Stem Cell Biology in Health and Disease

Stem Cell Biology in Health and Disease

Thomas Dittmar · Kurt S. Zänker Editors

Stem Cell Biology in Health and Disease

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Editors Dr. Thomas Dittmar Witten/Herdecke University Institute of Immunology Stockumer Str. 10 58448 Witten Germany e-mail: [email protected]

Prof. Dr. Kurt S. Zänker Witten/Herdecke University Institute of Immunology Stockumer Str. 10 58448 Witten Germany e-mail: [email protected]

ISBN 978-90-481-3039-9 e-ISBN 978-90-481-3040-5 DOI 10.1007/978-90-481-3040-5 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2009935391 © Springer Science+Business Media B.V. 2009 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

Within the last decade there has been a dramatic increase in the understanding and application of biological principles within stem cell therapies, which has made it necessary to produce a book which intends to summarize much of the body of knowledge concerning Stem Cell Biology in Health and Disease. Although some of the treatments have been suggested for many years, knowledge and technology have now progressed sufficiently to allow us to test many of the different concepts with human embryonic, induced pluripotent, organ-specific and resident, cancer and mesenchymal stem cells in animal models and clinical settings – alone or in combination with other therapies in cardiovascular and neurodegenerative diseases, in diabetes and against cancer. Studies on stem cells have been hampered in the past by the ethical and biological difficulties in preparing sufficient cell numbers in a reasonable characterized and pure form. In stem cell research we are now on the threshold of a revolution; a revolution that will have major ramification for human medicine. Giant strides in our understanding of stem cell biology and the elements that control the biological behavior of the different traits of stem cells have made it possible to intervene directly with regenerative life processes and to open a novel chapter in the fight against cancer. Chapter 1 shortly summarizes the historical hall marks of stem cell research in biology; Chapter 2 describes the hematopoietic stem and progenitor cells in clinical use; Chapter 3 describes the protocols to expand hematopoietic stem cells ex vivo; Chapter 4 highlights one important feature of hematopoietic stem/progenitor cells, namely cell migration; Chapter 5 opens the books on properties of mesenchymal stem cells for cancer cell therapy; Chapter 6 reviews intensively alternative embryonic stem cell sources to solve both ethical concerns and the allogeneic nature of human embryonic stem cells for therapeutic use; Chapters 7 and 8 describe the role of stem cell therapy in Multiple Sclerosis and Parkinson’s Disease; Chapters 9, 10 and 11 introduce novel perspectives on cancer stem cells stimulating a provocative discussion of the complexity of cancer origin, and their niches of existence either in a tumor mass or in chronically inflamed microenvironment, e.g. inflamed periodontium (Chapter 12); Chapters 13 and 14 directly address hematopoietic and solid cancer stem cells and Chapter 15 embarks on a novel role of the diversity of cancer stem cells in tumor relapse and metastases formation. Chapter 16 describes v

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new therapeutic approaches to eliminate cancer stem cells and Chapter 17 puts the focus on a molecular target family in cancer stem/progenitor cells - the ATP-binding cassette membrane transporters - which are promising therapeutic entities. Multiple key references are provided by the authors at the end of each chapter, and the reader is encouraged to consult these sources as well, because due to the limited space of a monograph the technical details cannot be presented in a survey of this type. Again, we would like to thank all distinguished authors for their valuable contributions to provide with this book a robust ground for the avalanche of discoveries that will deluge the field of stem cell research in the years to come. Summer 2009

Witten, (Germany) Thomas Dittmar Kurt S. Zänker

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thomas Dittmar and Kurt S. Zänker Part I

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Bone Marrow-Derived Stem Cells

2 Hematopoietic Stem and Progenitor Cells in Clinical Use – Transplantation and Mobilization . . . . . . . . . . . . . . . Michael Punzel

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3 Ex Vivo Expansion of HSPCs . . . . . . . . . . . . . . . . . . . . . Yaming Wei and Xin Ye

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4 Modulation of Hematopoietic Stem/Progenitor Cell Migration . . . Thomas Dittmar, Susannah H. Kassmer, Benjamin Kasenda, Jeanette Seidel, Bernd Niggemann, and Kurt S. Zänker

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5 Properties of Mesenchymal Stem Cells to Consider for Cancer Cell Therapy . . . . . . . . . . . . . . John Stagg and Sandra Pommey Part II

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Embryonic Stem Cells

6 Alternative Embryonic Stem Cell Sources . . . . . . . . . . . . . . Tomo Šari´c, Narges Zare Mehrjardi, and Jürgen Hescheler

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7 Cell Therapy in Parkinson’s Disease . . . . . . . . . . . . . . . . . R. Laguna Goya and R.A. Barker

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8 Transplantation of Stem Cells and Their Derivatives in the Treatment of Multiple Sclerosis . . . . . . . . . . . . . . . . . . . . Eric C. Larsen and Ian D. Duncan

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Part III Cancer Stem Cells 9 Cancer: A Stem Cell-based Disease? . . . . . . . . . . . . . . . . . James E. Trosko

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Contents

Stem Cell Niche Versus Cancer Stem Cell Niche – Differences and Similarities . . . . . . . . . . . . . . . . . Bruce C. Baguley and Graeme J. Finlay The Chronically Inflamed Microenvironment and Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hanchen Li, Calin Stoicov, Xueli Fan, Jan Cerny, and Jean Marie Houghton Does the Chronically Inflamed Periodontium Harbour Cancer Stem Cells? . . . . . . . . . . . . . . . . . . . . . . . . . . . Wolf-Dieter Grimm, Wolfgang H. Arnold, Sebastian Becher, Aous Dannan, Georg Gassmann, Stathis Philippou, Thomas Dittmar, and Gabor Varga

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Leukemia Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . Markus Müschen

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Cancer Stem Cells in Solid Tumors . . . . . . . . . . . . . . . . . . Melia G. Nafus and Alexander Yu. Nikitin

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“One for All” or “All for One”? – The Necessity of Cancer Stem Cell Diversity in Metastasis Formation and Cancer Relapse . Thomas Dittmar, Christa Nagler, Sarah Schwitalla, Kathrin Krause, Jeanette Seidel, Georg Reith, Bernd Niggemann, and Kurt S. Zänker

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Elimination of Cancer Stem Cells . . . . . . . . . . . . . . . . . . . A. Sagrera, J. Pérez-Losada, M. Pérez-Caro, R. Jiménez, I. Sánchez-García, and C. Cobaleda

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Potential Molecular Therapeutic Targets in Cancer Stem/Progenitor Cells: Are ATP-Binding Cassette Membrane Transporters Appropriate Targets to Eliminate Cancer-Initiating Cells? . . . . . . . . . . . . . . . . . . . . . . . . Murielle Mimeault and Surinder K. Batra

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors

Wolfgang H. Arnold Institute of Anatomy, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, [email protected] Bruce C. Baguley Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand, [email protected] R.A. Barker Cambridge Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge, CB2 2PY, UK; Department of Neurology, Addenbrookes Hospital, Cambridge, CB2 2QQ, UK; Edith Cowan University, Perth, Australia, [email protected] Surinder K. Batra Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA, [email protected] Sebastian Becher Department of Periodontology, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, basti [email protected] Jan Cerny Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected] C. Cobaleda Departamento de Fisiología y Farmacología, Edificio Departamental, Universidad de Salamanca, Campus Unamuno s/n, 37007-Salamanca, Spain, [email protected] Aous Dannan Department of Periodontology, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, [email protected] Thomas Dittmar Institute of Immunology, Faculty of Medicine, Witten/Herdecke University, Stockumer Str. 10, 58448 Witten, Germany, [email protected]

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Contributors

Ian D. Duncan Department of Medical Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA, [email protected] Xueli Fan Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected] Graeme J. Finlay Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand, [email protected] Georg Gassmann Department of Periodontology, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, [email protected] R. Laguna Goya Cambridge Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge, CB2 2PY, UK, [email protected] Wolf-Dieter Grimm Department of Periodontology, Faculty of Dental Medicine, Witten/Herdecke University, Alfred-Herrhausenstr. 50, 58448 Witten, Germany, [email protected] Jürgen Hescheler Institute for Neurophysiology, Center for Physiology and Pathophysiology, Medical Center; Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany, [email protected] JeanMarie Houghton Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected] R. Jiménez Departamento de Fisiología y Farmacología, Edificio Departamental, Universidad de Salamanca, Campus Unamuno s/n, 37007-Salamanca, Spain, [email protected] Benjamin Kasenda Department of Hematology and Oncology, University of Freiburg Medical Center, D-79106 Freiburg, Germany, [email protected] Susannah H. Kassmer Department of Laboratory Medicine, Yale Stem Cell Center, Yale University, New Haven, CT, USA, [email protected] Kathrin Krause Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected] Eric C. Larsen Department of Medical Sciences, University of Wisconsin-Madison, School of Veterinary Medicine, 2015 Linden Drive, Madison, WI 53706, USA, [email protected] Hanchen Li Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected]

Contributors

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Narges Zare Mehrjardi Institute for Neurophysiology, Center for Physiology and Pathophysiology, Medical Center, University of Cologne, 50931 Cologne, Germany; Department of Stem Cells, Royan Institute, Tehran, Iran, [email protected] Murielle Mimeault Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA, [email protected] Markus Müschen Leukemia Research Program, Childrens Hospital Los Angeles; Leukemia and Lymphoma Program, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90027, [email protected] Melia G. Nafus Department of Biomedical Sciences, Cornell University, Ithaca, New York, 14853, USA, [email protected] Christa Nagler Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected] Bernd Niggemann Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected] Alexander Yu. Nikitin Department of Biomedical Sciences, Cornell University, Ithaca, New York, 14853, USA, [email protected] M. Pérez-Caro OncoStem Pharma, Salamanca, Spain, [email protected] J. Pérez-Losada Departamento de Medicina, Facultad de Medicina, Universidad de Salamanca, Campus Unamuno s/n, 37007-Salamanca, Spain, [email protected] Stathis Philippou Institute of Pathology, Faculty of Medicine, Ruhr University Bochum, 44801 Bochum, Germany, [email protected] Sandra Pommey Department of Medicine, Immunology Research Centre, St. Vincent’s Hospital, University of Melbourne, Melbourne, Victoria, Australia, [email protected] Michael Punzel Institute of Transplantation Diagnostic and Cellular Therapeutics, Universitätsklinikum Düsseldorf, 40225 Düsseldorf, Germany, [email protected] Georg Reith Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected] A. Sagrera OncoStem Pharma, Salamanca, Spain, [email protected] I. Sánchez-García Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, CSIC/Universidad de Salamanca, Campus Unamuno s/n, 37007-Salamanca, Spain, [email protected]

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Tomo Šari´c Institute for Neurophysiology, Center for Physiology and Pathophysiology, Medical Center; Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany, [email protected] Sarah Schwitalla Second Department of Medicine, Klinikum rechts der Isar, Technical University of Munich, 81675 Munich, Germany, [email protected] Jeanette Seidel Medizinische Klinik II m. S. Hämatologie/Onkologie, Charité Campus Mitte, 10117 Berlin, Germany, [email protected] John Stagg Cancer Immunology Program, Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia, [email protected] Calin Stoicov Department of Medicine and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01536, USA, [email protected] James E. Trosko Department of Pediatrics/Human Development, College of Human Medicine, Michigan State University, East Lansing, Michigan 48824, MI, USA, [email protected] Gabor Varga Department of Oral Biology, Semmelweis University, Budapest, Hungary, [email protected] Yaming Wei Guangzhou Institute of Clinical Medicine, Guanzhou Municipal First People’s Hospital, Guangzhou Medical College, Guangzhou, China, [email protected] Xin Ye Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, China, [email protected] Kurt S. Zänker Institute of Immunology, Witten/Herdecke University, 58448 Witten, Germany, [email protected]

Chapter 1

Introduction Thomas Dittmar and Kurt S. Zänker

Within the past years our knowledge about stem cell biology in health and disease has changed dramatically. What rather sounded like Science Fiction 10–15 years ago, namely that e.g., stem cells from bone marrow or from adipose tissue can be used for regenerative medical approaches, or that it is possible to create donor specific stem cells (so-called induced pluripotent stem cells (iPS cells), exhibiting embryonic stem cell (ESC) properties) simply by transducing 2–4 transcription factors, has now become reality. Likewise, the knowledge that cancer tissues are hierarchically organized like normal tissues, namely comprising of a small amount of tumorigenic cancer stem cells (CSCs) and a huge mass of non-tumorigenic cancer cells will play a crucial role in the development of novel anti-cancer strategies. It is remarkable what has been achieved in the field of regenerative medicine within the past 10–15 years. In summary, this is an exciting story of what is possible in stem cell-based regeneration strategies, but it is also a story about a long and stony way with lots of unknown pitfalls. In 1999/2000 first data have been published demonstrating that bone marrowderived stem cells (BMDCs) can develop into hepatocytes [1, 2]. These original studies, being performed in rodents, were the first hints that stem cells of the bone marrow do not only give rise to cells of the blood lineage, but can also differentiate into cells of a different germ layer, a phenomenon, which has been referred to as “transdifferentiation” [3]. Till then (and to date), BMDCs were/are commonly used for bone marrow reconstitution after high-dose chemotherapy of patients with malignant hematopoietic disorders, such as multiple myeloma [4] or acute leukemias [5], or solid tumors [6]. The finding that BMDCs, and later on other types of adult stem cells, e.g., adipose-derived stem cells (ASCs) or neural stem cells (NSCs), are capable to transdifferentiate into various tissues, thereby restoring tissue integrity [7], offered perspectives for novel therapeutical approaches to heal various severe diseases, such as heart attack, liver cirrhosis, and neuronal degenerative disorders (stroke, T. Dittmar (B) Institute of Immunology, Faculty of Medicine, Witten/Herdecke University, Stockumer Str. 10, 58448, Witten, Germany e-mail: [email protected]

T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_1, 

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Parkinson Disease, etc.). Among adult stem cells, particularly BMDCs and ASCs raised (and still raise) great expectations for stem cell-based tissue regeneration strategies. Both stem cell types are easily accessible (BMDCs from bone marrow via aspiration or apheresis from mobilized donors, ASCs from liposuction) and possess an enhanced transdifferentiation capacity as verified by a plethora of excellent animal studies (for review see [7–9]). BMDCs can give rise to liver, skeletal muscle, gastric mucosa, and small intestinal epithelial cells [7]. The differentiation potential of ASCs includes adipocytes, cardiomyocytes, chondrocytes, endothelial cells, myocytes, neuronal-like cells, and osteoblasts [8]. However, there are some concerns about the overall pluripotency of adult stem cells. In contrast to ESCs and iPS cells, it is not possible to transdifferentiate adult stem cells functionally in certain tissues, like cardiomyocytes and dopaminergic neurons, in-vitro. In addition to that, even in vivo studies presented inconsistent data concerning the transdifferentiation capacity of adult stem cells. For instance, in 2001, Orlic and colleagues reported that transplanted adult bone marrow cells repaired myocardial infarcts in mice [10]. Examination of the infracted region after a period of 9 days following transplantation demonstrated that newly formed myocardium, comprising of proliferating myocytes and vascular structures, occupied about 68% of the infracted region [10]. Moreover, the functional competence of the left repaired ventricle was improved for several hemodynamic parameters [11] suggesting that efficient myocardial repair by application of BMDCs is conceivable. Only one year later, in 2002, Strauer et al. already reported about the repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans [12]. After standard therapy for acute myocardial infarction (AMI), 10 patients were transplanted with autologous BMDCs via a balloon catheter placed into the infarct-related artery during balloon dilation [12]. After 3 months of follow-up, patients of the cell therapy group showed a significantly decreased infarct region, a significantly increased infarction wall movement velocity, and a significant improvement in stroke volume index, left ventricular end-systolic volume and contractility [12]. At a first glance, these data might tell a successful “form bench to bedside” story. However, in 2004, two independent studies demonstrated that BMDCs do not undergo transdifferentiation into cardiomyocytes in myocardical infarcts [13, 14]. Murry and colleagues showed that only 1–3 cells per 100,000 cardiomyocytes were of bone marrow origin [14], which is in clear contrast to 68% as reported by Orlic et al. [10]. Likewise, data of Balsam and colleagues provided evidence that BMDCs rather adopted mature hematopoietic fates in ischemic myocardium than to transdifferentiate into cardiomyocytes [13]. Balsam and colleagues speculated that there may be differences in their anesthetic and/or surgical technique and that these may resulted in a different outcome [13], whereas Murry and colleagues assumed subtle differences in the protocols, e.g., differences in trace components in the stem cell preparation or different assays used to detect cardiomyogenic differentiation, which might explain the discrepant results [14]. In a long-term study Meyer and colleagues were able to show that a single dose of intracoronary bone marrow-derived HSPCs did not provide long-term benefit on left ventricular systolic function after acute

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myocardial infarction (AMI) as compared with a randomized control group [15]. Similar results were reported recently by Choi and colleagues demonstrating a lack of additional benefit of intracoronary transplantation of autologous peripheral blood stem cells in AMI patients [16]. However, both studies reported that after 6 months the left ventricular ejection fraction was significantly improved in the cell therapy group [15, 16], which may point to a stem cell specific effect. Further disadvantages of most adult stem cells are (i) that they do not remain in a stem cell state under in vitro conditions and (ii) that they can only expanded for limited passages. Both disadvantages omit long-term cultures of adult stem cells, which is in contrast to ESCs and iPS cells that could be cultivated nearly unlimited. For instance, bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) can be cultured for 5–7 days without a significant decrease of CD34/CD133 expression. Longer cultivation periods is associated with a decrease of these two HSPC marker molecules indicating induction of differentiation. To delay the autologous differentiation capacity of HSPCs, e.g., for ex vivo expansion approaches optimized culture medias have been developed, which mostly vary in the choice of supplemented cytokines. Using optimized culture conditions it is possible to expand HSPCs ex vivo without a noteworthy level of differentiation. On the other hand, these optimized culture condition might have different effects on the expanded cells. We have recently demonstrated that the stromal cell-derived factor-1α (SDF1α) induced migratory activity of cultivated murine HSPCs strongly depended on the used cytokine combinations [17]. For instance, cultivation of murine HSPCs in the presence of stem cell factor, thrombopoietin and Interleukin-11 yielded in the third highest expansion rate of all tested cytokines and cytokine combinations [17]. However, analysis of the migratory behavior revealed that these cells did not react to SDF-1α stimulation with an increased locomotory activity [17], which could be a severe side-effect if such cells would be used for HSPC transplantation for bone marrow reconstitution. In contrast to adult stem cells, ESCs remain in their stem cell state in vitro and can be propagated nearly unlimited. Moreover, these cells possess an unlimited differentiation capacity in vitro and in vivo. However, human ESCs are still a subject to controversial and ethical discussions since isolation of human ESCs prerequisites the destruction of a human embryo (or the killing of a putative human life). Another disadvantage of ESCs is that they could not be administered directly in degenerated tissues while this would result in teratoma formation (which nicely illustrates their unrestricted differentiation capacity). Thus, these cells could only be implanted after in vitro pre-differentiation. However, pre-differentiated ESCs exhibit an overall lesser survival rate when removed from culture and being transplanted. Ultimately, transplantation of pre-differentiated ESCs prerequisites immunosuppression of the patients to avoid the risk of graft rejection, which, however, is associated with other risks and concerns. The latter problem could be overcome by generating “patient/custom-made embryonic cell lines”, so-called therapeutic cloning. Even if this technique would be feasible one day the other two problems (ethical debate and risk of tumor formation) would remain.

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Within the past two to three years a novel embryonic stem cell-like type has emerged, so-called iPS cells. These cells can be generated by viral transfection of two or four transcription factors into adult stem cells or adult somatic cells, respectively [18, 19], which ultimately leads to a redirection of this cell types towards and embryonic-like, undifferentiated state. In fact, induced pluripotent stem cells possess several ESC characteristics, such as morphology, proliferation, gene expression, telomerase activity, epigenetic status, and the capacity of unrestricted differentiation. Like ESCs, the latter property is associated with teratoma formation in-vivo if iPS cells are transplanted undifferentiated. However, even if iPS cells will be pre-differentiated prior implantation, they might bear potentially tumorigenic risks since these cells were generated by using the proto-oncogene c-myc and viral vectors, which integrate randomly into the host genome. Whether human iPS cells, either generated without the use of c-myc [18, 20] or without viral integration [21] using plasmids, will find their way into clinical use has to be elucidated in future studies. Nonetheless, the benefit of such cells would be that they behave like ESCs, thus being capable to differentiate into various tissues, and “patient/custom-made iPS cells” can be generated, which supersedes immunosuppression. A severe side-effect of most, if not all, stem cells is their potential tumorinitiation capacity. It is well recognized that ESCs and iPS cells induce teratomas in-vivo if implanted in a undifferentiated state. Pre-differentiation of both ESCs and iPS cells could minimize this risk, whereby iPS cells might still bear potentially tumorigenic risks if such cells were generated by the use of the proto-oncogene c-myc and viral vectors, which integrate randomly into the host genome. With prolonged passage for >4 months, human ASCs have been observed to undergo malignant transformation, which was correlated with karyotypic abnormalities, tumor formation in immunodeficient mice [22], and epithelial-mesenchymal transition [23]. Nearly 4 years ago, Houghton and colleagues demonstrated that gastric cancer originates from BMDCs, which have been recruited and transformed malignantly by chronically inflamed gastric mucosa tissue [24]. In addition to gastric cancer there is compelling evidence that also other epithelial cancers, such as benign and malignant tumors of the skin, Kaposis sarcoma, and Barretts’ adenocarcinoma of the esophagus might originate from BMDCs (for review see [25]). The inherent tumorigenic capacity of stem cells points to another type of stem cells, which has gained much of attention within the last decade: cancer stem cells (CSCs) (for review see [26]). CSCs have been described as a rare population of cancer cells exhibiting stem cell properties such as self-renewing, differentiation, tissue reconstitution, and multiple drug resistance. Because of their tumor initiation capacity and resistance against cytotoxic drugs and radiation CSCs [27–29] have not only been linked to primary tumor formation, but also to metastases and cancer relapses. The knowledge that a tumor is organized hierarchically like normal tissue, namely comprising of a small number of stem cells, which give rise to differentiated cells, thereby maintaining tissue integrity and organ function, is of crucial interest for our understanding how to treat cancer in future times. The dilemma of current cancer therapies (conventional chemotherapy, radiation therapy, hormonal therapy, humanized monoclonal antibodies, and/or inhibitors) is that although most cancer

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patients respond to therapy, only few are definitely cured [30]; a matter, which applies to both solid tumors as well as hematological disorders. This phenomenon, which has been entitled as “the paradox of response and survival in cancer therapeutics” [30] has been compared to “cutting a dandelion off at ground level” [30, 31]. Current cancer therapies are designed to target highly proliferating tumor cells and determination of tumor shrinking concomitant with mean disease free survival of patients are commonly used as read-outs for the efficacy of the appropriate therapy. While such strategies eliminate the visible portion of the tumor, namely the tumor mass, they mostly fail to eliminate the unseen root of cancer, namely CSCs. Thus, elimination of the unseen root of cancer, CSCs, would mean to have a chance to cure disease. However, there is increasing evidence that both metastases and cancer relapses might be initiated by specific CSCs, referred to as metastatic CSCs (mCSCs) [32] and recurrence CSCs (rCSCs) [33]. Quite recently, Hermann and colleagues identified a specifically metastatic CSC subpopulation in pancreatic cancer [34], whereas Shafee et al. demonstrated that the cisplatin resistance of murine mammary CSCs was associated with genetic aberrations in the platinum resistant cells [35]. These findings suggest that different cancer stage specific CSCs exist, which might play a role in the development of anti-CSC strategies. Is it possible to eliminate distinct CSC subtypes with a single anti-CSC strategy or demand distinct CSC subtypes distinct anti-CSC strategies? The answer to this question can not be given yet since only a handful of data exist for mCSCs and rCSCs so far. In summary, it is remarkable what has been achieved in only 10–15 years in the field of stem cell biology in health and disease. Even if still some problems, being associated with stem cell-based regeneration strategies (e.g., choice of the stem cell type (adult stem cells, ESCs, or iPS cells), how to apply them (by injection, by infusion etc.), exist, we know from several animal studies that stem cell-based regeneration strategies are feasible and that it will be only a matter of time when such approaches will become reality in humans. Likewise, the knowledge that CSCs exist has changed our understanding of the disease cancer and will help us to develop novel anti-cancer strategies. There is a growing list of CSC specific target molecules/pathways, which might be used for selective CSC elimination or which could be used to drive CSCs from their stem cell state into a more differentiated state, thereby making these cells susceptible to conventional cancer therapy. So, we the scientists, physicians, and patients should be optimistic what the future will bring in the field of stem cell biology in health and disease. We are glad that so many internationally recognized experts accepted our invitation to contribute to this exciting book. We sincerely thank them all for their interest in this important topic and that they, despite other duties and responsibilities, found the possibility to present excellent and comprehensive overviews of the most important recent findings in their field of scientific engagement within this topic. We would also like to thank Cristina Aves dos Santos, Sara Huisman, and Peter Butler from Springer Publishers for their kind assistance and excellent collaboration on this project, as well as for giving the opportunity to realize this book project.

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We hope that this book may encourage new scientific approaches within the field of stem cell biology in health and disease as well as closer interdisciplinary collaborations on this fascinating and important issue in the future.

References 1. Petersen BE, Bowen WC, Patrene KD, et al. (1999) Bone marrow as a potential source of hepatic oval cells. Science 284: 1168–1170 2. Theise ND, Badve S, Saxena R, et al. (2000) Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation. Hepatology 31: 235–240 3. Eisenberg LM and Eisenberg CA (2003) Stem cell plasticity, cell fusion, and transdifferentiation. Birth Defects Res Part C Embryo Today 69: 209–218 4. Nau KC and Lewis WD (2008) Multiple myeloma: diagnosis and treatment. Am Fam Physician 78: 853–859 5. Niederwieser D, Gentilini C, Hegenbart U, et al. (2005) Allogeneic hematopoietic cell transplantation (HCT) following reduced-intensity conditioning in patients with acute leukemias. Crit Rev Oncol Hematol 56: 275–281 6. Banna GL, Simonelli M, and Santoro A (2007) High-dose chemotherapy followed by autologous hematopoietic stem-cell transplantation for the treatment of solid tumors in adults: a critical review. Curr Stem Cell Res Ther 2: 65–82 7. Dittmar T, Seidel J, Zaenker KS, et al. (2006) Carcinogenesis driven by bone marrow-derived stem cells. Contrib Microbiol 13: 156–169 8. Gimble JM, Katz AJ, and Bunnell BA (2007) Adipose-derived stem cells for regenerative medicine. Circ Res 100: 1249–1260 9. Mimeault M and Batra SK (2006) Concise review: recent advances on the significance of stem cells in tissue regeneration and cancer therapies. Stem Cells 24: 2319–2345 10. Orlic D, Kajstura J, Chimenti S, et al. (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410: 701–705 11. Orlic D, Kajstura J, Chimenti S, et al. (2001) Transplanted adult bone marrow cells repair myocardial infarcts in mice. Ann NY Acad Sci 938: 221–229; discussion 229–230 12. Strauer BE, Brehm M, Zeus T, et al. (2002) Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 106: 1913–1918 13. Balsam LB, Wagers AJ, Christensen JL, et al. (2004) Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 428: 668–673 14. Murry CE, Soonpaa MH, Reinecke H, et al. (2004) Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 428: 664–668 15. Meyer GP, Wollert KC, Lotz J, et al. (2006) Intracoronary bone marrow cell transfer after myocardial infarction: eighteen months follow-up data from the randomized, controlled BOOST (BOne marrOw transfer to enhance ST-elevation infarct regeneration) trial. Circulation 113: 1287–1294 16. Choi JH, Choi J, Lee WS, et al. (2007) Lack of additional benefit of intracoronary transplantation of autologous peripheral blood stem cell in patients with acute myocardial infarction. Circ J 71: 486–494 17. Kassmer SH, Niggemann B, Punzel M, et al. (2008) Cytokine combinations differentially influence the SDF-1alpha-dependent migratory activity of cultivated murine hematopoietic stem and progenitor cells. Biol Chem 389: 863–872 18. Kim JB, Zaehres H, Wu G, et al. (2008) Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454: 646–650 19. Takahashi K, Tanabe K, Ohnuki M, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872

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20. Yu J, Vodyanik MA, Smuga-Otto K, et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917–1920 21. Okita K, Nakagawa M, Hyenjong H, et al. (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science 322: 949–953 22. Rubio D, Garcia-Castro J, Martin MC, et al. (2005) Spontaneous human adult stem cell transformation. Cancer Res 65: 3035–3039 23. Rubio D, Garcia S, De la Cueva T, et al. (2008) Human mesenchymal stem cell transformation is associated with a mesenchymal-epithelial transition. Exp Cell Res 314: 691–698 24. Houghton J, Stoicov C, Nomura S, et al. (2004) Gastric cancer originating from bone marrowderived cells. Science 306: 1568–1571 25. Li HC, Stoicov C, Rogers AB, et al. (2006) Stem cells and cancer: evidence for bone marrow stem cells in epithelial cancers. World J Gastroenterol 12: 363–371 26. Wicha MS, Liu S, and Dontu G (2006) Cancer stem cells: an old idea – a paradigm shift. Cancer Res 66: 1883–1890; discussion 1895–1886 27. Eyler CE and Rich JN (2008) Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis. J Clin Oncol 26: 2839–2845 28. Rich JN (2007) Cancer stem cells in radiation resistance. Cancer Res 67: 8980–8984 29. Shervington A and Lu C (2008) Expression of multidrug resistance genes in normal and cancer stem cells. Cancer Invest 26: 535–542 30. Huff CA, Matsui W, Smith BD, et al. (2006) The paradox of response and survival in cancer therapeutics. Blood 107: 431–434 31. Blagosklonny MV (2005) Why therapeutic response may not prolong the life of a cancer patient: selection for oncogenic resistance. Cell Cycle 4: 1693–1698 32. Li F, Tiede B, Massague J, et al. (2007) Beyond tumorigenesis: cancer stem cells in metastasis. Cell Res 17: 3–14 33. Dittmar T, Nagler C, Schwitalla S (2009). Recurrence cancer stem cells - made by cell fusion? Med Hypotheses: doi: 10.1016/j.mehy.2009.05.044 34. Hermann PC, Huber SL, Herrler T, et al. (2007) Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1: 313–323 35. Shafee N, Smith CR, Wei S, et al. (2008) Cancer stem cells contribute to cisplatin resistance in Brca1/p53-mediated mouse mammary tumors. Cancer Res 68: 3243–3250

Chapter 2

Hematopoietic Stem and Progenitor Cells in Clinical Use – Transplantation and Mobilization Michael Punzel

Abstract It took exactly 100 years from the original discovery of the blood formation within the bone marrow until the first successful clinical bone marrow transplantation has been performed. Today, the transplantation of hematopoietic stem cells from various sources, such as bone marrow, mobilized stem cells as well as umbilical cord blood has become a routine procedure, reaching currently more than 10,000 transplantations per year in the allogeneic setting and over 40,000 autologous transplantations. Although, the number of transplantations is increasing every year, the field is constantly changing in terms of conditioning procedures and clinical indications. In addition, the increase in the availability of multiple graft sources for allogeneic transplantation, such as related or unrelated living donors versus frozen umbilical cord blood as well as the choice between mobilized peripheral blood versus steady state bone marrow is challenging not only for transplant physicians but also for the donors. This chapter provides an overview about the history of stem cell transplantation, current procedures and future developments in terms of donor selection and graft choices for hematopoietic stem cell transplantation. Keywords Hematopoietic stem/Progenitor cells · Bone marrow · Peripheral blood stem cells (PBSC) · Umbilical cord blood (UCB) · Stem cell transplantation · Stem cell mobilization · G-CSF · AMD3100 · Graft-versus-host-disease (GVHD) · CD34

Contents 2.1 Historical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Stem Cell Donors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Stem Cell Mobilization and Autologous Transplantation . . . . . . . . . . . . .

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M. Punzel (B) Institute of Transplantation Diagnostic and Cellular Therapeutics, Universitätsklinikum Düsseldorf, 40225 Düsseldorf, Germany e-mail: [email protected] T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_2, 

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2.4 Allogeneic Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2.1 Historical Aspects The origin of blood formation within the bone marrow was discovered in 1868 independently by Ernst Neumann [1] and by Giulio Bizzozero [2]. The German hematologist Arthur Pappenheim postulated in 1898 a monophyletic basophil mononuclear precursor for all blood cells, followed by the “common stem cell” concept of Alexander Maximow which suggested a common stem cell among the small blood lymphocytes [3, 4]. Although the interest in this field had been present since these initial observations, research efforts took another step after the first atomic bomb explosions in the wake of world war II in attempts to prevent the lethal effects of irradiation. One of the most important discoveries at that time was the observation that marrow failure and subsequent lethality of photon beam irradiation in mice could be reduced by shielding the spleen and femur with lead [5]. In the following years experimental evidence from animal experiments in rodents could demonstrate that intravenous infusion of bone marrow protected them from lethal irradiation [6]. Although there was a long controversy about the origin of the protective effects of marrow infusions, in the mid-1950s it was well accepted that not humoral factors but transplantable hematopoietic stem cells are responsible for marrow protection [7, 8]. In 1957 the pioneer of clinical stem cell transplantation, E. Donnall Thomas, published results on infusing unrelated bone marrow into six patients. Although all patients died and only one of them had transient engraftment, this particular report is considered as the seminal paper of modern hematopoietic stem cell transplantation. Thomas and colleagues showed for the first time that human bone marrow could be collected in significant quantities and could be administered safely after cryopreservation [9]. Two years later, Thomas s team performed the first successful bone marrow transplantation in a 3-year-old girl with leukemia using marrow donated from her identical twin. The girl did well for six months until her leukemia relapsed [10]. At this time it became evident, that alloreactivity is one of the most crucial factors for this therapeutic concept in two ways: On one hand the alloreactivity is directed against the tumor cells and protects the patient from relapse but on the other hand it caused fatal graft-versus-host disease (GVHD) if no identical twin has served as bone marrow donor. Doubts were raised if the “allogeneic barrier” could ever be passed since it turned out that the graft-versus-host (GVH) reaction in man was much more violent compared to inbred rodents [8]. The fatalities of allogeneic marrow infusions in the clinic setting caused most investigators to abandon such studies in the 1960s.

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However, under the impetus of accumulating knowledge of the human histocompatibility system, researchers laid the foundation for modern bone marrow transplantation. The Seattle group around ED Thomas developed matching strategies for bone marrow transplantations in dog experiments and related their results to the human leukocyte antigen (HLA)-system [11, 12]. Important knowledge to the field was added by Till and McCulloch in a series of experiments, which are generally considered as the beginning of the modern area of hematopoietic stem cell biology. Starting in 1961 the group demonstrated clonogenic colony formation of all hematopoietic lineages in the spleen (colonyforming-unit-spleen; CFU-S) in lethally irradiated mice after transplantation with bone marrow cells from healthy donor animals [13–15]. Thus, for the first time evidence was provided for the dose dependent, clonal repopulation, differentiation and self-renewing capacity of hematopoietic stem cells. The area of modern clinical bone marrow transplantation began in November 1968 when Robert Good from the University of Minnesota, USA carried out the first marrow transplantation in a 5-month-old boy with hereditary immunodeficiency that had killed 11 male members of his extended family with marrow from his 8-yearold matched sister [16]. Only 4 months later the Seattle group performed the first successful adult bone marrow transplantation in a patient with advanced leukemia using bone marrow from an HLA-matched sibling [17]. In the early years bone marrow transplantation was still restricted to patients with end-stage or refractory disease status and most patients were in poor condition at the time of transplantation, which resulted in a high proportion of deaths related to this therapy. Due to the myeloablative conditioning regimen that consisted of chemotherapy and total body irradiation various efforts had been made in the 1970s to decrease this transplant related mortality. On the one hand, a continuous improvement in the supportive therapy of blood cell substitution, antifungal, antimicrobiotic and antiviral chemoprophylaxis as well as nutritional supportive care could be achieved. On the other hand, the introduction of effective immunosuppressive agents in the GVHD-prophylaxis regimen, i.e. methotrexate and cyclosporine A improved the outcome of transplantation continuously [18, 19]. Important observations on the road to common practice for stem cell transplantation were published in the mid 1970s by the Seattle group: (i) Patients that were in better clinical condition at the time point of transplantation had a better long-term survival than those in poor condition, (ii) 75% of patients with advanced hematological disease relapsed after HSC-Tx, and (iii) the general proof of significant disease-free long-term survival in the first large cohort of patients with leukemia/lymphoma and aplastic anemia after failure of conventional therapy was encouraging to the field [17, 20]. Consequently, the number of patients referred for bone marrow transplantation at earlier stage of disease and in good clinical condition improved the field of allogeneic stem cell transplantation to full recognition as a clinical routine procedure in hematologic malignancies. As all subsequent studies confirmed the success of this treatment, E. Donnall Thomas received the Nobel Price for his pioneering work in clinical bone marrow transplantation in 1990.

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2.2 Stem Cell Donors While the number of transplants involving related donors increased continuously and proved to be successful, only 25–35% of patients had a matched sibling donor available. Further advances in histocompatibility typing technologies made it possible to include unrelated donors. In the beginning, serological matching for HLA-A, HLA-B and HLA-DR-loci and a non-reactive mixed lymphocyte culture (MLC) was required for donor selection and proved to be feasible in early clinical studies [21, 22]. In 1974 the initiative of recruitment of unrelated volunteers willing to donate bone marrow for anybody was started by Shirley Nolan in the United Kingdom in the search for bone marrow donors for her son, Anthony [23]. The Anthony Nolan Trust was the first active donor registry in the world. Today in almost every developed country registries with HLA-typed volunteers have been established, which have raised the chance for patients to find a suitable unrelated donor. Per November 2008 more than 12.5 [24] million donors have been registered world wide, of those more than 25% are registered in Germany [25]. This corresponds to more than 10% of all Germans between the age of 18–60 who have volunteered for a possible bone marrow donation. In Germany there are currently 29 national and local donor registries [25]. Since one third of all transplants worldwide requires a graft from a foreign country, searching all the national and local registries in the world step by step separately is virtually impossible and only at considerable expense and time [26]. Thus, several platforms and networks have been established to provide an easy accessible listing of all donors nationwide as well as worldwide. Beginning in 1988 the Bone Marrow Donors Worldwide (BMDW) database has been summarizing the data of most registries in the world [27]. The World Marrow Donor Association (WMDA) has defined policies and procedures for international data exchanges [26]. Since more than 95% of all unrelated transplants are facilitated through the pool of complete HLA-typed donors, it was of great importance that the number of donors which have been typed for HLA-A, -B and -DR increased up to 9.6 million. This relates to approximately 75% of all available donors worldwide [27]. However, due to the diversity of HLA-allele and haplotype frequencies in human populations, the vast majority of patients that can be provided with a full matched donor belong to the Northern European (Caucasian) ethnicity only. Therefore, many efforts have been undertaken to establish ethnic minority programs within most of the registries, i.e. within the largest single registry worldwide, the National Marrow Donor Program (NMDP) in the USA. This has resulted in a significant increase of donor availability especially for the Afro-American population within the NMDP [28]. Currently, the optimal choice for an unrelated donor is a full allele-match for HLA-class I (HLA-A, -B, -C) as well as two matched gene loci of HLA-class II (HLA-DRB1, -DQB1). This requires expensive high resolution DNA-typing. Challenges in terms of transplantation outcome still remain in undetected variations of the human major histocompatibility complex (MHC) as well as in non-genetic factors such as the disease status of the patient.

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During the 1980s, umbilical cord blood, which is collected from the umbilical cord and placenta of healthy newborns, has emerged as an alternative clinical source for hematopoietic stem cell transplantation. Elaine Gluckman performed in 1988 in Paris the first successful clinical transplantation in a six-year old boy suffering from Fanconi-anemia using umbilical cord blood (UCB) from a sibling [29]. In 1992, the first public UCB-bank was established in the New York Blood Center followed by institutions in many countries. In 1993, the first unrelated UCB-transplantation was performed at Duke University in the USA. Today, there are more than 330,000 UCB-units stored and available through the BMDW database and it is estimated that more than 14,000 unrelated UCB-transplantations have been performed so far [27, 30]. There are major differences between stem cell transplantations using grafts from adult donors or alternatively from UCB. UCB-transplants require fewer nucleated cells/kg body weight (>2.5×107 /kg) than bone marrow grafts (>2×108 /kg) and only 3 HLA-loci (HLA-A, -B, -DRB1) are relevant for transplantation at allelic level. Due to the lower alloreactivity of cord blood derived immune cells grafts with a limited HLA-disparity (1–2 allele mismatches) are suitable for transplantation [31, 32]. Over the last years it became evident that the nucleated cell dose, which correlates directly with the number of hematopoietic stem and progenitor cells in the UCB-transplant, is of higher priority than a full HLA-match [31–33]. This is significant since in the early years of UCB-banking many UCB-grafts were stored with only limited cell numbers [34, 35]. For this reason UCB-transplantations had been performed almost exclusively in children until the end of the last century [34, 35]. To overcome these limitations and to provide sufficient cell doses for adult patients novel graft selection strategies are under investigation. One attempt is the simultaneous transplantation of two UCB-units if the cell number of one single cord is insufficient, called “double cord blood” transplantation. Both of the two UCB-units must be matched to each other as well as to the patient appropriately, at least with 5/6 relevant alleles [36]. Another strategy has been the use of purified haploidentical stem and progenitor cells in conjunction with one UCB-unit. The haploidentical stem cells provide rapid engraftment and serve temporarily as “bridging cell unit” until the UCB engrafts and finally rejects the haploidentical cells from the patient’s relative [37, 38]. Based on these encouraging results and the increasing availability of suitable UCB-units in the BMDW-database, UCB-transplantation will become a valid alternative in the field of adult stem cell transplantation also for adults [31, 35, 39, 40].

2.3 Stem Cell Mobilization and Autologous Transplantation Encouraged by the rapid clinical development in the field of allogeneic bone marrow transplantation along with the feasibility of harvesting, processing, cryopreserving and reapplication of bone marrow cells, the concept of high dose chemotherapy

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with subsequent autologous transplantation has been proven safe and feasible for lymphohematologic malignancies as well as certain immune disorders [40]. Unlike allogeneic transplantations high dose chemo- and radiation therapy with autologous stem cell support can be performed in elderly patients as well without the significant mortality of transplantation related complications. Between 2002 and 2006 sixtytwo percent of all autograft recipients were older than 50 [41]. Since 1962 it has been known that peripheral blood leukocytes fully reconstituted lethally irradiated mice of the same genetic strain [42]. In humans, hematopoietic stem cells in the peripheral blood were reported in the early 1970s [43, 44]. An increase in the amount of human hematopoietic stem cells in the peripheral blood was observed after chemotherapy for the first time in 1976 [45]. The amount of hematopoietic stem and progenitor cells in the peripheral blood was determined by the number of colonies that could be generated in semisolid methylcellulose cultures. These colonies have been defined as Colony-Forming Units (CFUs) at different stages of maturation. The numbers of CFUs for granulocytes and macrophages (CFU-GM), CFUs for erythroid colonies (CFU-E) as well as the number of CFUs for more primitive CFU-GEMM (mixed colonies for granulocytes, erythroid cells and monocytes/macrophages) directly relates to the amount of vital stem and progenitor cells with repopulating capacity in the peripheral blood [43, 44, 46–48]. Thus, such colony assays are still in place as quality control measurement of cryopreserved stem cells. Finally, the technical development of cell separators made it possible to collect clinically relevant amounts of stem cells from the peripheral blood [49]. The disadvantage of time delay inherent in the methylcellulose assays lead to the application of immunophenotyping for stem and progenitor cell determination. One of the most important discoveries in the field was the establishment of the CD34-membrane glycoprotein as a surrogate marker for the clinical enumeration of human stem and progenitor cells for transplantation [50, 51]. Initial mobilization regimens and proof of principle for the feasibility of autologous transplantations were pioneered in 1979 by Goldman and colleagues in 6 patients with myeloproliferative disorders [52]. The first successful clinical transplantation after myeloablative radiochemotherapy with large numbers of chemotherapy mobilized peripheral blood stem cells (PBSC) being transplanted was performed in 1985 in Heidelberg, Germany. The rapid hematopoietic reconstitution within 9 days suggested an advantage over bone marrow and paved the way for the preferred use of mobilized PBSC as stem cell source today [53]. To et al. established the modern chemotherapy based mobilization regimen in the autologous transplantation setting as single infusion of cyclophosphamide (4 g/m2 ) that is still the gold standard, despite minor modifications [54–56]. The discovery and clinical development of human hematopoietic growth factors such as Granulocyte-colony stimulating-factor (G-CSF) and GranulocyteMacrophage colony-stimulating factor (GM-CSF) allowed the collection of larger amounts of hematopoietic stem cells compared to chemotherapy alone [57]. Since the mobilizing effect of G-CSF was better than GM-CSF the latter did not make it to a widespread clinical use. The addition of G-CSF to chemotherapy based mobilization regimens led to the favorable use of mobilized PBSC as autologous grafts [58].

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Today, the use of mobilized peripheral blood accounts for 90% of all autotransplants in children and for more than 95% in adults [41]. As minimal required cell dose, 1– 2×106 CD34+ cells per kg body weight have been established without any clinical benefit using CD34+ cell doses >8×106 /kg [55, 59]. Clinical indications and frequencies of high dose therapies with autologous stem cell transplantation have changed over the past decade. The number of autologous transplantations that is performed annually had risen from approximately 5,000 in early 1990 to almost 40,000 in 1999 worldwide [41]. This was mainly due to the introduction of high dose chemotherapy in solid tumors, such as malignant melanoma, small cell lung cancer, colon cancer and in particular breast cancer. The initial enthusiasm about preliminary results turned into disappointment after the first randomized studies did not show any significant survival differences compared to conventional treatment. The latter data together with the disclosure of scientific misconduct in one of the breast cancer trials [60] has virtually abandoned autologous transplantations in the treatment of most non-hematologic malignancies. However, high dose therapy in other diseases, such as multiple myeloma or systemic amyloidosis has emerged as preferred treatment modality and thus, the number of autologous transplantations is on the increase again since 2002. Today, multiple myeloma is the most common indication for high dose therapy and autologous transplantation with a 3-year survival probability of 68% [41]. Similar results could be obtained for relapsed diffuse large cell B-cell lymphoma (DLBCL) with a 3-year survival probability of 61% in chemosensitive disease as well as for relapsed or aggressive follicular lymphoma (FL) with a 3-year survival probability of 73% in chemosensitive disease [41]. Several major studies have shown the advantages of mobilized peripheral blood over bone marrow as stem cell source for autologous transplantation [61–63]. Patients that have received autologous mobilized PBSC-transplantations showed a more rapid granulocyte and platelet recovery, enhanced immune reconstitution and subsequently a reduced transplant related morbidity [64–66].

2.4 Allogeneic Transplantation The emergence of mobilized PBSC as preferred autologous stem cell source has sparked the use of G-CSF in healthy donors to obtain allogeneic PBSC-grafts with similar advantages as has been shown for the autologous setting [67, 68]. Studies that compared G-CSF-mobilized PBSC with bone marrow as graft source in related allogeneic HLA-identical transplantations demonstrated similar results for hematopoietic recovery as observed in the autologous setting: more rapid engraftment, less infectious complications and a lower transplantation related mortality were advantages of the PBSC-group [69–71]. Except one study, the rate of acute GVHD was not different in both graft sources but chronic GVHD was more frequent in patients that received PBSC-transplants [70–72]. Subsequently, the use of mobilized PBSC as preferred graft source for allogeneic transplantation has

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increased markedly in the last decade. With the exception of pediatric transplantation procedures, mobilized PBSC has been the most common source of allogeneic grafts from 2002 to 2006 in patients older than 20 years, with the use of PBSC twice as much as bone marrow [41]. The number of allogeneic grafts collected in Germany went over 3,000 in 2006, more than in any other country of the world. The data show that already 80% of these grafts were collected from mobilized PBSC and the numbers are rising [25]. Despite the preferred use of mobilized peripheral stem cells in allogeneic transplantations controversies still exist about long-term outcome from both adult graft sources [73]. Since most studies have demonstrated an increased risk of chronic GVHD in mobilized PBSC-transplantation, it is not yet clear whether this will result in higher late mortality or in a decrease of the relapse rate due to a prolonged graft versus malignancy effect. A meta-analysis of several randomized trials that compared the outcome of PBSC versus marrow as graft source in full matched sibling transplantations showed significant improvement in disease-free survival at 5 years (54–47%) which was associated with increased chronic GVHD (51–35%) and decreased relapse rate (24–32%) in favor of PBSC-grafts [74]. However, a recent study that had the longest follow up for matched sibling transplants so far could not confirm the improved 5-year disease-free survival from the metaanalysis after 6 years, despite confirmation of the increased chronic GVHD incidence [75]. Since the patient cohorts in both analysis were different, the advantage of mobilized PBSC in matched sibling transplants remains unclear. The first comprehensive analysis that compared bone marrow transplantations with mobilized PBSC allografts in matched sibling transplantations in the pediatric setting demonstrated a significant increased mortality of PBSC-transplants clearly attributed to the higher incidence of GVHD in the PBSC-group [76]. First data on long-term follow up in unrelated donor transplantations demonstrated an expected higher incidence in extensive chronic GVHD in the PBSC group (85 vs. 59%, p38 years, low baseline levels of CD34+ cells and single daily application instead of two applications per day were identified as predictors for poor mobilization [93–95]. In autologous patients that receive chemotherapy (cyclophosphamide) followed by G-CSF the mobilization failure rate is much higher and depends on previous chemotherapy, i.e. the cumulative dose of alkylating agents [92]. Recently, the better understanding of mechanisms in stem cell mobilization led to the discovery and subsequent clinical development of new mobilizing agents. The diversity and large number of hematopoietic growth factors, chemokines and cytotoxic agents that induces the release of hematopoietic stem and progenitor cells into the peripheral blood is somewhat surprising. Besides the clinically approved G-CSF and GM-CSF several cytokines, such as interleukin-3, interleukin-8, recombinant human growth hormone and stem cell factor, had been tested but did not make it to clinical use [84]. The introduction of a pegylated G-CSF molecule (Pegfilgrastim) with prolonged half-life into clinical use resulted in a more convenient single dose application but did not change the poor mobilization responses in some patients [96]. Clinical trials are currently underway to determine the efficacy of Pegfilgrastim as mobilizing agent in patients for autologous transplantations as well as in healthy donors.

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A specific CXCR-4 antagonist, called AMD3100, that reversibly inhibits the binding of SDF-1 to its receptor, is probably the most promising mobilizing agent of a new kind that has successfully passed clinical phase III studies and is expected to get clinical approval in Europe by 2009 [97–100]. Most importantly, in combination with G-CSF this drug allowed the mobilization of sufficient numbers of CD34+ cells into peripheral blood in poor mobilizers that previously failed G-CSF-mobilization [101, 102]. A single dose of AMD3100 causes a rapid and significant release of CD34+ cells from the bone marrow within 1 h. The number of peripheral progenitors peaks after 9 h and declines to baseline levels within 24 h, which allows stem cell harvest on the same day of application [84, 98, 103]. Although a single injection of AMD3100 results in a lower yield of CD34+ cells, it acts synergistically with G-CSF [101, 102]. Recently, two reports have demonstrated that a clinical grade antibody (natalizumab) approved to treat multiple sclerosis, was able to release clinically significant amounts of CD34+ stem and progenitor cells into peripheral blood by blocking VLA-4 [104, 105].

2.5 Outlook The increased availability of registered unrelated stem cell donors as well as suitable umbilical cord blood units has remarkably improved the outcome of allogeneic stem cell transplantations over the recent years and opens the perspective to choose from several available graft sources according to the specific conditions of each individual patient. This also includes the use of related haploidentical donors in various clinical settings. These donors are only partially HLA-matched relatives of the patients that are usually immediately available for transplantation workup. Based on initial results that have shown the feasibility of this approach despite the risk of graft failure and severe GVHD, modern concepts of haploidentical transplantations have incorporated reduced intensity conditioning (RIC) in the transplant procedure combined with high dose enrichment of CD34+ stem and progenitor cells [106–113]. Instead of purification of CD34+ cells by positive selection, the depletion of selected lymphocytes that leaves monocytes, Natural Killer cells (NKcells) and/or T-cell-subsets within the graft, has opened the perspective of targeted allogeneic immunotherapy by choosing stem cell donors that exhibit specific graft versus tumor/leukemia alloreactivity in the NK-cell repertoire but does not show significant graft versus host reactivity [112, 114–119]. The clinical introduction of AMD3100 and other possible mobilizing agents could change the field in many ways due to their different biological properties compared to G-CSF. Chemokine-receptor inhibitors release primitive hematopoietic cells into the blood stream that have differential cell cycle properties than G-CSF mobilized cells. The immunomodulatory effects of AMD3100 in the hematopoietic system differing from those observed after G-CSF treatment, the significant increase of circulating endothelial and angiogenic progenitor cells in the peripheral blood as

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well as additional (still unknown) properties open the exciting perspective of novel therapeutic approaches using mobilized peripheral stem cells. In addition, the release of stem cells from the niche by chemokine receptor inhibitors or antibodies against certain adhesion molecules, such as VLA-4, may lead to novel approaches to treat hematologic malignancies by releasing leukemic stem and progenitor cells from the niche into the peripheral blood that results in cell cycle entry and subsequently enhanced susceptibility to chemotherapy.

References 1. Neumann E (1868) Über die Bedeutung des Knochenmarks für die Blutbildung. Centralbl Med Wiss 44: 689. 2. Bizzozero G (1868) Sulla funzione ematopoetica del midollo delle ossa. Centralbl Med Wiss 6: 885. 3. Pappenheim A (1898) Abstammung und Entstehung der rothen Blutzellen. Arch Pathol Anat Physiol Klin Med 151: 89. 4. Maximow A (1909) Der Lymphozyt als gemeinsame Stammzelle der verschiedenen Blutelemente in der embryonalen Entwicklung und im postfetalen Leben der Säugetiere. Folia Haematol 8: 125. 5. Jacobson LO, Marks EK et al. (1949) Effect of spleen protection on mortality following x-irradiation. J Lab Clin Med 34: 1538–1543. 6. Lorenz E , Uphoff D et al. (1951) Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Natl Cancer Inst 12: 197–201. 7. Main JM and Prehn RT (1955) Successful skin homografts after the administration of high dosage X radiation and homologous bone marrow. J Natl Cancer Inst 15: 1023–1029. 8. van Bekkum DW (1970) Radiation chimeras. Transplant Proc 2: 479–482. 9. Thomas ED, Lochte HL, Jr. et al. (1957) Intravenous infusion of bone marrow in patients receiving radiation and chemotherapy. N Engl J Med 257: 491–496. 10. Thomas ED, Lochte HL, Jr. et al. (1959) Supralethal whole body irradiation and isologous marrow transplantation in man. J Clin Invest 38: 1709–1716. 11. Storb R, Epstein RB et al. (1968) Marrow grafts by combined marrow and leukocyte infusions in unrelated dogs selected by histocompatibility typing. Transplantation 6: 587–593. 12. Storb R, Rudolph RH et al. (1971) Marrow grafts between canine siblings matched by serotyping and mixed leukocyte culture. J Clin Invest 50: 1272–1275. 13. Till JE and Mc CE (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14: 213–222. 14. Becker AJ, Mc CE et al. (1963) Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197: 452–454. 15. Siminovitch L, McCulloch EA et al. (1963) The Distribution of Colony-Forming Cells among Spleen Colonies. J Cell Physiol 62: 327–336. 16. Gatti RA, Meuwissen HJ et al. (1968) Immunological reconstitution of sex-linked lymphopenic immunological deficiency. Lancet 2: 1366–1369. 17. Thomas ED, Storb R et al. (1975) Bone-marrow transplantation (second of two parts). N Engl J Med 292: 895–902. 18. Storb R, Epstein RB et al. (1970) Methotrexate regimens for control of graft-versus-host disease in dogs with allogeneic marrow grafts. Transplantation 9: 240–246. 19. Storb R, Deeg HJ et al. (1986) Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. N Engl J Med 314: 729–735.

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46. Udomsakdi C, Lansdorp PM et al. (1992) Characterization of primitive hematopoietic cells in normal human peripheral blood. Blood 80: 2513–2521. 47. Spitzer G, Verma DS et al. (1980) The myeloid progenitor cell – its value in predicting hematopoietic recovery after autologous bone marrow transplantation. Blood 55: 317–323. 48. Fauser AA and Messner HA (1979) Identification of megakaryocytes, macrophages, and eosinophils in colonies of human bone marrow containing neurtophilic granulocytes and erythroblasts. Blood 53: 1023–1027. 49. Weiner RS, Richman CM et al. (1977) Semicontinuous flow centrifugation for the pheresis of immunocompetent cells and stem cells. Blood 49: 391–397. 50. Civin CI, Strauss LC et al. (1984) Antigenic analysis of hematopoiesis. III. A hematopoietic progenitor cell surface antigen defined by a monoclonal antibody raised against KG-1a cells. J Immunol 133: 157–165. 51. Berenson RJ, Andrews RG et al. (1988) Antigen CD34+ marrow cells engraft lethally irradiated baboons. J Clin Invest 81: 951–955. 52. Goldman JM, Catovsky D et al. (1979) Cryopreserved peripheral blood cells functioning as autografts in patients with chronic granulocytic leukaemia in transformation. Br Med J 1: 1310–1313. 53. Korbling M, Dorken B et al. (1986) Autologous transplantation of blood-derived hemopoietic stem cells after myeloablative therapy in a patient with Burkitt’s lymphoma. Blood 67: 529–532. 54. To LB, Davy ML et al. (1989) Autotransplantation using peripheral blood stem cells mobilized by cyclophosphamide. Bone Marrow Transplant 4: 595–596. 55. To LB, Roberts MM et al. (1992) Comparison of haematological recovery times and supportive care requirements of autologous recovery phase peripheral blood stem cell transplants, autologous bone marrow transplants and allogeneic bone marrow transplants. Bone Marrow Transplant 9: 277–284. 56. To LB, Shepperd KM et al. (1990) Single high doses of cyclophosphamide enable the collection of high numbers of hemopoietic stem cells from the peripheral blood. Exp Hematol 18: 442–447. 57. Welte K, Platzer E et al. (1985) Purification and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor. Proc Natl Acad Sci USA 82: 1526–1530. 58. Stadtmauer EA, Schneider CJ et al. (1995) Peripheral blood progenitor cell generation and harvesting. Semin Oncol 22: 291–300. 59. Buscemi F, Indovina A et al. (1995) CD34+ cell subsets and platelet recovery after PBSC autograft. Bone Marrow Transplant 16: 855–856. 60. Bezwoda W (1999) Randomized, controlled trial of high dose chemotherapy (HD-VNVp) versus standard dose (CAF) chemotherapy for high risk, surgically treated, primary breast cancer, JCO, American Society of Clinical Oncology meeting (ASCO) Prcoeedings 1999 and related ASCO-statement letter February 4th, 2000. ASCO. 61. Beyer J, Schwella N et al. (1995) Hematopoietic rescue after high-dose chemotherapy using autologous peripheral-blood progenitor cells or bone marrow: a randomized comparison. J Clin Oncol 13: 1328–1335. 62. Hartmann O, Le Corroller AG et al. (1997) Peripheral blood stem cell and bone marrow transplantation for solid tumors and lymphomas: hematologic recovery and costs. A randomized, controlled trial. Ann Intern Med 126: 600–607. 63. Schmitz N, Linch DC et al. (1996) Randomised trial of filgrastim-mobilised peripheral blood progenitor cell transplantation versus autologous bone-marrow transplantation in lymphoma patients. Lancet 347: 353–357. 64. Bensinger W, Singer J et al. (1993) Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor. Blood 81: 3158–3163.

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65. Blume KG and Thomas ED (2000) A review of autologous hematopoietic cell transplantation. Biol Blood Marrow Transplant 6: 1–12. 66. Welte K, Gabrilove J et al. (1996) Filgrastim (r-metHuG-CSF): the first 10 years. Blood 88: 1907–1929. 67. Schmitz N, Dreger P et al. (1995) Primary transplantation of allogeneic peripheral blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor). Blood 85: 1666–1672. 68. Korbling M, Przepiorka D et al. (1995) Allogeneic blood stem cell transplantation for refractory leukemia and lymphoma: potential advantage of blood over marrow allografts. Blood 85: 1659–1665. 69. Champlin RE, Schmitz N et al. (2000) Blood stem cells compared with bone marrow as a source of hematopoietic cells for allogeneic transplantation. IBMTR Histocompatibility and Stem Cell Sources Working Committee and the European Group for Blood and Marrow Transplantation (EBMT). Blood 95: 3702–3709. 70. Cutler C, Giri S et al. (2001) Acute and chronic graft-versus-host disease after allogeneic peripheral-blood stem-cell and bone marrow transplantation: a meta-analysis. J Clin Oncol 19: 3685–3691. 71. Couban S, Simpson DR et al. (2002) A randomized multicenter comparison of bone marrow and peripheral blood in recipients of matched sibling allogeneic transplants for myeloid malignancies. Blood 100: 1525–1531. 72. Mohty M, Kuentz M et al. (2002) Chronic graft-versus-host disease after allogeneic blood stem cell transplantation: long-term results of a randomized study. Blood 100: 3128–3134. 73. Koca E and Champlin RE (2008) Peripheral blood progenitor cell or bone marrow transplantation: controversy remains. Curr Opin Oncol 20: 220–226. 74. Stem-Cell-Trialists’-Collaborative-Group (2005) Allogeneic peripheral blood stem-cell compared with bone marrow transplantation in the management of hematologic malignancies: an individual patient data meta-analysis of nine randomized trials. J Clin Oncol 23: 5074–5087. 75. Schmitz N, Eapen M et al. (2006) Long-term outcome of patients given transplants of mobilized blood or bone marrow: A report from the International Bone Marrow Transplant Registry and the European Group for Blood and Marrow Transplantation. Blood 108: 4288–4290. 76. Eapen M, Horowitz MM et al. (2004) Higher mortality after allogeneic peripheralblood transplantation compared with bone marrow in children and adolescents: the Histocompatibility and Alternate Stem Cell Source Working Committee of the International Bone Marrow Transplant Registry. J Clin Oncol 22: 4872–4880. 77. Ringden O, Remberger M et al. (1999) Peripheral blood stem cell transplantation from unrelated donors: a comparison with marrow transplantation. Blood 94: 455–464. 78. Remberger M, Ringden O et al. (2001) No difference in graft-versus-host disease, relapse, and survival comparing peripheral stem cells to bone marrow using unrelated donors. Blood 98: 1739–1745. 79. Remberger M, Beelen DW et al. (2005) Increased risk of extensive chronic graft-versus-host disease after allogeneic peripheral blood stem cell transplantation using unrelated donors. Blood 105: 548–551. 80. Garderet L, Labopin M et al. (2003) Patients with acute lymphoblastic leukaemia allografted with a matched unrelated donor may have a lower survival with a peripheral blood stem cell graft compared to bone marrow. Bone Marrow Transplant 31: 23–29. 81. Demetri GD and Griffin JD (1991) Granulocyte colony-stimulating factor and its receptor. Blood 78: 2791–2808. 82. Hernandez-Bernal F, Garcia-Garcia I et al. (2005) Bioequivalence of two recombinant granulocyte colony-stimulating factor formulations in healthy male volunteers. Biopharm Drug Dispos 26: 151–159. 83. Cashen AF, Lazarus HM et al. (2007) Mobilizing stem cells from normal donors: is it possible to improve upon G-CSF? Bone Marrow Transplant 39: 577–588.

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84. Nervi B, Link DC et al. (2006) Cytokines and hematopoietic stem cell mobilization. J Cell Biochem 99: 690–705. 85. Pelus LM (2008) Peripheral blood stem cell mobilization: new regimens, new cells, where do we stand. Curr Opin Hematol 15: 285–292. 86. Horowitz MM and Confer DL (2005) Evaluation of hematopoietic stem cell donors. Hematology Am Soc Hematol Educ Program 469–475. 87. Hernandez JM, Castilla C et al. (2005) Mobilisation with G-CSF in healthy donors promotes a high but temporal deregulation of genes. Leukemia 19: 1088–1091. 88. Nagler A, Korenstein-Ilan A et al. (2004) Granulocyte colony-stimulating factor generates epigenetic and genetic alterations in lymphocytes of normal volunteer donors of stem cells. Exp Hematol 32: 122–130. 89. Makita K, Ohta K et al. (2004) Acute myelogenous leukemia in a donor after granulocyte colony-stimulating factor-primed peripheral blood stem cell harvest. Bone Marrow Transplant 33: 661–665. 90. Bennett CL, Evens AM et al. (2006) Haematological malignancies developing in previously healthy individuals who received haematopoietic growth factors: report from the Research on Adverse Drug Events and Reports (RADAR) project. Br J Haematol 135: 642–650. 91. Shpilberg O, Modan M et al. (1994) Familial aggregation of haematological neoplasms: a controlled study. Br J Haematol 87: 75–80. 92. Moncada V, Bolan C et al. (2003) Analysis of PBPC cell yields during large-volume leukapheresis of subjects with a poor mobilization response to filgrastim. Transfusion 43: 495–501. 93. de la Rubia J, Arbona C et al. (2002) Analysis of factors associated with low peripheral blood progenitor cell collection in normal donors. Transfusion 42: 4–9. 94. Suzuya H, Watanabe T et al. (2005) Factors associated with granulocyte colony-stimulating factor-induced peripheral blood stem cell yield in healthy donors. Vox Sang 89: 229–235. 95. Lysak D, Koza V et al. (2005) Factors affecting PBSC mobilization and collection in healthy donors. Transfus Apher Sci 33: 275–283. 96. Molineux G, Kinstler O et al. (1999) A new form of Filgrastim with sustained duration in vivo and enhanced ability to mobilize PBPC in both mice and humans. Exp Hematol 27: 1724–1734. 97. Flomenberg N, Devine SM et al. (2005) The use of AMD3100 plus G-CSF for autologous hematopoietic progenitor cell mobilization is superior to G-CSF alone. Blood 106: 1867–1874. 98. Devine SM, Flomenberg N et al. (2004) Rapid mobilization of CD34+ cells following administration of the CXCR4 antagonist AMD3100 to patients with multiple myeloma and non-Hodgkin’s lymphoma. J Clin Oncol 22: 1095–1102. 99. Devine SM, Vij R et al. (2008) Rapid mobilization of functional donor hematopoietic cells without G-CSF using AMD3100, an antagonist of the CXCR4/SDF-1 interaction. Blood 112: 990–998. 100. Cashen A, Lopez S et al. (2008) A phase II study of plerixafor (AMD3100) plus G-CSF for autologous hematopoietic progenitor cell mobilization in patients with Hodgkin lymphoma. Biol Blood Marrow Transplant 14: 1253–1261. 101. DiPersio J, Micallef I et al. (2007) A Phase III, multicenter, randomized, double-blind, placebo controlled, comparative trial of AMD3100 (Plerixafor)+G-CSF vs. Placebo+G-CSF in Non-Hodgkin’s Lymphoma (NHL) patients for Autologous Hematopoietic Stem Cell (aHSC) transplantation. Blood 110: 601a. 102. Micallef I, Stiff P et al. (2007) Successful stem cell mobilization rescue by AMD3100 (Plerixafor) + G-CSF for patients who failed primary mobilization: rescue from phase III (3101-NHL) study. Blood 110: 602a. 103. Hendrix CW, Flexner C et al. (2000) Pharmacokinetics and safety of AMD-3100, a novel antagonist of the CXCR-4 chemokine receptor, in human volunteers. Antimicrob Agents Chemother 44: 1667–1673.

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Chapter 3

Ex Vivo Expansion of HSPCs Yaming Wei and Xin Ye

Abstract Transplantation of hematopoietic stem cells (HSCs, the cells that can give rise to all blood and most immune cell types) is a life-saving procedure for patients with hematopoietic malignancies, marrow failure syndromes, and hereditary immunodeficiency disorders. However, the wide application of this procedure is always limited either by availability of suitably HLA-matched adult donors or obtaining enough stem cell for a successful transplant. Over the past years, the results of ex vivo stem/progenitor cell expansion have been promising, numerous studies have described the effects of combinations of a variety hematopoietic growth factors on hematopoietic stem and progenitor cells (HSPCs) expansion in vitro. Most experimental evidence indicated that a combination of several cytokines such as stem cell factor (SCF), FLT-3/FLK-2 Ligand (Flt3-ligand), thrombopoietin (TPO) seems to be essential for progenitor amplification. Among these growth factors, SCF is unanimously agreed to be indispensable for stem and progenitor expansion and even shows to be a key factor for hematopoietic progenitor cell survival. With this cytokine cocktail, CD34+ cells can be expanded ex vivo about 10–1000-fold over pre-expanded values [1–3]. These kinds of expansion protocol provided sufficient numbers of hematopoietic progenitor cells to rapidly restore blood formation in patients undergoing high-dose chemotherapy or/and irradiation treatment. The development of ex vivo culture systems that facilitate the expansion of HSCs is crucial to stem cell research and clinical application. In this chapter, we describe the protocols to expand HSPCs ex vivo and analyze their population ability. This information is beneficial for successful use of stem cells in therapeutic studies. Keywords hematopoietic stem cell · progenitor cell · umbilical cord blood · ex vivo expansion · transplantation · cytokines · CD34

Y. Wei (B) Guangzhou Institute of Clinical Medicine, Guanzhou Municipal First People’s Hospital, Guangzhou Medical College, Guangzhou, China e-mail: [email protected] T. Dittmar, K.S. Zanker (eds.), Stem Cell Biology in Health and Disease, C Springer Science+Business Media B.V. 2009 DOI 10.1007/978-90-481-3040-5_3, 

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Contents 3.1 The Sources of HSPCs . . . . . . . . . . . . . . . 3.1.1 Bone Marrow HSPCs . . . . . . . . . . . 3.1.2 Peripheral HSPCs . . . . . . . . . . . . . 3.1.3 Umbilical Cord Blood HSPCs . . . . . . . 3.2 The Expansion of HSPCs . . . . . . . . . . . . . . 3.2.1 Cytokines . . . . . . . . . . . . . . . . 3.2.2 Ex Vivo Expansion of HSPCs . . . . . . . 3.2.3 Regulation of HSPCs Expansion . . . . . . 3.2.4 Free Radical Regulation on HSPCs Expansion 3.2.5 Megakaryocytic Progenitor Cells Expansion . 3.2.6 Red Cells Expansion . . . . . . . . . . . 3.2.7 T-Cell Expansion . . . . . . . . . . . . . 3.2.8 NK Cell Expansion . . . . . . . . . . . . 3.2.9 DC Expansion . . . . . . . . . . . . . . 3.2.10 HSPC Ex Vivo Expansion and Gene Therapy 3.3 Expansion Bioreactor . . . . . . . . . . . . . . . 3.4 The Application of Expanded HSPCs . . . . . . . . 3.4.1 Transplantation of HSPCs in Animal Model . 3.4.2 Transplantation of HSPCs in Human . . . . 3.5 The Future of HSPCs Expansion . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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28 28 29 29 31 31 31 34 35 36 36 37 38 39 41 41 44 44 47 48 48

3.1 The Sources of HSPCs Hematopoiesis in mammalian systems is initiated in the yolk sac (YS) and then migrates into embryo as the initial source of stem cells [4]. The bone marrow develops hematopoiesis at 11 week in human embryo [5]. After that time, bone marrow keeps the hematopoiesis capability in ones all life until the life ends or it is inhibited by disease or extra factors. All mature blood cells come from bone marrow stem/progenitor cells, include peripheral blood, and umbilical cord blood.

3.1.1 Bone Marrow HSPCs Hematopoietic stem/progenitor cells (HSPCs) reside in specific niches in the bone marrow and give rise to either more stem cells or maturing hematopoietic progeny depending on the signals provided in the bone marrow microenvironment. This microenvironment is comprised of cellular components as well as soluble constituents called cytokines. Therapeutic agents interrupt a stem and progenitor cell tethering to matrix molecules and stromal cells in the bone marrow environment. Stem cells and progenitor cells are released from their attachment to stromal cells

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when a cytokine-receptor axis such as SCF or CXCR4-stromal derived factor-1 is interrupted. The number of circulating peripheral blood HSCs can be increased in response to treatment with drugs and chemokines. Once released from its attachment, a stem cell undergoes transmural membrane migration through the opening in the basement membrane and endothelial lining of a blood sinus and enters the circulation. Bone marrow HSPCs were once used as the main source of clinical transplantation for a long time, but now, with the development of mobilization and collection of peripheral stem cells and cord blood, its application has become less and less.

3.1.2 Peripheral HSPCs The HSPCs in BM and blood are the ancestors of all mature blood cells. The initial report found that bovine fraternal twins sharing a common placenta and blood supply were each endowed with chimeric BM and lymphhematopoietic cells from its sibling after birth [6]. Experiments in 1940s, 1950s and 1960s demonstrated the existence of HSC in the circulation [7]. In the middle of 1980s, peripheral blood was found to be a stem cell resource to rescue patients following high-dose chemotherapy or chemoradiotherapy [8–11]. In the 1990s, with the realization that the yield of circulation primitive hematopoietic cells could be greatly increased during recovery from chemotherapy and/ or hematopoietic cytokines treatment, mobilized peripheral blood progenitor cells replaced BM as the source of stem cells for autologous and allogeneic transplantation [12, 13].

3.1.3 Umbilical Cord Blood HSPCs Umbilical cord blood (UCB) is a valuable source of the rare but precious primitive HSCs and progenitor cells, UCB stem cell transplantation (CBSCT) has approached significant success in treatment of lethal congenital or malignant disorders. UCB from sibling with more than one human leukocyte antigen (HLA) loci mismatches or unrelated partially mismatched donors has been increasingly used to reconstitute the hematopoietic system in patients after myeloablative therapy. UCB cells possess an enhanced capacity for progenitor cell proliferation and self-renewal in vitro. Moreover, CBSCT shows a relatively low incidence and severity of graft-versushost disease (GVHD) [14]. UCB has advantages of easy collection and storage, no risk to donors, low risk of transmitting infections, immediate availability and immune tolerance allowing successful transplantation despite HLA disparity. UCB is usually discarded, and it exists in almost limitless supply. Cord blood stem cells can be collected only once, an average 50–100 ml of blood containing stem cells is obtained from the umbilical cord and the placenta after birth. The predominant collection procedure currently practiced involves a relatively simple venipuncture, followed by gravity drainage into a standard sterile anti-coagulant-filled blood bag, using a closed system, similar to the one utilized on whole blood collection. After

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aliquots have been removed for routine testing, the units are cryopreserved and stored in liquid nitrogen. UCB banks have being established throughout the world. UCB units are collected for allogeneic unrelated and related HSC transplantation. In unrelated cord blood banks, donated UCB units are collected and stored for allogeneic use in patients who do not have an identified HLA matched relative. UCB banks report available units to national and international donor registries. The second model of UCB banking is referred to as family banking, where UCB is stored for the benefit of the donor or their family members. According to the National Marrow Donor Program (NMDP), more than 6,000 men, women and children are searching the NMDP registry on any given day. After more than one decade of clinical experience, it is currently accepted that UCB transplants, related and unrelated, are equivalent to or might compare favorably with bone marrow (BM) transplants, especially in children. Initial studies of long-term survival in children with both malignant and non-malignant hematologic disorders, who were transplanted with UCB from a sibling donor, demonstrated a comparable or superior survival rate to the children who received BM transplantation [15]. Since the first cord blood transplantation was performed in 1988 [16], the UCB transplantation program was established nearly all over the world. Up to 2005, two large groups from European and North American retrospective studies demonstrated that UCB is an acceptable alternative source of HSCs for adult recipients who lack HLA-matched adult donors, over 10,000 UCB transplant procedures in children and adults have been performed worldwide using UCB donors [17]. The greater the number of umbilical stem cells used, the better the prospects for healing will be. UCB cells are showing their unique qualities and potential, and consequently UCB banks might dramatically increase the scope of their clinical application [15]. UCB is anticipated to address needs in both transplantation and regenerative medicine fields. One factor that limits the use of UCB transplantation in adult patients is the relatively limited number of HSC that may be harvested from umbilical cord, resulting in a more time to engraftment and higher transplant related mortality, mainly due to the long aplasia period after transplantation and susceptibility to viral and fungal infections. To allow for multiple uses and also to increase the capacity for transplantation in adolescents and adults, researchers are developing methods to stimulate stem cells to divide and increase in number while retaining their primitive state. This has prompted intensive research on ex vivo expansion of UCB stem cells and UCB graft-engineering including accessory cells able to improve UCB engraftment and reconstitution and for tissue regenerative potential. Expanding the volume of stem cells would allow more patients to be treated, including adults. It would also allow families who have privately banked their cord blood stem cells to use them for multiple treatments and even potentially donate a portion of their cord blood sample to patients in need. The current strategies are focused on the development of much more efficient technologies for ex vivo expansion of HSPCs, such expanded stem cells have been proposed as elements suitable for cellular therapy and regenerative medicine.

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3.2 The Expansion of HSPCs Due to the low yield of HSC from typical sources, HSC transfusion has historically been most effective in children with limited applications and marginal success in adults. In recent years, the development of media used to expand and mature adult stem cells has greatly increased the number of candidates eligible as well as the success rate of adult stem cell therapy. In order to obtain sufficient numbers of cells for applications of this therapeutic approach in adults, ex vivo expansion has been utilized to ensure successful engraftment and minimize the short-term effects of neutropenia and thrombocytopenia. The following shows main cytokines and their functions which include media optimized for the expansion and maturation of various adult stem cell types.

3.2.1 Cytokines When the hematopoiesis system feels signal changes come from inflammation and cytopenia, increased levels of hematopoietic growth factors (HPGFs) induce in vitro mobilization and proliferation of HSC and hematopoietic progenitor cells, resulting in spatial and quantitative in vivo expansion of the hematopoietic tissue. HPGF are also known as colony stimulating factor (CSF), it was originally given to agents recognized to stimulate the growth of colonies containing differentiated myeloid cells from a single bone marrow-derived precursor cell plated in semisolid agar. CSF glycoproteins considered to include: granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF); macrophage colony-stimulating factor (M-CSF); interleukin-3 (IL-3); IL-5; erythropoietin (EPO), and TPO [18]. In later of 1990s, cytokines were introduced to ex vivo expand human umbilical cord blood HSPCs cells and to elucidate its capacities of self-renewal potential and reconstitution in mice. Exogenous administration of recombinant HPGFs, followed by collection and transplantation of autologous or allogeneic stem cells is routine for mobilization of stem cells. In animal experiments, recombinant SCF was injected into mice for 7 day inducing a 10-fold increase in HSPCs in the absolute number of HSC in total blood volume from a baseline value of 10–100, and a decrease in the number of HSPCs in bone marrow from 2,400 to 900, the overall increase in HSPC was three fold [19]. Most of these growth factors and cytokines have already been used both in research and clinical treatment. An overview of cytokines and its stimulated cells is given in Table 3.1.

3.2.2 Ex Vivo Expansion of HSPCs The expansion of UCB stem cells at differing stages of maturity has been successfully repeated in recent years. Depending on the composition of the experiment, an expansion fold of 10 to more than 1,000 has been achieved. What is most important

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Y. Wei and X. Ye Table 3.1 Cytokines and their target cells

Recombinant cytokines

Stimulated cells

Erythropoietin (EPO)

Erythroid progenitor cells. EPO receptors are lost during cell differentiation. Primitive hematopoietic (multi-lineage) progenitor cells that express the FLT-3 receptor. Myeloid and pro-B cells. Proliferation, differentiation, survival and activation factor for hematopoietic restricted granulocyte lineage cells. Neutrophils, myeloid leukemia cells, neutrophilic granulocytes. Growth, differentiation, and essential survival factor for granulocyte, macrophage and eosinophil lineage cells from progenitor stage to maturity. Most types of myeloid progenitor cells, mature monocytes, neutrophils, eosinophils, basiophils, dendritic cells and epithelial cells and osteoclasts. Multi-potential hematopoietic progenitor cells: macrophage, neutrophils, mast cells and megakaryocytes from bone marrow, and Stimulate T-cells and induce IgG secretion from activated B-cells. Wide range of cell types, such as fibroblasts, myeloid progenitor cells, T-cells, B-cells, and hepatocytes. Primarily targets macrophages and stimulates multiple responses, such as proliferation, cytokine and inflammatory modulator release, cytotoxicity and pinocytosis; osteoclast differentiation and placental trophoblasts. Broad activities on hematopoietic, pigment and primordial germ cell lineages, increase myeloid, erythroid, and lymphoid lineage colonies. Primary regulatory factor of growth and maturation of megakaryocytes and their progenitors for megakaryocytopoiesis and thrombopoiesis.

FLT-3/FLK-2 Ligand

Granulocyte Colony-Stimulating Factor (G-CSF)

Interleukin-3 (IL-3)

Interleukin-6 (IL-6)

Macrophage Colony-Stimulating Factor (M-CSF) Stem Cell Factor (SCF)

Thrombopoietin (TPO)

is not the expansion of all (both differentiated and undifferentiated) cells in the cord blood but the expansion of undifferentiated stem cells. Bone marrow stem cells were successfully expanded and then subsequently transplanted, the expanded stem cells regenerated the immune system in all case following chemotherapy. Mobilized peripheral blood (PB) is another important resource of HSPCs. Kawano et al. [20] assessed the efficacy of PB CD133+ cells in a coculture system (contained SCF, THP TPO and Flk-2/Flt3-ligand) using human telomerized stromal (HTS) cells. PB CD133+ cells proliferated efficiently above the stromal layer, while maintaining the characteristics of CD133+ cells, even after long-term hematopoietic-stromal interaction. In clinical trials, PB stem progenitor mobilization is always carried out and collected from donors, which is much more convenient, efficient, and economical than ex vivo expansion.

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UCB is a promising source of HSCs for allogeneic transplantation. Many published research indicates that CB stem cells can be successfully expanded under certain combination of cytokines and preserve the activity of transplanted cells [21, 22]. Others have evaluated the possibility of increasing the number of competitive repopulating units in a NOD-SCID murine recipient [23, 24]. Guenechea et al. [25] found that these cells retained their capacity to support long-term repopulation with delayed engraftment as compared to fresh cells, whereas Piacibello et al. [26] reported that CB CD34+ cells expanded for up to 10 weeks maintained their in vitro repopulating potential. Cord blood has also been proved to prompt the recovery of immune function in children who underwent CBT and this reconstitution was favored by the reduced incidence and severity of GVHD observed [27, 28]. Mohamed et al. [29] defined optimal conditions composed with SCF, GMCSF, IL-3, TPO for ex vivo expansion of CB stem cells, the culture expanded for 7 days was better than 11 days, if more cytokines added (IL-6 and Flt3L), the fold expansion of CD34+ cells were not significantly increased or even decreased, even apoptotic cells (CD95+ cells) were observed. Yao et al. [30] optimized another serum-free and cytokines-limited medium using statistic methodology for UBC-derived HSC expansion. After a 7-day culture, the average absolute fold expansions were CD133+ cells 21-fold, CD34+ CD133+ cells 20-fold, CD34+ CD38+ cells 723-fold, CD133+ CD38- cells 618-fold, CD34+ CXCR4+ cells 160-fold, CD133+ CXCR4+ cells 384-fold and long-term culture-initiating cells 8fold, respectively. In terms of telomere length and telomerase activity compared to adult HSCs, the expansion of human CB HSCs is instrumental in obtaining a large number of “good quality” cells, these expanded cells showed a high level of telomerase activity to maintain their telomere length and repopulated the lethally irradiated NOD/SCID mice in vivo [30, 31]. Madkaikar et al. [32] combined different cytokines and other support factors, assayed the mean CD34+ cell count, fold expansion, viability, clonogenic assays and immunophenotypic characterization at 7, 12 and 14 culture day, the maximum expansion was achieved using cytokines cocktail (SCF + IL-3 + GM-CSF) with stromal cell support, the mean CD34+ cell expansion on day 7 and 12 was 16.25- and 21.4-fold respectively, and the mean nucleated cell expansion was 15.1- and 21-fold, CFU-GEMM showed a 20.4-fold increase after 12 days. These cells can provide enough cells from a single cord blood unit to reduce the period of cytopenia after single unit cord blood transplantation. Wei et al. [33, 34] defined 7 groups of incubation conditions for ex vivo expansion and amplification CD34+ cell from UCB MNCs, all groups contained basic combination of SCF, IL-3, IL-6 for stimulating CD34+ cells expansion. Each test group showed significant increasing results of CD34+ cell either in percentage or expanded fold manner on 3, 7, 14 culture day compared to the decreasing in control group, which contains medium alone. The fold expansion rates of CD34+ cell numbers ranged from 10 to 50-fold as compared to the fresh UCB. The addition of IL-7, IL-2 or IL-4 into basic cytokine cocktails probably improved the expression of CD34 antigens on cells and increased CD34+ cell ratio respectively. On 7 expansion day, the least expansion of CD34+ cells number in basic

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combination with SCF, IL-3, IL-6 group was about 10 fold of that in fresh UCB, and enough for an adult transplantation. The expanded cells were able to engraft in the SCID recipients and reconstitute their hematopoiesis. Human hematopoietic cells could be detected in the marrow of the recipients 6 weeks after transplantation [35]. It seems possible to expand hematopoietic cells ex vivo efficiently and maintain concomitantly their self-renewal and hematopoietic reconstitution capacities by the combination of cytokines [36].

3.2.3 Regulation of HSPCs Expansion Although HSCs cycle and expand provide compelling evidence for a positive and dynamic regulation of HSC self-renewal [37, 38], the physiological regulators of HSC self-renewal and expansion remain largely unknown. However, besides cytokine genes commonly relevant to stem/progenitor cell expansion, there are many signal molecules involving in stem cell renew and regeneration. A few intrinsic cues – including the transcriptional repressor BMI-1 [39, 40], the protooncogene MYC [41], and the transcription factor C/EBP [42], overexpression of HoxB [43] promotes extensive HSC expansion ex vivo. TPO may act primarily to induce HSC apoptosis [44], LNK acts as a broad inhibitor of growth factors and cytokines TPO, KITL, EPO, IL-3, and IL-7 signaling pathways [45–49]. Uncovering the molecular mechanism underlying expansion of HSPCs is critical to extend current therapeutic applications. HOXB4 is known to be involved in stem cell maintenance and had shown some promise for stem cell expansion in mice. Zhang [50] showed that HOXB4 over-expression in populations of cells enriched for stem cells for 6–9 days prior to transplantation greatly improved their subsequent engraftment in radiated monkeys. Beslu et al. [51] proved HOXB4 gene can instruct stem cells into divide cell cycle and make more stem cells, these expanded cells cold reconstitute the monkeys’ immune and blood systems. AMD3100 is a small molecule initially developed as a highly potent and selective inhibitor of human immunodeficiency virus (HIV)-1 and HIV-2 replication. AMD3100 showed a binding-specificity to CXCR4, it can induce 1.5–3.1 fold WBC count, and 5-fold CD34+ increase in circulation as well as 18-fold CFU-GM [52–54]. SB-251353 is a truncated form of human chemokine GROB that binds specially to the CXCR2 receptor, SB-251353 combined with G-CSF could increase HSPCs in the circulation compared to G-CSF alone [55]. Some novel stem cell expansion factors were identified as part of pathways associated with mesodermal induction, or as factors produced by supportive stroma. These new factors showed their potential in CB HSC ex vivo expansion [56]. Okamoto [57] expanded ex vivo CD34+ CD133+ progenitor cells from human umbilical cord blood and analyzed gene expression changes using microarrays covering up to 55,000 transcripts. Several new genes and signaling pathways not previously associated with ex vivo expansion of CD133+ CD34+ cells were identified, most of which associated with cancer. Regulation of MEK/ERK and Hedgehog signaling genes in addition to numerous proto-oncogenes were detected during

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conditions of enhanced progenitor cell expansion. DOCK4 and SPARCL1 tumor suppressors, were confirmed down-regulation in CD133+ CD34+ cells. These findings suggest that there is a common source of stem cells and cancer stem cells, and some of them might be used both in stem/progenitor expansion and potential molecular targets for malignant treatment. Most of the hematopoietic cytokines promote either survival or differentiation or both in HSC ex vivo, whereas extracellular morphogens (Wnt, Notch, Hedgehog, bone morphogenetic protein 4, and Tie2/angiopoietin-1) signaling pathways, and intracellular mediators (phosphatase and tensin homolog and glycogen synthase kinase-3) have been signified a class of HSC regulators that support expansion of the HSC pool by a combination of survival and induced self-renewal in vivo, but these pathways alone does not result in substantive expansion of HSCs ex vivo. Bcl-2 gene family, which regulates cell apoptosis, may play an important role in inducing survival in HSCs both in vivo and ex vivo. Correctly understanding the effect of these unique signaling pathways and their relationship will be essential to achieve successful ex vivo expansion and make UCBT available to more patients, decrease engraftment times and allow more rapid immune reconstitution post transplant [58, 59].

3.2.4 Free Radical Regulation on HSPCs Expansion Series studies about reactive oxygen species (ROS) on stem cell expansion were investigated recently. Hypoxia favored the preservation of progenitor characteristics of HSPCs in bone marrow. Fan reported that NADPH oxidase activity and ROS generation were reduced in hypoxia with respect to normal oxygen tension. The NADPH oxidase inhibitor diphenyleneiodonium, or the ROS scavenger N-acetylcysteine could inhibit this procedure. Hypoxia effectively maintained biological characteristics of CD34+ cells through keeping lower intracellular ROS levels by regulating NADPH oxidase [60]. In another study, they investigated the effect of regulating intracellular ROS with antioxidants on the ex vivo expansion of cord blood CD34+ cells. The generation of ROS was increased markedly by the cytokine combination, and these ROS could be eliminated by antioxidant effectively. The percentage of CD34+ cells and CD34+ CD38- cells, the colony growth of colony-forming cells (CFC) and the re-expansion capability of CD34+ cells were enhanced by low concentration of antioxidant such as 2,000 U/mL SOD, 200 U/mL CAT or 2 mmol/L NAC. When increasing antioxidant to high concentration of 8,000 U/mL SOD, 1,000 U/mL CAT or 5 mmol/L NAC, the expansion of the cells was inhibited [61]. Copper (Cu) is known to generate oxidative stress in cells which in turn affects proliferation, differentiation and apoptosis. Prus [62] showed that Cu chelator tetraethylenepentamine (TEPA) reduces the free Cu content of HPCs and stimulates cord blood-derived CD34+ CD38- cells ex vivo expansion by lowering their oxidative stress. Srp et al. [63] reviewed oxygenation level as a physiological regulator of HSC maintenance: very low oxygen concentrations (0.1%) enable the preservation of the quiescent (G0 ) stem cell pool; low oxygen concentrations (1%) are compatible with the proliferation of primitive stem cells (self-renewal)

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but inhibit their differentiation; moderately low oxygen concentrations (3%) allow a balance between differentiation and self-renewal, permitting the simultaneous amplification of progenitors and the maintenance of stem cell activity; and very high oxygen concentrations (20–21%), like those in the air, enhance the differentiation of primitive stem cells, abrogating their self-renewal capacity.

3.2.5 Megakaryocytic Progenitor Cells Expansion Megakaryocytic progenitor cells (MKPC) infusion is a treatment selection for thrombocytopenia after HSC transplantation. Xia et al. [64] described some culture system composed of various cytokine combinations (TPO, SCF, Flt3-ligand, IL-1, IL-3, IL-6) on ex vivo expansion of megakaryocytic progenitors from CD34+ cells of peripheral blood. The content of CD41+ cells increased 94-fold at day 5 and 131-fold at day 10 and then decreased obviously, the CFU-MK were 93 and 121 respectively at day 5 and 10. The cytokine combination TPO/FL/IL-6/IL-3 was optimal for expansion ex vivo of megakaryocytic progenitors from mobilized PB. Boyer [65] designed a two-phase culture strategy to induce megakaryocyte (MK) differentiation from CD34+ -enriched CB cells. They optimized two functionally divergent cocktails to significantly increase the final yield of both MKs and HPC. Lin et al. [66] reviewed growth factors, including TPO, megakaryocyte growth and development factor (MGDF), IL-1, IL-3, IL-6, IL-11, platelet-derived growth factor (PDGF), and serotonin (5-HT) on the regulation of megakaryocyte/platelet development, and the efficient conditions for the expansion of the MK progenitors from HSPC. TPO alone could produce a high proportion of MK progenitors but a low total cell count. IL-1β, IL-3, IL-6 and Flt3-ligand improved the expansion outcome. PDGF also enhanced the ex vivo expansion of CD61+ CD41+ cells and CD34+ cells in combination with TPO, IL-1β, IL-3, IL-6 and Flt3-ligand, as well as engraftment of human stem and progenitor cells in NOD/SCID mice, but without promoting their in vitro maturation. The combination of three to five cytokines produced more efficient expansions of hematopoietic stem and MK progenitors.

3.2.6 Red Cells Expansion It is difficult in obtaining adequate supplies of all blood components, especially great numbers of red blood cells (RBCs). Douay et al. [67] described a methodology permitting the massive ex vivo production of mature human RBCs having all the characteristics of native adult RBCs from hematopoietic stem cells of diverse origins: blood, bone marrow, or cord blood. This protocol allows both the massive expansion of HSPCs and their complete differentiation to the stage of perfectly functional mature RBCs. The levels of amplification obtained 1×105 to 2×106 are compatible with an eventual transfusion application. Even if this is a considerable advance in blood transfusion, we do not think we can afford its luxury in clinical practice.

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3.2.7 T-Cell Expansion Potential advantages of using CB relate to the high proportion and quality of HSPCs. CBSCT requires less stringent HLA matching and results in less GVHD. This may be attributed to the immature neonatal immune system, which shows more tolerant to alloantigens, compared with the corresponding cells from adult. More studies have been reported that cord blood T-lymphocytes are immature in phenotype and function and that little cytotoxic is generated after allogeneic transplantation [68–70]. Since mature T-cells play an important role in GVHD pathogenesis and GVL effect process. Interleukins are essential for immune cells [71–73]. Immune cells decreased in expanded cells supplemented with SCF, Flt3-ligand, G-CSF without adding interleukins, even no T, B, NK cell [74] were detected when incubating over 3 weeks. Ballen et al. [75] incubated the cord blood with a cytokine mixture of IL-3, IL-6, IL-11 and SCF, and resulted in increased survival of irradiated NOD-SCID recipients posttransplantation of the expanded cord blood. Robinson [76] reported that ex vivo combination of IL-2, IL-12, anti-CD3, and IL-7 significantly enhances the proliferation, activation, maturation, and cytotoxic potential of UCB T-cells of both fresh and thawed UCB MNC. The four cytokine combination significantly induced expression of CD45 RO in both the CD4+ and CD8+ T-cells expressing CD25 respectively and increased the production of IFN-γ. The combination also significantly increased the killing of K562 target cells. For an adoptive immunotherapy of cancer, autoimmunity, and infectious disease, Skea et al. [77] developed a new method involving the use of a conditioned medium (XLCM) that consistently results in levels of UCB T-cell expansion. From initiation of the UCB or adult PB low-density LDMNC/XLCM cultures up to approximately 2 weeks, the cultures were dominated by CD4+ T-lymphocytes. By 4 weeks, more than 80% of the cultured cells bear the CD8+ phenotype, it permits the selective expansion of different T-lymphocyte subsets from a single source. Li et al. [78] has proved that CB CD34+ cells were cultured for 5 days in the presence of human cytokines and the murine stromal cell line HESS-5, and transplanted into irradiated NOD/SCID mice, functional capacity of B cells marker CD19+ cells appeared at 6 weeks. Enrichment of donor grafts with CB T-cells expanded ex vivo might facilitate improved T-cell immune reconstitution post-transplant. We studied UCB-derived T-cell amplification under the cytokines combinations. The CD3+ T-cells could be expanded to higher level in the combination 50 ng/mL SCF + 2 ng/mL IL3, 20 ng/mL IL-6 cocktail with 5 ng/mL IL-7 or 10 ng/mL IL-2, or 10 ng/mL IL-4. Moreover, if IL-2 or IL-4 concentration were increased to 5 times, more effective expansion of CD3+ cells exhibited [78]. D’Arena et al. [67] compared the difference between HUCB and adult PB lymphocytes in their immunophenotypic profile. Significant differences in percentage were found between cord and adult T-cells, respectively (CD3+ : 59.9 vs. 74.9%), CD3- CD16+ and/or CD56+ NK cells (23.8 vs. 10.8%) and CD3+ CD16+ and/or CD56+ cytotoxic T-lymphocyte subset (0.3 vs. 10.7%). There was no difference in CD4/CD8 ratio (1.7 vs. 1.6%) between the two groups. Szabolcs et al. [79] and Guo et al. [80] utilized FACS

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to characterize surface and intracellular protein expression on lymphocyte subsets from fresh unmanipulated UCB and adult PB, similar results were acquired. Parmer [81] successfully expanded CB T-cells using paramagnetic microbeads covalently linked to anti-CD3 and anti-CD28 Ab in the presence of 200 IU/mL IL-2. A mean 100-fold expansion (range 49–154) of total nucleated cells was observed in the CD3+ magnetically enriched fraction. The expanded CB T-cells retained a naive and/or central memory phenotype and contained a polyclonal TCR diversity. This in particular showed that HUCB T-lymphocytes appeared to be phenotypically immature. Cycling UCB T-cells retain a naive immunophenotype that may represent homeostatic expansion rather than antigen-driven proliferation. Although still in its infancy, human CB progenitor cells hold considerable potential for in vitro expansion and to transplant the adult recipients with genetic inherited diseases, cancer and some immunodeficiencies [82].

3.2.8 NK Cell Expansion NK cells are important as the first line of the host defense, and as one of the final effectors cells in resistance to tumor, metastases, and viral infections. Allogeneic NK cells are known to show a high cytotoxic activity against HLA-nonidentical residual leukemia or tumor cells and relapse, and to reinduce remission after bone marrow transplantation, but its application has been limited by the inability to obtain sufficient numbers of pure NK cells. It is possible to effectively expand cord bloodderived CD56+ cells ex vivo, while maintaining their high lymphokine activated killer activity. NK cells can be induced by various stimuli, in particular IL-2, from bone marrow, cord blood and peripheral blood purified CD34+ stem cells and exhibit similar phenotype and functions [83, 84]. Cytokine IL-2 has been proved to enable to activate in vitro antitumor cytotoxic of HSCs even at a low-dose [85]. Li et al. [86] isolated NK cells from human peripheral blood and cultured them in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium) combinations with IL-2, IL-12, IL-15 for 15 days, 50.5 and 52.4-fold cells were expanded in IL-2 + IL-15 and IL-2 + IL-15 + IL-12 group respectively. All expanded cells showed over 94% CD3- CD56+ NK cells purity, and a significantly higher cytotoxicity were observed compared to starting population. Koehl et al. [87] purified and activated CD56+ CD3- NK cells with IL-2, a five-fold expansion of NK cells was observed, which showed a highly increased lytic activity against the MHC-I deficient K562 cells and a medium cytotoxicity against patients’ leukemic cells. UCB is a rich source of cytotoxic CD56+ cells including fetal NK cells (CD16- CD56+ ) with high lytic capabilities. NK T-cells in human CB are very small populations (