islet transplantation would supercede whole pancreas transplantation, which was associated with appalling morbidity and mortality rates in that particular time. While the initial attempts at islet transplantation appeared to be safe, these efforts were largely ineffective. Of seven patients transplanted with dispersed pancreatic tissue into the peritoneal cavity or via the portal vein, no patient achieved insulin independence, although some were able to reduce insulin requirements for limited periods (56). The first C-peptide negative Type 1 diabetic to achieve sustained insulin independence by one year after islet transplantation occurred in 1978 in Zurich, Switzerland

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after single donor-to-recipient transplantation of non-purified islet tissue embolized to the spleen, simultaneous with kidney transplantation (57). Despite a number of anecdotal reports since 1979, only 35 Type 1 diabetic patients have attained insulin independence after islet allograft transplantation, according to data registered with the International Islet Transplant Registry, as of December 1997 (58). Activity in clinical islet transplantation could be subdivided into five categories: (i) islet autografts in patients undergoing total pancreatectomy; (ii) islet allografts after total pancreatectomy; (iii) islet allografts in Type 1 diabetic patients; (iv) fetal islet allografts or xenografts in Type 1 diabetics; and (v) islet allografts in Type 2 diabetics. Success may be judged in terms of patient survival, graft survival (C-peptide production), attainment of insulin independence, effect upon glycemic control (glycosylated HbA1c), overall quality of life, and impact upon secondary diabetic complications.

ISLET AUTOGRAFTS The remarkable success of islet autotransplantation had a major impact on overall progress and attitudes towards islet transplantation established beyond doubt the concept of insulin independence after islet transplantation in the clinical setting. Indeed, the literature suggested that, after total pancreatectomy for chronic pancreatitis and intraportal infusion of purified or unpurified pancreatic digest, approximately 50% of patients will be rendered independent of insulin. The first islet autotransplant following pancreatectomy for chronic pancreatitis was carried out in Minnesota in 1977 (59), and over the subsequent 20 years a world experience has been accrued in 189 patients in 22 centers (7,58). Most patients underwent total or near-total pancreatectomy for intractable pain in chronic pancreatitis without pancreatic duct dilatation. Pyzdrowski et al. reported a limited series of intraportal islet autografts in whom all recipients became insulin independent after transplantation, with documentation of functional intrahepatic islets on liver biopsy staining positive for insulin, glucagons, and somatostatin, and with evidence of intrahepatic insulin secretion on hepatic vein catheterization (60). Reviewing the experience of 69 islet autografts reported to the Islet Transplant Registry, 80% of patients became insulin independent for longer than one week, and 61% maintained insulin independence beyond one year (58). The longest follow-up of insulin independence in islet autografts was more than 13 years (61,62). The best predictor of insulin independence in islet autografts is the number of islets transplanted, with a transplant mass exceeding 300,000 islets associated with an insulin independence rate of 74% at two years post-transplant (63). Farney et al. further showed in a series of 29 islet autografts that 21% of patients lost graft function between 3 and 24 months after intraportal islet embolization where a median of 148,000 islets were

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transplanted, but if a median of 384,500 islets were given there were no late graft failures beyond 2 years, with a maximal follow-up of over 12 years (64). Most centers have used non-purified pancreatic digest for islet autotransplantation because the fibrotic and atrophic nature of grafts scarred by chronic pancreatitis typically yields low tissue volume (usually 5 mls or less). There is also concern that further purification of an already marginal islet transplant mass may render the exercise futile. While complications of portal vein thrombosis, disseminated intravascular coagulopathy, and fatality have been described after islet autotransplantation previously, the risks have been minimized in recent years by systemic heparinization and better characterization of the dispersed grafts (7,30). An accepted approach was to Ficoll-purify pancreatic digests exceeding 15 ml in volume to further lower the risk of portal vein thrombosis (7). The introduction of low-endotoxin collagenase (Liberase) may also be critical in minimizing the acute risk of physiological perturbations associated with infusion of non-purified islet preparations. While many different sites have been tried for islet autotransplantation, the optimal site appears to be through portal venous embolization. Attempts to embolize to the spleen led to significant life-threatening complications of splenic infarction, rupture, and even gastric perforation (26).

ISLET ALLOGRAFTS AFTER PANCREATECTOMY A unique series of nine islet allografts were completed at the University of Pittsburgh in 1989 in patients undergoing abdominal exenteration with multi-visceral resection for malignancy, followed by cluster transplantation of liver, kidney, and bowel (65). Islets were isolated from a single multivisceral donor pancreas in the majority of cases, and infused intraportally after liver reperfusion. Over 50% of recipients achieved and maintained insulin independence until their demise from recurrent malignancy. The series represented an unusual opportunity to complete islet allografts in the absence of an autoimmune diabetes background, which may have contributed to the preservation of the functional reserve of these grafts. Other major factors contributing to the success of the cluster–islet transplantation experience included: (i) embolization of non-purified islet preparations; and (ii) the use of steroid-free immunosuppression (high dose tacrolimus monotherapy), which represented the first experience with less diabetogenic immunosuppression (66).

ISLET ALLOGRAFTS IN TYPE 1 DIABETES A total of over 447 attempts to treat Type 1 diabetes with islet allografts were reported to the Islet Transplant Registry between 1974 and 2000, 394

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of which occurred within the most recent decade (53). Mainstay immunosuppression was largely based on the combination of glucocorticoids, cyclosporine, and azathioprine, with anti-lymphocyte serum induction (67). The majority of these grafts were combined islet–kidney transplants, since it was felt inappropriate to initiate new immunosuppression in islet-alone recipients who would not have otherwise required therapy to sustain another solid organ kidney or liver graft. Under these protocols, fewer than 10% of patients were able to discontinue insulin therapy for longer than one year, although 28% had sustained C-peptide secretion at one year post transplant (54). These disappointing results contrasted with the success of islet autografts and partial success of islet allografts in non-diabetic pancreatectomized recipients where glucocorticoid-free immunosuppression was combined with unpurified islet preparations (66). A key question remained unanswered: were the previous poor results of islet allografts in Type 1 diabetic recipients a result of poor control of alloimmune pathways, or did they reflect recurrence of autoimmune diabetes? Insulin independence was only rarely achievable under glucocorticoid and cyclosporine-based immunosuppression. C-peptide secretion diminished to zero over time in most cases, suggesting islet graft loss from acute rejection or possible recurrence of autoimmune diabetes. Results of whole pancreas transplantation indicate that stable graft function is achievable over time, even with lower dose maintenance immunosuppression, suggesting that prevention of autoimmune destruction might be more readily achieved than prevention of alloimmune rejection. Autoimmune recurrence after whole pancreas transplantation only appeared to be a challenge when no immunosuppression was given, as occurred in a livingdonor hemi-pancreas transplant between identical twins, where autoimmune recurrence led to graft loss within two months. Detailed analysis identified four “common characteristics” associated with improved success (cold ischemia < 8 hours; transplant mass > 6,000 IE/ Kg; intraportal delivery; and ALG/ATG induction, but not OKT3) (55,68); 29% of this subgroup were independent of insulin, and 46% had HbA1c levels of less than 7%, which in the context of the DCCT trial suggests that tight glycemic control afforded by islet transplantation might slow progression of secondary diabetic complications (69). In the late 1990s, results of clinical islet transplantation in patients with T1DM improved under cyclosporine, glucocorticoid, and azathioprine immunosuppression, together with anti-IL2 receptor induction and antioxidants. Combined data from the Giessen and Geneva (GRAGIL consortium) groups reported a 50% rate of C-peptide secretion and 20% insulin independence rate at one year (70,71). Islets were cultured for a mean of two days, and mean islet implant mass was 9,000 IE/ kg, derived from single donors in half of cases. Two of ten patients achieved insulin

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independence after single-donor islet infusions, but it took 6–8 months to achieve independence, and both were recipients of shipped islets from a central islet isolation site (70). The University of Milan further reported experience with two immunosuppressant protocols in Type 1 diabetic islet after kidney recipients [anti-lymphocyte serum (ALS) þ cyclosporine þ azathioprine þ prednisone in the first Era (1989–1996) vs. anti-thymocyte globulin (ATG) þ cyclosporine þ mycophenolate þ metformin together with antioxidants in the second Era (1998–2001)] (72). Rejection rates were low in both eras (3/21 vs. 3/20 in era one vs. era two, respectively). Rates of insulin independence were enhanced from 33% to 59% with the elimination of prednisone and addition of mycophenolate and metformin. Over 50% of patients maintained insulin independence beyond one year with the newer protocol, possibly as a result of more effective immunosuppression coupled with anti-inflammatory, less diabetogenic, and improved insulin action with the newer protocol. The recent era of the Edmonton Protocol and subsequent protocol modifications is outlined in subsequent chapters of this book. LIVING DONOR ISLET TRANSPLANTATION The early era of clinical islet allotransplantation was fraught with difficulties as outlined above. In an attempt to overcome the poor islet viability and yields obtained from these early cadaveric islet isolations, Dr. David Sutherland and colleagues in Minnesota attempted two living donor islet transplants, the first of which took place on September 27th, 1977 (73). Islets were isolated from an HLA-identical sister and transplanted intraportally without purification into her diabetic brother, a recipient of a previous kidney transplant from a different sibling. The recipient became C-peptide positive for six weeks, but never achieved insulin independence. A further attempt at living donor islet transplantation was carried out by the same group on July 12th, 1978. This recipient achieved only temporary insulin independence during the third week post-transplant, then promptly rejected the islets during an episode of renal rejection. Both of these early attempts, while heroic, were largely considered as technical failures. The Minnesota Group then turned their attention to living donor segmental vascularized pancreas grafts, and have reported a high rate of success now in approximately 150 cases, including recent experience with laparoscopic, minimally invasive retrieval surgery in the living donor (74,75). The first successful living donor islet transplant, with sustained attainment and maintenance of insulin independence, was carried out in Kyoto, Japan by Matsumoto and colleagues in January, 2005 (76,77). This was a motherto-daughter transplant in which the recipient had chronic pancreatitis in the absence of autoimmunity. Insulin independence was maintained for at least 7 months, and the early results are encouraging.

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FETAL ISLET ALLOGRAFTS OR XENOGRAFTS IN TYPE 1 DIABETES A surprisingly large number of attempts to transplant fetal or neonatal islet allografts and xenografts has occurred mainly in the Far East in human Type 1 diabetic recipients. In fact, the number of fetal and neonatal islet transplants actually exceeds the number of adult islet allografts by a factor of ten. A total of 3,185 cases have been published or registered since 1977, but the true cumulative total is now estimated to exceed 5,000 (55,58). Access to human fetal tissue is clearly more readily available in China and Eastern Europe, where over 96% of these transplants have been carried out. Turchin et al. reported on their experience in 1,500 human fetal and neonatal porcine islet transplants carried out in Kiev, and found a reduction in hypoglycemic episodes (78). Insulin independence after human fetal or neonatal dispersed pancreas tissue transplantation was reported by Hu et al. in 48 recipients from 54 hospitals in China, with delayed progression of microvascular secondary complications in patients with good graft function after 29 months of followup (79,80). Insulin independence after human fetal islet transplantation has been reported in 9 further recipients in other centers (81–83). Unfortunately, despite this extensive experience, these apparently successful outcomes must be interpreted with caution, as the majority of grafts have been poorly characterized in terms of transplant mass and pre-transplant C-peptide negativity. Tuch et al. recovered human fetal islet grafts with persistent b-cells in three patients 9–14 months after transplantation, but could not demonstrate immunoreactive C-peptide in peripheral blood and found histological changes of islet rejection (84). Groth et al. detected porcine C-peptide in the urine from 200 to 400 days after transplantation in four of ten patients transplanted with fetal porcine islet clusters, but could not document C-peptide in serum (85). Some investigators used non-human xenogeneic islet tissue derived from bovine, porcine, and rabbit sources, with implantation to a variety of sites, including muscle, spleen, bone marrow, and even direct intracerebral implantation (55,86). Most of the transplants were performed without adjuvant immunosuppression. Based on current evidence, human fetal islet transplants are not protected from autoimmune attack (87). The issues of rejection (88), immaturity of the human fetal pancreas, and ethical issues surrounding recovery of human fetal tissue remain significant challenges for this approach in the cure of diabetic patients.

PANCREAS TRANSPLANTATION While this book is largely about islet transplantation, it is important that the progress achieved in islet transplantation is placed in context with the now excellent results attainable in whole pancreas transplantation. Dramatic improvement in outcome has occurred in clinical vascularized pancreas transplantation since the first procedure was carried out by Kelly and Lillehei

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at the University of Minnesota in 1966 (89). The earliest attempts met with dismal mortality rates in excess of 60% and graft survival of only 3% at one year, related to uncontrolled sepsis from failure of duodenal anastomotic healing in the face of high dose steroids (90,91). In 1983, two crucial developments immediately enhanced the success of the procedure. First, the introduction of cyclosporine provided enhanced immunologic potency and reduced sepsis with better tissue healing through its steroid sparing potential; secondly, both Cory and Sollinger described techniques for bladder drainage of pancreatic exocrine secretions, which provided better immune monitoring through urinary amylase assessment, a lower anastomotic leak rate with reduced gram negative sepsis (92–94). Subsequent improvement in outcome led to endorsement of simultaneous pancreas-kidney transplantation as recommended treatment of the Type 1 diabetic presenting in non-reversible renal failure (95). Up till 2000, pancreas transplantation was currently the only treatment of Type 1 diabetes that consistently restores sustained endogenous secretion of insulin responsive to near-normal levels, leading to correction of HbA1c, far surpassing the impact of intensive insulin management achievable in the DCCT trial (96,97). Currently, there have been more than 25,000 whole pancreas transplants performed worldwide, the majority of which have been carried out as simultaneous pancreas-kidney (SPK) grafts, although numbers of solitary pancreas transplants are increasing. At the time the Edmonton Protocol moved forward in 2000, the actuarial survival of patients and of functional pancreas grafts (with complete insulin independence) was 94% and 89% at one year and 81% and 67% at five years, respectively, according to registry data (98). The results of pancreas-alone grafts were inferior to simultaneous pancreas-kidney grafts. However, pancreas-alone transplantation led to excellent outcomes in carefully selected individuals under tacrolimus-based immunosuppression, with one-year graft survival at 80% to 90%, with corresponding patient survival as high as 95% (99–101). Compared with kidney-alone transplantation in Type-1 diabetics, patient survival improved by at least 10% by five years and by up to 59% at ten years following combined transplantation (102,103). Freedom from insulin, blood glucose monitoring, and dietary restriction improves the overall quality of life for the diabetic undergoing successful pancreas-kidney transplantation, but scores generally fail to match those of the general non-diabetic healthy population by one year posttransplant (104–106). Quality of life improvement is particularly evident in patients with hypoglycemic unawareness, brittle diabetes, or gastroparesis (107). Recent advances in surgical technique, immunosuppression, and posttransplant monitoring have had major impact in reducing the morbidity of patients undergoing simultaneous pancreas-kidney and solitary pancreas transplantation. A recent return to enteric exocrine drainage by graft duodenojejunal anastomosis has dramatically reduced complications of urinary tract infection, urethritis, urethral stricture, and metabolic acidosis, and therefore the need to perform enteric conversion in up to 33% of cases (108–111).

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THE HISTORY OF ISLET TRANSPLANTATION BY PAUL E. LACY PHILADELPHIA, PENNSYLVANIA, U.S.A. DECEMBER 2ND, 2004 Paul E. Lacy passed away on February 15th, 2005. Approximately three months before he passed away, he gave his final, remarkable public lecture where most of the leaders in the islet transplant field were in attendance. The lecture was recorded, and a verbatim extract is provided below.

Paul E. Lacy giving his final lecture at the NDRI Meeting in Philadelphia, on December 2nd 2004, three weeks before his death.

I was asked whether I would be using PowerPoint for this lecture but they forgot that when I started we were still using the magic lantern and I haven’t progressed much beyond that. What I have planned is an overview of islet transplantation. I will not be going in to details, and I will not be mentioning some people not because their work wasn’t great because it was great, but I’m just trying to give a feeling of how it all came about. You can’t transplant islets unless you know how to isolate them. There was a man named Moskalewski in the mid-1960s who reported you could take the guinea pig pancreas, incubate it with collagenase, chop it and let it settle in a cylinder and in saline, and the islets would accumulate at the bottom. Well I tried it. Yes you could. You could get some islets from the guinea pig pancreas. So we tried it on the rat, which is the one we used for research on diabetes. With that we got a few but not very many. So we worked on the process for quite a while, and then came upon the idea that the islets are not connected to the pancreatic ducts. So we simply injected a salt solution into the pancreatic ducts, which blew

A Historical Perspective on Experimental and Clinical Islet Transplantation

up the acinar tissue, separated it from the islets, then we incubated with collagenase, chopped it, and there were hundreds of islets. You could see them under the dissecting microscope, you could hand-pick individual islets so they were absolutely pure. Now this made it possible to do fundamental studies on insulin formation, storage, and release in many laboratories, and it was wonderful. It did raise the curiosity question, and it was curiosity, what would happen if you transplanted these islets into diabetic rats in an inbred strain where you wouldn’t have to worry about rejection? We tried it. It worked. It reversed the diabetes. Young David Scharp came along at that time—he was a postdoctoral fellow training in surgery—and he looked at the different sites one used to transplant the islets. He found that the liver was the most ideal in terms of the smallest number of islets needed to reverse diabetes, and you put them in by injecting into the portal vein that drained into the liver. They were large enough to be trapped in the liver, small enough that they didn’t damage the liver. So that then opened up other questions as to what would happen with the complications of diabetes as they occur in the rodent. Konrad Federlin in Germany led the way in this area, and many others as well, and demonstrated that it would prevent these complications if you transplanted the islets ahead of time, and would reverse the early complications almost entirely in rats. So now it was a question of what you do now? Well, I thought it would be a perfect opportunity to see could you transplant islets in rats without having to give continuous immunosuppression. And at time there was a theory, a theory proposed by Snell, in which he suggested that maybe passenger leukocytes, antigen-presenting cells, carried along with the transplant, they were responsible for initiating rejection. So if you wanted to prevent rejection, you got rid of the passenger leukocytes. Pure theory, very little to support it. But it was one, as we had pure islets, we thought we could test. So, with Joe Davey, who was Head of Immunology at our institution at the time, we started. And we thought that with pure islets, that had no lymph nodes, no ducts, no nothing, they were absolutely pure, maybe we could just use those and that that would work. No it didn’t. When you transplanted between strains they were rejected. So if the theory was correct that meant there were still passenger leukocytes in the islets that we had to get rid of. So we tried different approaches, one of them was to inject silica, sand, into the donor animals, remove the islets hoping it would kill off macrophages. It helped a little, not very much. Then we read of Kevin Lafferty’s original publication, in which he stated he could take the mouse thyroid, incubate it in 95% oxygen for three weeks, then transplant it from one strain of mouse to another, no immunosuppression. He suggested he was killing the passenger leukocytes within the thyroid. We tried it immediately. Unfortunately, within three days the islets were dead. They could not tolerate the high oxygen concentration.

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I happened to see an article by Terasaki in California. He had been shipping lymphoid spleen cells across the country at room temperature. He found that when they went from California to New York that they would not stimulate an immune reaction. Well that was exactly what we wanted. So I said OK, let’s try it. So we took islets in a Petri dish in tissue culture, rat islets, and put them on the top of the desk and left them at room temperature. Looking at them each day, they didn’t disintegrate, they were there, they were intact, so we said, “what the heck, let’s transplant them, and give an injection of an antibody to lymphocytes at the time we transplanted.” We did. It worked. It completely prevented rejection without the need for any further immunosuppression. Now the immunologists were a little suspicious of that, thinking that maybe you mixed up the white rats! So we knew you couldn’t mix up rats and mice, so we tried it, rat islets into mice across the species barrier. Same procedure, it worked. No question. Now the question was what to do next, because that raised the possibility that if you ever transplanted islets into diabetic patients, that maybe you could use this method or a modification of the method to produce tolerance in the recipient, and that tolerance was specific for the tissue you put in. You could put in other tissue and it would be rejected. That would be ideal for the child. So David Scharp and I talked about what to do. We decided to go on and now try to isolate human islets. Many others had gone on with this, and used syringes and needles to disrupt the collagenase-digested pancreas and obtained some islets. Others used perfusion to disrupt the pancreas and obtained some islets, but it needed to be improved. So we used the old cow pancreas as the model for the human, because it is big and it has lots of collagen within it. But one of the problems with the collagenase digestion is that the collagen breaks down and you have “goo” and that traps the islets. So, my wife said, “Why don’t you use Velcro?” Well that was way long time ago. I didn’t know what Velcro was! So as you know it has little hooks, and we put the pancreas on the hooks and sure enough it held it in place and held back the “goo”, but it was a little traumatic and it held back the islets as well. So David and I talked and we thought about it. And then we thought you need a way of chopping the digested pancreas as rapidly as possible. Well, if you think about it, you chop normally with a pair of scissors as we did in the rat, but we can’t do that with the human pancreas or with the old cow pancreas. So it brought to mind my mother’s hand-operated meat grinder that was in the attic. We brought it in, changed the force plate on it, put the pancreas through, and sure enough there were islets. Now to separate those initially we used a flour sifter. You see we moved from the sewing room to the kitchen for these elegant tools! The wire that goes around on the flour sifter was a little hard on the islets, so David then developed a way to separate them on Ficoll gradients by centrifugation.

A Historical Perspective on Experimental and Clinical Islet Transplantation

Now that brought us to the point where we had to have human pancreases. Lee Ducat saved the day, as we could only get maybe one or two human pancreases locally. No way could we develop a method with just those few pancreases. She established a network called the National Diabetes Research Interchange (NDRI), obtained the pancreases, and we got underway. About this time also the elegant work of Ray Rajotte was done, in which he cryopreserved the islets in animals and in man. So that made it possible, even though we were getting few islets, that you could bring together different isolations, cryopreserve them, and give them all then to a patient, which we did. The patients had no C-peptide, no indication of insulin secretion prior to transplantation even with stimulation. After transplantation, they did not come off insulin, but they had C-peptide. There was no question there was function there, but it was not good enough. About that time a young man came to my laboratory to learn about islet transplantation, and that was Camillo Ricordi. The day after he arrived, we took him around to look at apartments, and on the way there he saw a jeep, “Is that a jeep?” “Yes, that’s a jeep.” “We don’t sell those in Italy.” He kept yapping about the jeep, and just couldn’t get it out of his system. I don’t think he heard a thing about the apartments. For the next three days he went from dealership to dealership, and finally he bought a jeep, and was delighted with it. It took a few more days till he got it all out of his system. Then he began working in the lab and it was elegant. He developed a way to gently digest a pancreas and get islets. We had an extra human pancreas that David and I knew we couldn’t get islets out with the meat grinder method we were using, so we gave it to him and he got islets. We dropped everything and turned to this method, which is now used in nearly all laboratories in the world. Now having many many islets, hundreds of thousands, it was possible to transplant into patients, and they would come off insulin. This was done in many laboratories. In coming off insulin, you followed them and a year later only about 15% were still off insulin. No one had any idea what was going on. But there was a young man in Edmonton, Canada, James Shapiro and his associates, had an idea what to do. They tried it. It worked. I am very very proud of him for what he’s done because he rejuvenated the area and the field of islet transplantation. Now, at this time, as he will tell you, 85% of the patients are still off insulin one year later, not 15%, which is marvelous. From my standpoint there are two problems that still remain. One of them is to be able to transplant the islets without the need for continuous immunosuppression. Approaches for this are to try to induce tolerance in the recipient as I told you about in the rat, another is encapsulation of the islets to protect them from the immune system, and remarkable advances have been made in both those areas.

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The second major problem from my standpoint is the source of islets, the continued supply of islets. How are you going to do this? You will hear about stem cells to make islets, making islets in vitro from duct cells and other cells. This is an approach that I think very well will work, and it will need more effort and more time to do it. Another approach is to use xenografts of islets, pig islets. Carl Groth was the first to transplant pig islets into diabetic patients. There is now lots of work being done in that area. Now, finally I have no doubt it will be possible to do all these things, and it will be possible to transplant islets into a diabetic child without continuous immunosuppression. For me this journey has been a delightful one. Of course there have been peaks, and of course there have been terrible valleys that have been deep and wide, but it has been a wonderful, wonderful journey. I feel so very privileged to have been a part of a therapeutic approach from the bench to the bedside of the patient. I also feel privileged in having worked with so many, many, wonderful people, and for having established friendships that I shall always cherish. Thank you.

Paul E. Lacy, M.D. (1924–2005)

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Williams P. Notes on diabetes treated with extract and by grafts of sheep’s pancreas. British Medical Journal 1894; 2:1303. Minkowski O. Weitere Mitteilungen u¨ber den diabetes mellitus nach extirpation des pankreas. Berl Klin Wochenschr 1892; 29:90. von Mering J, Minkowski O. Arch Exp Pathol Pharmakol 1889; 26:371. Pybus F. Notes on suprarenal and pancreatic grafting. Lancet 1924:550. Banting F. Extract from Banting’s Notebook 2am October 31st. Academy of Medicine notebook, Archives of Toronto University, Canada 1920 . Bliss M. The discovery of insulin. Toronto:McClelland and Stewart Limited, 1982. Robertson GS, Dennison AR, Johnson PR, London NJ. A review of pancreatic islet autotransplantation. Hepatogastroenterology 1998; 45(19):226. Bretzel RG, Hering BJ, Federlin KF. Islet cell transplantation in diabetes mellitus—from bench to bedside. Exp Clin Endocrinol Diabetes 1995; 103 (Suppl 2):143. Bensley RR. Studies on the pancreas of the guinea pig. Am J Anat 1911; 12:297. Hellerstro¨m C. A method for the microdissection of intact pancreatic islets of mammals. Acta Endocrinol 1964; 45:122. Moskalewski S. Isolation and culture of the islets of langerhans of the guinea pig. Gen Comp Endocrinol 1965; 5:342. Lacy P, Kostianovsky M. Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 1967; 16:35. Lindall A, Steffes M, Sorenson R. Immunoassayable insulin content of subcellular fractions of rat islets. Endocrinology 1969; 85(2):218. Scharp DW, Kemp CB, Knight MJ, Ballinger WF, Lacy PE. The use of ficoll in the preparation of viable islets of langerhans from the rat pancreas. Transplantation 1973; 16(6):686. Younoszai R, Sorensen R, Lindall A. Homotransplantation of isolated pancreatic islets. Diabetes 1970; 19(suppl 1):406. Ballinger WF, Lacy PE. Transplantation of intact pancreatic islets in rats. Surgery 1972; 72(2):175. Reckard CR, Ziegler MM, Barker CF. Physiological and immunological consequences of transplanting isolated pancreatic islets. Surgery 1973; 74(1):91. Kemp C, Knight M, Scharp D, Ballinger W, Lacy P. Effect of transplantation site on the result of pancreatic islet isografts in diabetic rats. Diabetologia 1973; 9:486. Menger MD, Wolf B, Hobel R, Schorlemmer HU, Messmer K. Microvascular phenomena during pancreatic islet graft rejection. Langenbecks Arch Chir 1991; 376(4):214. Vajkoczy P, Menger MD, Simpson E, Messmer K. Angiogenesis and vascularization of murine pancreatic islet isografts. Transplantation 1995; 60 (2):123. Menger MD, Vajkoczy P, Beger C, Messmer K. Orientation of microvascular blood flow in pancreatic islet isografts. J Clin Invest 1994; 93(5):2280.

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22. Mirkovitch V, Campiche M. Successful intrasplenic autotransplantation of pancreatic tissue in totally pancreatectomized dogs. Transplantation 1976; 21: 265. 23. Warnock GL, Rajotte RV, Procyshyn AW. Normoglycemia after reflux of islet-containing pancreatic fragments into the splenic vascular bed in dogs. Diabetes 1983; 32(5):452. 24. Griffin SM, Alderson D, Farndon JR. Comparison of harvesting methods for islet transplantation. Br J Surg 1986; 73(9):712. 25. Gores PF, Najarian JS, Stephanian E, Lloveras JJ, Kelley SL, Sutherland DE. Transplantation of unpurified islets from single donors with 15- deoxyspergualin. Transplant Proc 1994; 26(2):574. 26. White SA, London NJ, Johnson PR, et al. The risks of total pancreatectomy and splenic islet autotransplantation. Cell Transplant 2000; 9(1):19. 27. Shapiro AM, Lakey JR, Rajotte RV, et al. Portal vein thrombosis after transplantation of partially purified pancreatic islets in a combined human liver/islet allograft. Transplantation 1995; 59(7):1060. 28. Walsh TJ, Eggleston JC, Cameron JL. Portal hypertension, hepatic infarction, and liver failure complicating pancreatic islet autotransplantation. Surgery 1982; 91(4):485. 29. Froberg MK, Leone JP, Jessurun J, Sutherland DE. Fatal disseminated intravascular coagulation after autologous islet transplantation. Hum Pathol 1997; 28(11):1295. 30. Mehigan DG, Bell WR, Zuidema GD, Eggleston JC, Cameron JL. Disseminated intravascular coagulation and portal hypertension following pancreatic islet autotransplantation. Ann Surg 1980; 191(3):287. 31. Horaguchi A, Merrell RC. Preparation of viable islet cells from dogs by a new method. Diabetes 1981; 30(5):455. 32. Noel J, Rabinovitch A, Olson L, Kyriakides G, Miller J, Mintz DH. A method for large-scale, high-yield isolation of canine pancreatic islets of Langerhans. Metabolism 1982; 31(2):184. 33. Gray DW, McShane P, Grant A, Morris PJ. A method for isolation of islets of Langerhans from the human pancreas. Diabetes 1984; 33(11):1055. 34. Rajotte RV, Warnock GL, Evans MG, Ellis D, Dawidson I. Isolation of viable islets of Langerhans from collagenase-perfused canine and human pancreata. Transplant Proc 1987; 19(1 Pt 2):918. 35. Warnock GL, Kneteman NM, Evans MG, Dabbs KD, Rajotte RV. Comparison of automated and manual methods for islet isolation. Can J Surg 1990; 33(5):368. 36. van Suylichem PT, Wolters GH, van Schilfgaarde R. Peri-insular presence of collagenase during islet isolation procedures. J Surg Res 1992; 53(5):502. 37. Ricordi C, Finke EH, Lacy PE. A method for the mass isolation of islets from the adult pig pancreas. Diabetes 1986; 35(6):649. 38. Gray DW, Warnock GL, Sutton R, Peters M, McShane P, Morris PJ. Successful autotransplantation of isolated islets of Langerhans in the cynomolgus monkey. Br J Surg 1986; 73(10):850. 39. Warnock GL, Cattral MS, Rajotte RV. Normoglycemia after implantation of purified islet cells in dogs. Can J Surg 1988; 31(6):421.

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40. Lakey JR, Warnock GL, Shapiro AM, et al. Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion. Cell Transplant 1999; 8(3):285. 41. Ricordi C, Lacy PE, Scharp DW. Automated islet isolation from human pancreas. Diabetes 1989; 38Suppl 1:140. 42. Ao Z, Lakey JR, Rajotte RV, Warnock GL. Collagenase digestion of canine pancreas by gentle automated dissociation in combination with ductal perfusion optimizes mass recovery of islets. Transplant Proc 1992; 24(6):2787. 43. Toomey P, Chadwick DR, Contractor H, Bell PR, James RF, London NJ. Porcine islet isolation: prospective comparison of automated and manual methods of pancreatic collagenase digestion. Br J Surg 1993; 80(2):240. 44. Ricordi C, Rastellini C. Automated method for pancreatic islet separation. In:Ricordi C, ed. Methods in Cell Transplantation. Austin, Tx:RG Landes, 1995:433. 45. Lake SP, Bassett PD, Larkins A, et al. Large-scale purification of human islets utilizing discontinuous albumin gradient on IBM 2991 cell separator. Diabetes 1989; 38 Suppl 1:143. 46. Lakey JR, Cavanagh TJ, Zieger MA. A prospective comparison of discontinuous EuroFicoll and EuroDextran gradients for islet purification. Cell Transplant 1998; 7(5):479. 47. Brandhorst H, Brandhorst D, Brendel MD, Hering BJ, Bretzel RG. Assessment of intracellular insulin content during all steps of human islet isolation procedure. Cell Transplant 1998; 7(5):489. 48. Gill JF, Chambers LL, Baurley JL, et al. Safety testing of Liberase, a purified enzyme blend for human islet isolation. Transplant Proc 1995; 27(6):3276. 49. Linetsky E, Selvaggi G, Bottino R, et al. Comparison of collagenase type P and Liberase during human islet isolation using the automated method. Transplant Proc 1995; 27(6):3264. 50. Linetsky E, Bottino R, Lehmann R, Alejandro R, Inverardi L, Ricordi C. Improved human islet isolation using a new enzyme blend, liberase. Diabetes 1997; 46(7):1120. 51. Lakey JR, Cavanagh TJ, Zieger MA, Wright M. Evaluation of a purified enzyme blend for the recovery and function of canine pancreatic islets. Cell Transplant 1998; 7(4):365. 52. Lakey JR, Warnock GL, Rajotte RV, et al. Variables in organ donors that affect the recovery of human islets of Langerhans. Transplantation 1996; 61 (7):1047. 53. Brendel M, Hering B, Schulz A, Bretzel R. International Islet Transplant Registry Report. University of Giessen, Germany , 1999:1. 54. Bretzel RG, Brandhorst D, Brandhorst H, et al. Improved survival of intraportal pancreatic islet cell allografts in patients with type-1 diabetes mellitus by refined peritransplant management. J Mol Med 1999; 77(1):140. 55. Hering B, Ricordi C. Islet transplantation for patients with Type 1 diabetes: results, research priorities, and reasons for optimism. Graft 1999; 2(1):12. 56. Najarian JS, Sutherland DE, Matas AJ, Steffes MW, Simmons RL, Goetz FC. Human islet transplantation: a preliminary report. Transplant Proc 1977; 9(1): 233.

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57. Largiader F, Kolb E, Binswanger U, Illig R. [Successful allotransplantation of an island of Langerhans]. Schweiz Med Wochenschr 1979; 109(45):1733. 58. Brendel MD, Hering BJ, Schultz AO, Bretzel RG. Islet Transplant Registry Newsletter No. 8, 1998. 59. Najarian JS, Sutherland DE, Matas AJ, Goetz FC. Human islet autotransplantation following pancreatectomy. Transplant Proc 1979; 11(1):336. 60. Pyzdrowski KL, Kendall DM, Halter JB, Nakhleh RE, Sutherland DE, Robertson RP. Preserved insulin secretion and insulin independence in recipients of islet autografts. N Engl J Med 1992; 327(4):220. 61. The international islet transplant registry report. Newsletter No. 7 1996; 6(1):1. 62. Robertson RP, Lanz KJ, Sutherland DE, Kendall DM. Prevention of diabetes for up to 13 years by autoislet transplantation after pancreatectomy for chronic pancreatitis. Diabetes 2001; 50(1):47. 63. Sutherland DE, Gores PF, Hering BJ, Wahoff D, McKeehen DA, Gruessner RW. Islet transplantation: an update. Diabetes Metab Rev 1996; 12(2):137. 64. Farney AC, Hering BJ, Nelson L, et al. No late failures of intraportal human islet autografts beyond 2 years. Transplant Proc 1998; 30(2):420. 65. Tzakis AG, Ricordi C, Alejandro R, et al. Pancreatic islet transplantation after upper abdominal exenteration and liver replacement. Lancet 1990; 336(8712):402. 66. Ricordi C, Tzakis AG, Carroll PB, et al. Human islet isolation and allotransplantation in 22 consecutive cases. Transplantation 1992; 53(2):407. 67. Boker A, Rothenberg L, Hernandez C, Kenyon NS, Ricordi C, Alejandro R. Human islet transplantation: update. World J Surg 2001; 25(4):481. 68. Hering B, Brendel M, Schultz A, Schultz B, Bretzel R. International Islet Transplant Registry. Newsletter 1996; 6(7):1. 69. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of longterm complications in insulin dependent diabetes mellitus. N Engl J Med 1993; 329:977. 70. Oberholtzer J, Benhamou P, Toso C, et al. Human islet transplantation network for the treatment of type 1 diabetes: first (1999-2000) data from the Swiss-French GRAGIL Consortium. Americal Journal of Transplantation 2001; 1(1):182. 71. Oberholzer J, Triponez F, Mage R, et al. Human islet transplantation: lessons from 13 autologous and 13 allogeneic transplantations. Transplantation 2000; 69(6):1115. 72. Maffi P, Bertuzzi F, Guiducci D, et al. Per and peri-operative management influences the clinical outcome of islet transplantation. Americal Journal of Transplantation 2001; 1(1 Suppl 1):181. 73. Sutherland DE, Goetz FC, Najarian JS. Living-related donor segmental pancreatectomy for transplantation. Transplant Proc 1980; 12(4 Suppl 2):19. 74. Gruessner RW, Sutherland DE, Drangstveit MB, Bland BJ, Gruessner AC. Pancreas transplants from living donors: short- and long-term outcome. Transplant Proc 2001; 33(1-2):819. 75. Tan M, Kandaswamy R, Sutherland DE, Gruessner RW. Laparoscopic donor distal pancreatectomy for living donor pancreas and pancreas-kidney transplantation. Am J Transplant 2005; 5(8):1966.

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76. Matsumoto S, Okitsu T, Iwanaga Y, et al. Follow-up study of the first successful living donor islet transplantation. Transplantation 2006; 82(12):1629. 77. Matsumoto S, Okitsu T, Iwanaga Y, et al. Insulin independence after livingdonor distal pancreatectomy and islet allotransplantation. Lancet 2005; 365 (9471):1642. 78. Turchin I, Tronko M, Komissarenko V. Experience of 1.5 thousand transplantations of b-cell cultures to patients with diabetes mellitus. Fifth International Congress on Pancreas and Islet Transplantation. Miami, Florida, 1995. 79. Hu YF, Cheng RL, Shao AH, et al. The influences of islet transplantation on metabolic abnormalities and diabetic complications. Horm Metab Res 1989; 21(4):198. 80. Hu YF, Gu ZF, Zhang HD, Ye RS. Fetal islet transplantation in China. Transplant Proc 1992; 24(5):1998. 81. Chastan P, Berjon JJ, Gomez H, Meunier JM. Treatment of an insulindependent diabetic by homograft of fetal pancreas removed before the tenth week of pregnancy: one-year follow-up. Transplant Proc 1980; 12(4 Suppl 2): 218. 82. Bojko N, Chooklin S, Perejaslov A. Results of clinical islet transplantation after allotransplantation of pancreatic islet cell cultures to diabetic patients. Acta Diabetologica 1997; 34:148(abstract). 83. Valente U, Ferro M, Barocci S, et al. Report of clinical cases of human fetal pancreas transplantation. Transplant Proc 1980; 12(4 Suppl 2):213. 84. Tuch BE, Sheil AG, Ng AB, Trent RJ, Turtle JR. Recovery of human fetal pancreas after one year of implantation in the diabetic patient. Transplantation 1988; 46(6):865. 85. Groth CG, Korsgren O, Tibell A, et al. Transplantation of porcine fetal pancreas to diabetic patients. Lancet 1994; 344(8934):1402. 86. Komissarenko VP, Turchin IS, Komissarenko IV, Efimov AS, Benikova EA. Transplantation of an islet cell culture of human and animal fetal pancreases as a treatment method in diabetes mellitus. Vrach Delo 1983(4):52. 87. Sundkvist G, Bergqvist A, Weibull H, et al. Islet cell antibody reactivity with human fetal pancreatic islets. Diabetes Res Clin Pract 1991; 14(1):1. 88. Djordjevic PB, Brkic S, Lalic NM, et al. Human fetal pancreatic islet transplantation in insulin-dependent diabetics:possibilities of early detection of transplant destruction. Glas Srp Akad Nauka [Med] 1994; 44:83. 89. Kelly WD, Lillehei RC, Merkel FK, Idezuki Y, Goetz FC. Allotransplantation of the pancreas and duodenum along with the kidney in diabetic nephropathy. Surgery 1967; 61(6):827. 90. Bartlett S. Pancreatic transplantation after thirty years: still room for improvement. J Am Coll Surg 1996; 183:408. 91. Sutherland D, Kendall D, Goetz F, Najarian J. Pancreas transplantation in humans. In: Flye MW, ed. Principles of organ transplantation. Philadelphia: W.B. Saunders Company, 1989. 92. Nghiem DD, Gonwa TA, Corry RJ. Metabolic effects of urinary diversion of exocrine secretions in pancreatic transplantation. Transplantation 1987; 43(1):70.

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93. Cook K, Sollinger HW, Warner T, Kamps D, Belzer FO. Pancreaticocystostomy: an alternative method for exocrine drainage of segmental pancreatic allografts. Transplantation 1983; 35(6):634. 94. Sollinger HW, Knechtle SJ, Reed A, et al. Experience with 100 consecutive simultaneous kidney-pancreas transplants with bladder drainage. Ann Surg 1991; 214(6):703. 95. American Diabetes Association. Position statement: pancreas transplantation for patients with diabetes mellitus. Diabetes Care 1992; 15:1668. 96. Ryan EA. Pancreas transplants: for whom? Lancet 1998; 351(9109):1072. 97. Stratta RJ. Vascularised pancreas transplantation. BMJ 1996; 313(7059):703. 98. Sutherland DE. Pancreas transplantation. J Diabetes Complications 2001; 15 (1):10. 99. Bartlett ST, Schweitzer EJ, Johnson LB, et al. Equivalent success of simultaneous pancreas kidney and solitary pancreas transplantation. A prospective trial of tacrolimus immunosuppression with percutaneous biopsy. Ann Surg 1996; 224(4):440. 100. Gruessner RW, Sutherland DE, Najarian JS, Dunn DL, Gruessner AC. Solitary pancreas transplantation for nonuremic patients with labile insulindependent diabetes mellitus. Transplantation 1997; 64(11):1572. 101. Sutherland DE, Gruessner RW, Dunn DL, et al. Lessons learned from more than 1,000 pancreas transplants at a single institution. Ann Surg 2001; 233(4):463. 102. Tyden G, Bolinder J, Solders G, Brattstrom C, Tibell A, Groth CG. Improved survival in patients with insulin-dependent diabetes mellitus and end-stage diabetic nephropathy 10 years after combined pancreas and kidney transplantation. Transplantation 1999; 67(5):645. 103. Kumar A, Newstead CG, Lodge JP, Davison AM. Combined kidney and pancreatic transplantation. Ideal for patients with uncomplicated type 1 diabetes and chronic renal failure. BMJ 1999; 318(7188):886. 104. Gross CR, Limwattananon C, Matthees BJ. Quality of life after pancreas transplantation: a review. Clin Transplant 1998; 12(4):351. 105. Matas AJ, McHugh L, Payne WD, et al. Long-term quality of life after kidney and simultaneous pancreas-kidney transplantation. Clin Transplant 1998; 12 (3):233. 106. Adang EM, Kootstra G, Engel GL, van Hooff JP, Merckelbach HL. Do retrospective and prospective quality of life assessments differ for pancreaskidney transplant recipients? Transpl Int 1998; 11(1):11. 107. Kendall DM, Rooney DP, Smets YF, Salazar Bolding L, Robertson RP. Pancreas transplantation restores epinephrine response and symptom recognition during hypoglycemia in patients with long-standing type I diabetes and autonomic neuropathy. Diabetes 1997; 46(2):249. 108. Kuo PC, Johnson LB, Schweitzer EJ, Bartlett ST. Simultaneous pancreas/ kidney transplantation—a comparison of enteric and bladder drainage of exocrine pancreatic secretions. Transplantation 1997; 63(2):238. 109. Newell KA, Bruce DS, Cronin DC, et al. Comparison of pancreas transplantation with portal venous and enteric exocrine drainage to the standard technique utilizing bladder drainage of exocrine secretions. Transplantation 1996; 62(9):1353.

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110. Sollinger HW, Sasaki TM, D’Alessandro AM, et al. Indications for enteric conversion after pancreas transplantation with bladder drainage. Surgery 1992; 112(4):842. 111. Sollinger HW, Ploeg RJ, Eckhoff DE, et al. Two hundred consecutive simultaneous pancreas-kidney transplants with bladder drainage. Surgery 1993; 114(4):736.

2 Hypoglycemia in Type 1 Diabetes: The Need for a New Approach Stephanie A. Amiel King’s College School of Medicine, King’s College, London, U.K.

INTRODUCTION When blood glucose concentrations fall too low to support normal brain function, confusion, abnormal behaviors, coma, and seizures may ensue. It is not surprising that hypoglycemia, low blood glucose, ranks with blindness and renal failure as a fear for patients with Type 1 diabetes (1) and is widely believed to be a major limitation to the ability to achieve targets for glycemic control (2). The major trial that demonstrated unequivocally the link between lower mean blood glucose concentrations and reduced risk of longterm vascular complications (The Diabetes Control and Complications Trial, or DCCT) showed a three-fold increase in hypoglycemic episodes severe enough to render the patient incapable of self-management in those randomly assigned to receive intensive diabetes therapy (3). The negative associations shown between risk of hypoglycemia and mean glycated hemoglobin (HbA1c, a measure of mean blood glucose concentrations over about 2 months) are compatible with the logical assumption that running blood glucose concentrations lower increases the risk of overshooting into frank hypoglycemia (4). In the DCCT, there was also a direct association between the use of intensive insulin therapy and the risk of severe hypoglycemia, irrespective of HbA1c achieved (Fig. 1) (5). Although more modern methods of diabetes management can achieve improved mean blood glucose concentrations without increasing the risk of severe hypoglycemia (6–8), expectations have also increased. Patients and professionals now expect to achieve the more rigorous glucose targets needed in the fight against long-term complications but, at the same time, are far less accepting 29

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Rate per 100 patient years

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Figure 1 The rate of severe hypoglycemia from the Diabetes Control and Complications Trial, plotted against HbA1c. Note that as HbA1c rises, the risk of severe hypoglycemia increases, but the risk is higher during intensified therapy for any given HbA1c. Source: From Ref. 3.

of the lifestyle restrictions imposed on people using insulin therapy than before. Despite major improvements in treatment regimens (6–9), the introduction of designer insulins with (slightly) more physiological action profiles (e.g., 10,11) and advances in home blood glucose monitoring (12,13), severe hypoglycemia remains a major problem in the appropriate management of insulin-deficient diabetes mellitus. Furthermore, avoiding intensified insulin therapy and not maintaining near-normoglycemia does not provide protection against severe hypoglycemia; all insulin deficient diabetic patients are at risk. DEFINITIONS OF HYPOGLYCEMIA Definitions of hypoglycemia remain controversial. Professional fear of hypoglycemia has led the American Diabetes Association recently to define hypoglycemia in diabetes treatment as any blood glucose less than 4 mmol/L, which means that healthy people often fall into this category (14)! It is true that the healthy body’s own mechanisms for maintaining blood glucose concentrations are activated as soon as blood glucose concentrations start to fall, with a reduction in endogenous insulin secretion and, at an arterialized plasma glucose of around 3.6 mmol/L, an increase in pancreatic glucagon secretion, together mediating an increase in endogenous glucose production primarily

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from the liver as a defense against a more profound glucose fall (15). A slightly greater hypoglycemia is required to trigger adrenergic and autonomic activation, leading to subjective awareness of a low blood glucose concentration. More profound hypoglycemia still triggers growth hormone and cortisol responses (15,16). The effects of the neuro-humoral responses are to increase endogenous glucose production, both directly and indirectly (by provision of additional substrate for gluconeogenesis), and to reduce glucose uptake by “inessential” peripheral tissues, such as muscle and fat, diverting available glucose to the brain and heart. In healthy individuals, these mechanisms are so efficient that severe hypoglycemia, with clinically significant cognitive dysfunction, does not occur, at least outside extremes of behavior, such as marathon running. In experimental studies, where the circulating glucose concentration is forced down, usually by insulin infusion, cognitive dysfunction is first detectable at an arterialized plasma glucose concentration of around 3 mmol/L (17). The initial cortical dysfunction will be manifest as slowed or less accurate performance of specific cortical function tasks, but has been interpreted as relevant to the sort of impairment that increases the risk of driving and other accidents. Dangerously impaired performance can be demonstrated in driving simulations at these sorts of glucose concentrations (18). An increasing range of cortical functions becomes impaired as plasma glucose declines further (19–21). Furthermore, although personality traits persist at the level of hypoglycemia used in ethical research studies (22), mood changes, including an increase in feelings of anger, have been demonstrated (23). Because of the probable relevance of slowed reaction times on formal testing to clinically important events, such as impaired intellectual performance and slowed reactions while undertaking potentially dangerous activities, a blood glucose of less than 3 mmol/L is often used to define clinically significant hypoglycemia in research studies of new therapies. For therapeutic purposes, it is probably useful to differentiate between the lower end of the target glucose range, which should be over 4 mmol/L (24), and that which constitutes hypoglycemia, requiring immediate intervention. A clinical definition for the latter may be set at 20 and possibly 21) (12,35,40,41) Donor normoglycemia ( 14 7 – 14 (days) Points 3 2 0 5 5 Amylase / Allocated >5 15 – 2x 2 – 5x Lipase (U/L) Points Normal Normal Normal Normal 5 2 1 5 Vasopressors Allocated Moderate High Low Points 4 0 2 4 Allocated Normal Normal High Blood High Points Treated Tresod Glucose 4 2 3 1 4 Procuring Allocated Edmonton Distant Points Team 2 9 9 Social Allocated Drug Abuse Promiscocus Jail Time Other Behavior History Points –1 –1 –1 –1 4 Medical Allocated Hypertension Alcohol Arrests Transfusion Other abuse >5 min History Points –5 3 –1 –1 –2 5

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Figure 3 (A) The donor variables assessment form. (B) The pancreas physical properties assessment form. Source: From Ref. 34.

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whether to accept or decline the pancreas. Another benefit of the scoring system is that it is a quick and efficient way to monitor and evaluate trends in the quality of donor organs (34). Besides the scoring system, there are absolute donor rejection criteria including clinical or active viral hepatitis (A, B, or C), acquired immune deficiency syndrome (AIDS) or human immunodeficiency virus (HIV) seropositivity, human T-lymphocyte virus (HTLV) type I/II, active viral encephalitis or encephalitis with unknown origin, Creutzfeldt-Jakob disease, rabies, treatment for active tuberculosis, septicemia, dementia, malignancy, diabetes mellitus Type 1 or 2, and serious illness of unknown etiology. Donors with a history of high-risk behaviors are also excluded. CHALLENGES IN PANCREAS DIGESTION The next major advancement in islet isolation technology was in 1988 when Ricordi et al. introduced a tissue dissociation chamber (25). Several stainless steel marbles are added into the shaking chamber, which also contains the pancreas pieces. A stainless steel 500 µm screen is inserted and the system is sealed. The Liberase-HITM solution (Roche) collected from the perfusion unit is added into the dissociation system (Fig. 4, 5) (35). A major obstacle to successful human pancreatic dissociation has been the low enzymatic activity of the bacterial collagenase preparations. The introduction of Liberase HI has helped to eliminate some of the lot-to-lot and intra-lot variability of enzyme effectiveness, and the need for pre-isolation screening. Liberase digestion consistently yields large numbers of islets without compromising functional viability and has become the “gold standard” for islet isolation (36). The chamber is shaken gently vertically with 2 to 3 cm

Figure 4

Continuous palsatile perfusion of enzyme in a perfusion unit.

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Figure 5

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Ricordi’s dissociation chamber.

of deflection (100 times/min) to agitate the pancreas. The shaking continues until dithizone-stained free islets are seen in the samples taken at 2-minute intervals. When the integrity and number of islets are acceptable, the enzyme is diluted and cooled. A new method has been reported recently, in which the pancreas remains uncut and is kept intact during collagenase intraductal injection. A large filtration chamber to accommodate whole pancreas, low concentration of collagenase (Liberase HI) for digestion, and large plastic containers for large-scale islet purification are used to improve islet isolation outcome (37). A major obstacle to successful human and canine pancreatic dissociation has been the low enzymatic activity of the bacterial collagenase preparations. Endogenous proteases and their respective inhibitors within donor pancreas have critical roles in the islet isolation process due to their effects on collagenase proteolysis, digestion times, islet yield, and functional viability. Endogenous pancreatic enzyme activity of the donor pancreas increases during the digestion phase. High trypsin levels are associated with poor islet yields and adverse viability and functional outcomes (38). Trypsin is believed to act through the proteolysis of collagenase (39). PefablocTM [4-(2-aminoethyl)-benzene sulfonyl fluoride, hydrochloride] (Roche Molecular Biochemicals, Mannheim, Germany), a broad-spectrum serineprotease inhibitor, has been used successfully in pig and human islet isolations (40). We have previously shown that Pefabloc supplementation during the isolation phase can improve islet recovery from human pancreata with prolonged cold ischemia times (41). There was no significant difference in the enzymatic activity digestion time with or without Pefabloc, suggesting that other non-serine proteases or other mechanisms may be altering

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collagenase activity (42). Recently, significantly higher islet yield in porcine islet isolation has been reported by trypsin inhibition but reduced collagenase inhibition, in comparison to UW solution, with a new designed “M-Kyoto” solution containing a cytoprotective against stress (trehalose) with different Naþ and Kþ concentrations and osmolality in addition to a trypsin inhibitor (ulinastatin) (43). The optimal combination of enzymes necessary to maximize the isolation of large numbers of high-quality islets has yet to be determined. The slightest amount of hydration of the Liberase during storage can reduce enzyme function (44). This hydration activates the proteases, which then degrade the higher molecular weight collagenase, resulting in poor yields, viability, and functional outcomes. We are currently evaluating the extent of degradation of collagenase that occurs during storage. Human islet isolation outcomes remain highly variable despite considerable efforts to manufacture highly purified and standardized collagenase blends. The heterogeneity of collagenase preparations and the immense variability between human donor pancreata continue to hamper a process that is inherently difficult to control (45). It has been clarified that the ratio between collagenase class I (ccI) and class II (ccII) is of significant relevance for releasing of islets from pancreatic tissue and optimizing islet yield and viability (46). The variability in collagenase blends has been considered as the most important determinant of the success or failure in isolated islet yields, and this variation in potency has been observed between, and even within, lots of Liberase HI (47). Recently, Hughes et al. reported that collagen VI is a major component of the islet-exocrine interface of the adult pancreas, the content being more than double that of collagen I or IV. Their results may facilitate the design of new collagenases, targeting major substrates such as collagen VI in order to improve clinical islet isolation (48). A better understanding of the characteristics and specific activities of each component in the collagenase blends will allow more specific and selective cleavage of the islets from the surrounding extra cellular matrix. CHALLENGES IN ISLET PURIFICATION Currently, the purification of islets from exocrine tissue is performed by continuous Ficoll gradients using a refrigerated COBE 2991 cell processor (49). It is believed that there are several advantages to transplanting highly purified islets, including improved engraftment, increased safety, and reduced graft immunogenicity (50). Despite overwhelming success in animal models, implantation of unpurified human pancreatic preparations (which may contain greater than 90% exocrine tissue) has been plagued with serious complications: wedge splenic infarction, splenic capsular tear, disseminated intravascular coagulation (DIC), systemic hypertension, portal vein thrombosis, sequelae of portal hypertension including bleeding

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esophageal varices, hepatic infarction, liver failure, and even death. This increase in portal pressure is believed to be the direct result of embolization of large volumes of unpurified tissue into the liver (51). The aforementioned studies demonstrated that transplantation of dispersed human pancreatic tissue was unsafe, suggesting that some form of purification was necessary to decrease the complications, improve islet engraftment, and reduce graft immunogenicity. In 1989, Lakey et al. developed a method for large-scale purification of human islets suitable for safe transplantation (52). Originally designed to process bone marrow and to remove the cryoprotectant from banked blood, the COBE 2991 cell processor permitted rapid, large volume Ficoll gradient processing of a single pancreas within a sterile, self-contained disposable system. Unfortunately, this method still produces significant b-cell stress as demonstrated by zymogen degranulation and loss of insulin content (53). The most common method of islet purification remains density gradient centrifugation (54). Density-dependent elutriation or isopyknic separation of tissue separates individual cells as they migrate and settle within the density gradient according to their specific gravity. Lacy and Kostianovsky were able to separate rodent islets from digested exocrine tissue by differential density elutriation using discontinuous sucrose gradients although the islets were unresponsive to hyperglycemic challenge in vitro (55). This observation was more likely the result of hyperosmolar injury from cellular and islet dehydration rather than dissociation-induced trauma. The replacement of sucrose with Ficoll, a high molecular weight polymer of sucrose (40 kD), permitted the recovery of functionally viable islets (56,57). When Ficoll powder is dissolved in EuroCollins (EC) solution (Euro-Ficoll), hypertonic exposure of the exocrine tissue reduces cell swelling and enhances the islet/exocrine density differential, thereby improving islet recovery (58). Other continuous and non-continuous density gradients have been tested with varying degrees of success: bovine serum albumin (BSA), dextran, hypaque-Ficoll, metrizamide, percoll, and sodium diatrizoate (59,60). The inability to produce consistent highly purified human islet preparations has hindered the development of islet transplantation as a realistic treatment option for patients with Type 1 diabetes (61). Gores et al. have suggested that until specific tolerance protocols are a reality, more effort should be directed at modifying the host’s immune response while using impure preparations to maximize islet yield (62). Crude or partially purified pancreatic homogenates have been used to maximize islet engraftment mass (63,64). Thrombotic complications are believed to be secondary to the thromboplastins released from the digested exocrine tissue. Mehigan et al. found that the addition of heparin and aprotinin (Trasylol) to the tissue preparation at the time of transplantation could ameliorate the risk of DIC (65). We have demonstrated that highly purified islet preparations,

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small packed cell volumes (PCV) 4,000 IE/kg

Figure 3

180 Days posttransplant

Edmonton Protocol.

insulin independence from a single donor was achieved with around 7000 IE/kg (29). In a 5-year follow-up data review at the University of Alberta, it would appear that there is no predictable relationship between the number of islets and insulin independence, as other factors including islet quality, viability, engraftment, and function are equally important (Fig. 4) (26). In spite of the high percentage of insulin-independent patients at 1 year following transplant, this quickly starts to decline thereafter, with 40–50%

1.2

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r = -0.73, P-value 90%. Islet fractions

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are separately cultured until the day of transplantation or shipment. Nonendocrine cells can produce noxious cytokines and chemokines that may contribute to loss of islet mass and function during culture. To minimize this, islet fractions with high and low purity are separately cultured until the day of transplantation. Less pure islet preparations are generally cultured at a density calculated using the following formula for number of islets per flask:   Purity % IEQ per flask ¼ 20;000 IEQ 100 The seeding density of islet preparations during culture plays an important role in islet survival. After isolation, most islet cells will be hypoxic and significantly damaged. Oxygen diffusion in the media will be critical for islet survival during culture. The typical flask loading is equivalent to 115–133 IEQ/cm2 of surface area. Given a uniform islet size of 150 µm diameter (87), this translates to a 1.2–2.4% flask surface utilization. When the cell density exceeds the above cutoff, cells begin to experience anoxia and cell death in the core regions. When the cell number is increased, the anoxic tissue fraction increases precipitously to approximately 65% at a seeding density of approximately 500 IEQ/cm2. Culture Temperature Human islet preparations are cultured in humidified incubators (95% air, 5% CO2). There is no consensus regarding the standardization of incubating temperature for human islet preparation culture. At our facility, islets are cultured at 37 ˚ C for the first 24 hours and at 22 ˚ C for the remaining 24–48 hours until transplantation (6,18,21). Culture at 37 ˚ C for the first 24 hours may be helpful for the recovery of islet cells from the damage during isolation. Culturing islet preparations at 22 ˚ C until transplantation might favor reduction of exocrine cells, therefore improving the purity of the final preparations for transplantation. Preparation of Human Islets Cultures The human islet preparation is cultured in a humidified incubator (95% air, 5% CO2) in supplemented Miami-Modified Media (MM1, Table 1) containing 0.5% HSA. Preparation of the islet suspension for plating begins by adding enough MM1 to the transfer container (i.e., 250 ml conical bottle or 100 ml beaker) containing the islet preparations so that the number of islets to be cultured in each flask is re-suspended in 10 ml of medium. For instance, if 5 flasks are to be cultured, the islet preparation should be re-suspended in a total volume of 50 ml. Islet cells will be gently resuspended by using wide mouth pipettes, and 2 ml of the islet cell suspension are aspirated immediately from the containers and transfered into each

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culture flask. In order to distribute the islets evenly between flasks, a total of 10 ml of re-suspended islets is transferred into a given flask in five 2 ml aliquots. The transfer container is then rinsed with an additional 10 ml of culture medium that are then distributed in 2 ml aliquots at a time to each flask before adding further medium to a final volume of 30 ml per flask. The caps are then replaced on all the flasks and closed in the vented position. The caps are gas-permeable so gas exchange will occur. Each flask is labeled in order to identify islet batch number, date, number of islets plated, and the fraction number (denoting endocrine purity). All flasks are then transferred into the 37 ˚ C CO2 incubator where they are kept for the first 24 hours of culture. Flasks are inspected after the first 24 hours before replacing existing with fresh media. After inspection, the flasks are placed in a Class II Biological Safety Cabinet (BSC) and tilted at a 45 ˚ angle in order to allow islets to settle down for approximately 5 minutes. Twenty ml of culture media are removed aseptically and replaced with fresh MM1. After completion of the media change, flasks are transferred into the 22 ˚ C CO2 incubator for another 24–48 hrs prior to transplant. SHIPPING HUMAN ISLETS BETWEEN CENTERS Transfer of knowledge and expertise required for acquiring sufficient number and quality of islets for a Clinical Islet Transplant Program (CITP) has been the most difficult challenge for a newly established Islet Cell Processing Center (ICPC). The need for specific infrastructures, dedicated personnel, and acquisition of specialized expertise in islet cell isolation and purification has substantial economical impact and may require long-term commitment for a center starting a CITP (5,7). This is intrinsic to the technology required for human islet cell isolation and culture. In addition, islet cell products are regulated by the FDA as an Investigational New Drug under the somatic cell therapy category, which mandates implementation of current Good Manufacturing Practices (cGMP) with high demand for quality control and quality assurance, and use of standardized protocols for cell processing and characterization (83–85). Recent clinical multi-center trials have highlighted the critical role of established center experience in the success rate of the CITP (88,89). This observation, together with the burden of demanding regulations for the production of clinical cell products for transplantation have favored increasing interest in the implementation of regional ICPCs that isolate high quality human islet cells for transplantation in clinical trials at a remote CITP. This approach has proven successful to maximize the success rate of islet isolation while containing the costs of setting up a fully operational cGMP facility in recent clinical trials. This strategy has enabled successful clinical trials both in United States and Europe with excellent outcomes (28,39–41,90–93). The need to ship islets between distant centers requires optimization of shipping

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conditions that allow maximal preservation of islet mass, viability, potency, and sterility. This will be of utmost importance in favorable outcomes of clinical islet transplantation trials, but also in improve distribution of human islet preparations to researchers at distant institutions without altering islet cell quality and physiology for further understanding of human islet cell biology. Very few data are currently available regarding islet shipping protocols at different centers, suggesting that optimization and standardization are still not well defined. In the Group Rhin-Rhone-Alpes-Geneve pour la Transplantation d’Ilots de Langerhans (GRAGIL) experience, islets are transported by an ambulance to the remote transplant centers where they are transplanted within a few hours loaded in a syringe or infusion bag utilized for islet implantation (40,41). The University of California Islet Transplant Consortium reported shipping islets in the infusion bag to the remote centers within hours of isolation, after culture, or cryo-preserved prior to shipment (90,91). Although the Minneapolis-Seattle collaboration have reported shipment of islet preparations for clinical transplantation, the detailed protocol for shipment was not published (93). We have recently established partnerships between our center and distant CITPs where islets are shipped for transplant by charter jet (28,39). To comply with FDA regulations, islet cell products are assessed before (at the islet cell processing facility) and after shipment (at the remote CITP) to confirm that adequate quality (i.e., viability and sterility) and mass of islets are available for transplantation (7,39). In the recently published series of islet transplant alone performed using the consortium concept, islets were isolated at our center from pancreata procured at the remote center, the Methodist Hospital at the Baylor’s College of Medicine in Houston (9). Isolated islets were shipped to the remote partner CITP where product release and quality control were repeated before intrahepatic islet transplantation into patients with Type 1 diabetes (28,39,94). The clinical results obtained in our islet consortium experience are quite promising (6,28,39): insulin independence has been achieved consistently using sequential islet infusions with data that are comparable to those obtained without shipping islets at our Center (6,28,39) and those reported by other CITPs (30). Herein we summarize the protocol developed at our center for the shipment of clinical-grade human islet shipment that has been recently utilized in the ongoing clinical trials with remote sites (28,39,94). Shipping Bag The most critical issue in islet shipment is prevention of hypoxia in transit, which could impair islet cell functional integrity and mass. In order to address this issue, at our center islets are shipped using sterile Lifecell Tissue Culture bags (PL-732 polyolefin lifecell; Nexell Therapeutics, Inc., Irvine,

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Gas-permeable shipping bag and stainless steel racks.

California, U.S.A.) (Fig. 1), which are gas-permeable and made from the same material used for platelet product storage and shipment (95). Oxygen diffusion from ambient air into the gas-permeable bags utilized in our ongoing clinical trial may play a role in maintaining quality of human islet cell products during shipment. Availability of sufficient oxygen diffusion to islet preparations during shipment at 25 ˚ C using polyolefin bags may achieve a peri-islet oxygen partial pressure that never reaches a level that would create diffusion limitations to the core regions of the islets, therefore helping preserve islet cell health over time. On the day of shipment, the islet preparation for transportation is transferred into the Lifecell Tissue Culture bag under aseptic conditions in a BSC. Layers with different purities are generally kept separately, and those with comparable purity are collected from the culture flasks and pooled into a 250 ml conical bottle. After two centrifugation steps (1,000 rpm for 1 minute) aimed at replacing all medium, samples are obtained prior to loading the islet preparation in the shipping bags for product release assessment and quality assurance (including cell counts, determination of overall purity, and final volume of tissue). In a BSC, a 60 ml syringe without plunger is connected to the corresponding luer-lock found at the end of the tissue culture bag tubing. The syringe is used as a funnel to load the tissue into the shipping bag, and it is kept vertical by a metal stand. Final islet density and medium volume in the bag are calculated according to the bag size utilized and the purity of the cell product (Table 2). Loading of the bags is completed by adding culture medium without bicarbonate. The bags are not filled to capacity, so that the height of the media is kept constant in all bag sizes (0.733 cm) in order to prevent impairment of oxygen diffusion to the tissue. The same medium used during culture (Table 1) is also utilized for

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Table 2

Human Islet Density into Different Size Gas-Permeable Shipping Bags

Bag volume (L) 0.3 1.5 3.0

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Total IEQ/bag

Maximum volume (ml)

Bag surface area (cm2)

Media height (cm)

47,880  purity/100 79,800  purity/100 200,000  purity/100

132 219 550

180 300 750

0.733 0.733 0.733

shipment, with the only exception being absence of bicarbonate buffer in the shipping formulation to prevent pH alterations in the absence of controlled CO2 during shipment. After loading the cell product and culture medium into the bags, the bag is heat-sealed. Shipping Container Currently utilized shipping containers consist of large coolers for transport (Fig. 2). The bottom of each large insulated cooler is lined with absorbent material, and stainless steel racks are assembled into it. Each cooler can carry a maximum of three shipping bags (one per shelf). One shipping bag is placed on each shelf, ensuring that it is lying flat and not hanging off the

Figure 2 Shipping containers consist of large coolers for transport. The bottom of the large insulated cooler is lined with absorbent material and stainless steel racks are assembled into it. Each cooler can carry a maximum of three shipping bags (one per shelf). One shipping bag is placed on each shelf, ensuring that it is lying flat and not hanging off the edge of the shelf. This will allow for maximal exposure of the bag surfaces to favor gas exchange.

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edge of the shelf. This favors optimal gas exchange. The container is sealed and shipped to the remote CITP by airplane (28,39). Temperature Control In order to stabilize temperature between 20–24 ˚ C inside the shipping container during transport, coolant packs (Blood/Platelet CoolantTM; model 1290 SEBRA, Tucson, Arizona, U.S.A.) are included. The temperature is logged using a portable Internal/External Temperature Logger computer system (StowAway XTI Logger; ONSET Computer Corp., Bourne, Massachusetts, U.S.A.) designed to log real-time temperatures from months to years at intervals of the user’s choosing (seconds to hours). This allows for proper recording of all temperatures to which the human islet cell product has been exposed during shipment. The electronic data are subsequently uploaded on a computer using dedicated software. Islet Collection and Assessment at the Remote Site Once the shipping container has been received at the remote site, under aseptic conditions and in a BSC, the contents of the bags are transferred to 250 ml conical bottles and pooled for transplantation. Standard product release and quality assurance testing are repeated (assessment of islet numbers, purity, viability, sterility, etc.) to ascertain that adequate islets for transplantation are available and that the shipping did not affect the quality of the cell product (96).

CONCLUSIONS The results of clinical islet transplantation have dramatically improved in recent years thanks to the introduction of more efficient techniques in pancreatic processing and more effective immunosuppressive strategies. Islet transplantation is becoming an increasingly promising option for the treatment of patients with Type 1 diabetes. Improved islet culture and shipping methods have greatly contributed to the steady evolution of this therapeutic approach. The results obtained in the ongoing clinical trials with islets isolated and cultured at our centralized, regional ICPC and transplanted at distant CITPs after shipment have confirmed the feasibility of the islet consortium, which we are currently extending to other centers. Our constant efforts to optimize culture and shipping conditions may be of assistance in further improving the success rate of islet transplantation in the future. This would facilitate increases in the number of transplants and benefit larger cohorts of patients with diabetes. We strongly encourage the implementation of such an approach between centers because of the

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substantial benefits in pancreas utilization and in attainment of consistently high quality islet preparations for transplantation. ACKNOWLEDGMENTS This work has been possible thanks to: the National Institutes of Health/ National Center for Research Resources (U42 RR016603, M01RR16587), the Juvenile Diabetes Research Foundation International (#4-2000-946 and 2003), the National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases (5 R01 DK55347, 5 R01 DK056953), the State of Florida, the American Diabetes Association, and continuous support by the Diabetes Research Institute Foundation (www.diabetesresearch.org). REFERENCES 1.

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63. Bertuzzi F, Saccomanno K, Socci C, et al. Long-term in vitro exposure to high glucose increases proinsulin-like-molecules release by isolated human islets. J Endocrinol 1998; 158(2):205–11. 64. Brendel MD, Kong SS, Alejandro R, Mintz DH. Improved functional survival of human islets of Langerhans in three-dimensional matrix culture. Cell Transplant 1994; 3(5):427–35. 65. Fraga DW, Sabek O, Hathaway DK, Gaber AO. A comparison of media supplement methods for the extended culture of human islet tissue. Transplantation 1998; 65(8):1060–6. 66. Matsumoto S, Goel S, Qualley S, Strong DM, Reems JA. A comparative evaluation of culture conditions for short-term maintenance < ( 24 hr) of human islets isolated using the Edmonton protocol. Cell Tissue Bank 2003; 4(2/4):85–93. 67. Marzorati S, Melzi R, Nano R, et al. In vitro modulation of monocyte chemoattractant protein-1 release in human pancreatic islets. Transplant Proc 2004; 36(3):607–8. 68. Eizirik DL, Korbutt GS, Hellerstrom C. Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the b-cell function. J Clin Invest 1992; 90(4):1263–8. 69. Straus DS. Growth-stimulatory actions of insulin in vitro and in vivo. Endocr Rev 1984; 5(2):356–69. 70. Clayton H, Turner J, Swift S, James R, Bell P. Supplementation of islet culture medium with insulin may have a beneficial effect on islet secretory function. Pancreas 2001; 22(1):72–4. 71. Clark SA, Chick WL. Islet cell culture in defined serum-free medium. Endocrinology 1990; 126(4):1895–903. 72. Hoorens A, Pipeleers D. Nicotinamide protects human b-cells against chemically-induced necrosis, but not against cytokine-induced apoptosis. Diabetologia 1999; 42(1):55–9. 73. Eizirik DL, Sandler S, Welsh N, Bendtzen K, Hellerstrom C. Nicotinamide decreases nitric oxide production and partially protects human pancreatic islets against the suppressive effects of combinations of cytokines. Autoimmunity 1994; 19(3):193–8. 74. Andersen HU, Jorgensen KH, Egeberg J, Mandrup-Poulsen T, Nerup J. Nicotinamide prevents interleukin-1 effects on accumulated insulin release and nitric oxide production in rat islets of Langerhans. Diabetes 1994; 43(6):770–7. 75. Aikin R, Rosenberg L, Paraskevas S, Maysinger D. Inhibition of caspasemediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans. J Mol Med 2004; 82(6):389–97. 76. Johansson H, Lukinius A, Moberg L, et al. Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation. Diabetes 2005; 54(6):1755–62. 77. Moberg L, Johansson H, Lukinius A, et al. Production of tissue factor by pancreatic islet cells as a trigger of detrimental thrombotic reactions in clinical islet transplantation. Lancet 2002; 360(9350):2039–45. 78. Piemonti L, Leone BE, Nano R, et al. Human pancreatic islets produce and secrete MCP-1/CCL2: relevance in human islet transplantation. Diabetes 2002; 51(1):55–65.

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79. Bertuzzi F, Marzorati S, Maffi P, et al. Tissue factor and CCL2/monocyte chemoattractant protein-1 released by human islets affect islet engraftment in type 1 diabetic recipients. J Clin Endocrinol Metab 2004; 89(11):5724–8. 80. Chen MC, Proost P, Gysemans C, Mathieu C, Eizirik DL. Monocyte chemoattractant protein-1 is expressed in pancreatic islets from prediabetic NOD mice and in interleukin-1 b-exposed human and rat islet cells. Diabetologia 2001; 44(3):325–32. 81. Luca G, Nastruzzi C, Basta G, et al. Effects of anti-oxidizing vitamins on in vitro cultured porcine neonatal pancreatic islet cells. Diabetes Nutr Metab 2000; 13(6):301–7. 82. Burkart V, Gross-Eick A, Bellmann K, Radons J, Kolb H. Suppression of nitric oxide toxicity in islet cells by alpha-tocopherol. FEBS Lett 1995; 364(3): 259–63. 83. Weber DJ. FDA regulation of allogeneic islets as a biological product. Cell Biochem Biophys 2004; 40(3 Suppl):19–22. 84. Weber DJ, McFarland RD, Irony I. Selected Food and Drug Administration review issues for regulation of allogeneic islets of langerhans as somatic cell therapy. Transplantation 2002; 74(12):1816–20. 85. Wonnacott K. Update on regulatory issues in pancreatic islet transplantation. Am J Ther 2005; 12(6):600–4. 86. Latif ZA, Noel J, Alejandro R. A simple method of staining fresh and cultured islets. Transplantation 1988; 45(4):827–30. 87. Ricordi C. Quantitative and qualitative standards for islet isolation assessment in humans and large mammals. Pancreas 1991; 6(2):242–4. 88. Shapiro AM, Ricordi C. Unraveling the secrets of single donor success in islet transplantation. Am J Transplant 2004; 4(3):295–8. 89. Shapiro AM, Ricordi C, Hering B. Edmonton’s islet success has indeed been replicated elsewhere. Lancet 2003; 362(9391):1242. 90. Brunicardi FC, Atiya A, Stock P, et al. Clinical islet transplantation experience of the University of California Islet Transplant Consortium. Surgery 1995; 118(6):967–71; discussion 971–2. 91. Brunicardi FC. Clinical islet transplantation: a consortium model. Transplant Proc 1996; 28(4):2138–40. 92. Rabkin JM, Leone JP, Sutherland DE, et al. Transcontinental shipping of pancreatic islets for autotransplantation after total pancreatectomy. Pancreas 1997; 15(4):416–9. 93. Rabkin JM, Olyaei AJ, Orloff SL, et al. Distant processing of pancreas islets for autotransplantation following total pancreatectomy. Am J Surg 1999; 177 (5):423–7. 94. Goss JA, Soltes G, Goodpastor SE, et al. Pancreatic islet transplantation: the radiographic approach. Transplantation 2003; 76(1):199–203. 95. Lemoli RM, Tafuri A, Strife A, Andreeff M, Clarkson BD, Gulati SC. Proliferation of human hematopoietic progenitors in long–term bone marrow cultures in gas-permeable plastic bags is enhanced by colony-stimulating factors. Exp Hematol 1992; 20(5):569–75. 96. Ichii H, Sakuma Y, Pileggi A, Fraker C, et al. Shipment of human islet for transplantation. Am J Transplantation 2007; 7(4):1010–1020.

15 Development and Application of Contemporary Immunosuppression in Human Islet Transplantation Dixon B. Kaufman Department of Surgery, Division of Transplantation, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, U.S.A.

INTRODUCTION An effective and safe immunosuppression protocol for successful islet transplantation will prevent a variety of cytodestructive auto- and alloimmune host responses without inhibiting the secretion or peripheral action of insulin and, of course, without imparting detrimental effects on recipient well-being. Perfection in this realm does not exist. Nevertheless, important strides have been made over the past several years that have resulted in meaningful breakthroughs, positioning the field of islet transplantation into pivotal licensing studies to establish it as a truly therapeutic treatment option for treatment of diabetes in selected patients. This chapter reviews the development of the current state-of-the-art immunosuppressive approach to islet allotransplantation. Many similarities exist between the immunosuppressive properties needed for successful solid organ transplants and islet transplantation. However, there are several considerations pertinent to islet transplantation that differentiate it from solid organ transplants. First, the effectiveness of the anti-rejection properties in preventing a variety of cytodestructive host immune responses must be nearly perfect because the marginal mass of islets that ultimately engraft has virtually no excess capacity to tolerate b-cell injury without reaching the tipping point causing metabolic dysfunction. The islets also appear to have essentially null capacity to regenerate if 269

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injured. Because surrogate serum markers of very early islet graft rejection do not exist, there is currently no timely prompt to the clinician to guide decision-making regarding application of anti-rejection therapy for the precise purpose of interrupting the immune destructive processes. These challenges have not held back the field, and a brief glance back sheds some light on what has been accomplished and why, and provides some direction on where the field is heading. THE EVOLUTION OF MODERN IMMUNOSUPPRESSION FOR CLINICAL ISLET TRANSPLANTATION Prior to 2000 the success rate of islet transplantation was relatively low. Discerning the technical from the immunological causes of failure was difficult. At that time the technical methods for manufacturing a consistently potent islet product were becoming quite sophisticated but were still in development. The use of pre-transplant culture was not in practice, and there were no validated measures of islet potency that could be quantified prior to transplant that would correlate with clinical success or failure. Despite these shortcomings, a relatively high success rate of islet autotransplantation (1) was being accomplished and suggested that the technical aspects of islet isolation were not holding the field back. Rather, the failure of islet allotransplants appeared to be largely due to inadequacies of the immunosuppression method (2). The immunosuppression approach at that time was strongly influenced by the renal solid organ perspective. Islet transplants performed in the 1990s placed greater emphasis on application to Type 1 diabetic patients with nephropathy that were to receive the islets simultaneously with, or after, a previous kidney transplant. The immunosuppression armamentarium consisted of the same agents that were used to maintain function of the kidney grafts. Unfortunately, those agents were toxic to the b-cells and insufficient in potency to minimize destructive host immune responses. For example, chronic corticosteroid maintenance therapy was an important component of the immunosuppressive approach. Also, the potency of the combination of tacrolimus and sirolimus was unknown. In fact, concerns that the properties of those two agents would mutually exclude their combination held back the field. So, the many failures of islet transplants at that time were due to the routine practice of implanting an impotent islet mass and using toxic and marginally effective immunosuppressive therapy. A few key studies in large animal pre-clinical models helped shape the things that were to emerge as true breakthroughs in human pilot studies. The benefits of induction therapy were shown to have a promising effect on outcome in functional large animal models of islet allotransplantation (3,4). With respect to maintenance immunotherapy, the diabetogenic side effects of chronic corticosteroid therapy were shown to be particularly deleterious

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in the presence of a marginal islet mass. Even short courses of low-dose prednisone therapy immediately post-transplant were shown to significantly shorten the durability of islet function in dogs that underwent total pancreatectomy and pancreatic islet autotransplantation (3,5,6). Calcineurin inhibitor therapy has become the backbone of virtually all immunosuppressive protocols since 1983. No significant deleterious effects of cyclosporine monotherapy on islet graft metabolic function were found (7). However, the combination of steroids and calcineurin inhibitors was especially detrimental to islet function, as demonstrated in dogs with stable long-term islet autograft function (8). Of particular importance was the report in 1996 that sirolimus had a beneficial metabolic impact in canine islet autograft recipients (9). Sirolimus was associated with increased glucose clearance, increased total and stimulated insulin release in response to glucose, and increased fasting plasma insulin level, as well as reduced insulin clearance. Studies in the pre-clinical pig islet allotransplant model strongly suggested efficacy of sirolimus, combined with low-dose calcineurin inhibitors, in preventing islet allograft rejection (10). In summary, the large animal models indicated that induction therapy in combination with a sirolimus-based maintenance regimen was of benefit and that corticosteroids were deleterious. In the clinical arena, McAlister, et al. (11) reported on a successful new maintenance immunosuppressive approach in 32 kidney, pancreas, and liver recipients that were transplanted from April 1998 through May 1999 with low-dose tacrolimus (trough concentration 3–7 ng/mL) and sirolimus (trough concentration 6–12 ng/mL) combination immunosuppression. THE EDMONTON IMMUNOSUPPRESSION PROTOCOL Investigators from the University of Alberta developed a steroid-free version of this protocol for human islet transplantation that had remarkable results. The Edmonton Protocol was born. It consisted of induction therapy with the IL-2 receptor antagonist, daclizumab. High-dose sirolimus combined with relatively low-dose tacrolimus was used for maintenance therapy (12). No glucocorticoids were given at any time. Daclizumab induction therapy was given intravenously at a dose of 1 mg/kg every 14 days for a total of 5 doses over a 10-week period, thus allowing an extended period for a supplemental islet transplant procedure. If the second islet transplant procedure occurred more than 10 weeks after the first, the course of daclizumab was repeated. Sirolimus was dosed to achieve and maintain trough levels of 12 to 15 ng/mL for the first 3 months and of 7 to 10 ng/mL thereafter. Tacrolimus was administered at an initial dose of 1 mg twice daily, then adjusted to maintain a trough concentration at 12 hours of 3 to 6 ng/mL. Type 1 diabetic islet allograft recipients reliably achieved and maintained freedom from the need for exogenous insulin after

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transplantation of an adequate mass of islets prepared from 2–4 donor organs, suggesting that the Shapiro et al. protocol protected against alloimmune and autoimmune reactivity. For any type of immunosuppression, efficacy and toxicity must be balanced. A recent analysis of adverse events observed in the first 31 Edmonton islet transplant recipients treated with daclizumab, sirolimus, and reduced-dose tacrolimus (follow-up, up to 3 years) revealed dyslipidemia in 62% and mouth ulceration in 81% (13). A significant increase in serum creatinine was only seen in two recipients who had elevated creatinine levels pre-transplant. Opportunistic infections and malignancies were not noted in any of the recipients followed for up to 3 years. The consistently successful outcomes reported by the University of Alberta group have been duplicated at other institutions (14–17). Immunosuppression with daclizumab, sirolimus, and reduced-dose tacrolimus has evolved as the gold standard for Type 1 diabetic islet transplant recipients. A few modifications of the induction therapy involving the use of a T-cell depleting agent, anti-thymocyte globulin (rabbit), in combination with TNF-α blockade are showing promising results.

INDUCTION AND ANTI-CYTOKINE AGENTS Rabbit anti-thymocyte globulin (Thymoglobulin; Genzyme, Cambridge, Massachusetts, U.S.A.) was approved by the FDA in 1999 for the treatment for acute renal graft rejection. It is a polyclonal IgG antibody generated by rabbits immunized with human thymocytes. Rabbit anti-thymocyte globulin contains cytotoxic antibodies directed against antigens expressed on human T lymphocytes. The rationale for anti-thymocyte globulin induction immunosuppression includes prevention of autoimmune recurrence in transplanted islets via deletion of autoreactive memory cells, prophylaxis of islet allo-rejection, avoidance of the use of calcineurin inhibitors in the immediate post-transplant period, and attenuation of non-specific inflammatory responses to transplanted islets, thereby maximizing engraftment and functional survival. Rabbit anti-thymocyte globulin has shown a consistent safety profile, with most adverse events being manageable and reversible; the most common events are fever, chills, and leukopenia. While rare, the most severe events include allergic or anaphylactoid reactions and serum sickness. As with all immunosuppression, administration of rabbit anti-thymocyte globulin may be associated with an increased risk of infection and development of malignancy (especially of the skin and lymphoid system). Published results of the use of anti-thymocyte globulin in clinical and experimental islet transplantation are limited to relatively small cohorts. Hirshberg et al. (4) described the successful role of rabbit anti-thymocyte

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globulin and sirolimus in reducing rejection of islet allografts in primates. Hering et al. (14) described a beneficial role of rabbit anti-thymocyte globulin induction (6 mg/kg) in 8 consecutive patients. They received an average of 7271 ± 1035 IE/kg from a single donor pancreas, and all achieved insulin independence and were protected against recurrence of hypoglycemia (14). Etanercept is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) TNF receptor linked to the Fc portion of human IgG1. Etanercept inhibits binding of both TNF-α and TNF-b to cell surface TNF receptors, rendering TNF biologically inactive (18,19). It is FDA-approved for use in severe rheumatoid arthritis, juvenile arthritis, ankylosing spondylitis, and psoriatic arthritis. The basic premise behind the treatment is that peri-transplant administration of etanercept will interfere with the biological activity of TNF-α released early post-transplant as part of the activation of the innate immune response. Blockade of TNF-α in the early post-transplant period is expected to lessen early islet loss. TNF-α is known to be cytotoxic to human islet b-cells (20). In murine models, selective inhibition of TNF-α in the peritransplant period has promoted reversal of diabetes after marginal-mass islet isografts (21). Experience with anti-TNF-α therapies in clinical islet transplantation has been limited. Nineteen islet transplant recipients have received etanercept in the peri-transplant period for the purpose of enhancing engraftment and functional survival of transplanted human islets at the University of Minnesota, University of California San Francisco (UCSF), and the University of Miami. Etanercept was administered as follows: 50 mg IV at 1 hr prior to transplant, 25 mg subcutaneous on days þ3, þ7, and þ10 post-transplant. In patients transplanted at the University of Minnesota, etanercept was combined with anti-thymocyte globulin, and in two patients at UCSF, etanercept was combined with hOKT3 (Ala-Ala) for induction immunotherapy. At the University of Miami, etanercept was combined with daclizumab (n ¼ 4) or alemtuzumab (n ¼ 3) induction immunotherapy. No adverse events related to etanercept were encountered in these 19 patients. The Minnesota/UCSF pilot study was promising with all four patients each receiving islets from a single donor pancreas. One patient became insulin-independent and the other three achieved markedly improved glycemic control on substantially reduced exogenous insulin doses. At the University of Miami, one of the three subjects that received alemtuzumab (Campath; Genzyme) and etanercept became insulin-independent after receiving islets from a single donor. The follow-up on the first 8 Minnesota patients is more complete and insulin independence was achieved in all with islets prepared from a single pancreas as described above.

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MODERN IMMUNOSUPPRESSION FOR CLINICAL ISLET TRANSPLANTATION Figure 1 shows an algorithm of a contemporary immunosuppressive regimen for islet transplantation. In this scenario, the isolated islets are determined to meet preliminary release criteria for transplantation 2 days prior to infusion. They are then placed in culture. During the culture period, anti-thymocyte globulin is administered to the recipient. The 48-hour leadtime allows for resolution of possible antibody-induced cytokine release prior to islet infusion. Tacrolimus-based maintenance immunotherapy (usually in combination with sirolimus) is also initiated 48 hours prior to infusion. Chronic corticosteroids are not administered. Guidelines for contemporary immunosuppression medications are outlined below. For the initial islet infusion, rabbit anti-thymocyte globulin is administered as induction therapy two days prior to islet infusion. A total of 6 mg/kg is given. The dose is 0.5 mg/kg on day 2, 1.0 mg/kg on day 1, and 1.5 mg/kg on days 0, þ1, and þ2. Premedications include: n n n n

Acetaminophen (paracetamol) 650 mg ½ hr before and midway through ATG infusion. Diphenhydramine 50 mg p.o. ½ hr before and midway through ATG infusion. Methylprednisolone 1 mg/kg i.v. one hour prior to and midway through the first ATG infusion only (i.e. on day –2). Pentoxifylline 400 p.o three times daily to be initiated one hour prior to the first ATG infusion and to be continued through day þ7.

Tacrolimus-based maintenance immunosuppression (tacrolimus / sirolimus) Antilymphocyte globulin 6 mg/kg over 4-6 days beginning day -2 Etanercept day 0, 3, 7, 10 Daclizumab day 0 and q 2 weeks x 4 (re-transplants)

-2

-1

0

+14

+28

+42

Days post transplant Islet isolation

Islet transplant

Islet culture (~ 48 hours)

Figure 1

Islet transplant immunosuppression protocol.

+56

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For anti-inflammatory therapy, etanercept (Enbrel) may be administered at a dose of 50 mg IV on day 0 (1 hr prior to transplant), and 25 mg subcutaneous on days þ3, þ7, and þ10 post-transplant. For maintenance therapy, sirolimus is administered at an initial dose 0.2 mg/kg p.o. on day 2 relative to islet transplant, followed by 0.1 mg/kg four times daily. The daily dose will be adjusted to achieve a whole blood 24-hr trough concentration of 10–15 ng/mL for the first 3 months and 8–12 ng/mL thereafter. Tacrolimus is administered at an initial dose 0.015 mg/kg p.o. twice daily on day þ1 to achieve a whole blood 12-hour trough concentration of 3–6 ng/mL. If subsequent islet infusions are required, then the immunosuppressive regimen will be identical to the regimen for the initial islet transplant with the following exceptions. Daclizumab is used instead of rabbit antithymocyte globulin for all subsequent islet transplants. The first dose is 2 mg/kg and is given within two hours prior to islet transplant. Doses 2–5 are at 1 mg/kg and are given every 2 weeks starting on day 14 after the subsequent transplant. If a third transplant is deemed necessary and performed between 30 and 70 days after the second transplant, no additional doses of daclizumab are required. If a third islet transplant is performed more than 70 days after the second transplant, all five doses of daclizumab should be repeated.

THE NORTH AMERICAN EXPERIENCE OF IMMUNOSUPPRESSION FOR ISLET TRANSPLANTATION The Collaborative Islet Transplant Registry (CITR) has collected data on 203 islet transplantation alone (ITA) recipients. The immunosuppressive regimens used are described in Tables 1 and Table 2. The most commonly Table 1 Islet Alone Recipients—Induction Immunosuppression Regimen at Time of First Islet Infusion Total

N 194

% 100.0

Daclizumab alone Anti-thymocyte globulin and daclizumab Alemtuzumab and daclizumab Basiliximab alone Anti-thymocyte globulin alone hOKT3-1(Ala-Ala) alone Alemtuzumab alone None

150 11 9 9 8 3 3 1

77.3 5.7 4.6 4.6 4.1 1.5 1.5 0.5

Source: From the Collaborative Islet Transplant Registry, Annual Report, 2006:86.

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Table 2

Islet Alone Recipients—Maintenance Immunosuppression Regimen at Time of First Islet Infusion

Total

N 203

% 100.0

Sirolimus þ tacrolimus Sirolimus þ tacrolimus þ MMF Sirolimus þ MMF Sirolimus þ tacrolimus þ MMF þ methylprednisolone Sirolimus þ MMF þ methylprednisolone Neoral cyclosporine þ everolimus Neoral cyclosporine þ methylprednisolone Neoral cyclosporine þ methylprednisolone þ everolimus Missing information on immunosuppression

176 3 1 7 1 1 1 4 9

86.7 1.5 0.5 2.5 0.5 0.5 0.5 2.0 4.4

Source: From the Collaborative Islet Transplant Registry, Annual Report, 2006:85.

used class of induction agent was an IL-2 receptor antagonist. Daclizumab was used in virtually all of those cases. T-cell depleting agents were not commonly used. However, rabbit anti-thymocyte globulin is being more commonly used following the report by Hering et al. (14) on the success with single donor islet infusions to achieve insulin independence. The most commonly used maintenance agents are tacrolimus and sirolimus in nearly 90% of cases. Corticosteroids are infrequently used. In approximately 25% of cases, anti-inflammatory agents that suppress cytokines (usually TNF) are administered in the early post-transplant course (Table 3).

IMMUNOSUPPRESSION FOR ISLET-AFTER-KIDNEY TRANSPLANTATION Individuals with Type 1 diabetes and a successful kidney allograft are particularly appropriate candidates for an islet after kidney (IAK) Table 3 Islet Alone Recipients—Anti-Inflammatory Agents Used at Time of First Islet Infusion Total

N 203

% 100.0

Infliximab Etanercept 15-deoxyspergualin Infliximab þ etanercept None Missing information on immunosuppression

17 24 5 2 146 9

8.4 11.8 2.5 1.0 71.9 4.4

Source: From Collaborative Islet Transplant Registry, Annual Report, 2006:85.

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transplant procedure because they are already committed to life-long chronic immunosuppression. Thus, the nominal risk of the islet transplant procedure itself is minimal compared to the risk of initiating new immunosuppression in non-uremic patients with diabetes in addition to the transplant procedure (22–24). Also relevant is the preliminary evidence from uncontrolled studies suggesting that transplanted islets in patients who have already received a kidney transplant may provide beneficial vascular and renal effects (25,26). Candidates for IAK transplants are distinguished from ITA recipients in several respects. First, recipients that have a successful kidney transplant and are prescribed an immunosuppressive regimen that differs from the corticosteroid-free protocol described by the Edmonton group may be required to convert to the Edmonton immunosuppression protocol prior to islet transplantation. There are at least two situations where approaches to kidney transplant immunosuppression have implications with respect to preparation for a subsequent IAK transplant. The first scenario includes renal transplant recipients with Type 1 diabetes that were initially prescribed a “conventional” immunosuppression regimen including corticosteroids at the time of kidney transplantation prior to any consideration of an IAK procedure. A typical immunosuppression protocol might entail cyclosporine, azathioprine (or mycophenolate mofetil), and prednisone. This situation in which immunosuppression was initially prescribed in consideration of the kidney transplant only is referred to as a “casual approach.” In this circumstance the patient may undergo immunotherapy conversion to that resembling the Edmonton Protocol of tacrolimus and sirolimus without corticosteroids, prior to the IAK transplant. The issue of converting a patient on a cyclosporine-based maintenance therapy to tacrolimus is relatively straightforward, as is the situation of conversion from an anti-metabolite (azathioprine or mycophenolate mofetil) to sirolimus. A typical immunotherapy conversion protocol starts with a conversion from cyclosporine to tacrolimus. Tacrolimus 12-hr trough concentration target levels are 4–6 ng/mL. After a period of 3 months, if renal function is stable, then the anti-metabolite (azathioprine or MMF) is converted to sirolimus. Sirolimus 24-hr trough concentration target levels are 6–8 ng/mL. After another 3-month period of observation the corticosteroids are slowly weaned off. Usually, patients require only 2.5–7.5 mg per day of prednisone. If the dose is 7.5 mg, then an immediate cut to 5 mg per day is made when the tacrolimus is started. The last 5 mg is tapered off over a period of 3 months after successful conversion to tacrolimus and sirolimus. It is prudent that baseline measures of kidney transplant function (e.g., 24 hour collection for creatinine clearance and protein) are obtained prior to immunosuppression conversion to fully determine any subtle changes in renal transplant function.

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It is important that patients are informed that some corticosteroid withdrawal protocols are associated with an increased risk of acute rejection and renal allograft loss. Virtually all of the reports of corticosteroid withdrawal failure, however, are reported in recipients maintained on cyclosporine and either azathioprine or MMF (27–29). That is not to say that successful steroid withdrawal protocols have not been described in kidney transplant recipients on cyclosporine (Neoral; Novartis, East Hanoever, New Jersey, U.S.A.) and MMF (30). Recent reports with the combination of cyclosporine (Neoral) and sirolimus appear promising as well (31). There is also published experience with steroid withdrawal in patients receiving tacrolimus-based immunosuppression, but it is relatively limited (32,33). In short, the benefits of the subsequent IAK transplant must outweigh the risks of withdrawing corticosteroids and precipitating an acute renal allograft rejection episode. The other scenario includes renal transplant recipients with Type 1 diabetes that are prescribed an immunosuppression regimen in which the corticosteroids are avoided or immediately rapidly eliminated following the kidney transplantation in anticipation of the subsequent islet transplant. This approach is referred to as “expectant immunosuppression.” Rapid corticosteroid elimination (avoidance) appears to be associated with less risk of graft loss from rejection compared to the slow steroid withdrawal protocols described above. We have previously reported that the combined use of tacrolimus and MMF with an IL-2 receptor antagonist allows corticosteroids to be withdrawn within 3 days of renal transplantation (34). The risk of a renal allograft rejection episode is approximately 13% (85–90% of occurring within three weeks of transplantation), and all episodes were reversible with appropriate anti-rejection therapy. No increase in risk of graft loss was observed versus the conventional chronic corticosteroid therapy approach. Others have described success with rapid corticosteroid elimination protocols in kidney transplantation. Matas et al. (35) have described a rapid steroid elimination protocol utilizing cyclosporine (Neoral), MMF, and anti-thymocyte globulin (rabbit). Cole et al. (36) reported on the efficacy of a steroid-free protocol using cyclosporine (Neoral), MMF, and daclizumab. In both studies, no clinically significant increase in risk of graft loss or rejection ensued compared to historical control groups. One of the criticisms of steroid avoidance protocols is that the long-term results are not known. The long-term outcome of steroid avoidance has been addressed by Birkeland (37). A five-year follow-up of 100 kidney transplant recipients indicated that renal allograft survival was not compromised by omitting chronic steroid exposure. Application of these immunosuppression options for IAK recipients would be advisable in programs with established successful steroid-sparing protocols already in place in the renal transplant program. A coordinated approach to renal transplant immunotherapy with subsequent medical

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conversion including possible corticosteroid withdrawal in preparation for islet transplantation requires integration of the kidney and islet transplant programs. A functionally integrated program enables consideration of whole pancreas transplantation for some individuals with an existing kidney allograft. Transplantation of the whole pancreas after a kidney transplant does not require corticosteroid withdrawal. Maintenance agents are often converted to a tacrolimus/MMF regimen, although this is not an absolute requirement. The second distinguishing feature is that an IAK candidate already has a functioning renal transplant, and maintenance of optimal kidney transplant function becomes paramount. This difference requires consideration of treatment protocols for islet transplantation to take a backseat to those needed to maintain optimal kidney transplant function. Conflicts involving approaches to immunosuppression could arise when application of tacrolimus and sirolimus immunosuppression suited for islet transplantation unexpectedly compromises renal allograft function due to nephrotoxicity. For example, an individual who has enjoyed good and stable kidney transplant function for years while receiving cyclosporine, azathioprine, and prednisone for immunosuppression is converted to tacrolimus and sirolimus at levels appropriate for the subsequent islet transplantation begins to demonstrate deteriorating renal function. In such a case, it may not be possible to achieve the target levels of tacrolimus and sirolimus that have been established in the Edmonton Protocol. Moving ahead with the subsequent islet transplantation in the face of “sub-therapeutic” immunosuppression may result in inferior outcomes and risk of worsening renal function. Third, IAK transplant recipients would be pre-immunosuppressed prior to islet transplantation. This may result in more efficient islet engraftment such that insulin independence could be achieved with a mass of islets significantly less than the threshold of approximately 8–10,000 islet equivalents/kilogram body weight, which usually requires two or more cadaveric donors. The Edmonton group and others have observed that insulin can often be stopped almost immediately following the second islet transplant. There are at least two potential contributory factors differing from the first transplant: the greater overall mass of islets and the preimmunosuppressed state. Since both may play a role, this raises the theoretical possibility that the pre-immunosuppressed state could result in more efficient early islet survival such that insulin independence may be achieved with a mass of islets significantly less than the threshold of approximately 8–10,000 islet equivalents/kilogram body weight, increasing likelihood of single donor success. The caveat is that the patient selection criteria for IAK transplants may not necessarily be identical to those applied in ITA candidate selection. Body mass index (generally 155, and >260 days, respectively, although the first developed simian CMV and SV40 viremia without disease. Thus, while there remain concerns that the aggressive level of immunosuppression used might limit this strategy in human patients, there is now evidence from both these studies that pig islet xenografts in these nonhuman primate diabetic models can indeed provide a “cure” of the disease. Finally, taking a very different approach, the last recent study transplanted neonatal pig islets after microencapsulation in alginatepolyornithine-alginate into the intraperitoneal spaces of eight diabetic cynomolgus macaque monkeys (23). All animals received a second transplant 3 months after the first. There was no immunosuppression given and two animals died of causes not related to the transplants. In the final analysis the

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exogenous insulin requirement of the transplanted animals compared with the control group was decreased by a mean of 43% with equally good blood glucose control. While the potential of biomaterial encapsulation of islets to prevent immune rejection and avoid a need for long term immunosuppression has been discussed for many years with evanescent success reported, this latest iteration of the technology must be considered promising. The potential of using encapsulation to reduce or eliminate the requirement for long term immunosuppression for islet xenografts is an area that merits continued work, particularly when taken in the context of the concerns raised regarding the intensity and toxicity of the two successful immunosuppressive strategies already reviewed.

POTENTIAL RISK OF PORCINE ENDOGENOUS RETROVIRUS One focus of work in my own group has been on the potential risk of crossspecies transmission of porcine endogenous retrovirus (PERV) from pig donor tissues to immunosuppressed human patients [reviewed in (24)]. While a complete review of this area is not intended here, it is important to briefly update the reader. First, a collaboration led by Dr. Clive Patience and including our group cloned the human PERV receptor and identified two new genes that appear to have arisen by gene duplication (25). Interestingly, orthologs of the human PERV receptor, now called HuPAR, are found all the way down the evolutionary tree to C. elegans, xenopus, and drosophila, consistent with the conclusion that this receptor family are critical cellular molecules. By inference from several other known retrovirus receptors, the HuPAR may be cellular nutrient or electrolyte transporters. This is further supported by the fact that we believe by structural analysis that they are multi-membrane–spanning proteins. Second, we reported that non-human primate cell lines and a number of primary cells could not be infected with PERV (26). Indeed, non-human primate cells have two different defects blocking PERV infection, one at the level of viral entry and the other at the level of viral assembly. In this situation, the potential of productive PERV infection in a non-human primate is extremely unlikely. In contrast, many human cells and cell lines studied have no such defects to productive PERV infection (24). The obvious conclusion is that all the evidence accumulating in different non-human primate models that have been cited as demonstrating no evidence of PERV infection are on one hand true but on the other hand not predictive of the situation that should be encountered in human patients. Thus, we are disappointed that all the latest papers cited above each add comments about finding no PERV infection in the non-human primate models without acknowledging at least the potential that these results are not relevant in the context of risk assessment.

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In parallel, it was recently reported that PERV could be pseudotyped by endogenous murine retrovirus (27). The model involved pig and human hematopoietic cell and tissue transplantation to create stable, long-lived pig/ human/mouse chimeras. In this case, it turned out that endogenous mouse retroviruses could package the PERV genomes and effectively carry them into the transplanted human cells. In this case, it might appear that PERV is directly infecting the human cells (and indeed this might be the case), but it is equally possible that the PERV infection of the human cells was due to the presence of the murine retrovirus, a situation obviously not present in any human patients. We followed by reporting a similar result using a simpler pig tissue transplantation model that more efficiently demonstrated the pseudotyping phenomenon (28). Thus, the previously published studies suggesting PERV infection of human cells as a risk issue for xenotransplantation based on using immunodeficient mice transplanted with human cells and pig tissues are all open to question due to this property of murine retrovirus. It is possible that the infection of the human cells was still due to PERV, but that conclusion cannot be proven and the value of this chimeric human/mouse model for these studies must be re-evaluated critically. However, it is very important to emphasize that none of these results have any impact on our original publication raising the issue of potential PERV risk to humans by demonstrating that immunodeficient mice transplanted with pig islets are infected in multiple tissue compartments by PERV (29). Pseudotyping by mouse endogenous retrovirus cannot happen to the mouse cells in the transplanted animal. Finally, we recently developed a mouse transgenic for one of the two human PERV receptors, HuPAR2, and did a series of experiments where we injected infectious PERV-containing supernatants and then evaluated the potential of infection at multiple times and in many tissue compartments (30). In this context, it is important to note that our original studies with mouse cell lines demonstrated that expression of the human PERV receptor resulted in productive PERV infection (25). Thus, we demonstrated that there are no viral entry or assembly defects in the mouse, in contrast to our findings in non-human primate cells (26). In the more recent studies, the human PERV receptor transgenic mice were productively infected with PERV in many tissue compartments including brain, kidney, liver, and bone marrow. Interestingly, we have not documented any clinical disease or tumors in these animals, though animals injected in the neonatal stage demonstrated significant growth retardation. Ongoing studies are now evaluating the immune response to PERV in these animals, including neutralizing antibodies and PERV-specific CTL as well as the impact of immunosuppression. Thus, at present, it would appear that animals, including humans, that express a functional PERV receptor will be productively infected with PERV if exposed to a sufficiently infectious dose of virus. That does not necessarily

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mean that a PERV infection will cause any disease and that the human immune system will not clear PERV infection through natural immune mechanisms. We also have no evidence to suggest that there is any risk of spreading PERV from an infected animal to a healthy littermate in a way that would support concerns of a general public health risk from PERV. It is also still possible that certain strains of pigs can be selected and bred that have a reduced level of PERV produced of the type infectious for human cells. Unfortunately, all known pigs have multiple copies of PERV in their genomes and the nature of retroviruses is to readily recombine, mutate, and adapt to enhance their infectious range and capacity. Nonetheless, we continue to believe that cautious, closely monitored clinical studies in xenotransplantation should be supported and that the possible risk of PERV simply be incorporated into the informed consent procedure. Obviously, there is also a compelling need to support ongoing research in this area. FINAL THOUGHTS The emphasis in this chapter has been on areas where the experience with pig islet xenotransplantation is different from that of human islet allograft transplantation. Nonetheless, it is important to end by reminding the reader that the major challenges of pig islet xenotransplantation are the same: effective isolation and functional islet preparations, strategies to reduce immediate post-transplant cell death, safe strategies to manage the immune response, and alternative strategies to enhance islet cell engraftment and identify the ideal site for transplantation. Moreover, the potential that stem cells (adult or embryonic) or gene-modified cells could become islet cell alternatives is of equal importance in shaping the future of islet xenotransplantation. Thus, all the elements discussed in the previous chapters on human islets are largely relevant here. Finally, it must be noted that the potential of genetically engineering the pig donors to incorporate strategies for enhanced islet isolation, survival, engraftment, resistance to rejection, proliferation, etc. create opportunities for building on the next advances in human islet transplantation and the molecular mechanisms of islet injury to a new generation of strategies for successful islet xenotransplantation. In this way, too, the futures of human islet allograft transplantation and pig islet xenotransplantation are interlinked. REFERENCES 1. 2.

MacKenzie DA, Hullett DA, and Sollinger HW. Xenogeneic transplantation of porcine islets: an overview. Transplantation 2003; 76(6):887–91. Rood PP, et al. Pig-to-non-human primate islet xenotransplantation: a review, Buhler LH, Bottinom, of current problems. Cell Transplant 2006; 15(2): 89–104.

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O’Connell PJ, Lew AM, Cowan PJ, et al. Islet xenotransplantation: progress towards a clinical therapy. Current Opinion in Organ Transplantation 2006; 11:174–179. Yonekawa Y, Matsumoto S, Okitsu T, et al. Effective islet isolation method with extremely high islet yields from adult pigs. Cell Transplant 2005; 14(10):757–62. Hayek A. In search of endocrine progenitor/stem cells in the human pancreas. Pediatr Diabetes 2004; 5(2):70–4. Ta M, Choi Y, Atouf F, et al. The defined combination of growth factors controls generation of long-term replicating islet progenitor-like cells from cultures of adult mouse pancreas. Stem Cells, 2006; 24(7):1738–1749. Ebihara A, Kondo K, Ohashi K, et al. Comparative clinical pharmacology of human insulin (Novo) and porcine insulin in normal subjects. Diabetes Care 1983; 6(1):17–22. Bucher P, Gang M, Morel P, et al. Transplantation of discordant xenogeneic islets using repeated therapy with anti-CD154. Transplantation 2005; 79(11): 1545–52. Mirenda V, Golshayan D, Read J, et al. Achieving permanent survival of islet xenografts by independent manipulation of direct and indirect T-cell responses. Diabetes 2005; 54(4):1048–55. Safley SA, Kapp LM, Tucker-Burden C, et al. Inhibition of cellular immune responses to encapsulated porcine islet xenografts by simultaneous blockade of two different costimulatory pathways. Transplantation 2005; 79(4):409–18. Rayat GR and Gill RG. Indefinite survival of neonatal porcine islet xenografts by simultaneous targeting of LFA-1 and CD154 or CD45RB. Diabetes 2005; 54(2):443–51. Adams AB, Shirasugi N, Jones TR, et al. Development of a chimeric antiCD40 monoclonal antibody that synergizes with LEA29Y to prolong islet allograft survival. J Immunol 2005; 174(1):542–50. Wang G, Feng Y, Hao J, et al. Induction of xenogeneic islet transplantation tolerance by simultaneously blocking CD28-B7 and OX40-OX40L costimulatory pathways. Sci China C Life Sci 2005; 48(5):515–22. Yang TY, Chen JP, Ku Kw, et al. Survival prolongation of microencapsulated allogeneic islet by nanosized nordihydroguaiaretic acid. Transplant Proc 2005; 37(4):1828–9. Schneider S, Felin PJ, Brunnenmeier F, et al. Long-term graft function of adult rat and human islets encapsulated in novel alginate-based microcapsules after transplantation in immunocompetent diabetic mice. Diabetes 2005; 54 (3):687–93. Thomas FT, Ricordi C, Contreras JL, et al. Reversal of naturally occuring diabetes in primates by unmodified islet xenografts without chronic immunosuppression. Transplantation 1999; 67(6):846–54. Valdes-Gonzalez RA, Dorantes LM, Ganbay GN, et al. Xenotransplantation of porcine neonatal islets of Langerhans and Sertoli cells: a 4-year study. Eur J Endocrinol 2005; 153(3):419–27. Shamekh R, El-Badri NS, Saporta S, et al. Sertoli cells induce systemic donorspecific tolerance in xenogenic transplantation model. Cell Transplant 2006; 15(1):45–53.

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19. Dufour JM, Hamilton M, Rajotte RV, et al. Neonatal porcine Sertoli cells inhibit human natural antibody-mediated lysis. Biol Reprod 2005; 72(5): 1224–31. 20. Isaac JR, Skinner S, Elliot R, et al. Transplantation of neonatal porcine islets and sertoli cells into nonimmunosuppressed non-human primates. Transplant Proc 2005; 37(1):487–8. 21. Hering BJ, Wijkstrom M, Graham ML, et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed non-human primates. Nat Med 2006; 12(3):301–3. 22. Cardona K, Korbutt G.S., Milas Z, et al. Long-term survival of neonatal porcine islets in non-human primates by targeting costimulation pathways. Nat Med 2006; 12(3):304–6. 23. Elliott RB, Escobar L, Tan PL BB, et al. Intraperitoneal alginate-encapsulated neonatal porcine islets in a placebo-controlled study with 16 diabetic cynomolgus primates. Transplant Proc 2005; 37(8):3505–8. 24. Van der Laan L and Salomon D. Cross-species transmission of porcine endogenous retroviruses in xenotransplantation: a PERVerted reality? In: Sher L and Grinyo J, Eds. Current Opinion in Organ Transplantation. SpringerVerlag: Berlin, 2001:51–58. 25. Ericsson TA, Takeuchi Y, Templin C, et al. Identification of receptors for pig endogenous retrovirus. Proc Natl Acad Sci USA 2003; 100(11):6759–64. 26. Ritzhaupt A, Laan LJ, Salomon DR, et al. Porcine endogenous retrovirus infects but does not replicate in non-human primate primary cells and cell lines. Journal of Virology 2002; 76(22):11312–11320. 27. Yang YG, Wood JC, Lan P, et al. Mouse retrovirus mediates porcine endogenous retrovirus transmission into human cells in long-term humanporcine chimeric mice. J Clin Invest 2004; 114(5):695–700. 28. Martina Y, Kurian S, Cherqui S, et al. Pseudotyping of porcine endogenous retrovirus by xenotropic murine leukemia virus in a pig islet xenotransplantation model. Am J Transplant 2005; 5(8):1837–47. 29. Van der Laan LJ, Lockey JC, Griffeth BC, et al. Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice. Nature 2000; 407(6800):90–4. 30. Martina Y, Marcucci KT, Cherqui S, et al. Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. Journal of Virology 2006; 80(7):3135–46.

17 Approaches to b-Cell Regeneration and Neogenesis Susan C. Campbell Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle, U.K.

Wendy Macfarlane School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, U.K.

INTRODUCTION Loss of b-cell mass is the critical convergence point in the development of both Type 1 and Type 2 diabetes. Consequently, restoration of b-cell mass is the fundamental underlying premise of many current therapeutic approaches to the treatment of both forms of the disease. Improvement in the success of islet transplantation procedures has provided critical proof of the principal that the restoration of b-cell mass can reverse diabetes and restore normal insulin secretion. Hence, many research laboratories worldwide are engaged in the development of new sources of insulin-producing cells, utilizing a broad range of molecular approaches. It is clear that in a healthy individual, a fine balance is constantly maintained between b-cell apoptosis and b-cell neogenesis. In patients with diabetes, this critical balance is lost, with the resultant decrease in b-cell mass contributing to the eventual loss of normoglycemic control. This chapter reviews our current understanding of the events regulating b-cell regeneration and neogenesis, with a view to potential therapeutic intervention for the improvement of b-cell mass and function in patients with diabetes.

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REGULATION OF PANCREATIC b-CELL MASS Healthy b-cell mass is maintained and regulated both during development and in adults in response to a number of nutritional, hormonal, and environmental changes. The adaptive nature of b-cell mass allows the body to respond to changes in the cellular environment, such as prolonged high glucose concentrations (1) or peripheral insulin resistance (2), as well as fundamental changes in whole-body physiology, such as pregnancy (3), obesity (4), and pancreatic tissue damage (5). The initial events determining b-cell mass occur during the earliest development stages, when pancreatic development begins in the endodermal region of the primitive foregut. b-cell development is regulated by the hierarchical expression of a specific complement of transcription factor proteins, reviewed in (6). Beginning in cells expressing the homeodomain transcription factor PDX1 (pancreatic/duodenal homeobox 1) and repressing sonic hedgehog, both endocrine and exocrine cells of the adult pancreas arise from endodermal cells expressing PDX1 (7) and Ptf1a (8). Proliferation of progenitor cells is stimulated by the fibroblast growth factor family of proteins (9) and greatly influenced by the production of specific inductive stimuli from the surrounding developing mesenchyme (10). Transient expression of the paired domain transcription factor Pax4 in ngn3expressing cells drives the formation of b-cells, with the final b-cell transcription factor complement being driven by PDX1, Pax6, Nkx2.2, and Nkx6.1 (6). This is the point at which b-cell expansion begins. In rat models, the most rapid expansion of b-cells occurs in late gestation, with a doubling of b-cell mass occurring every day from day 16 onwards (11). In humans, a similar expansion of b-cell mass is observed in week 20 of gestation (12). The source of b-cell expansion has been the subject of much study, but it would appear that both in rats and in humans, the percentage of b-cells that are replicating is extremely low (90%) may produce a more dramatic

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growth pattern, with both b-cell replication and neogenesis being observed (22,23). When 90% pancreatectomy in rats was combined with the destruction of residual b-cells by streptozocin, this did not affect the capacity of the pancreas to regenerate, supporting the hypothesis that, under severe pancreatectomy conditions, neogenesis of b-cells is the dominant compensatory growth mechanism, not b-cell replication (24). Treatment of animal models with streptozocin alone has also been used to study the process of islet and b-cell neogenesis. Treatment of rodent models with the DNA-alkylating compound streptozocin results in the destruction of the b-cells of the pancreas (25). The regenerative capacity of the pancreas, as discussed above, is at its peak during the early stages of neonatal development. After 5 days, the compensatory response of the pancreas to streptozocin is muted, indicating that the early pancreas potentially contains more precursor cells that can be utilized to expand b-cell mass (26). In adult rodent models, little compensation is seen and the proliferative and differentiation capacity of the islets appears low (27). However, in studies using both streptozocin and the free radical generator alloxan, treatment with a low dose of the toxic effector, or treatment of only part of the pancreas with the toxic effector, does produce a neogenic response (28). When part of the pancreas is treated with alloxan in isolation, this part does not regenerate. However, in a response similar to that seen during pregnancy or in response to obesity, neogenesis of b-cells from pre-existing duct cells is observed in the remaining portion of the pancreas, attempting to compensate for the loss of functional b-cell mass. Combination of these toxic effector molecules with stimuli such as GLP-1, EGF, or gastrin, provokes a much more robust neogenic response. For example, combination of gastrin and EGF in response to alloxan administration in a recent study provoked a 30% increase in b-cell growth rate per day, leading to the doubling of b-cell number in only 3 days (29). This increase in b-cell mass has been attributed to neogenesis from precursor cells within the pancreas, as no increase in b-cell replication was observed. Indeed, transient co-expression of insulin and CK19 in some cells in the regenerating pancreas would support the transdifferentiation of ductal cells as the major source of neogenic b-cells in this model. In combination with the chemical induction of islet and b-cell neogenesis, several animal models utilize the ability of the pancreas to recover from physical assault. Islet neogenesis has been shown to occur following cellophane wrapping of the pancreas in rodent models (30). This model stimulates the proliferation of cells in the pancreatic ducts, eventually resulting in an increase in islet number and b-cell mass (31). Partial duct obstruction using this method was shown in streptozocin-treated animals to result in islet cell regeneration, again suggesting that neogenic b-cells were arising not from existing b-cells by replication, but by transdifferentiation of ductal cells in the adult pancreas. Brief occlusion of the main pancreatic duct by a simple

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“squeezing” technique has been shown to produce an increase in duct cell proliferation and islet mass in a similar manner to cellophane wrapping (32), again implicating ductal cells as the key precursors in the neogenesis of new b-cells (33). The production of new b-cells from ductal cells is also observed in an elegant transgenic mouse model utilizing the expression of interferon γ (IFN-γ). Transgenic mice expressing the IFN-γ gene under the control of the b-cell specific insulin gene promoter develop diabetes through b-cell destruction within 6–8 weeks after birth (34). As in the chemically induced diabetes described above, new b-cells in this model of diabetes were derived from the expansion and differentiation of ductal cells in the pancreas. This model of islet regeneration is particularly elegant, as the compensatory growth processes generating the replacement b-cells seem to mirror the normal development processes driving embryonic b-cell formation. The many animal models of islet neogenesis and b-cell regeneration currently being studied point to the ductal cells of the pancreas as the main source of precursor cells able to transdifferentiate to b-cells and hence provide compensatory b-cell expansion under conditions of b-cell stress. However, while it is clear that ductal cells may play this role in neogenic islets, several other cell types are currently under investigation for their ability to generate functional b-cells. These include the pancreatic acinar cells, liver cells, and the intestinal K cells, as discussed below.

NEW β-CELLS THROUGH TRANSDIFFERENTIATION Transdifferentiation is a coordinated alteration in gene expression and cellular phenotype that allows the conversion of one adult cell type into another adult cell type. The plasticity of adult cells is only now becoming clear. As discussed above, it is the plasticity of the ductal cells of the pancreas that many now believe drives the formation of new b-cells. However, significant literature now suggests that the generation of functional b-cells from adult precursor cells is not limited to the ductal cells, or merely to cells of the pancreas. In this section we review the use of alternative adult cell types in the derivation of new insulin-producing cells, including pancreatic acinar cells, and the cells of the liver and gut (Fig.2). The acinar cells of the pancreas are certainly a potential source of neogenic b-cells (35). In several of the animal models described above, the expansion of ductal cells and the transdifferentiation to new b-cells is preceded by an initial acino-ductal transdifferentiation (36). That is, the starting population of cells undergoing transdifferentiation from duct to b cell is thought to be comprised of mature ductal cells augmented with newly formed ductal cells generated from the acinar tissue. In IFN-γ mice (37) and in the duct ligation model described above (38), there is also evidence of direct transdifferentiation of acinar cells to b-cells, since a

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β-Cells ES cells

Differentiation, transcription factor expression Differentiation?

Bone marrow

PDX1 expression

Liver, K-cells Acinar

Transdifferentiation Ductal

Differentiation β-Cells

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Figure 2 New sources of b-cells. A number of potential sources of new b-cells have been proposed, including replication of pre-existing b-cells; differentiation of progenitor cells within the ductal cells of the pancreas; and transdifferentiation of adult pancreatic acinar cells, either directly to b-cells or by initial acino-ductal transdifferentiation. New sources of b-cells are not limited to cells of the pancreas, and the capability of a number of alternative non-pancreatic cells is being investigated: transdifferentiation of liver cells, through the transgenic expression of key transcription factors such as pancreatic/duodenal homeobox 1 (PDX-1); transdifferentiation of intestinal cells (K cells) and bone marrow cells; and finally through differentiation of embryonic stem cells (ES) cells.

transient subpopulation of cells exists that co-express insulin with the acinar marker amylase. The transdifferentiation potential of the acinar cells can also be enhanced by expression of key developmental transcription factors such as PDX1, again demonstrating the extraordinary plasticity of the cells of the pancreas, and suggesting a further possible source of neogenic b-cells in the pancreas (36). However, this plasticity is not limited to the cells of the pancreas, as liver cells have also been shown to be capable of transdifferentiation to insulin-producing cells. In general, cells that are capable of interconversion/transdifferentiation arise from adjacent regions of the developing embryo. Hence some of the early regulatory events and stimuli driving the formation of these cells are common to both adult cell types. Such is the case for the liver and pancreas, which are both derived from the endoderm of the developing embryo, and which share some adult characteristics, such as glucose sensitivity and pathways of protein processing and secretion. Transdifferentiation of liver to pancreas (and of pancreas to liver) is a phenomenon naturally observed in rare human disease states, as well as in well-characterized animal models (39). Hepatic foci have been observed in the pancreas of humans, rodents, and simian models. Indeed, the appearance of hepatic cells in the adult pancreas has been studied for over twenty years. Whether the hepatic cells observed in the pancreas in vivo arise directly from transdifferentiation of adult pancreatic cells remains

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controversial; however, in vitro studies with transformed cell lines have shown that the transdifferentiation from liver to b-cell, and from b-cell to liver, is possible, given the right experimental conditions, or the transgenic expression of key transcription factors such as PDX1 (40). Several of the hepatic nuclear factor family of transcription factors have also been shown to be critically important to the transdifferentiation of liver to pancreas and vice versa. These include HNF1, FoxA2, HNF4, and HNF6 (41,42). The C/EBP, b and, γ mRNAs have also been shown to increase during the regeneration of the pancreas in copper-deprived animal models of the transdifferentiation process (42). Although transdifferentiation of liver to pancreas occurs in disease states such as hepatic cirrhosis (43), and in several animal models and in vitro systems (39), the role of these transdifferentiation events in normal b-cell turnover and adult pancreatic b-cell function has not been established, and it seems unlikely that these events have a major role to play in the normal processes of b-cell regeneration and neogenesis. Just as liver cells emerge from the embryonic endoderm closely associated with pancreas development, so the gut cells are subjected to similar developmental stimuli. Recent studies have investigated the potential of gut K cells to produce functional insulin-secreting cells (44). Intestinal K cells have been successfully engineered in vitro to express insulin and to secrete the protein in a glucose-responsive manner (45). Gut cells, like liver cells, have the functional advantage in that they contain the appropriate secretory pathway components to regulate protein secretion. Hence, studies with liver and gut cells have proven the most successful in terms of generating glucose-responsive insulin-secreting cells by transdifferentiation. However, while these approaches have proven extraordinarily valuable in increasing our understanding of the transdifferentiation process, it seems unlikely that either cell type contributes to b-cell neogenesis in vivo. Hence, the value of these cells in terms of novel therapeutic approaches to the treatment of diabetes remains limited. Such approaches would require extensive in vitro manipulation of isolated adult cells, followed by transplantation of modified cells. While this is certainly a possibility in the long term, it seems more likely at present that successful generation of insulin-producing cells from pancreas-derived material (ductal and islet cells) will provide a more practical approach in the short-term. Current approaches to islet transplantation could certainly be enhanced by the augmentation of purified adult islets with islet-derived cells capable of transdifferentiation into b-cells. In terms of the augmentation of islet function through transdifferentiation of ductal precursor cells, or in terms of the improvement of b-cell mass and neogenesis rates in a patient with Type 2 diabetes, one candidate approach appears the most attractive at present. This approach is the augmentation of islet function by the intestinal polypeptide GLP-1. Current progress in GLP-1 research is reviewed below.

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GLP-1 GLP-1 is an incretin hormone produced and released from the L cells of the intestine (46). The insulinotropic actions of GLP-1 include the augmentation of insulin secretion and the effective lowering of circulating blood glucose concentrations (47). In b-cells, duct cells, and acinar cells, GLP-1 binds to and activates a specific GLP-1 receptor present on the cell membranes (48). Knock-out of GLP-1 receptors in transgenic mice produce defective islet function, resulting in defective regulation of blood glucose concentrations in these animals (49). In pancreatic b-cells and acinar cells, activation of the GLP-1 receptor is known to result in intracellular signaling events involving protein kinase A and changes in intracellular c-AMP levels. Characterization of these events in acinar and b-cells, and the recent finding that duct cells also express abundant functional GLP-1 receptors, indicate a key role for GLP-1 in the regulation of normal islet function (48). In addition, GLP-1 is known to regulate the expression of several key transcription factor proteins in adult b-cells, including the critical homeodomain protein PDX1 (50). However, of greatest interest in the present context is the potential role of GLP-1 in the neogenesis and regeneration of b-cells. The potential of GLP-1 to stimulate b-cell neogenesis is the subject of intense study at the present time. In early studies, investigation of the function of GLP-1 was hampered by the extremely short half-life of the protein (51). GLP-1 released from the intestinal L cells is very rapidly degraded by the dipeptidase enzyme DPPIV. The undesirably short half-life of GLP-1 has resulted in investigation of DPPIV inhibitors (52), GLP-1 agonists with longer half-lives such as Exendin 4 (Ex4) (53), and shorter fragments of active GLP-1, such as the functional 7-36 Amide (54) form of the peptide. Ex4 has proven of particular interest, since this has been extensively studied in the transdifferentiation of ductal cells to b-cells. Using the in vitro transformed ductal cell line Capan-1, Ex4 was shown to promote transdifferentiation to an endocrine phenotype (55). This process occurred through stimulation of the expression of the key transcription factors PDX1 and FoxA2, both of which are known to be critical in the normal development of b-cells in the embryo (7), and in the transdifferentiation process from alternative adult cell types. FoxA2 binds to and regulates the PDX1 gene promoter, events which are critical in the initiation of PDX1 gene expression and differentiation towards a b-cell phenotype. These early studies indicated that administration of GLP-1 activated both adenylate cyclase and MAP kinase signaling pathways, ultimately producing increased PDX1 gene expression and the resultant emergence of an insulin-producing b-cell phenotype (55). These data complement extensive findings suggesting that GLP-1 and Ex4 both stimulate PDX1 gene expression in ductal cells (56), with PDX1 gene expression being upregulated during the regeneration of the pancreas (57). However, even more compelling evidence comes from

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recent animal studies that firmly implicate GLP-1 in the process of b-cell neogenesis. The potential role of GLP-1 in stimulating the process of b-cell neogenesis was originally demonstrated in vivo in lean 20-day-old normoglycemic mice (58). Since these initial studies, a substantial number of studies have confirmed that GLP-1 and Ex4 exert major trophic effects on the cells of the pancreas, increasing b-cell neogenesis and boosting b-cell mass. Using the longer half-life of Ex4, recent studies have shown that administration of this GLP-1 receptor agonist to streptozotocin-treated rats results in the increased appearance of small clusters of insulin-producing cells in the ducts of these animals, and contributes to the restoration of normal blood glucose control in this model of Type 1 diabetes (48). Treatment of the animals with Ex4 resulted in a prolonged and sustained improvement in glucose tolerance, and this was shown to occur not only through the generation of neogenic b-cells from duct cells, but additionally to result in part through an increase in b-cell mass. No change was observed in the levels of b-cell apoptosis, suggesting that the effects of Ex4 were not a result of a shift in the balance between apoptosis and b-cell neogenesis, but rather a simple positive effect of increasing b-cell number and mass (48). These interesting findings indicate that Ex4 can increase b-cell neogenesis and b-cell mass in diabetic animals, restoring islet function and allowing the control of circulating blood glucose concentrations. Of most clinical relevance, these results were also observed in isolated human pancreatic ducts, where treatment with Ex4 again resulted in the increased appearance of insulin-positive clusters of cells (48). It has been reported that the insulin secretory response to glucose in islets following transplantation, a measure of functional b-cell mass, correlates significantly with the number of ductal cells originally present in the transplanted islets (59). Hence the finding of abundant GLP-1 receptors on human ductal cells is consistent with a key role for GLP-1 in the b-cell neogenesis process. These findings complement many recent animal studies. GLP-1 was found to be a potent stimulator of b-cell neogenesis following partial pancreatectomy (60). Interestingly, GLP-1 receptor knock-out mice showed much poorer glucose tolerance following partial pancreatectomy, identifying a significant defect in b-cell mass regeneration in these animals compared to wild-type animals with an intact GLP-1 response (60). In obese hyperglycemic ob/ob mice, Ex4 increases b-cell mass and improves b-cell function, restoring normal glycemic control (58). In other mouse models of diabetes, GLP-1 has been shown to increase b-cell mass through neogenesis of b-cell from ductal cells expressing PDX1 (61), and GLP-1 administration even delays the onset of diabetes in the db/db mouse model (62). These recent studies add to a growing weight of evidence suggesting that GLP-1 and its analogues may be key regulators of b-cell regeneration and neogenesis. From the original studies in normoglycemic mice, through

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study of pancreatectomized animals, GLP-1 receptor knock-out mice, aging mice (63), neonatal streptozotocin-treated animals (64), the Goto-Kakizaki (GK) rat model (65), in vitro analysis of isolated cell lines, right through to the recent identification of abundant GLP-1 receptors on rat and human ductal cells, the weight of data now suggests a potentially exciting therapeutic role for GLP-1 administration in the promotion of b-cell neogenesis, and the restoration of b-cell mass and GLP-1 may represent one of the best therapeutic candidates for the improvement of b-cell mass and function in patients with diabetes. ENHANCING β-CELL MASS AND NEOGENESIS GLP-1 represents an exciting potential therapeutic agent to increase b-cell neogenesis and b-cell mass in the treatment of diabetes. However, several other approaches to the enhancement of b-cell neogenesis and mass are currently under investigation. Two of the major alternative approaches under current investigation are combination therapy through treatment of islets with gastrin and EGF, and the enhancement and protection of b-cell mass through administration of thiazolidinediones. It remains possible that a combination of these approaches may prove beneficial in enhancing the function of b-cells in patients with diabetes, or following islet transplantation. Combined treatment of streptozocin-treated rats with gastrin and EGF has been reported to not only increase b-cell mass, but to reduce hyperglycemia (66). In alloxan-treated animals, the same combination also resulted in restored normoglycemia through induction of b-cell neogenesis from ductal cells (67). Hence, further studies have begun to investigate the combined effects of these stimuli on b-cell regeneration and islet function. Culture of isolated human islets (containing ductal cells) with gastrin and EGF resulted in a significant increase in functional b-cell mass (68). This increase was related to increased expression of the transcription factor PDX1 and resulted in increased production of both insulin and C-peptide from the expanded b-cell mass in vitro. When human islets were transplanted into NOD/SCID mice, an immunocompromised mouse model of type 1 diabetes, administration of gastrin and EGF in combination increased functional b-cell mass and insulin secretion, as well as increasing the insulin secretory response of the transplanted islets to oral glucose challenge (68). Hence, recent studies indicate that combination of gastrin and EGF increases b-cell mass and function in isolated human islets in vitro and in vivo, through induction of b-cell neogenesis from ductal cells. Combination of gastrin and EGF, or the use of GLP-1 analogues, has the potential to generate an increased b-cell mass in vivo and in vitro in isolated and subsequently transplanted islets. A third therapeutic approach currently under intense scrutiny is the potential protection and enhancement of neogenic b-cell function through administration of thiazolidinediones

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such as rosiglitazone. Rosiglitazone is a commonly prescribed antihyperglycemic drug prescribed to those with Type 2 diabetes (69). Initially characterized as a modulator of adipocyte cell function, the agonistic actions of rosiglitazone on the nuclear hormones receptor peroxisome proliferator activator gamma (PPAR) are known to result in decreased hepatic glucose output, increased peripheral glucose uptake, and alterations in adipocyte metabolism. However, recent studies indicate that in addition to these wellcharacterized effects, rosiglitazone acts directly on the pancreatic b-cells (70). Rosiglitazone administration has been shown to stimulate PDX1 gene expression in b-cells in culture, as well as increasing the expression and nuclear localization of the PDX1 and FoxA2 proteins, both of which are well-characterized regulators of b-cell growth and development. These key development transcription factors have also been repeatedly implicated in the process of b-cell neogenesis and increases in b-cell mass. Consistent with these observations of the direct effects of rosiglitazone on b-cell function, and the success of rosiglitazone in improving (71) and protecting b-cell function in vivo (72), administration of rosiglitazone has also been shown to enhance the function of transplanted islets (73). This raises the interesting possibility that administration of rosiglitazone may perform three roles in patients undergoing islet transplantation. First, rosiglitazone may enhance b-cell function and increase b-cell mass; second, systemic effects of rosiglitazone are known to improve insulin sensitivity and decrease hyperglycemia through targeting liver, muscle, and adipocyte cell function; and last, rosiglitazone may protect not only the transplanted islets, but the neogenic b-cells emerging from transplanted ductal material. Hence, mounting evidence supports the idea that rosiglitazone administration may have an overall beneficial effect on b-cell neogenesis and function, both in vivo and following transplantation of isolated islets. In summary, the augmentation of transplanted islets with increased neogenic potential may well be achieved through stimulation with GLP-1 (Ex4) in combination with rosiglitazone. This would promote b-cell neogenesis and an increase in b-cell mass while endowing the neogenic b-cells with an element of protection and promoting increased function. Only time will tell whether this type of combined therapy can improve islet graft function, and in the long term, potentially also improve islet function of patients with Type 2 diabetes. As described in the sections above, an increasingly large volume of work indicates that the plasticity of the adult cells of the pancreas can be utilized to generate new sources of insulin-producing cells. The optimal regeneration of b-cell function, and the promotion of b-cell neogenesis, may require a combination of one or more of these approaches. In the longer term, limitations on the amount of available donor islet tissue may result in a drive towards the successful generation of functional b-cells through transdifferentiation of other adult cell types such as liver cells. Finally, a worldwide effort is underway to generate functional b-cells

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from embryonic stem cells in culture (74) and from bone-marrow derived stem cells (75). Successful generation of functional b-cells from embryonic stem cells would generate a tissue bank of insulin-producing cells for the treatment of large numbers of patients with diabetes. However, as reviewed here, the events controlling the emergence of a mature b-cell phenotype, and the balance between proliferation and apoptosis of the b-cells, are extremely complex. Hence, differentiation of embryonic stem cells to a fully functional and self-renewing b-cell phenotype is certainly a challenging prospect. Current stem-cell approaches for the generation of new b-cells are the subject of the chapter that follows. REFERENCES 1.

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14. Bouwens L, De Blay E. Islet morphogenesis and stem cell markers in rat pancreas. J Histochem Cytochem 1996; 44:947–951. 15. Van Assche FA, Gepts W, Aerts L. Immunocytochemical study of the endocrine pancreas in the rat during normal pregnancy and during experimental diabetic pregnancy. Diabetologia 1980; 18:487–491. 16. Van Assche FA, Aerts L, De Prins F. A morphological study of the endocrine pancreas in human pregnancy. Br J Obstet Gynaecol 1978; 85:818–820. 17. Allen SR. Gestational diabetes: a review of the treatment options. Treat Endocrinol 2003; 2(5):357–65. 18. Greenberg AS, Obin MS. Obesity and the role of adipose tissue in inflammation and metabolism. Am J Clin Nutr 2006; 83(2):461S–465S. 19. Kloppel G, Lohr M, Habich K, Oberholzer M, Heitz PU. Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv Synth Pathol Res 1985; 4:110–125. 20. Dor Y, Brown J, Martinez OI, Melton DA. Adult pancreatic b-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004; 429:41–46. 21. Gao R, Ustinov J, Korsgren O, Otonkoski T. In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells. Diabetologia 2005; 48(11):2296–304. 22. Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE. A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 1993; 42:1715–1720. 23. Dor Y, Brown J, Martinez OI, Melton DA. Adult pancreatic b-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004; 429:41–46. 24. Finegood DT, Weir GC, Bonner-Weir S. Prior streptozotocin treatment does not inhibit pancreas regeneration after 90% pancreatectomy in rats. Am J Physiol Endocrinol Metab 1999; 276:E822–E827. 25. Szkudelski T. The mechanism of alloxan and streptozotocin action in B cells of the rat pancreas. Physiol Res 2001; 50(6):537–46. 26. Wang RN, Bouwens L, Klo¨ppel G. Beta cell growth in adolescent and adult rats treated with streptozotocin during the neonatal period. Diabetologia 1996; 39:548–557. 27. Ahren B, Sundkvist G. Long-term effects of alloxan in mice. Int J Pancreatol 1995; 17:197–201. 28. Waguri M, Yamamoto K, Miyagawa JI, et al. Demonstration of two different processes of b-cell regeneration in a new diabetic mouse model induced by selective perfusion of alloxan. Diabetes 1997; 46:1281–1290. 29. Rooman I, Bouwens L. Combined gastrin and epidermal growth factor treatment induces islet regeneration and restores normoglycaemia in C57Bl6/J mice treated with alloxan. Diabetologia 2004; 47:259–265. 30. Rafaeloff R, Pittenger GL, Barlow SW, et al. Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J Clin Invest 1999; 99:2100–2109. 31. Rosenberg L, Clas D, Duguid. Trophic stimulation of the ductal/islet cell axis: a new approach to the treatment of diabetes. Surgery 1990; 108:191–197.

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32. Woodroof CW, de Villiers C, Page BJ, van der Merwe L, Ferris WF. Islet neogenesis is stimulated by brief occlusion of the main pancreatic duct. S Afr Med J 2004; 94(1):54–7. 33. Ferris WF, van der Merwe L, Campbell SC, Macfarlane WM. Glucocorticoid administration and brief occlusion of the main pancreatic duct are likely to increase islet mass by a similar mechanism. Pancreas 2005; (2):132–7. 34. Sarvetnick NE, Gu D. Regeneration of pancreatic endocrine cells in interferon-γ transgenic mice. Adv Exp Med Biol 1992; 321:85–9. 35. Bouwens L. Transdifferentiation versus stem cell hypothesis for the regeneration of islet b-cells in the pancreas. Microsc Res Tech 1998; 43(4):332–6. 36. Rooman I, Heremans Y, Heimberg H, Bouwens L. Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro. Diabetologia 2000; 43(7):907–14. 37. Wang RN, Kloppel G, Bouwens L. Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 1995; 38(12): 1405–11. 38. Gu D, Arnush M, Sarvetnick N. Endocrine/exocrine intermediate cells in streptozotocin-treated Ins-IFN-gamma transgenic mice. Pancreas 1997; 15(3): 246–50. 39. Shen CN, Horb ME, Slack JM, Tosh D. Transdifferentiation of pancreas to liver. Mech Dev 2003; 120(1):107–16. 40. Koizumi M, Doi R, Toyoda E, et al. Hepatic regeneration and enforced PDX-1 expression accelerate transdifferentiation in liver. Surgery 2004; 136(2):449–57. 41. Rao MS, Yukawa M, Omori M, Thorgeirsson SS, Reddy JK. Expression of transcription factors and stem cell factor precedes hepatocyte differentiation in rat pancreas. Gene Expr 1996; 6:15–22. 42. Dabeva MD, Hurston E, Sharitz, DA. Transcription factor and liver-specific mRNA expression in facultative epithelial progenitor cells of liver and pancreas. Am J Pathol 1995; 147:1633–1648. 43. Wolf HK, Burchette JL, Garcia, JA, Michalopoulos G. Exocrine pancreatic tissue in human liver: a metaplastic process? Am J Surg Pathol 1990; 14: 590–595. 44. Fujita NY, Cheung AT, and Kieffer TJ. Harnessing the gut to treat diabetes, Pediatr Diabetes 2004; 5(Suppl 2):57–69. 45. Cheung AT, Dayanandan B, Lewis JT, et al. Glucose-dependent insulin release from genetically engineered K cells. Science 2000; 290:1959–1962. 46. Kieffer TJ, Habener JF. The glucagon-like peptide. Endocr Rev 1999; 20: 876–913. 47. Gutniak M, Orskov C, Holst JJ, Ahren B, Efendis S. Antidiabetogenic effect of glucagon-like peptide-1 (7-36) amide in normal subjects and patients with diabetes mellitus. N Engl J Med 1992; 326:1316–1322. 48. Xu G, Kaneto H, Lopez-Avalos MD, Weir GC, Bonner-Weir S. GLP-1/ exendin-4 facilitates b-cell neogenesis in rat and human pancreatic ducts. Diabetes Res Clin Pract 2006; 73(1):107–10. 49. Flamez D, Gilon P, Moens K, et al. Altered cAMP and Ca2þ signaling in mouse pancreatic islets with glucagon-like peptide-1 receptor null phenotype. Diabetes 1999; 48(10):1979–86.

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50. Campbell SC, Macfarlane WM. Regulation of the pdx1 gene promoter in pancreatic b-cells. Biochem Biophys Res Commun 2002; 299(2):277–84. 51. Deacon CF, Johnsen AH, Holst JJ. Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo. J Clin Endocrinol Metab 1995; 80:952–957. 52. Holst JJ. Treatment of type 2 diabetes mellitus with agonists of the GLP-1 receptor or DPP-IV inhibitors. Expert Opin Emerg Drugs 2004; 9(1):155–66. 53. Nielsen LL, Young AA, Parkes DG. Pharmacology of exenatide (synthetic exendin-4): a potential therapeutic for improved glycemic control of type 2 diabetes. Regul Pept 2004; 117(2):77–88. 54. Nauck MA, Kleine N, Ørskov C, Holst JJ, B. Willms, Creutzfeldt W. Normalization of fasting hyperglycaemia by exogenous glucagon-like peptide 1 (7–36 amide) in type 2 (non-insulin-dependent) diabetic patients, Diabetologia 1993; 36:741–744. 55. Zhou J, Pineyro MA, Wang X, Doyle ME, Egan JM. Exendin-4 differentiation of a human pancreatic duct cell line into endocrine cells: involvement of PDX-1 and HNF3beta transcription factors. J Cell Physiol 2002; 192(3): 304–14. 56. Stoffers DA, Kieffer TJ, Hussain MA, et al. Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein PDX-1 and increase islet size in mouse pancreas. Diabetes 2000; 49:741–748. 57. Sharma A, Zangen DH, Reitz P, et al. The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 1999; 48:507–513. 58. Edvell A, Lindstrom P. Initiation of increased pancreatic islet growth in young normoglycemic mice (Umea þ/γ).Endocrinology 1999; 140(2):778–83. 59. Street CN, Lakey JR, Shapiro AM, et al. Islet graft assessment in the Edmonton Protocol: implications for predicting long-term clinical outcome. Diabetes 2004; 53(12):3107–14. 60. De Leon DD, Deng S, Madani R, Ahima RS, Drucker DJ, Stoffers DA. Role of endogenous glucagon-like peptide-1 in islet regeneration after partial pancreatectomy. Diabetes 2003; 52:365–371. 61. Stoffers DA, Kieffer TJ, Hussain MA, et al. Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increases islet size in mouse pancreas. Diabetes 2000; 49:741–748. 62. Wang Q and Brubaker PL. Glucagon-like peptide-1 treatment delays the onset of diabetes in 8 week-old db/db mice. Diabetologia 2002; 45:1263–1273. 63. Perfetti R, Zhou J, Doyle ME, Egan JM. Glucagon-like peptide-1 induces cell proliferation and pancreatic-duodenum homeobox-1 expression and increases endocrine cell mass in the pancreas of old, glucose-intolerant rats. Endocrinology 2000; 141(12):4600–5. 64. Tourrel C, Bailbe D, Meile MJ, Kergoat M, Portha B. Glucagon-like peptide1 and exendin-4 stimulate b-cell neogenesis in streptozotocin-treated newborn rats resulting in persistently improved glucose homeostasis at adult age. Diabetes 2001; 50(7):1562–70. 65. Tourrel C, Bailbe D, Lacorne M, Meile MJ, Kergoat M, Portha B. Persistent improvement of type 2 diabetes in the Goto-Kakizaki rat model by expansion

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18 Stem Cell Approaches for b-Cell Replacement Enrique Roche Instituto de Bioingenieria, Universidad Miguel Hernandez, Elche, Alicante, Spain

Bernat Soria CABIMER, Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Andalucia, Spain

INTRODUCTION Type 1 diabetes mellitus is a degenerative pathology caused by autoimmune destruction of pancreatic b-cells, the only cell type that in the adult organism is responsible for bioactive insulin production and secretion (1). Insulin is a key hormone in glucose and fatty acid homeostasis, enhancing uptake of these nutrients by peripheral tissues and thereby controlling their circulating levels. The function of insulin cannot be mimicked by other hormones, and, without insulin replacement, Type 1 diabetes inevitably leads to ketoacidosis and death. Daily injections have changed the fate of the disease, but the maintenance of correct glucose levels requires very well trained and motivated patients in order to diminish secondary complications, such as neuropathy, nephropathy, retinopathy, and cardiovascular disease (1). Up to 0.5% of the population in developed countries is affected by Type 1 diabetes and the incidence is increasing. This disorder appears mainly, but not exclusively, in children and young people. Indeed, recent evidence suggests that 5–15% of patients initially diagnosed with Type 2 diabetes in fact have a more slowly progressive form of diabetes, termed latent autoimmune diabetes in adults, or LADA (2). The underlying pathogenesis of Type 1 diabetes mellitus remains unclear, although it is believed that several environmental factors are capable of activating autoimmune mechanisms in genetically 311

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predisposed individuals. The HLA-DR3, in addition to HLA-DR4 in Caucasian people, is predictive of disease (3,4). Markers of islet autoimmunity include antibodies against b-cell proteins, such as surface antigens (anti-islet), glutamate decarboxylase, and tyrosine phosphatase (3,4). The pathology displays two phases: insulinitis, corresponding to islet leukocyte infiltration, and overt diabetes, corresponding to b-cell destruction. Unfortunately, in most cases Type 1 diabetes mellitus is diagnosed once more than 90% of the b-cell mass has been destroyed. Recent successes in islet transplantation have confirmed the huge potential advantages of cell therapy for diabetes in comparison to exogenous insulin injection and whole organ transplantation (5). Two major hurdles remain: the need to refine immunosuppression or immunoisolation approaches (6) and the limited number of cadaveric pancreata (7). Even in Spain, with the highest rate of organ donation in the world, only a maximum of 1500 pancreata would be available each year. This will never be sufficient for the needs of more than 100,000 Spanish people with Type 1 diabetes. This strongly suggests that new sources of islets for replacement protocols have to be found. In this context, embryonic and adult stem cells offer interesting possibilities meriting further exploration. Stem cells are defined by the potential to undergo symmetric cell divisions for self-renewal and asymmetric cell divisions for lineage commitment, for example, to form differentiated insulin-producing cells (8). Stem cells may thus offer an important and unlimited source for b-cell replacement protocols (9,10). In addition, emerging technologies, such as nuclear transfer (11–15), oocyte parthenogenesis (16,17), and cell reprogramming (18,19) by employing host-derived cells may circumvent immune rejection.

STEM CELLS Stem cells can be classified as embryonic (ESCs) and adult (ASCs). ESCs are obtained from the inner cell mass of the blastocyst, a structure formed during embryonic development at day 6 in humans and day 3.5 in mice (20). ASCs are present within tissues of adult organisms with a key role in turnover and/or repopulation (21). Although both cell types are capable of self-renewal commitment to specific cell fates, ESCs demonstrate greater plasticity (pluripotency) in terms of differentiation with potential to generate almost any cell type present in the adult organism, including the germinal lineage. ASCs appear to be committed to a more limited repertoire of cell fates (multipotency). However, the possibility that ASCs may be able to overcome cell commitment restrictions by transdifferentiation may widen possibilities for autologous transplantation (22). In this chapter, the

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potential for generation of insulin-producing cells from both ESCs and ASCs is considered further.

ADULT STEM CELLS ASCs are an attractive option representing a good alternative for cell replacement protocols in terms of immune compatibility. However, limited capacity for proliferation out from their in vivo niches with inherent commitment to specific cell fates may constrain their clinical applications (23). To achieve b-cell neogenesis, two sources of ASCs have to be considered: pancreatic and extrapancreatic stem cells.

Pancreatic Stem Cells In vitro experiments support the hypothesis that ASCs reside within the pancreatic duct with the potential to differentiate in vitro into islet-like structures, and that some ductal cells are thus islet progenitors (24,25). Culture of isolated ductal tissue from donor human pancreas under specific conditions enables formation of cell aggregates, termed cultivated human islet buds (CHIBs), that expressed (at the protein level) islet-specific hormones, including insulin and glucagon, in addition to transcription factors, such as Pdx1. A 2.5-fold increase in insulin secretion was attained in the presence of 20 mM glucose in comparison to 5 mM glucose, although this required relatively long 24-hour incubation of CHIBs (24). Another group reported isolated islet pluripotent stem cells (IPSCs) budding from pancreatic ducts isolated from pre-diabetic non-obese diabetic (NOD) mice. These IPSCs displayed increased insulin release in response to high glucose concentration (17.5 mM), and partially reversed hyperglycemia on transplantation under the kidney capsule in NOD mice (25). However, the low amount of insulin produced by IPSCs in vitro does not equate with the improvement in hyperglycemia in transplanted animals, suggesting that an as-yet uncharacterized maturation processes occurred in vivo. These reports indicate that duct epithelium obtained from cadaveric pancreata may have true potential for cell replacement protocols in Type 1 diabetes. Both CHIBs and IPSCs seem to arise from specific pluripotent cells residing within duct epithelium. Some groups believe nestin, a protein present in neurofilaments, to be a specific marker for these cells. In addition, the expression of nestin has also been reported in cells derived from islets called nestin-positive islet-derived progenitors (NIPs). NIPs isolated from rat and human islets proliferate in vitro and express endo- and exocrine pancreatic markers as well as hepatic genes (26). However, data obtained from transgenic mice indicate that nestin-positive cells seem to participate only in islet microvasculature formation (27). Therefore, whether nestin is a

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marker for islet precursors remains a matter of considerable debate. Nevertheless, the presence of islet stem cells outside ductal tissue and possibly within islets themselves remains. The Melton group have proposed that new b-cells in the adult are derived solely from replication of pre-existing b-cells and not from stem cell precursors (28). However, the experimental design that supports this conclusion does not exclude the existence of a pancreatic stem cell population. Although adult b-cells present a low rate of BrdU incorporation, it seems clear that we are not born with a mass of b-cells for the rest of our life. This suggests that these cells display a low turnover or neogenesis that could be significantly activated under specific circumstances, such as pancreatectomy or chemical insult (e.g.. streptozocin injection). However, the key molecular mechanisms underlying this process remain unclear. All these studies continue to suggest that meaningful generation of new b-cells for transplantation can be achieved from adult pancreata ex vivo or possibly even in situ in the individual with diabetes. At present, however, biosynthesized insulin remains too low for successful clinical transplantation. Moreover, it will be important to demonstrate physiological minute-tominute glucose-responsive insulin secretion. Identification of true islet progenitors and the development of small molecules to stimulate in vivo differentiation are key ongoing challenges in this field.

Extrapancreatic Stem Cells The constraints imposed by limited pancreatic tissue turnover and differentiation could be overcome by using alternative sources of ASCs. In this context, bone marrow contains a stem cell population that displays a broad plasticity, extending beyond blood and bone renewal. Dr Verfaillie’s group have isolated a multipotent cell side population from bone marrow, called Multipotent Adult Progenitor Cells (MAPCs). MAPCs have been isolated, cultured in vitro and differentiated to ectoderm, mesoderm, and endoderm lineages (29). The possibility of obtaining insulin-positive cells from bone marrow stem cells is still a matter of debate. One report claimed pancreas repopulation using highly purified bone marrow stem cells (30). Furthermore, isolated human bone marrow mesenchymal cells transfected with DNA constructs encoding for key transcription factors involved in endocrine pancreas development and cultured in islet-conditioned medium were capable of insulin gene expression. On the other hand, other laboratories failed to reproduce the results of the first report (31,32). In recent experiments progenitors circulating in peripheral blood have been coaxed to acquire an islet cell phenotype (33) and restored blood glucose control when implanted in streptozocin-diabetic immunocompromised mice. To this end, isolated circulating monocytes were dedifferentiated by adding

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interleukin-3 and macrophage colony stimulating factor to the culture medium. Subsequent exposure to epidermal and hepatic growth factors and nicotinamide resulted in insulin-containing cells (34). Liver, like the pancreas, is derived from definitive endoderm and could provide ASCs as an alternative source for bioengineering insulin-producing cells. In this context, it has been described that oval hepatic stem cells are capable of differentiation into cells positive for insulin I and II and other islet-specific markers, such as Pdx1, Pax-4, Pax-6, Nkx2.2, Nkx6.1, glucagon, pancreatic polypeptide, and the glucose transporter GLUT-2. Transplantation of these cells under the renal capsule of streptozocindiabetic NOD-SCID mice resulted in recovery of euglycemia, although graft removal for subsequent analysis and proof of restoration of the diabetic phenotype has not been reported (35). Hepatocytes share many phenotypic characteristics with b-cells, expressing key metabolic enzymes that are common to both cell types. This suggests that these cells could be interesting candidates for ectopic insulin production. This can be achieved by expressing key transcription factors essential for b-cell function such as Pdx1 or NeuroD combined with betacellulin (36,37). Hepatic expression of such proteins induces activation of the insulin gene within liver cells. Taken together, these studies offer interesting possibilities for the use of liver biopsies to generate functional and autotransplantable islet-like structures for cell replacement protocols in Type 1 diabetes. Alternatively, in situ transdifferentiation of liver cells to an insulin-secreting phenotype or b-cell neogenesis within the liver may ultimately provide a novel therapeutic strategy.

EMBRYONIC STEM CELLS ESCs provide another potential source of new b-cells. Mouse ESCs are maintained in the undifferentiated state by addition of Leukemia Inhibitory Factor (LIF), a cytokine of the interleukin-6 family, to the culture medium or by culturing on feeder layers of mitotically inactivated fibroblasts. In contrast, human ESCs do not respond to human LIF and even when cultured on feeder layers spontaneously initiate differentiation processes (38). Compared to mouse ESCs, it is thus more difficult to maintain human ESCs in an undifferentiated state in vitro. Differentiation towards specific lineages requires transfer from growth as an adherent monolayer to cell culture in suspension in the absence of LIF. Under these conditions, cells form aggregates called embryoid bodies (EBs) (38) in which markers from the three embryonic layers (ectoderm, mesoderm, and endoderm) as well as primitive endoderm are detected at different times in culture. The molecular and biophysical determinants that activate differentiation programs in EBs are still unknown, but it has been

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proposed that gradients in nutrient, oxygen, and growth factor concentrations across the EB, as well as intercellular connections, are instrumental in this process (39). Insulin expression together with detection of other pancreatic hormones has been observed during spontaneous differentiation in EBs at 10–23 d (40). However, the fraction of insulin positive cells is less than 2% of the total number of cells (41), and the total amount of insulin produced is too low to contemplate clinical transplantation trials. As stated above, the b-cell is derived from endoderm and acquires its phenotype through the expression of specific proteins, most critically insulin. In this sense, it has been accepted that insulin is the distinctive protein of pancreatic b-cells, and bioengineering strategies are targeted towards expression of this hormone. This has been the rationale behind the vast majority of culture condition manipulation protocols designed to obtain insulin-producing cells from ESCs. However, the intracellular amount of insulin obtained with these strategies is 1000 times lower than the insulin content detected in mature b-cells (42–44). The explanation may be related to the origin of the insulinpositive cells detected in the culture plates after differentiation protocols. In addition to the islet b-cell, the insulin gene is expressed in neuroectoderm-derived tissues and in primitive endoderm (45,46). The role of the hormone in these tissues has not yet been fully clarified. Recent work suggested that insulin is working as a remodeling factor in the initial stages of nervous system development when insulin-like growth factors (IGFs) are absent (47). Rodents have two insulin genes (insulin I and II). Insulin I gene is exclusively expressed in pancreatic b-cells, while insulin II is mainly expressed in yolk sack and specific neurons (46). In this context, the expression pattern of both genes may reveal important differences that should be considered in bioengineering protocols. For instance, insulin II gene is not regulated by glucose; its protein product is not fully processed, yielding thereby proinsulin from neuroectodermal tissues, and the amount of peptide produced is 1000 times lower than the amount of insulin detected in islets (48). Therefore, the presence of ectodermal-derived insulin-positive cells obtained in bioengineering protocols is a possibility that should be taken into account. This can be tested by determining the expression of insulin II gene in mouse-derived cell lines. However, humans only express one insulin gene, and complementary markers would be necessary in order to determine the exact origin of insulin-positive cells obtained from human ESCs (Fig. 1). It might thus be proposed that commitment to definitive endoderm should be integral to new protocols designed to obtain clinically useful insulin-producing cells from ESCs. Expression of insulin I and II should be used as markers at least in rodent cell lines, but appropriate markers for human cell lines must be further defined. Alternatively, based on the relative ease of generating neuroectoderm in vitro, strategies to increase the production of correctly processed insulin in cells of this origin should be considered (Fig. 2).

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Neuroectoderm ESCs -Oct3/4 -Nanog -Esg1

-Insulin II -NF-200 -GFAP -Nestin -Otx2

Primitive -Insulin II -Amnionless endoderm -Pthr1 Definitive -Insulin I -Cxcr4 endoderm -Glucagon

317 -Pdx1 -Isl1 -Pax6

-AFP -Foxa2 -Foxa3

Figure 1 Tentative scheme listing some markers detected by reverse transcriptase polymerase chain reaction (RT-PCR) that could allow distinguishing insulin-positive cells obtained in bioengineering protocols from embryonic stem cells (ESCs). Note that some markers are shared by different cell lineages. Markers for pluripotentiality of ESCs are listed as well. Source: From Refs. 39,70.

Figure 2 Strategies proposed to obtain insulin-producing cells from embryonic stem cells (ESCs). Late passages of ESCs (A) give rise to neuroectodermal insulin-positive cells (B) (39). However, these cells need to be manipulated in order to improve insulin production and correct hormone processing. (A) Transmission image of R1-ESCs passage 18. (A) and (B) images were captured at magnification X20 (Bars: 100 µm). Alternative strategies have to consider early passages of ESCs and embryoid bodies (EBs) commitment to definitive endoderm. Under these conditions different strategies could be adopted (see text for more details).

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IN VITRO DIFFERENTIATION PROTOCOLS A definitive protocol for in vitro differentiation to obtain insulin-secreting cells from ESCs or ASCs is still to be perfected. The various approaches tested so far by several laboratories can be classified into two categories: spontaneous differentiation combined with selection strategies and directed differentiation employing coaxial and reprogramming technology.

Spontaneous Differentiation Spontaneous differentiation combined with gating selection technology allows isolation of cell populations that specifically express the insulin gene (Fig. 2). To this end, particular DNA constructs are used in order to confer resistance to antibiotics under the control of b-cell specific promoters (49,50). This simple selection method has been applied to obtain other cell types, such as cardiomyocytes and neuronal-like cells (51–53). The strategy to obtain insulin-secreting cells from murine ESCs (49) consisted of a double-selection protocol based on the resistance to two antibiotics: hygromycin and neomycin. Expression of the hygromycin resistance gene under the control of the constitutive promoter of the phosphoglycerate kinase gene enabled selection of transfected single clonal cell lines. Expression of the neomycin resistance gene under the control of the regulatory regions of the insulin gene allowed selection of insulin-producing cells. The main disadvantage of this strategy is the inability to discriminate between ectodermal- and endodermal-derived insulin producing cells. One alternative would be the derivation of progenitor cells that could subsequently be differentiated to insulin-producing cells. Gating technology employing promoter elements of genes that are functionally active during intermediate stages of embryonic development provides an approach to specific derivation of islet precursor cells. In this sense, an alternative gating protocol has been developed (50) using the Nkx6.1 gene promoter controlling resistance to neomycin. Nkx6.1 is a transcription factor involved in the expansion of b-cell precursors during islet development (54). Selection with neomycin, together with specific culture conditions (presence of nicotinamide, anti-sonic hedgehog, and conditioned media from pancreatic buds), resulted in a pure population of murine cells coexpressing insulin, b-cell specific transcription factors, glucokinase, GLUT-2, and Sur-1. The obtained cells secreted insulin appropriately in response to increasing concentrations of extracellular glucose. Subsequent transplantation under the kidney capsule restored normoglycemia for 3 weeks in streptozocin-diabetic mice with reversion to hyperglycemia once the graft was removed. This new strategy offers still low but reproducible amounts of intracellular insulin content which will require further augmentation prior to any consideration of clinical transplantation trials.

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Directed Differentiation The directed differentiation approach includes both coaxial and cell reprogramming strategies (Fig. 2). Coaxial approaches are based on the design of specific culture media to drive ESCs or ASCs to insulin-positive cells. Cell reprogramming is dependent on introduction of specific proteins into the target cell either directly (cell extracts or purified proteins) or by using specific gene constructs. Coaxial Protocols A number of protocols have been developed based on the idea that nestinpositive cells could be precursors of neuronal and endocrine pancreatic cells (42). These in vitro differentiation protocols included incubation in ITSFn medium (DMEM/F12 containing insulin, transferrin, selenium, and fibronectin) and N2 medium [DMEM/F12 containing B27 supplement, insulin, transferrin, selenium, progesterone and putrescine supplemented with basic fibroblast growth factor (bFGF)]. N2 medium allowed expansion of nestin-positive cells in serum-free conditions. Withdrawal of bFGF from this medium and addition of nicotinamide in the last stage favored expression of the insulin gene (49). Insulin biosynthesis, however, was very low, and subsequent studies were performed in order to increase the number of insulin-positive cells and hormone content. Insulin content was modestly increased by addition of phosphoinositide-3-kinase inhibitors (LY294002 or wortmannin) or glucagon-like peptide-1 (GLP-1) in the last stage of the protocol (55,56). Addition of keratinocyte and epidermal growth factors (KGF and EGF, respectively) during the expansion phase of nestin-positive cells and inclusion of gating selection strategies also resulted in modest improvements (57,58). Interestingly, this nestin-selection strategy has been used for obtaining insulin-positive cells from human ESCs (59). The protocol employs culture medium containing low glucose concentration in the final incubation step. However, the co-expression of insulin with other pancreatic endocrine genes, such as glucagon and somatostatin, suggested that the final cells were still not fully differentiated (60). Although improvements to this protocol are necessary, the feasibility of using human ESCs to obtain insulin-producing cells has been confirmed, raising new hopes for a future diabetes therapy. Coaxial methodology has been applied to putative ASCs, including pancreatic duct derived insulin positive cells (24). Expansion, differentiation, and maturation of ductal cells was performed in DMEM/F12 serum-free medium containing ITS (Insulin þ Transferrin þ Selenium), KGF (to favor ductal tissue expansion), nicotinamide, 8 mM glucose, and MatrigelTM (a commercial, mouse-derived, growth-factor-rich, extracellular cell matrix that allows formation of CHIB structures; BD Biosciences, San Jose, California, U.S.A.) (25).

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ASCs from other organs, such as bone marrow or liver, have also been used in coaxial protocols to obtain insulin-producing cells (30,35,61–63). For instance, rat oval cells can be isolated and maintained undifferentiated in serum-free medium containing low glucose (5 mM) and LIF. Differentiation to insulin-containing structures can be performed by adding 10% serum, increasing glucose concentration (23 mM), and removing LIF from the culture medium. Coaxial approaches from mouse or human ESCs require rigorous application of optimized characterization criteria. In this context, insulin immunodetection is not a sufficient demonstration of endogenous hormone production. Notably, it has been reported that insulin can be sequestered from the culture medium into the cells (64), inhibiting at the same time de novo synthesis of the hormone (65). Exogenous insulin is present in serum and as a specific additive in the culture media used to select nestin-positive cells and to obtain CHIBs. This observation suggests that C-peptide matching the concentration of secreted insulin, together with the presence of insulin mRNA, should be used as more robust criteria to confirm intracellular insulin biosynthesis. Cell Reprogramming Certain proteins include membrane transduction domains that allow translocation across the plasma membrane and access to the intracellular compartment. Pdx1 posses an antennapedia-like domain that could be instrumental in reprogramming protocols. This domain was responsible for permeation of this transcription factor from the extracellular medium into pancreatic islets and ductal cells, inducing insulin gene expression (66). However, this domain is not present in all proteins and alternative permeabilization strategies would thus be required to transduce cells. Streptolysin O has been used to transiently permeabilize cellular membranes and thereby introduce protein extracts into target cells. The presence of key transcription factors and chromatin remodeling proteins in the extract may enable reprogramming by modulating the expression of a specific set of genes (67). Using this methodology, fetal rat fibroblasts have been reprogrammed to express the insulin gene following transduction with whole-cell extracts obtained from an insulinoma cell line (INS-1E) (68). These strategies therefore offer promise for induction of insulin gene expression in target cells without requiring DNA constructs overcoming the ethical issues posed by genetic manipulation. On the other hand, the use of specific gene constructs does enable constitutive or regulated expression of key proteins, such as transcription factors that are instrumental in b-cell function. Expression of Pax4 under the control of the constitutive CMV-promoter in transfected ESCs cultured following the nestin-selection strategy resulted in insulin expression (43).

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These DNA-based strategies have been used to transdifferentiate adult cells including hepatocytes. Expression of Pdx1 or NeuroD transgenes in hepatic cells resulted in induction of the insulin gene (36,37). In a similar way, human bone marrow mesenchymal stem cells were able to express insulin gene after infection with adenoviral vectors encoding key transcription factors in islet development (Pdx1, Hlxb9, and FoxA2) with subsequent incubation in islet-conditioned medium (69). CONCLUSIONS Although major further refinements are required in all protocols, a reasonable body of evidence confirms that stem cells could provide a potential source of transplantable tissue for replacement protocols in the treatment of Type 1 diabetes. The goal now has to be sharply focused on obtaining a final cell product that mimics as closely as possible the phenotype and function of mature b-cells in order to assure appropriate restoration of lost function in the recipient. Aside from insulin expression, this bioengineered cell will have to fulfill several criteria in order to accomplish a correct function in the transplanted organism. These can be classified according to three groups of proteins that work in coordination in mature pancreatic b-cells: 1. 2. 3.

The glucose-sensing machinery The regulated secretory pathway The insulin biosynthetic and processing apparatus

However, this work represents just the first part. The second step will be transplantation and this is likely to pose new challenges to scientists (7). Some of these are to: n n n

n n n

select appropriate animal models to test the function of bioengineered cells before human trials; establish the best site for implantation in order to assure implant survival, and nutrient/oxygen supply; solve the problem of immune rejection by making customized tissues through gene manipulation, nuclear transfer, and oocyte parthenogenesis; enhance implant survival by placing it in well irrigated sites and considering anti-apoptotic and anti-necrotic approaches; design biosafety strategies that allow elimination of the graft in case of non-function or tumor formation; and harness in vivo differentiation processes controlling phenotypic changes in situ.

In conclusion, we are beginning to decipher the potential of stem cells in diabetes therapy. There is still much to do before new bioengineered b-cells

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from either ASCs or ESCs will be created suitable for clinical trials. The emerging technology will help to achieve this goal, but we need to further investigate the basic biology of stem cells and the determinants that control self-renewal and differentiation in order to establish reliable protocols.

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18. Western PS, Surani MA. Nuclear reprogramming—alchemy or analysis? Nat Biotechnol 2002; 20:445–446. 19. Pomerantz J, Blau HM. Nuclear reprogramming:A key to stem cell function in regenerative medicine. Nat Cell Biol 2004; 9:810–816. 20. Smith AG. Embryo-derived stem cells: Of mice and men. Annu Rev Cell Develop Biol 2001; 17:435–462. 21. Fuchs E, Tumbar T, Guasch G. Socializing with the neighbors: Stem cells and their niche. Cell 2004; 116:769–778. 22. Wagers AJ, Weissman IL. Plasticity of adult stem cells. Cell 2004; 116: 639–648. 23. Potten CS, Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 1990; 110: 1001–1020. 24. Bonner-Weir S, Taneja M, Weir GC, et al. In vitro cultivation of human islets from expanded ductal tissue. Proc Natl Acad Sci USA 2000; 97:7999–8004. 25. Ramiya VK, Maraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JG. Reversal of insulin-dependent diabtes using islets generated in vitro from pancreatic stem cells. Nat Med 2000; 6:278–282. 26. Zulewski H, Abraham EJ, Gerlach MJ, et al. Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine and hepatic phenotypes. Diabetes 2001; 50:521–533. 27. Treutelaar MK, Skidmore JM, Dias-Leme CL, et al. Nestin-lineage cells contribute to the microvasculature but not endocrine cells of the islet. Diabetes 2003; 52:2503–2512. 28. Dor Y, Brown J, Martı´ nez OI, Melton DA. Adult pancreatic b-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004; 429:41–46. 29. Jiang Y, Jahagirdar SN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418:41–49. 30. Ianus A, Holz GG, Theise ND, Hussain MA. In vivo derivation of glucosecompetent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 2003; 111:843–850. 31. Choi JB, Uchino H, Azuma K, et al. Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic b-cells. Diabetologia 2003; 46: 1366–1374. 32. Lechner A, Yang Y-G, Blacken RA, Wang L, Nolan AL, Habener JF. No evidence for significant transdifferentiation of bone marrow into pancreatic b-cells in vivo. Diabetes 2004; 53:616–623. 33. Runhke M, Ungefroren H. Nussler A, et al. Reprogramming human peripheral blood monocytes into functional hepatocyte and pancreatic isletlike cells. Gastroenterology 2005; 128:1774–1786 34. Runhke M, Ungefroren H, Nussler A, et al. Reprogramming human peripheral blood monocytes into functional hepatocyte and pancreatic isletlike cells. Gastroenterology 2005; 128(7):1774–1786. 35. Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci USA 2002; 99:8078–8083.

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36. Ferber S, Halkin A, Cohen H, et al. Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocininduced hyperglycemia. Nat Med 2000; 6:568–572. 37. Kojima H, Fujimiya M, Matsumura K, et al. NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and reverses diabetes in mice. Nat Med 2003; 9:596–603. 38. Smith AG. Culture and differentiation of embryonic stem cells. J Tissue Cult Meth 1991; 13:89–94. 39. Roche E, Sepulcre P, Reig JA, Santana A, Soria B. Ectodermal commitment of insulin-producing cells derived from mouse embryonic stem cells. FASEB J 2005; 19:1341–1343. 40. Soria B, Skoudy A, Martı´ n F. From stem cells to b-cells: new strategies in cell therapy of diabetes mellitus. Diabetologia 2001; 44:407–415. 41. Shiroi A, Yoshikawa M, Yokota H, et al. Identification of insulin-producing cells derived from embryonic stem cells by zinc-chelating dithizone. Stem Cells 2002; 20:284–292. 42. Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, McKay R. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 2001; 292:1389–1394. 43. Blyszczuk P, Czyz J, Kania G, et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulinproducing cells. Proc Natl Acad Sci USA 2003; 100:998–1003. 44. Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, Tzukerman M. Insulin production by human embryonic stem cells. Diabetes 2001; 50:1691–1697. 45. Alpert S, Hanahan D, Teitelman G. Hybrid insulin genes reveal a developmental lineage for pancreatic endocrine cells and imply a relationship with neurons. Cell 1988; 53:295–308. 46. Melloul D, Marshak S, Cerasi E. Regulation of insulin gene transcription. Diabetologia 2002; 45:309–326. 47. Vicario-Abejo´n C, Yusta-Boyo MJ, Ferna´ndez-Moreno C, de Pablo F. Locally born olfactory bulb stem cells proliferate in response to insulin-related factors and require endogenous insulin-like growth factor-I for differentiation into neurons and glia. J Neurosci 2003; 23:895–906. 48. Herna´ndez-Sa´nchez C, Mansilla A, de la Rosa EJ, Pollerberg GE, Martı´ nezSalas E, de Pablo F. Upstream AUGs in embryonic proinsulin mRNA control its low translation level. EMBO J 2003; 22:5582–5592. 49. Soria B, Roche E, Berna G, Leo´n-Quinto T, Reig JA, Martı´ n, F. Insulinsecreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 2000; 49:157–162. 50. Leo´n-Quinto T, Jones J, Skoudy A, Burcin M, Soria B. In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells. Diabetologia 2004; 47:1442–1451. 51. Klug MG, Soonpa MH, Koh GY, Field LJ. Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 1996; 98:216–224. 52. Li M, Pevny L, Lovell-Badge R, Smith A. Generation of purified neural precursors from embryonic stem cells by lineage selection. Curr Biol 1998; 8:971–974.

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53. Mu¨ller M, Fleischmann BK, Selbert S, et al. Selection of ventricular-like cardiomyocytes from ES cells in vitro. FASEB J 2000; 14:2540–2548. 54. Chakrabarti SK, Mirmira RG. Transcription factors direct the development and function of pancreatic b-cells. Trends in Endocrinol Metab 2003; 14:78–84. 55. Hori Y, Rulifson IC, Tsai BC, Heit JJ, Cahoy JD, Kim SK. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells. Proc Natl Acad Sci USA 2002; 99:16105–16110. 56. Bai I, Meredith G, Tuch BE. Glucagon-like peptide enhances production of insulin in insulin-producing cells derived from mouse embryonic stem cells. J Endocrinol 2005; 186:343–352. 57. Moritoh Y, Yamato E, Yasui Y, Miyazaki S, Miyazaki J. Analysis of insulinproducing cells during in vitro differentiation from feeder-free embryonic stem cells. Diabetes 2003; 52:1163–1168. 58. Miyazaki S, Yamato E, Miyazaki J. Regulated expression of Pdx1 promotes in vitro differentiation of insulin-producing cells from embryonic stem cells. Diabetes 2004; 53:1030–1037. 59. Segev H, Fishman B, Ziskind A, Shulman M, Itskovitz-Eldor J. Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells 2004; 22:265–274. 60. Polak M, Bouchareb-Banaei L, Scharfmann R, Czernichow P. Early pattern of differentiation in the human pancreas. Diabetes 2000; 49:225–232. 61. Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002; 418:41–49. 62. Hess D, Li L, Martin M, et al. Bone marrow-derived stem cells initiate pancreatic regeneration. Nat Biotechnol 2003; 21:763–770. 63. Kim SK, Hebrok M. Intercellular signals regulating pancreas development and function. Gen Develop 2001; 15:11–127. 64. Rajagopal J, Anderson WJ, Kume S, Martinez OI, Melton DA. Insulin staining of ES cell progeny from insulin uptake. Science 2003; 299:363. 65. Paek H-J, Morgan J-R, Lysaght MJ. Sequestration and synthesis: The source of insulin in cell clusters differentiated from murine embryonic stem cells. Stem Cells 2005; 23:862–867. 66. Noguchi H, Kaneto H, Weir GC, Bonner-Weir S. Pdx1 protein containing its own antennapedia-like protein transduction domain can transducer pancreatic duct and islet cells. Diabetes 2003; 52:1732–1737. 67. Collas P, Ha˚kelien A-M. Teaching cells new tricks. Trends Biotechnol 2003; 21:354–361. 68. Ha˚kelien A-M, Gaustad KG, Collas P. Transient alteration of cell fate using a nuclear and cytoplasmic extract of an insulinoma cell line. Biochem Biophys Res Commun 2004; 316:834–841. 69. Moriscot C, De Fraipont F, Richard M-J, et al. Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro. Stem Cells 2005; 23:594–604. 70. Yasunaga M, Tada S, Torikai-Nishikawa S, et al. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nat Biotech 2005; 23:1542–1550.

19 Diabetes Gene Therapy Peter S. Chapman Matthew J. Ryan Veterinary Hospital, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A.

James A. M. Shaw Diabetes Research Group, Institute of Cellular Medicine, University of Newcastle, Newcastle upon Tyne, U.K.

UNDERLYING CONCEPTS AND POSSIBILITIES Gene therapy encompasses transfer of any prophylactic or therapeutic gene to human or animal cells resulting in subsequent expression in patients. Transfer of DNA can be achieved in situ by direct delivery to the host cells of the individual to be treated or performed ex vivo in cells that are subsequently transplanted. Cells or organs to be genetically manipulated prior to transplantation may be harvested and returned to the patient. Alternative sources include living or deceased human donors, animal donors, and proliferative transformed cell lines. Cell lines may be of embryonic or adult human or animal origin. The DNA sequence encoding the transgene can be incorporated into plasmid or viral vectors. In addition to employing naked plasmid DNA or a viral vector alone, approaches to achieve and enhance successful gene transfer in situ and ex vivo include plasmid complexation with a liposomal transfection agent or a wide range of other adjuvants in addition to electroporation. A wide range of targeting strategies in addition to natural tropisms associated with various viral vectors have been harnessed to attain a degree of selectivity in targeting delivery to specific organs following intravenous injection. Expression restricted to specific organs or cell types can be achieved by employing tissue-specific promoters to initiate and regulate transgene expression. These promoters may enable physiological regulation at the level of transcription. Alternatively, strong viral promoters may enable efficient constitutive expression regardless of cell type. Incompletely understood mechanisms that 327

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may have evolved as a defense against pathogenic viral expression in eukaryotic cells may, however, lead to downregulation of viral promoters [reviewed in (1)]. Promoters have been further engineered to enable pharmacological regulation of transcription following administration or withdrawal of a specific small molecule ligand (2). Regulators include tetracycline or related antibiotics, FK506 analogues, and progestogens. Each system has potential advantages and disadvantages but there has been little experience of any regulated gene therapy approach in humans to date. Viral Vectors The vast majority of clinical gene therapy trials to date (3) have been undertaken in individuals with malignant disease (67% of ongoing trials) and have employed viral vectors (70% of ongoing trials). Efficient gene delivery to a wide range of mammalian cells has been confirmed with viral vectors, but their inherent survival properties, including chromosomal insertion potentially at a site leading to oncogenic transformation, remain concerning. In addition, the mammalian immune system has evolved specific strategies to eliminate viral infection with associated risk of immune rejection and inflammatory response. The ubiquity of previous viral exposure in clinical subjects in contrast to experimental animals maintained in a specific pathogen-free environment may in particular lead to unpredicted responses. A further potential danger is re-establishment of replication competency in vivo and symptomatic viral infection. Successful gene transfer with first-generation retroviral vectors requires actively dividing cells and results in long-term expression due to insertion of the transgene into chromosomal DNA. Clinical success has been realized employing retroviral vectors to transduce host T cells in individuals with severe combined immune deficiency. Specific targeting of the vector to the promoter of an oncogene, LMO2, has, however, led to two cases of leukemic transformation in the first 10 individuals treated (4). More recently, HIV-derived lentiviral vectors have been developed. These retroviruses are able to target non-dividing cells due to an ability to traverse the intact nuclear membrane. There remain concerns regarding decreased expression over time and generation of infective wild-type HIV. Liver tumors have also been reported in pre-clinical trials (5). Adenoviral vectors can be used to transduce non-dividing, enddifferentiated cells. Risk of oncogenic transformation is low, as the vector is not incorporated into the host chromosome. A profound acute inflammatory response may be induced, however, particularly with first generation vectors. This can lead to particularly short-lived expression and has resulted in at least one death in a Phase 1 clinical study (6). Recombinant adenoassociated viral vectors have shown particular promise for successful clinical translation over the last 10 years. These are

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non-pathogenic and can mediate long-term transgene expression particularly following in vivo gene transfer. At least 20 clinical trials are underway or completed using adenoassociated viral vectors derived from AAV-2, the most common human serotype (7). These have confirmed favorable safety profile but level of expression and tissue selectivity may remain suboptimal. Neutralizing antibodies may reduce efficiency in individuals previously exposed to AAV Serotype 2. More recently, adenoassociated viral vectors have been derived from a range of other naturally occurring serotypes. AAV-1 and AAV-7 transduce skeletal muscle efficiently and AAV-8 has been employed to successfully transfer genes to liver (7). Efficient ex vivo transduction of human pancreatic islets prior to transplantation in rodent models has been confirmed using a double stranded AAV-2 vector without eliciting an immune response (8). Longterm expression in mice following in vivo administration of adenoassociated viral vectors has been confirmed following intraperitoneal and intravenous delivery. Direct cannulation and injection of the pancreatic exocrine duct with AAV-6 serotype-derived vectors appears particularly promising enabling transduction of virtually all islets (although not all cells within the core zone of each islet) minimizing viral load and extrapancreatic dissemination (9). Engineering of adenoassociated viral vectors is ongoing to further enhance tissue specificity and efficiency of gene transfer. Plasmid Vectors The excellent safety profile of plasmid DNA has led to its use in 17% of all clinical trials (3). Low efficiency of gene transfer has, however, been a major limitation. Long-term transgene expression can be achieved in skeletal muscle following simple percutaneous intramuscular plasmid injection. This approach is particularly appropriate for constitutive delivery of therapeutic proteins directly into the systemic circulation (2). Efficiency has been considerably enhanced by in situ electroporation or other adjuvant approaches so that therapeutically active circulating levels can now be achieved in large animal models of comparable weight to human patients (10). Therapeutically meaningful transfection efficiency has also been attained in liver following systemic or hepatic vein delivery of plasmid vector accompanied by hydrodynamic pressure to enhance uptake (11) or complexation with an adjuvant agent such as polyethyleneimine (PEI) (12). Plasmid-mediated gene transfer to pancreas has been attained following retrograde injection of the exocrine duct (13). Gene Therapy for Diabetes The possibilities for employing gene therapy approaches in the treatment of diabetes are seemingly limitless. Careful consideration must, however, be

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given to the potential risks and benefits in comparison to alternative conventional or novel interventions. Approaches considered include: 1. 2.

3. 4.

Prevention of disease progression and maintenance of b-cell function in Type 1 diabetes and islet transplant recipients Gene therapy approaches to insulin replacement, including derivation of conditionally transformed b-cell lines, b-cell transdifferentiation/ neogenesis, and insulin gene transfer to non-b-cells Gene transfer with non-insulin glucose-lowering genes, including insulin sensitizers and anti-obesity gene therapy Gene therapy targeted at diabetic complications

PREVENTION OF DISEASE PROGRESSION AND MAINTENANCE OF b-CELL FUNCTION IN TYPE 1 DIABETES AND ISLET TRANSPLANT RECIPIENTS Strategies for the prevention of Type 1 diabetes can be classified as: Primary prevention targeted at genetically predisposed high risk populations prior to serological evidence of established islet autoimmunity; Secondary prevention in those with islet specific antibodies evidencing established autoimmune b-cell destruction prior to clinical hyperglycemia; Tertiary prevention following presentation with diabetes aimed at attenuating or reversing further b-cell loss. Previous and current clinical Type 1 diabetes prevention trials have recently been reviewed (14). It is unlikely that gene therapy will have a role in primary prevention, as genetic screening in the absence of established disease will remain insufficient for absolute prediction of progression to diabetes given the need for an additional environmental trigger. Prior to the onset of overt clinical Type 1 diabetes, there is a long period of subclinical disease characterized by the presence of an autoimmune process specifically targeted at islet b-cells (15). Pre-clinical diagnosis of individuals with a very high likelihood of progression to overt disease is possible through presence of circulating antibodies to specific b-cell antigens, including insulin, glutamic acid decarboxylase, and the IA-2 phosphoprotein, in genetically predisposed individuals with or without evidence of subtle abnormalities in first phase insulin secretion in an intravenous glucose tolerance test. This provides a window for secondary prevention. Immunomodulation may also have a role following clinical disease onset when at least 10% of b-cell function remains, although studies in the NOD mouse model of autoimmune Type 1 diabetes have demonstrated the increased difficulty in reversing the autoimmune process once diabetes is established (16).

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The potential for recurrent b-cell autoimmunity in addition to alloimmune response following islet transplantation has been demonstrated in animal models and may play a role in loss of long-term graft function in clinical patients (17). Any successful immunoprophylactic strategy may have a further application in islet transplant recipients, and there are clearly specific opportunities for ex vivo gene transfer to islets pre-transplantation.

Antigen-Specific Tolerization Approaches There is increasing evidence for incomplete thymic deletion of islet b-cell specific T-cell precursors and defective peripheral tolerance leading to T-cell expansion, differentiation and initiation of a Type 1 immune process (18). Gene therapy approaches mediating autoantigen expression in the thymus; by circulating antigen presenting cells; or systemically may restore immune tolerance. Transgenic expression of a proinsulin cDNA under the control of the MHC Class II promoter resulted in intrathymic expression and prevention of diabetes onset in NOD mice (19). Although gene transfer to thymus following direct injection has been attained in animal models with the potential for tolerance induction (20), further studies are required to explore feasibility, safety, and efficacy of this approach for clinical translation. Injection of hematopoietic stem cells derived from the transgenic MHC promoter-driven proinsulin expressing cells resulted in prevention of spontaneous autoimmune diabetes in NOD mice (21). This provides initial proof of principle for ex vivo gene transfer to autologous cells derived from peripheral blood with subsequent reinjection. However, methods for lowrisk, efficient, and stable gene transfer to hematopoietic cells require further refinement. Moreover, the published studies required recipient bone marrow ablation, clearly unacceptable for individuals with early Type 1 diabetes or following islet transplantation. Islet autoantigens can also be expressed in muscle following direct intramuscular injection of naked plasmid DNA with uptake enhanced by in situ electroporation. Prevention of diabetes in NOD mice has been reported employing plasmids encoding the immunodominant epitope of insulin (a portion of the B chain) (22) or glutamic acid decarboxylase downstream of a signal peptide sequence mediating systemic secretion (23).

Antigen-Independent Approaches Antigen-independent gene therapy approaches to attenuating the initial autoimmune diabetogenic process or preventing recurrence in transplanted islets include expression of immunoregulatory cytokines including IL-10, IL-4, and TGF-b following in situ gene transfer to muscle or directly to

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pancreas [reviewed by Giannoukakis et al. (18)]. Alternatively autologous dendritic cells or donor islets can be engineered to express these factors ex vivo prior to transplantation (24). Direct transduction of islets to prevent antigen-presenting cell activation is an alternative approach. Examples include expression of IL-1 receptor antagonist gene to prevent IL1b b-cell toxicity and APC activation (25) and expression of a cytotoxic T-lymphocyte-associated protein 4 gene to block the second signal necessary for T-cell activation by APCs (26). Downregulation of CD40, CD80, and CD86 co-stimulatory molecule expression in harvested dendritic cells by antisense oligodeoxyribonucleotides with reimplantation by intradermal injection has recently been approved by the FDA as a Phase 1 clinical trial in individuals with Type 1 diabetes (27). An approach particularly being explored for xenograft transplantation is modification of islet cellular antigens to prevent recognition by the host immune system. Transgenic pigs have been engineered in which the alphagalactosidase transferase gene has been knocked out (28). This prevents expression of α-galactosidase, a trigger of hyperacute rejection, on the endothelial cell surface of transplanted islets. Transgenic pigs (29) and porcine islets in vitro (30) have also been engineered to express the human complementary regulatory protein to attenuate complement activation. Successful transplantation in non-human primate models of diabetes without immune rejection has been reported.

Attenuation of Apoptosis The continued presence of b-cells undergoing apoptosis even after 50 years of Type 1 diabetes (31) has highlighted the potential importance of novel anti-apoptotic therapies in preventing or reversing Type 1 diabetes and preventing loss of islet graft function over time. There is huge scope for gene therapy to have a role through in situ gene transfer to pancreas or ex vivo gene transfer to transplanted islets. Potential anti-apoptotic genes include Bcl-2, heat shock proteins, and an inhibitor of Caspase-8 activation (I-FLICE). Gene transfer strategies for cytoprotection of b-cells have recently been reviewed (32).

Enhanced Islet Engraftment It appears that less than a third of potential b-cell function is achieved following clinical islet transplantation due to poor engraftment and specifically inadequate early revascularization (33). Enhanced graft angiogenesis, insulin staining intensity, and function following transplantation under the renal capsule in immunodeficient mice with streptozocin-induced diabetes

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has been demonstrated following ex vivo vascular endothelial growth factor gene transfer to murine islets employing an adenoviral vector (34).

Augmentation of b-Cell Function Within Intact Islets Gene therapy may also be employed to directly enhance b-cell mass and function in transplanted islets. Hepatocyte growth factor has been shown to induce b-cell proliferation in culture, to increase insulin secretion, and to prolong survival in transplant models. Adenoviral hepatocyte growth factor gene transfer to rat islets improved transplant outcomes following transplantation into the portal vein of an allogeneic rat strain with streptozocin-induced diabetes using daclizumab, tacrolimus, and sirolimus immunosuppression to mimic the Edmonton Protocol (35). There are a wide range of other factors with proven potential for enhancing b-cell mass and/ or function that could be delivered to isolated islets prior to transplantation employing viral vectors or non-viral transfection. These include growth factors such as epidermal growth factor, gastrin, and IGF-1 (36); key b-cell transcription factors such as PDX1 (37) and PAX4 (38); and the incretin hormone GLP-1 (39) or one of its analogues/homologues. In situ gene transfer to pancreas following endoscopic retrograde cannulation of the pancreatic duct with one or more of these factors may ultimately have a role in restoration of b-cell function in non-transplant recipients with Type 1 diabetes (40).

GENE THERAPY APPROACHES TO INSULIN REPLACEMENT It has been made clear throughout this book that severe limitations in the number of suitable donor organs will restrict availability of islet transplantation to a small percentage of those with Type 1 diabetes. It is hoped that approaches to prevent b-cell loss or restore endogenous insulin secretion will ultimately preclude the need for transplantation altogether. Augmentation of islet function, enhancement of engraftment, and prevention of immune rejection employing one or more of the approaches described in this and other chapters may enable consistent long-term insulin independence from a single donor organ. Further approaches to generate tissue banks of insulin-secreting cells sufficient for all suitable recipients with diabetes are, however, required. Detailed phenotyping of newly derived cells beyond expression of the insulin gene and positive insulin immunocytochemical staining will be imperative before any consideration of clinical trials (41). Physiological insulin expression, biosynthesis, processing, and secretion by the normal b-cell is briefly outlined below as a benchmark for comparison with genetic engineering approaches to insulin replacement. Some key components of a normal functional b-cell are illustrated in Figure 1.

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Glucose

GK

Metabolism

Glucose

G-6-P

SAPK2

K+ ATP:ADP + + + +

P Cytoplasm

PDX-1

Membrane depolarisation

Nucleus Insulin granules

2+

Ca

Ca-gated insulin secretion

P insulin mRNA

Constitutive secretory pathway

Glucose-responsive insulin gene expression Endoplasmic reticulum

Golgi apparatus

Secretory vesicles

Figure 1 The physiological b-cell. Key components of a normally functional pancreatic b-cell are highlighted, including GLUT2 and GK expression constituting the “glucose sensor,” metabolism-induced PDX1 translocation to the nucleus and binding to the insulin gene promoter to mediate “glucose-responsive insulin gene expression,” and closure of the ATP-dependent potassium channel leading to membrane depolarization and calcium influx mediating “Ca-gated insulin secretion” from storage granules within the regulated secretory pathway. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate; GK, glucokinase; GLUT2, glucose transporter 2.

Physiological Glucose-Responsive Insulin Gene Expression Insulin synthesis in the b-cell is dependent on transcription and translation of the human insulin gene located on the short arm of chromosome 11. Regulation of insulin gene transcription is dependent on promoter sequences located upstream of the transcriptional start site and the interaction of these discrete elements with a number of b-cell transcription factors. Key cis-acting elements include conserved C, E, and A elements in addition to the Z region in the human gene (42). Glucose uptake into the b-cell is facilitated by the high Km glucose transporter (GLUT2). This allows rapid equilibration of intracellular with circulating glucose levels. Glucose is then phosphorylated by glucokinase, which also has a high Km (11mmol/L) and is the key enzyme regulating flux through the glycolytic pathway (43). Glucose phosphorylation in response to raised glucose within the b-cell mediates PI3K and SAPK2 phosphorylation. This results in PDX1 modification including phosphorylation by activated PDX-1-kinase (44), enabling translocation of PDX-1 to the nucleus where it binds the four ‘A-boxes’ of the insulin gene promoter directly upregulating gene

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transcription (45). Insulin biosynthesis is further regulated at the level of translation, which is undoubtedly glucose-responsive in the physiological b-cell, although the underlying mechanisms remain incompletely understood (46). Physiological Proinsulin Biosynthesis and Processing Insulin is synthesized as a larger precursor, preproinsulin. The N-terminal signal peptide sequence targets newly synthesized proinsulin to the endoplasmic reticulum through a ubiquitous mechanism involving prepeptide cleavage by signal peptidase (47). Proinsulin is transferred through the trans-Golgi network where it is packaged into immature clathrincoated secretory granules that develop into insulin storage granules (48). The b-cell in common with other neuro-endocrine cells contains a regulated secretory pathway in addition to the ubiquitous constitutive pathway for protein secretion (49). Greater than 99% of newly synthesized proinsulin enters the regulated secretory pathway where two specific endoproteases PC2 and PC3 are uniquely expressed (50). These are responsible for posttranslational processing to mature insulin, which is completed by removal of two basic arginine residues by ubiquitously expressed carboxypeptidase E (51). Physiological Insulin Secretion Physiological glucose homeostasis is tightly controlled predominantly by minute-to-minute variation in b-cell secretion of pre-formed insulin via the regulated secretory pathway. GLUT2-mediated glucose uptake and glucokinase-mediated phosphorylation regulates flux through the glycolytic pathway. Glucose metabolism increases the adenosine triphosphate (ATP)/ adenosine diphosphate (ADP) ratio, leading to closure of ATP-dependent potassium channels and membrane depolarization, opening voltage-gated calcium channels, which, in turn, stimulates exocytosis of pre-formed insulin storage granules (52). Derivation of Proliferative b-Cells The limited capacity for b-cell proliferation within islets or as a single phenotype in cell culture has led to generation of a number of transformed b-cell lines. Sources include a radiation-induced rat insulinoma (RinM5F) (53), isolated hamster islets transformed with SV40 virus, and b-cell tumors induced in transgenic mice by SV40 large T-antigen expression (bTC and MIN6). Formation of proliferative cell lines is, however, associated with dedifferentiation altering the underlying phenotype. Insulin content may be

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decreased. Expression of the specific glucose transporter (GLUT2) and glucose phosphorylating enzyme (glucokinase), which together constitute the glucose sensor, tend to decrease with concomitant increase in more ubiquitous GLUT1 and hexokinase expression (54). This results in induction of insulin secretion at sub-physiological glucose concentrations with potential for dangerous hypoglycemia. There have been extensive efforts to iteratively engineer these cell lines to stably express human insulin, GLUT2 and glucokinase (55). Further manipulation would, however, be required to ablate expression of endogenous animal insulin, hexokinase, and GLUT1, and this approach has been largely superseded in recent years. Generation of human b-cell lines from insulinoma tissue or by oncogenic transformation of human islets has proved much more challenging. Cells with at least limited capacity to replicate in culture have been derived from patients with persistent hyperinsulinemic hypoglycemia of infancy, which is characterized by inappropriate constitutive insulin secretion and often treated by pancreatic resection. Stable transfection of one PHHI-derived cell line with the two components of the dysfunctional KATP channel (SUR-1 and Kir6.2) in addition to the key b-cell transcription factor PDX1 resulted in restoration of insulin secretion in response to changes in glucose concentration within the physiological range (56). Unregulated tumorigenic proliferation is a further hurdle to clinical transplantation of b-cell lines. Efrat and colleagues, however, pioneered a conditional b-cell immortalization approach in which transgenic mice expressing SV40 large T antigen under the control of a tetracyclineresponsive promoter were crossed with transgenic mice expressing a tetracycline-repressible transcriptional repressor downstream of the rat insulin promoter (57). Derived b-cells (designated bTCtet cells) proliferated well in vitro and led to normalization of hyperglycemia following intraperitoneal injection in mice with streptozocin-induced diabetes. Continued proliferation in vivo led to fatal hypoglycemia. Implantation together with a slow release tetracycline pellet attenuated further proliferation, with maintenance of euglycemia for up to 4 months. Proliferative b-cells have been successfully derived from human islets by over-expression of dominant oncogenes but this has been associated with dedifferentiation (58). Reversible immortalization of human islet-derived b-cells has been reported following transfection with lox-P flanked SV40 large T-antigen and human telomerase reverse transcriptase cDNAs (59). Non-tumorigenic clones were selected and infected with an adenoviral crerecombinase vector enabling removal of immortalization genes. Characterization following formation of three-dimensional cell aggregates in vitro confirmed b-cell specific gene expression, insulin storage within secretory granules with insulin content 40% of control islets, and glucose/ alternative secretagogue-stimulated insulin secretion. Sub-renal capsule

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transplantation in mice with steptozocin-induced diabetes normalized glucose levels for 30 weeks with no evidence of oncogene expression or tumor formation. Transdifferentiation/Neogenesis Approaches The potential for generating new end-differentiated non-tumorigenic b-cells following proliferation, differentiation, and selection of embryonic or adult stem cells is considered in detail in Chapter 18. Transdifferentiation approaches may ultimately enable derivation of fully functional b-cells from hepatocytes or non-endocrine pancreatic cells in situ or ex vivo as explored in Chapter 17. Neogenesis and transdifferentiation gene therapy strategies have included transduction with fluorescent marker or antibiotic resistance genes under the control of b-cell specific promoters to enable cell selection and sorting, in addition to transfection with key b-cell transcription factors (PDX1) (60), NGN-3 (61), MafA (62), and differentiation factors (GLP1). Insulin Gene Transfer to Non-b-Cells The ultimate goal of physiological glucose responsive insulin secretion may not be attainable by genetic manipulation of non-b-cells, given the highly specialized mechanism by which insulin gene transcription and secretion of fully-processed insulin is regulated by glucose uptake and metabolism in the normal pancreatic b-cell. However, the potential for long-term insulin replacement following in situ gene delivery without the need for transplantation and avoiding both alloimmune and autoimmune rejection merits further study. This may provide a cost-effective therapy with sufficient utility for the growing millions of individuals currently reliant on insulin injections, many of whom have sub-optimal control with associated risk of long-term complications and severe hypoglycemia. Ex Vivo Insulin Gene Transfer to Neuro-Endocrine Cells Neuro-endocrine cells express the regulated secretory pathway, together with the specific endoproteases, PC2 and PC3, necessary for cleavage of proinsulin to bioactive insulin. Transfection of the mouse pituitary corticotrophin cell line (AtT20) with an insulin gene construct resulted in secretion of fully processed insulin via the regulated secretory pathway, induced by membrane depolarization in the presence of calcium (63). Secretion was not glucose-responsive despite endogenous glucokinase expression. Cotransfection with GLUT2 resulted in insulin secretion in response to glucose, but in the sub-physiological range with maximum stimulation

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occurring at glucose concentration of 10 µmol/L (64). Intraperitoneal injection of insulin over-expressing AtT20 cells delayed onset of hyperglycemia induced by streptozocin by 2 weeks (65,66). Subsequent hyperglycemia may have been due to insulin resistance caused by co-secretion of ACTH. Downregulation of diabetogenic hormone expression would thus be necessary before clinical use of ex vivo engineered neuro-endocrine cells could be considered.

In Situ Insulin Gene Transfer to Neuro-Endocrine Cells K cells are endocrine cells located in the stomach, duodenum, and jejunum of mammals. They synthesize the hormone glucose-dependent insulinotropic polypeptide (GIP), which is produced and secreted in response to food ingestion in a similar way to regulation of b-cell insulin secretion according to changes in blood glucose. GIP is a potent stimulator of insulin secretion (67). K cells express PC2 and PC3 within the regulated secretory pathway in addition to glucokinase but not GLUT2 (68). The GIP promoter has been sub-cloned upstream of a human proinsulin construct and employed to stably transfect GTC-1 cells, a GIP expressing K-cell line. These cells synthesized, stored, and secreted mature insulin in response to nutrient stimulation (68,69). Transgenic expression of this construct in mice resulted in K-cell–specific insulin expression and protection from hyperglycemia induced by treatment with streptozocin in comparison to control animals (69). Meal-stimulated secretion of mature processed insulin has also been achieved in transgenic mice in which the human insulin gene has been expressed in gastric G cells under the control of the gastrin promoter (70). Specific and safe targeted gene transfer to K or G cells in vivo, possibly following oral or endoscopic delivery would, however, be required for successful clinical translation (71).

Insulin Gene Transfer to Non-Endocrine Cells Non-endocrine cells lack the regulated pathway necessary for minuteto-minute insulin storage and secretion in addition to proinsulin processing. Insulin gene transfer would thus be expected to mediate constitutive secretion of unmodified proinsulin with less than 20% of the biological activity of insulin. Several groups have engineered mutant preproinsulin constructs in which PC2 and PC3 cleavage sites at A-chain/C-peptide and B-chain/ C-peptide junctions have been altered to form tetrabasic consensus sites enabling recognition and cleavage by furin, a protease expressed ubiquitously in the trans-Golgi network of eukaryotic cells (72).

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An alternative approach has been explored in which insulin A- and B-chains were expressed linked by a peptide much shorter than the 35 residue C-peptide, a short-turn–forming heptapeptide (Gly-Gly-Gly-ProGly-Lys-Arg). The recombinant modified insulin showed much higher biological activity than proinsulin, but lower activity than human insulin, both with regard to receptor binding and glucose uptake activity, without requiring enzymatic processing (73). Transfection of the human insulin gene into a monkey kidney fibroblast cell line resulted in the expression of proinsulin but not mature, active insulin, due to the lack of PC2 and PC3 protease (74). Transfection of murine NIH 3T3 fibroblasts with a retrovirus expressing a furin-cleavable human preproinsulin gene enabled secretion of fully-processed biologically active insulin (75). In vivo implantation studies have, however, been complicated by hypoglycemia due to unregulated cell proliferation with increasing insulin secretion.

Hepatic Insulin Gene Therapy Insulin gene therapy directed to liver has several potential advantages. Hepatocytes share a glucose-sensor comparable to that in pancreatic b-cells expressing both GLUT2 and glucokinase (76). Transfection of host hepatocytes in situ can be achieved following simple intravenous injection of viral vectors or plasmid DNA pre-complexed with PEI or alternative adjuvant agent (76). Host cell transduction following injection of pharmaceutical grade vector has particular utility and cost-effectiveness for widespread clinical utilization. It avoids the need for cell culture and transplantation in addition to circumventing alloimmune rejection. Uptake and incorporation into a stable end-differentiated tissue also prevents tumorigenic proliferation with associated risk of hypoglycemia. Hepatocytes lack the regulated secretory pathway for regulated secretion of pre-formed and fully processed insulin but do benefit from endogenous genes, which are physiologically upregulated by glucose and downregulated by insulin. This offers the potential for glucose-responsive transcriptional regulation by cloning the insulin transgene downstream of regulatable promoters, including those derived from L-type pyruvate kinase, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and GLUT2. A number of groups have explored this approach in vitro and in vivo employing furin-cleavable or single chain insulin (73) transgenes (77). The intrinsic time delay required for insulin gene transcription and translation in response to elevated glucose leads to potential for persistent hyperglycemia early after a meal with later risk of hypoglycemia. The most physiological results have been attained employing three copies of the L-PK promoter glucose-responsive element in combination with an inhibitory element

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from the insulin-like growth factor–binding protein gene in an adenoviral vector delivered to the portal vein of rats with streptozocin-induced diabetes (78).

Muscle-Targeted Insulin Gene Therapy Constitutive secretion of fully processed human insulin for up to 5 weeks has been obtained following simple injection of naked plasmid DNA encoding a furin-cleavable insulin construct into accessible skeletal muscle in rodents (79). Efficiency has been enhanced by use of in situ electroporation (80). Glucose lowering without dangerous hypoglycemia has been confirmed in mice with streptozocin-induced diabetes (81,82). Transcriptional regulation of proinsulin secretion has been confirmed following oral tetracycline administration, offering the potential for regulating basal constitutive insulin replacement (83). The promoter of the gene encoding rat transforming growth factor-b expressed upstream of a furin gene construct has been used to achieve glucose-regulated processing of transgenically expressed furin-cleavable human proinsulin in smooth muscle cells transplanted into diabetic BB rats (84). An elegant approach to storage and rapid ligand-mediated secretion of pre-formed insulin in non-endocrine cells has been developed (85). This is dependent on conditional aggregation within endoplasmic reticulum mediated by mutated FK506 binding protein domains expressed upstream of a furin cleavage site and the insulin transgene. Administration of a nonimmunosuppressive rapamycin analogue results in disaggregation and protein delivery to the trans-Golgi network, where furin cleaves off the FK506 domains and processes proinsulin secreted by the constitutive secretory pathway. Pharmacokinetics closely mimicking physiological postprandial insulin secretion have been demonstrated in transfected fused human muscle fibers in vitro (2).

GENE TRANSFER WITH NON-INSULIN GLUCOSE-LOWERING GENES Insulin Sensitizers Enhancement of insulin action by over-expression of genes that increase glucose uptake and storage by liver and muscle may provide new therapeutic options for insulin-resistant Type 2 diabetes. Over-expression of hepatic glucokinase has been shown to normalize fasting blood glucose in rodent models of insulin-deficient (86) and insulin-resistant (87) diabetes. The potential for excessive glycogen and fat accumulation in liver in addition to an increase in circulating free fatty acids and triglycerides following unregulated glucokinase over-expression is, however, a concern (88).

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A second potential target in liver is the glycogen-targeting subunits of protein phosphatase-1. These proteins target protein phosphatase-1 to the glycogen particle and bind differentially to glycogen synthase, glycogen phosphorylase, and phosphorylase kinase, serving as molecular scaffolds (89). A prominent member of this family is “protein targeting to glycogen” (PTG), which is expressed in a wide range of tissues (90). It has been demonstrated that glucose can be diverted to glycogen in normal rodents by the over-expression of PTG (91). A novel subunit, GM∆C, has been developed with a unique combination of glycogenic potency and responsiveness to glycogenolytic signals (92). Adenoviral hepatic over-expression of GM∆C prevented glucose intolerance in rats fed a high fat diet. Transgenic mice engineered to express liver glucokinase in the muscle demonstrated increased insulin sensitivity (93) and did not develop obesity, insulin resistance, or hyperglycemia on being fed a high fat diet (94). Transplantation of glucokinase-expressing myotubes attenuated hyperglycemia in mice with streptozocin-induced diabetes (95). In situ adenoviral glucokinase vector delivery to muscle also increased glucose uptake (96). Although this improved insulin sensitivity in obese rats, it did not prevent or delay the appearance of hyperglycemia and hyperinsulinemia (97). Transgenic mice over-expressing the muscle glucose transporter GLUT4 demonstrated improved glucose tolerance, which was not further enhanced by concomitant over-expression of hexokinase II (98). Most recently, intramuscular injection of streptozotocin into diabetic mice with adenoassociated viral vectors constitutively expressing insulin and hepatic glucokinase resulted in restoration of fasting and fed normoglycemia for more than four months (99). It is envisaged, however, that successful clinical translation would necessitate glucokinase gene transfer to a major proportion of total skeletal muscle to sufficiently enhance whole body glucose uptake.

Anti-Obesity Gene Therapy Increasing understanding of the neuro-endocrine modulators of appetite and weight has uncovered a wide range of novel targets for gene therapy, offering the potential for weight loss in obesity with enhanced insulin sensitivity and improved glucose tolerance in Type 2 diabetes. Decreased food intake and a reduction in body weight with lower insulin levels and maintained normoglycemia has been attained in non-diabetic mice following electroporation-enhanced plasmid-mediated leptin gene transfer to muscle (100). The leptin gene has also been transferred directly to brain by intracerebroventricular injection of an adenoassociated viral vector in an attempt to circumvent systemic pleiotropic effects of leptin [reviewed in (101)]. Normalization of insulin resistance with improved glucose tolerance

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and decreased levels of circulating fatty acids has also been demonstrated in mice with diet-induced obesity after muscle-targeted gene transfer of an adiponectin gene (102).

GENE THERAPY TARGETED AT DIABETIC COMPLICATIONS Finally, there are many potential gene therapy applications targeted at the long-term complications of diabetes that are beyond the scope of this chapter. Examples include pro-angiogenic therapy for atherosclerotic vascular disease (103). Clinical trials have recently been undertaken in which VEGF expression vectors were delivered intramuscularly to individuals with diabetes complicated by critical limb ischemia (104). In another trial, an adenoviral VEGF construct was injected directly into the myocardium of patients with severe angina (105). Results of these trials remain preliminary but safety and initial outcome data are encouraging. Gene therapy approaches for diabetic retinopathy directly targeted to the eye within the blood-retinal barrier have recently been reviewed (106). As a circulating therapeutic protein, erythropoietin is an attractive candidate for replacement following muscle-targeted gene delivery (107). This may ultimately have a role in treatment of anemia secondary to diabetic nephropathy. Similarly, muscle-targeted gene transfer of neurotrophic or neuroprotective factors in addition to delivery to the dorsal root ganglion employing herpes simplex virus-derived vectors is being explored (108).

CONCLUSION In recent years, it has often been stated that gene therapy has failed to realize its initial promise and is in many ways being superseded by alternative approaches including stem cell–derived transplantation. On the contrary, clinical success with a wide range of gene therapy approaches is accruing, and there have been dramatic recent strides in gene delivery avoiding the toxicity of early generation vectors. There is huge potential for gene transfer approaches to complement islet transplantation and b-cell replacement therapy following ex vivo transduction prior to implantation. There is also considerable as yet untapped future potential for in situ delivery of genes to individuals with diabetes without the need for transplantation or immunosuppression. Therapeutic genes may prevent diabetes progression, maintain b-cell function, enable replacement of circulating insulin and noninsulin glucose lowering peptides, or directly target long-term complications. Moreover, these approaches offer sufficient utility for cost-effective implementation in the huge number who may benefit globally with Type 1 and potentially also Type 2 diabetes.

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97. Jimenez-Chillaron JC, Telemaque-Potts S, Gomez-Valades AicG, Anderson P, Newgard CB, Gomez-Foix AM. Glucokinase gene transfer to skeletal muscle of diabetic Zucker fatty rats improves insulin-sensitive glucose uptake. Metabolism 2002; 51(1):121–6. 98. Lombardi A, Moller D, Loizeau M, Girard J, Leturque A. Phenotype of transgenic mice overexpressing GLUT4 and hexokinase II in muscle. FASEB J 1997; 11(13):1137–44. 99. Mas A, Montane J, Anguela XM, et al. Reversal of Type 1 Diabetes by Engineering a Glucose Sensor in Skeletal Muscle 10.2337/db05-1615. Diabetes 2006; 55(6):1546–53. 100. Wang XD, Liu J, Yang JC, Chen WQ, Tang JG. Mice body weight gain is prevented after naked human leptin cDNA transfer into skeletal muscle by electroporation. J Gene Med 2003; 5(11):966–76. 101. Kalra SP, Kalra PS. Gene-transfer technology: a preventive neurotherapy to curb obesity, ameliorate metabolic syndrome and extend life expectancy. Trends in Pharmacological Sciences 2005; 26(10):488–95. 102. Park JH, Lee M, Kim SW. Non-viral adiponectin gene therapy into obese type 2 diabetic mice ameliorates insulin resistance. Journal of Controlled Release 2006; 114(1):118–25. 103. Yla-Herttuala S. An update on angiogenic gene therapy: vascular endothelial growth factor and other directions. Curr Opin Mol Ther 2006; 8(4):295–300. 104. Kusumanto YH, van Weel V, Mulder NH, et al. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial. Hum Gene Ther 2006; 17(6):683–91. 105. Stewart DJ, Hilton JD, Arnold JM, et al. Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121) (AdVEGF121) versus maximum medical treatment. Gene Ther 2006. 106. Ting JHY, Martin DK. Basic and Clinical Aspects of Gene Therapy for Retinopathy Induced by Diabetes. In: Current Gene Therapy:193–214. 107. Prudhomme GJ, Glinka Y, Khan AS, Draghia-Akli R. ElectroporationEnhanced Nonviral Gene Transfer for the Prevention or Treatment of Immunological, Endocrine and Neoplastic Diseases. In: Current Gene Therapy:243–73. 108. Mata M, Chattopadhyay M, Fink DJ. Gene therapy for the treatment of sensory neuropathy. Expert Opin Biol Ther 2006; 6(5):499–507.

Index

Absolute neutrophil count (ANC), 165 Acetaminophen, 274 Addison’s disease, 67 Adenosine diphosphate (ADP), 255 Adenosine triphosphate (ATP), 32, 83, 100–102, 255 Adjuvant therapies, 160 Adult stem cells (ASCs), 312, 313–317 extrapancreatic stem cells, 314–317 pancreatic stem cells, 313–314 Alanine-amino transferase (ALT), 163 Alemtuzumab (Campath 1H), 153–154 dose, 154 mechanism of action, 153–154 side effects, 154 Allografts, islet after pancreatectomy, 11 fetal islet allografts in, 14 rejection, 2 in Type 1 diabetes, 11–13 Alloislet transplantation, 197–199 Alpha cell function, 198 Anemia, 163 Angiogenesis, 5 Angiotensin converting enzyme inhibitors (ACEIs), 170 Anticoagulation, 159 Antigen-independent approaches, for diabetes, 331–332 Antigen-specific tolerization approaches, for diabetes, 331 Anti-insulins, 33 Anti-obesity gene therapy, 341–342 Antiplatelet agents, 66 Aphthous ulcers, 166

Apoptosis, attenuation of, 332 Arginine stimulation test, 182–183 Aspartate-amino transferase (AST), 163 Aspects and challenges of islet isolation, 115–129 challenges in islet assessment, 124–126, See also under Islet assessment islet culture, 126–128: vitamins on islets, 127–128 islet purification, 122–124 pancreas digestion, 120–122: low enzymatic activity, 121 donor selection, 118–120 donor variables assessment form, 119 pancreas physical properties assessment form, 119 Assessment, islet challenges in, 124–126 islet viability, 126 islet yield, 124–126 ATG (Thymoglobulin), 152–153 Atovaquone (Mepron), 160 Autografts, islet, 10–11 Autoimmune disease, 67 Autoimmune recurrence, 12 Autoislet recipients, metabolic studies, 195–197 Avitene, 240 Azathioprine, 277–278

Beta (b) cells Beta score in islet graft function assessment, 181

351

352 [Beta (b) cells] function in Type 1 diabetes, maintenance, 330–333 regeneration and neogenesis, 293–305, See also separate entry: enhancing, 303–305 GLP-1, 301–303 pancreatic b-cell mass, regulation of, 294–296 regulatory mechanism of, 32–33 replacement, stem cell approaches for, 311–321, See also Stem cell approaches Blood glucose concentration and insulin secretion, link between, 32 Body mass index (BMI), 84, 168, 295 islet isolation and, 118–119 Bromocriptine, 162

Calcineurin inhibitor therapy, 271 California Transplant Donor Network (CADN), 222 Carbamazepine, 162 Cardiovascular disease and risk factors, 66 antiplatelet agents, 66 Care of islet transplant recipient, 147–174, See also Immunosuppressive management and Complications Carrel patch, 91 Catheter placement, 136 Cell reprogramming, 320–321 Celsior solution, 82, 99, 109 Centers for Medicare and Medicaid Services (CMS), 284 Ceropithecus aethiops, 285 Challenges in new islet transplant program setting, 203–213 Associated Research Program, 212–213 clinical islet transplantation, 209–212 ethical approval, 210 islet transplant procedure, 211–212 multidisciplinary islet transplant team, establishing, 209–210 patient recruitment, 210–211 experimental context, 203–204 islet isolation, 206–209 clean room facilities requirement, 207 personnel requirement, 209

Index [Challenges in new islet transplant program setting islet isolation] technical challenges, 207 pancreas procurement, 204–206 Cimetidine, 162 Cisapride, 162 Clarithromycin, 162 Clinical islet transplantation (CIT), 57–64, 258 challenges in, 209–212, See also under Challenges clinical assessment of individuals seeking, 73–74 contraindications to, 61–62 age, 61 alcohol, 62 drugs, 62 infection and neoplasia, 62 obesity and insulin resistance, 61 psychiatric disease, cognitive impairment, & non-compliance, 62 smoking, 62 current outcomes of, 229–244, See also individual entry goals of, 58 indications for, 58–61 glycemic lability, 60 hypoglycemia and hypoglycemia unawareness, 59–60 Type 1 diabetes, 59 modern immunosuppression for, 274–275, See also under Contemporary immunosuppression unrealistic expectations and misconceptions, 69–70 Clinical trials setting in islet cell transplantation, factors to consider, 215–227 center infrastructure, 216–218 budget, 217–218 personnel, 216–217: clinical personnel, 217 islet isolation personnel, 217 consenting, 223 data and safety monitoring board, 226–227 data management, 223–224 informed consent, key elements, 224 key elements to be included, 220–221 project management, 216, 218–219 protocol development, 219

Index [Clinical trials setting in islet cell transplantation, factors to consider project management] training by experienced islet cell transplantation centers, 219 recruitment, 221–223 regulatory issues, 225–227 institutional review board (independent ethics committee), 225–226 Investigational New Drug (IND) application, 225 regulatory, 216 space, 218 Standard Operating Procedures (SOPs), 224–225 UNOS and local OPO, 219–225 Clotrimazole, 162 Coaxial protocols, 319–320 Cold swelling, 101 Collaborative Islet Transplant Registry (CITR), 275 Concomitant medications anti-inflammatory therapy, 158–159 Etanercept, 158–159, See also separate entry Connaugh Medical Research Laboratories (CMRL), 253 Contemporary immunosuppression in human islet transplantation animal pre-clinical models, 270 development and application of, 269–280 Edmonton immunosuppression protocol, 271–272 guidelines for, 274 induction and anti-cytokine agents, 272–273 for islet-after-kidney transplantation, 276–280 modern immunosuppression for, 274–275 clinical islet transplantation, 270–271 north American experience, 275–276 Continuous glucose monitoring systems (CGMS), 60 Core-to-mantle perfusion, 5 Cultivated human islet buds (CHIBs), 313 Culture and transportation of human islets between centers, 251–263 cultured islet preparations advantages, 252 development methods, 252

353 [Culture and transportation of human islets between centers] human islet culture, 252–258, See also separate entry practicality, 252 safety, 252 shipping human islets between centers, 258–262 islet collection and assessment at the remote site, 262 shipping bag, 259–261 shipping container, 261–262 temperature control, 262 Current Good Manufacturing Practices (cGMP), 258 Current outcomes of clinical islet transplantation, 229–244 culture, 233–234 efforts to improve outcomes, 230–234 enzyme preparation, 232–233 future directions, 244 improving engraftment, 244 tolerance induction, 244 insulin independence, 234–236 islet isolation, 232 long-term diabetes complications, prevention of, 238–240 metabolic control, 236–238 new strategies to improve, 242–244 islet-after-kidney transplantation, 243–244 living-related donation (LRD), 243 single donor islet transplantation, 242–243 pancreas preservation and transportation, 232 pancreas procurement, 231 post-transplant complications, 240–242 immunosuppression-related, 241 procedure-related, 240–241 Custodial, 82, 99 Cyclosporine (CyA) (Neoral), 157, 162, 278 dose, 157 mechanism of action, 157 side effects, 157 Cytochrome P-450 enzymes, 160–161 CYP3A, 161 CYP3A4 iso-enzyme, 161 Cytomegalovirus (CMV), 160, 171–172, 241 cytomegalovirus prophylaxis, 65

354 Daclizumab (Zenapax), 148–151, 152, 275 dose, 151 mechanism of action, 148–151 side effects, 151–153 Danazol, 162 Dapsone, 160 Delavirdine, 162 Density gradient centrifugation, 123 Department of Health and Human Services (DHHS), 226 Diabetes Diabetes Control and Complications Trial (DCCT), 29–30 diabetes gene therapy, 327–342, See: Gene therapy for diabetes diabetic rodents, transplantation studies in, 4 and pancreas, link between, 3 treatment by transplantation, 45–46 Digital image analysis (DIA), for islet volume determination, 125 Diltiazem, 162 Diphenhydramine 50, 274 Disseminated intravascular coagulation (DIC), 122 Donor selection for islet isolation, 118–120 Dyslipidemia, 168

Edema, 169 Edmonton Protocol, 148, 163, 234–235, 240, 252 Efavirenz, 162 Electrolyte disorders, 169 hypomagnesemia, 169 hypophosphatemia, 169 Embryonic stem cells (ESCs), 312, 315–317 insulin-producing cells from, 317 Endocrine replacement therapy, 3 Enhanced islet engraftment, 332–333 Enzymatic activity and pancreas digestion, 121 Edmonton immunosuppression protocol, 271–272 Epstein Barr virus (EBV), 242 Erythromycin, 162 Etanercept (Enbrel), 152–153, 158–159, 273, 275 dose, 159

Index [Etanercept (Enbrel)] mechanism of action, 158 side effects, 159 Ethidium bromide (EB), 126 Euro-Collins solution, 7, 123 Euro-FicollTM, 7, 123 Ex vivo insulin gene transfer to neuroendocrine cells, 337–338

Female reproductive system, alterations of, 172 Fetal calf serum (FCS), 127 FicollTM, 4, 7, 11 FK binding protein (FKBP), 155 Fluconazole, 162 Fluorescein diacetate (FDA), 126 Food and Drug Administration (FDA), 256, 286 Function after transplantation, of islet, 194–199 alloislet transplantation, 197–199 metabolic studies of autoislet recipients, 195–197

Ganciclovir, 65 Gastrointestinal disorders, 167 Gastroparesis, 65 Gatifloxacin, 162 Gene therapy for diabetes, 327–342 anti-obesity gene therapy, 341–342 b-cell function in Type 1 diabetes, maintenance, 330–333 antigen-independent approaches, 331–332 antigen-specific tolerization approaches, 331 attenuation of apoptosis, 332 augmentation within intact islets, 333 enhanced islet engraftment, 332–333 concepts and possibilities, 327–330 viral vectors, 328–329, See also separate entry disease progression prevention, 330–333 strategies for, 330: primary prevention, 330 secondary prevention, 330 tertiary prevention, 330

Index [Gene therapy for diabetes] gene transfer with non-insulin glucoselowering genes, 340–342, See also Non-insulin glucose-lowering genes to insulin replacement, 333–340, See also separate entry plasmid vectors, 329 targeted at diabetic complications, 342 Gift of Hope Organ & Tissue Donor Network (ILIP), 222 Gift of Life Donor Program (PADV), 222 Glucagon-like peptide-1 (GLP-1), 301–303 Glucagons glucagon secretion and blood glucose, link between, 33 Glucose phosphorylation, 32 Glucose potentiation of arginine-induced insulin secretion (GPAIS), 195–196, 198 Glucose tolerance tests, 198 Glycemic lability, 60 Glycemic variability, assessment, 181–182 Glycosuria, 2 Good Manufacturing Practice (GMP), 128 Graft, islet, monitoring and imaging, 179–188 Beta Score in, 181 islet function, monitoring, 180–184 glucose stability, 180 glycemic variability, assessment, 181–182 hypoglycemia, burden of, 181–182 hypoglycemic events, 180 overall function, 180 stimulation tests, 180 islet graft biopsy, 184 islet graft imaging, 185–187 molecular monitoring, 184–185 stimulated insulin/C-peptide secretion assessment of, 182–184: arginine stimulation test, 182–183 mixed meal stimulation test, 183 Grapefruit, 162

Health Resources and Services Administration (HRSA), 284 Hematological abnormalities, 163–166 adverse events by frequency of appearance, 164–165 Anemia, 163

355 [Hematological abnormalities] Leukopenia, 163 oral ulcers, 166–167 Thrombocytopenia, 165 Hepatic disorders, 67 Hepatic insulin gene therapy, 339–340 Herpes Simplex Virus (HSV), 167 Histidine-tryptophan-ketoglutarate (HTK) solution (Bretschneider), 82, 103, 105 Human islet culture, 252–258 culture density, 256–257 culture temperature, 257 human islet culture at the University of Miami, 253–258 optimal culture conditions, 253 preparation, 257–258 at the University of Miami, 253–258 Hypercholesterolemia, 238 Hypertension, 168–169 HYPO score, 59, 239 Hypoglycemia burden of, 181–182 counter-regulatory neuro-endocrine response to, 33 definitions of, 30–31 hypoglycemia unawareness, mechanisms of, 35–36 coping strategies, 44 insulin secretion and absence of hypoglycemia, 31–33, See also Insulin new tools and devices, 44–45 treatment by transplantation, 45–46 individual risk, assessing, 37–39 islet transplantation and, 46–47 occurrence, reason for, 34–35 patient selection and assessment, 59–60 predisposing factors for, 39 problems of reversibility, 41–44 protection against hypoglycemia, strategies for, 39–41 risk of, biochemical associations, 40 diabetes related, 40 disorders of food absorption, 40 drugs and exercise, 40 endocrine diseases, 40 reduced insulin clearance, 40 symptoms of, 34 in Type 1 diabetes, 29–48

356 Hypomagnesemia, 169 Hypophosphatemia, 169 Hypothermia metabolic changes during, 100–101 Hypoxia metabolic changes in, 101–103

Immune thrombocytopenic purpura (ITP), 166 Immunosuppression, See also Contemporary immunosuppression; individual entries Immunosuppression, maintenance, 155–158 Cyclosporine (CyA) (Neoral), 157, See also separate entry Mycophenolate Mofetil, 157, See also separate entry Mycophenolate sodium (Myfortic), 158, See also separate entry Sirolimus, 155, See also separate entry Tacrolimus, 156, See also separate entry Immunosuppressive management and complications, 147–174 adjuvant therapies, 160 alternative induction therapies, 153–155 Alemtuzumab, 153, See also separate entry rabbit anti-thymocyte globulin, 154–155, See also separate entry anticoagulation, 159 concomitant medications, 158–159, See also separate entry drug metabolism and interactions, 160–161 female reproductive system, alterations of, 172 frequency of adverse events related to, 152–153 high frequency (61% or greater), 152 low frequency (10% or less), 153 medium frequency (11% to 60%), 152 gastrointestinal disorders, 167 immunosuppressive therapy-related complications, 161–173 elevation in liver function tests, 163 hematological abnormalities, 163–166, See also separate entry maintenance immunosuppression, 155–158, See also Immunosuppression

Index [Immunosuppressive management and complications] malignancy, 173 retinopathy, 173 neurological disorders, 168 prophylactic therapies, 159–160, See also separate entry protocols for, 148 current immunosuppressive protocols, 149–150: flow chart, 151 renal disorders, 168–172, See also separate entry standard induction therapy, 148–153, See also separate entry weight loss, 172 In situ insulin gene transfer to neuroendocrine cells, 338 In vitro differentiation protocols, 318–321 directed differentiation, 319–321 cell reprogramming, 320–321 coaxial protocols, 319–320 spontaneous differentiation, 318 Independent Ethics Committee (IEC), 222 Indiana Organ Procurement Organization (INOP), 222 Infectious diseases, 170–171 Instant blood mediated inflammatory reaction (IBMIR), 244 Institutional or Independent Ethics Committee (IEC), 226 Institutional Review Board (IRB), 222, 225–226 Insulin anti-insulins, 33 independence, 9, 234–236 after islet allograft transplantation, 10 insulin gene transfer to non-b-cells, 337 to non-endocrine cells, 338–339 insulin-like growth factors (IGFs), 316 as normoglycemia controller, 31–33 replacement, gene therapy approaches to, 333–340 ex vivo insulin gene transfer to neuroendocrine cells, 337–338 hepatic insulin gene therapy, 339–340 in situ insulin gene transfer to neuroendocrine cells, 338 insulin gene transfer to non- b-cells, 337

Index [Insulin replacement, gene therapy approaches to] insulin gene transfer to non-endocrine cells, 338–339 muscle-targeted insulin gene therapy, 340 neogenesis approaches, 337 physiological glucose-responsive insulin gene expression, 334–335 physiological insulin secretion, 335 physiological proinsulin biosynthesis and processing, 335 proliferative b-cells, derivation, 335–337 transdifferentiation, 337 secretion and absence of hypoglycemia, 31–33 sensitizers, 340–341 Insulinoma cell line (INS-1E), 320 Intraportal embolization, 5 Intrasplenic autotransplantation, 5 Intravenous glucose tolerance test (IVGTT), 183 Investigational New Drug (IND) application, 225–226 Ischemia reperfusion injury, 102 Islet cell infusion, 138–140 Islet Cell Processing Center (ICPC), 258 Islet cell transplant (ICT), 135 Islet culture, challenges in, 126–128 Islet equivalents (IEQ), 124 Islet graft, See: Graft, islet Islet pluripotent stem cells (IPSCs), 313 Islet purification, challenges in, 122–124 Islet quantification sheet, 125 Islet-after-kidney (IAK) transplantation, 243–244 immunosuppression for, 276–280 patients, 210 Islets of Langerhans, 194 Isolation, islet, 232 aspects and challenges of, 115–129, See also Aspects and challenges automated method for, 233 donor selection for, 118–120 history of, 116–118 pancreas preservation for, 99–110, See also Pancreas preservation with magnetic microspheres, 124 Itraconazole, 162

357 Ketoconazole, 162

Lability Index (LI), 181 Lacy, Paul E., 17–21 Latent autoimmune diabetes in adults (LADA), 311 Leukemia Inhibitory Factor (LIF), 315 Leukopenia, 163 LiberaseTM, 7, 9, 120 Life Alliance Organ Recovery Agency (FLMP), 222 Life Center Northwest Donor Network (WALC), 222 LifeGift Organ Donation Center (TXGC), 222 LifeLink of Georgia (GALL), 222 LifeNet (VATB), 222 Lifeshare of the Carolinas (NCCM), 222 LifeSource Upper Midwest Organ Procurement Organization (MNOP), 222 Liver function tests, 163 Living donor islet transplantation, 13 Living-related donation (LRD), 243 Los Angeles Preservation Solution 1 (LAP-1), 108–109

Macaca mulatta, 285 Macacca fascicularis, 285 Macrophage chemoattractant protein-1 (MCP-1), 256 MAGE (Mean Amplitude of Glycemic Excursions), 60, 181 Magnetic resonance imaging (MRI), 185 Malignancy, 173 Mammalian target of rapamycin (mTOR), 155 Mass after islet transplantation, 193–200, See also Metabolic measures Mean amplitude of glycemic excursions (MAGE), See: MAGE Metabolic measures of islet function function after transplantation, 194–199 and mass after islet transplantation, 193–200 recommended clinical monitoring

358 [Metabolic measures of islet function recommended clinical monitoring] for islet transplant recipients, 199–200 Methylprednisolone, 162, 274 Metoclopramide, 162 Miami-Modified Medium-1 (MM1) for human islet culture, 254–255 Mid-South Transplant Foundation (TNMS), 222 Minnesota/UCSF pilot study, 273 Mixed meal stimulation test, 183, 198 M-Kyoto solution, 122 Molecular monitoring, 184–185 Moxifloxacin, 162 Multi-organ harvesting, 87–88 Multipotent Adult Progenitor Cells (MAPCs), 314 Muscle-targeted insulin gene therapy, 340 Mycophenolate Mofetil (MMF) (Cellcept), 152–153, 157 dose, 158 mechanism of action, 157 side effects, 158 Mycophenolate sodium (Myfortic), 158 Myocardial perfusion imaging (MPI), 66

Nafcillin, 162 Nalgene jar, 86, 88, 93 National Institute of Health (NIH), 226 National Organ Transplant Act (NOTA), 219 Nefazadone, 162 Nelfinavir, 162 Neogenesis, 293–305, 337 animal models of, 296–298 new b-cells through transdifferentiation, 298–300 sources, 299 Neoplasia, 62 Neoral, 155, 278 Neovascularization, 5 Nephropathy, 63–64 Nestin-positive islet-derived progenitors (NIPs), 313 Neurological disorders, 168 Neuropathy, 65 autonomic neuropathy, 65 diabetic neuropathy, 65 Nevirapine, 162

Index New England Organ Bank (MAOB), 222 New Jersey Organ and Tissue Sharing Network (NJTO), 222 New York Organ Donor Network (NYRT), 222 Nicardipine, 162 Nicotinamide adenine dinucleotide (NAD), 255 Non-insulin glucose-lowering genes, gene transfer with, 340–342 insulin sensitizers, 340–341 North American experience of immunosuppression for islet transplantation, 275–276 Nursing coordinator’s perspective in clinical trials setting for islet cell transplantation 215–227, See also Clinical trials

OneLegacy (CAOP), 222 Oral glucose tolerance test (OGTT), 183 Organ Procurement Organizations (OPOs), 218, 284 Organ Procurement and Transplantation Network (OPTN), 219 at the University of Wisconsin (WIUW), 222

Packed cell volume (PCV), 138 Pancreas and diabetes, link between, 3 digestion, 117 challenges in, 120–122 pancreas preservation for islet isolation, 99–110 current preservation solutions, 103–109: EuroCollins solution (EC), 103 membrane stabilizers, 104 buffers, 104 energy substrates, 104 FRIs & ORSSs, 104 electerolytes, 104 additives, 104 Histidine-tryptophanketoglutarate solution (Bretschneider), 105 University of Wisconsin solution, 105–106 twolayer method (TLM), 106–108 Los Angeles Preservation Solution 1 (LAP-1), 108–109 superoxide dismutase (SOD), 108

Index [Pancreas pancreas preservation for islet isolation] metabolic changes: during hypothermia, 100–101 in hypoxia, 101–103 pancreas procurement, 204–206, 231 donor organs allocation for research, 206 models, 204 whole pancreas vs. islet transplant allocation, 205–206 pancreatic b-cell mass regulation of, 294–296 pancreatic stem cells, 313–314 extrapancreatic stem cells, 314–317 pancreatic tissue fragments, transplant, 2 transplantation, 14–16, 115 pancreatic tissue dissociating methods, 117 Pancreatic islet transplantation pancreas procurement for, surgical aspects of, 81–94 back-table preparation and cold storage, 92–93 dissection of pancreas prior to crossclamping, 88–91 donors selection, 83–86: criteria, 84–85 exposure for multi-organ harvesting, 87–88 general surgical principles in, 93 perfusion and cross-clamping and retrieval, 91–92 preparation prior to procurement, 86–87 technique, 83–93 Panel reactive antibody (PRA), 236 Parenchymal track embolization, 140–141 Avitene paste, 140–141, 143 gelatin-sponge, 140 stainless steel or platinum embolization coils, 140 thrombin saturated Gelfoam collagen paste, 140 Tisseal, 140 Patient selection and assessment, 57–77, See also Clinical islet transplantation diabetes complications and other comorbidities, 62–67 autoimmune disease, 67 autoimmune thyroid disease, 67 cardiovascular disease and risk factors, 66 hepatic disorders, 67

359 [Patient selection and assessment diabetes complications and other comorbidities] nephropathy, 63–64 neuropathy, 65, See also individual entry retinopathy, 63 endocrinologist’s perspective, 57–77 islet transplantation assessment, approach to, 67–76, See also under Islet transplantation overall purpose of, 57 Pentamidine Isethionate (Pentam), 160 Pentoxifylline, 274 Percutaneous portal vein access choice of access, 135–136 equipment, 136 islet cell infusion, 138–140 set-up, 140 parenchymal track embolization, 140–141, See also separate entry post-procedural management and followup, 144 pre-procedure requirements and contraindications, 136–137 procedure, 137–138 aseptic technique, 137 sedation, 137 stiffened micropuncture set, 138 procedure-related complications, 141–144 radiological aspects, 135–144 subsequent transplants and secondary interventions, 144 ultrasound, 136 Perfluorocarbon (PFC), 83, 93 Peroxisome proliferator activator gamma (PPAR), 304 Phenobarbital, 162 Phenytoin, 162 Physiology, islet, 194 alpha cell, 194 delta cell, 194 insulin secretion, 194 Islets of Langerhans, 194 Pig islet xenotransplantation, 283–290 in primates, 285–288 Ceropithecus aethiops, 285 Macaca mulatta, 285 Macacca fascicularis, 285 organ shortage and fiscal reality, 283–284

360 [Pig islet xenotransplantation] porcine endogenous retrovirus, potential risk of, 288–290 Plasmid vectors, 329 Pneumocystis carinii prophylaxis, 65, 159–160 Atovaquone (Mepron), 160 Dapsone, 160 Pentamidine Isethionate (Pentam), 160 Trimethoprim-Sulfamethoxazole (Bactrim), 159 Poly adenosine diphosphate ribose polymerase (PARP) inhibition, 255 Polyethyleneimine (PEI), 329 Polyuria, 2 Porcine endogenous retrovirus (PERV), 288–290 Portal embolization, 5 Portal vein thrombosis (PVT), 141–142 Positron-emission tomography (PET), 185–187 Post-transplant amnesia and moving goal posts, 70–71 Post-transplant lymphoproliferative disorders (PTLDs), 242 Prograf, 148 Proliferative b-cells, derivation, 335–337 derived b-cells, 336 proliferative b-cells, 336 Prophylactic therapies, 159–160 Pneumocystis carinii prophylaxis, 159–160, See also separate entry Proteinuria, 169–170 Public Health Service Act (PHS Act), 225

Rabbit anti-thymocyte globulin (ATG, Thymoglobulin), 154–155 dose, 155 mechanism of action, 154 side effects, 155 Rapamune, 148 Renal disorders, 168–172 cytomegalovirus, 171–172 edema, 169 electrolyte disorders, 169 hypertension, 168–169 infectious diseases, 170–171 kidney function alterations, 169 proteinuria, 169–170 skin manifestations, 170

Index Retinopathy, 63, 173 Reverse transcription polymerase chain reaction (RT-PCR), 184 Reversibility, problems of, 41–44 Ricordi continuous digestion chamber, 7, 121 Rifabutin, 162 Rifampin, 162 Rifapentine, 162 Ritonavir, 162

Secretory Unit of Islet Transplant Objects (SUITO), 181 Semi-automated dissociation chamber, 6 Simultaneous islet and kidney (SIK) patients, 210 Simultaneous pancreas-kidney (SPK) grafts, 15, 64 Single donor islet transplantation, 242–243 Sirolimus (Rapamune), 65, 152, 155, 238, 275, 277 dose, 156 mechanism of action, 155 side effects, 156 Skin manifestations, 170 Southwest Transplant Alliance (TXSB), 222 SPIO labeling, 185 Standard induction therapy, 148–153 Daclizumab, 148–151, See also separate entry Standard Operating Procedures (SOPs), 221, 224–225 Stem cell approaches for b-cell replacement, 311–321 adult stem cells (ASCs), 312–317, See also separate entry embryonic stem cells (ESCs), 312, 315–317, See also separate entry in vitro differentiation protocols, 318–321, See also separate entry Superoxide dismutase (SOD), 108 Super-paramagnetic iron oxide (SPIO) labeling, 185

Tacrolimus (Prograf) (FK506), 152–153, 156, 277 dose, 156

Index [Tacrolimus (Prograf) (FK506)] mechanism of action, 156 side effects, 156–157 Thrombocytopenia, 165 Thymoglobulin, 272 Tiseel, 240 Transdifferentiation, 337 new b-cells through, 298–300 Transhepatic catheterization, 136 Transplantation, diabetes treatment by, 45–46 Transplantation, islet, See also Pancreatic islet transplantation assessment, approach to, 67–76 clinical assessment, 72 patient expectations, goals, and aspirations, 68–69 post-transplant amnesia and moving goal posts, 70–71 social, financial, and psychological considerations, 71–72 suggested investigations and additional assessments, 75–76 transplant endocrinologist’s role, 67–68 unrealistic expectations and misconceptions, 69 waiting list and reassessment, 72–76 categories of, 10 components of, 204–213 pancreas procurement, 204–206, See also separate entry early clinical trials of, 9–10 experimental and clinical, 1–21 British Medical Journal article, 1 historical perspective, 1–21 The Lancet article, 3 fetal islet allografts in type 1 diabetes, 14 hypoglycemia AND, 46–47 islet allografts after pancreatectomy, 11, See also Allografts islet autografts, 10–11 Islet Transplantation Activity (1999–2006), 231 islet transplantation alone (ITA), 275 living donor islet transplantation, 13 pancreas transplantation, 14–16 Paul E. Lacy’s lecture on, 17–21 setting, challenges in, 203–213, See also under Challenges

361 [Transplantation, islet] transplant recipient, care of, 147–174, See also Immunosuppressive management and complications Trimethoprim-Sulfamethoxazole (Bactrim), 65, 159 Tumor necrosis factor (TNF- ), 158 Two-layer method (TLM), 106–108, 232 Type 1 diabetes (T1DM), 57–77 hypoglycemia in, 29–48, See also Hypoglycemia

United Network of Organ Sharing (UNOS), 218 University of Wisconsin (UW) solution, 105–106

Verapamil, 162 Vesicular monoamine transporter 2 (VMAT2), 187 Viral vectors, 328–329 adenoviral vectors, 328 recombinant adenoassociated viral vectors, 328 retroviral vectors, 328 Vitamin E (Trolox), 256 Vitamins on islets, 127–128 Voriconazole, 162

Washington Regional Transplant Consortium (DCTC), 222 Weight loss, 172 World Health Organization (WHO), 128

Xenograft pancreas tissue, 2

Zenapax, 148 Zetia, 168