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Characterization of mouse hematopoietic stem cells primed to actively self-renew by NUP98-HOXA10hd fusion.. Sekulovic, Sanja 2011

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CHARACTERIZATION OF MOUSE HEMATOPOIETIC STEM CELLS PRIMED TO ACTIVELY SELF-RENEW BY NUP98-HOXA10hd FUSION GENE  by SANJA SEKULOVIC B.Sc., The University of Belgrade, 2001 M.Sc., The University of British Columbia, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Medical Genetics)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  July, 2011 © Sanja Sekulovic, 2011  Abstract  High-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Recently, it has been demonstrated that engineered NUP98-HOXA10hd expression stimulates >1,000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin-Sca-1+c-kit+ cells. In this thesis I coupled such ability of engineered NUP98-HOXA10hd expression, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. I discovered that NUP98HOXA10hd stimulates comparable expansions of HSCs from both sources at near unit efficiency in cultures initiated with single cells. The clonally expanded HSCs showed preservation of normal proliferation kinetics in vitro and consistent balanced contributions long-term to the lymphoid and myeloid lineages in vivo without evidence of leukemogenic transformation. Preservation of a normal proliferating HSC phenotype allowed their re-isolation in large numbers at 25% purity. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process. Although there is growing excitement about the prospect of in vitro expansion of HSCs and their use to enhance the safety and application of transplant-based therapies, deleterious consequences of such manipulations remain unknown. Thus, I further examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing  ii  ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. I observed ~10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert-/HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type or Tert-/HSCs, suggesting that HSC replication post-transplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity upon prolonged self-renewal stimulation.  iii  Preface  A version of chapter 2 has been submitted for publication entitled “Ontogeny stage-independent  and  high-level  clonal  expansion  in-vitro  of  mouse  hematopoietic stem cells stimulated by an engineered NUP98-HOX fusion transcription factor”. The work presented was designed primarily by me with intellectual input from Maura Gasparetto, Clay Smith, Corinne Hoesli, James Piret, Connie Eaves, and Keith Humphries. Transduction of highly purified bone marrow and fetal liver cells, cell culture, transplantation and analysis of transplanted mice was primarily performed by me. The staining and repurification strategy of in vitro expanded HSCs was jointly discovered and optimized by Maura Gasparetto and myself. Assistance with purification of fresh HSCs was provided by Maura Gasparetto and David Kent, assistance with transplantation, peripheral blood sampling and staining was provided by Christy Brookes, and assistance with viral integration analysis was provided by Adrian Wan and Patty Rosten. A microfluidic cell culture, time-lapse imaging and analysis were performed by Veronique Lecault, a graduate student from the labs of Dr. James Piret and Dr. Carl Hansen, at Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada. Factorial experiments were designed together with Corinne Hoesli, a graduate student from the lab of Dr. James Piret, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada. Experiments were  iv  performed and analyzed by me and factorial effects calculated and figure 2.5 generated by Corinne Hoesli. I generated the figures (except figures 2.2B and 2.5) and tables and primarily prepared the manuscript, with significant input from Connie Eaves and Keith Humphries. The manuscript was edited by Clay Smith and James Piret.  A version of chapter 3 has been submitted for publication entitled “Prolonged self-renewal activity unmasks telomerase control of telomere homeostasis and function of mouse hematopoietic stem cells”. This article was co-first-authored with Vala Gylfadottir, a former graduate student from the lab of Dr. Fabio Rossi, The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada. They designed, performed and analyzed longitudinal and serial transplantation studies with non-manipulated wild-type and Tert-/- bm cells. This data is presented in figure 3.1. I designed serial transplantation studies with NUP98-HOXA10hdtransduced wild-type and Tert-/- bm cells with significant input from Peter Lansdorp, Connie Eaves and Keith Humphries. Transduction of bone marrow cells, cell culture, transplantation and peripheral blood analysis was primarily preformed by me. I also designed, performed and analyzed competitive repopulating unit and colony forming cell assays. Assistance with transplantation, peripheral blood sampling and staining was provided by Christy Brookes. Flow-fluorescent in situ hybridization method for measuring the average length of telomere repeats in bone marrow samples was preformed and data  v  analyzed by Irma Vulto, a senior research technician from the lab of Dr. Peter Lansdorp. Staining and flow cytometry for γ-H2AX was performed together with Maura Gasparetto. I generated the figures and primarily prepared the manuscript, with significant input from Connie Eaves and Keith Humphries. The manuscript was edited by Fabio Rossi and Peter Lansdorp.  Animal research conducted in this thesis has been approved by UBC Animal Care Committee (Animal Care Certificate number: A09-0009).  vi  Table of Contents Abstract......................................................................................................................... ii Preface ......................................................................................................................... iv Table of Contents....................................................................................................... vii List of Tables ................................................................................................................ x List of Figures ............................................................................................................. xi List of Abbreviations ................................................................................................. xii Acknowledgements ................................................................................................... xv Dedication.................................................................................................................. xvi CHAPTER 1  Introduction.......................................................................................... 1  1.1. Thesis overview ............................................................................................... 1 1.2. General overview of hematopoiesis ................................................................. 3 1.2.1 Hierarchical model of the mouse hematopoietic system ......................... 3 1.2.2 The significance of HSCs ........................................................................ 4 1.2.3 The evidence for the existence of HSCs ................................................. 6 1.2.4 Potential applications of ex vivo expansion of HSCs in clinical and basic research settings.............................................................................. 8 1.3 HSCs............................................................................................................... 11 1.3.1 Ontological sources of HSCs ................................................................. 11 1.3.2 Functional characterization of HSCs...................................................... 13 1.3.3 Phenotypic characterization of HSCs .................................................... 18 1.4 Regulation of HSC self-renewal process ........................................................ 20 1.4.1 Extrinsic regulation of HSC self-renewal................................................ 21 1.4.1.1 Niche signals .............................................................................. 21 1.4.1.2 Control of HSC self-renewal by growth factors .......................... 24 1.4.2 Intrinsic regulation of HSC self-renewal ................................................. 25 1.4.2.1 Intracellular signalling molecules................................................ 26 1.4.2.2 Cell cycle regulators ................................................................... 27 1.4.2.3 Epigenetic factors ....................................................................... 28 1.4.2.4 Hematopoietic transcription factors ............................................ 30 1.4.2.5 HOX transcription factors ........................................................... 31 1.4.3 Maintenance of genomic integrity in HSCs............................................ 34 1.4.3.1 Telomere, loss of telomeric DNA and telomerase ...................... 34 1.4.3.2 Telomere maintenance in HSCs ................................................ 36 1.5 In vitro expansion of HSCs ............................................................................. 38  vii  1.5.1 Demonstrated strategies for mouse HSC expansion ............................ 38 1.5.1.1 HOXB4 as a stimulator of HSC self-renewal in vitro .................. 39 1.5.1.2 NUP98-HOX fusions are powerful stimulators of HSC self-renewal in vitro................................................................................. 39 1.5.2 Demonstrated strategies for human HSC expansion ............................ 42 1.6 Thesis objectives ............................................................................................ 43 CHAPTER 2  Ontogeny stage-independent and large magnitude clonal expansion in vitro of HSCs stimulated by an engineered NUP98-HOX fusion transcription factor ...................... 49  2.1 Introduction ..................................................................................................... 49 2.2 Materials and methods.................................................................................... 50 2.2.1 Mice ....................................................................................................... 50 2.2.2 Flow cytometry ....................................................................................... 51 2.2.3 Viral transduction and cell culture .......................................................... 51 2.2.4 Microfluidic cultures ............................................................................... 52 2.2.5 Transplantation and analysis of transplanted mice ................................ 53 2.2.6 Factorial design experiments ................................................................. 54 2.2.7 Viral integration analysis ........................................................................ 55 2.3 Results ............................................................................................................ 56 2.3.1 Efficient high-level expansion of HSCs in single cell cultures of NUP98-HOXA10hd-transduced CD45+lin-Rho-SP or E-SLAM cells ................................................................................................................. 56 2.3.2 The in vitro growth kinetics of NUP98-HOXA10hd-transduced HSCs is not perturbed..................................................................................... 57 2.3.3 Proliferation phenotype of NUP98-HOXA10hd-expanded HSCs allows their isolation at high purity .................................................................. 58 2.3.4 Factorial analysis of the effect of different conditions on NUP98-HOXA10hd-transduced HSC expansion in vitro ................................ 60 2.3.5 NUP98-HOXA10hd induces the in vitro expansion of fetal HSCs............................................................................................................... 61 2.3.4 NUP98-HOXA10hd-transduced HSCs that undergo high level expansion in vitro continue to expand in vivo ................................................. 62 2.4 Discussion....................................................................................................... 63 CHAPTER 3  Prolonged self-renewal activity unmasks telomerase control of telomere homeostasis and function of mouse HSCs ................................................................................................... 84  3.1 Introduction ..................................................................................................... 84 3.2 Materials and methods.................................................................................... 87 3.2.1 Mice ....................................................................................................... 87 3.2.2 Transduction of mouse bone marrow cells ............................................ 87 3.2.3 Transplantation ...................................................................................... 88 3.2.4 Assessment of HSC frequencies ........................................................... 90  viii  3.2.5 Peripheral blood analysis ....................................................................... 90 3.2.6 Colony forming cell (CFC) assays ......................................................... 91 3.2.7 Flow-FISH .............................................................................................. 91 3.2.8 Flow cytometry for γ-H2AX .................................................................... 92 3.3 Results ............................................................................................................ 93 3.3.1 Telomerase deficiency affects telomere homeostasis under conditions of prolonged self-renewal stimulation ............................................ 93 3.3.2 Primitive hematopoietic cells lacking telomerase activity exhibit signs of enhanced DNA damage ......................................................... 98 3.4 Discussion....................................................................................................... 99 CHAPTER 4  Conclusion and recommendation for future work ....................... 112  4.1 Potential cellular mechanisms of NUP98-HOXA10hd-mediated HSC expansion ............................................................................................. 112 4.2 Potential molecular mechanisms of NUP98-HOXA10hd-mediated HSC expansion ............................................................................................. 117 4.2.1 The role of NUP98 fusion partner ........................................................ 120 4.2.2 The role as a transcription factor ......................................................... 121 4.3 Potential therapeutic applications ................................................................. 122 REFERENCES........................................................................................................... 124 APPENDICES............................................................................................................ 150 Appendix A: Effect of NUP98-HOXA10hd on individual HSC clones of CD45+lin-Rho-SP phenotype ................................................................. 150 Appendix B: Average peripheral blood lineage contribution to donor-derived compartment of representative recipients shown in figure 2.1C (top panel) ............................................................................................. 151 Appendix C: Effect of NUP98-HOXA10hd on individual HSC clones of E-SLAM phenotype ............................................................................... 152 Appendix D: Average peripheral blood lineage contribution to donor-derived compartment of representative recipients shown in figure 2.1C (bottom panel) ....................................................................................... 153 Appendix E: Design matrix for the two-level factorial experiment .............................. 154 Appendix F: NUP98-HOXA10hd effect on telomere length of wild-type and Tert-/- bone marrow cells during in vitro culture..................................... 155  ix  List of Tables Table 2.1 – Summary of all highly purified HSC clones tested and average HSC expansion in vitro achieved in response to forced expression of NUP98-HOXA10hd ............................................................................... 70 Table 2.2 – Calculated day 10 HSC frequencies and HSC content estimates of total or various FACS-purified subpopulations of NUP98-HOXA10hdtransduced and in vitro cultured bone marrow cells (as shown in figure 2.3C) .......................................................................... 75 Table 2.3 – Calculated day 10 HSC frequencies and HSC content estimates of total or various FACS-purified subpopulations of NUP98-HOXA10hdtransduced and in vitro cultured bone marrow cells (as shown in figure 2.4B) .......................................................................... 78  x  List of Figures Figure 1.1 – Hierarchical model of hematopoiesis ...................................................... 46 Figure 1.2 – Key regulators of HSCs .......................................................................... 47 Figure 1.3 – Clustered Hox gene organization............................................................ 48 Figure 2.1 – NUP98-HOXA10hd promotes multi-log expansion in vitro of individually purified adult bm HSCs ........................................................ 67 Figure 2.2 – The in vitro cell division kinetics of highly purified HSCs transduced either with control or NUP98-HOXA10hd vector ..................................... 71 Figure 2.3 – The CD150+CD48- subset of day 10 NUP98-HOXA10hd-transduced and expanded population contains less than half of the total HSCs generated in vitro.................................................................................... 73 Figure 2.4 – Proliferation phenotype of NUP98-HOXA10hd-expanded HSCs............ 76 Figure 2.5 – Growth factor effects on NUP98-HOXA10hd-induced HSC expansion in vitro .................................................................................... 79 Figure 2.6 – NUP98-HOXA10hd induces the in vitro expansion of fetal HSCs .......... 80 Figure 2.7 – The progeny of in vitro expanded NUP98-HOXA10hd-transduced HSC clones continue to expand in vivo and re-expand in vitro .............. 82 Figure 3.1 – Mouse HSCs have a reservoir of telomeres sufficient to sustain their self-renewal during several cycles of serial transplantation ................. 104 Figure 3.2 – NUP98-HOXA10hd effect on telomere maintenance and reconstitution activity of wild-type and Tert-/- HSCs .............................. 106 Figure 3.3 – Absence of telomerase activity blunts NUP98-HOXA10hd-induced self-renewal of myeloid progenitors and HSCs in vitro .......................... 109 Figure 3.4 – Primitive Tert-/- hematopoietic cells express elevated levels of γ H2AX .................................................................................................... 110  xi  List of Abbreviations  Abd-B  Abdominal B  AGM  Aorta gonad mesonephros  ALT  Alternative lengthening of telomeres  AML  Acute myeloid leukemia  ANG1  Angiopoietin 1  APC  Allophycocyanine  Bm  Bone marrow  CB  Cord blood  CD  Cluster of differentiation  CDK  Cyclin dependant kinse  CFU-S  Colony forming unit - spleen  ChIP  Chromatin immunoprecipitation  CLP  Common lymphoid progenitor  CMP  Common myeloid progenitor  CRU  Competitive repopulation unit  DKC  Dyskeratosis congenita  DMEM  Dulbecco’s modified Eagle’s medium  Dpc  Day postcoitus  E  Embryonic day  FACS  Fluorescence-activated cell sorting  xii  FBS  Fetal bovine serum  FGF-1  Fibroblast growth factor-1  FISH  Fluorescent in situ hybridization  FITC  Fluorescein isothiocyanate  FL  Fetal liver  GFP  Green fluorescence protein  GM  Granulocyte/Moncyte  GVHD  Graft versus host disease  Gy  Gray  Hd  Homeodomain  Ho  Hoechst 33342  HSC  Hematopoietic stem cell  IGF2  Insulin-like growth factor 2  IL  Interleukin  L  Ligand  LDA  Limiting dilution assay  Lin  Lineage markers  LSC  Leukemic stem cell  LSK  Lin-Sca1+c-kit+  LTRC  Long-term repopulating cell  Mpb  Mobilized peripheral blood  MPP  Multipotent progenitor cells  NA10hd  NUP98-HOXA10hd  xiii  NUP98  Nucleoporin 98  Pb  Pheripheral blood  PE  Phycoerythrin  PI  Propidium iodide  Rho  Rhodamine 123  RU  Repopulation unit  SCF  Stem cell factor  SP  Side population  STRC  Short-term repopulating cell  Tert  Telomerase reverse transcriptase  TGF-β  Transforming growth factor beta  TPO  Thrombopoietin  WBC  White blood cell  xiv  Acknowledgements  I would first and foremost like to thank my supervisors Keith Humphries and Connie Eaves for their fervent support and selfless guidance throughout this project. I also truly appreciate the wisdom and motivation they provided at every step along the way and freedom they gave me to pursue several different aspects of this project. Thank you to my committee members, Fabio Rossi and Jamie Piret, for their guidance, contribution and assistance with this thesis. Also, thank you to Clay Smith and Peter Lansdorp for their valuable academic contribution and for being great mentors, even without official roles. I would like to thank Hide Ohta for providing invaluable initial leads pointing us towards the high potency of engineered hox fusion genes to stimulate murine HSC expansion in vitro and for teaching me the assays for hematopoietic cells. Thank you to my dearest friends Suzan Imren and Maura Gasparetto for their constant encouragement in both academic and personal endeavors throughout my time in the lab. Finally, I send my thanks to past and present members of the Humphries and the Eaves lab whom I have had the pleasure sharing this experience with (Eric, Bob, Florian, Michael, Tobias, Patty, Michelle, Christy, Adrian, Courteney, David, and Claudia), for keeping the work environment enjoyable and for their support on this project.  xv  To my parents, my sister and my husband, who believed in me, encouraged me and put up with me at every step along the way.  xvi  CHAPTER 1 Introduction  1.1  Thesis overview  Hematopoiesis is the life-long process of blood cell development. Establishment and maintenance of the blood system relies on hematopoietic stem cells (HSCs)1. They reside as a rare population in the bone marrow (bm) of adult mammals and are capable of self-renewal and differentiation to all blood cell lineages. These processes are prerequisites for life-long hematopoiesis and their deregulation may lead to severe clinical consequences. HSCs are also highly valuable for their ability to reconstitute the hematopoietic system when transplanted and thus enable transplantation-based therapies for a variety of genetic disorders, acquired states of bm failure and cancers2-4. Given these pivotal roles of HSCs, much effort has been directed at developing tools for their detection and characterization, and in understanding and ultimately exploiting the mechanisms underlying their behaviour (i.e. selfrenewal). A longstanding major goal has been to develop methods for achieving significant expansion of HSC numbers both in vitro and in vivo and thereby improve the safety and application of stem cell based transplantation therapies and to hasten hematopoietic recovery after chemotherapy4. Attaining this goal has proven challenging given the still limited understanding of the mechanisms controlling HSC proliferation and self-renewal. Indeed until recently, in vitro  1  culture of HSCs under a wide range of conditions resulted in hematopoietic differentiation or death, resulting in overall loss of long-term repopulating HSCs5. It was shown previously that several members of the Hox transcription factor family are potent regulators and/or modulators of primitive hematopoietic cell function, including the self-renewal process6. Moreover, our recent findings demonstrated the marked stimulation of HSC expansion by a series of novel, engineered Nucleoporin98-Homeobox (Nup98-Hox) fusion genes with multi-log absolute increases in HSC numbers occurring in short-term cultures7,8. Building on these initial observations of the potent stem-cell stimulatory effects of such Nup98-Hox fusions, I focussed my attention on one such fusion between Nup98 and the homeodomain encoding region of HoxA10 (NUP98HOXA10hd) and exploited its potent ability to stimulate >1000-fold HSC expansion in vitro8. This fusion gene coupled with methods to purify HSCs and to rigorously measure HSC number and function was then used to probe the ability to stimulate the self-renewal behaviour of single highly purified HSCs from both fetal and adult sources. I further extended this strategy to generate and reisolate large numbers of highly purified HSCs in vitro, by determining the phenotype of transduced and expanded HSCs. Lastly, I examined the possible consequences of such increased and prolonged HSC self-renewal on their overall genomic integrity and function. In the following sections, the key concepts, assays and regulatory mechanisms of HSC function, along with some examples of recently  2  demonstrated strategies for HSC expansion in vitro that guided and enabled the research undertaken, are briefly reviewed.  1.2  General overview of hematopoiesis  1.2.1 Hierarchical model of the mouse hematopoietic system A current view of hematopoiesis is that of a hierarchically organized system, with a rare population of HSCs giving rise to progeny that progressively lose selfrenewal potential and successively become more and more restricted in their differentiation capacity9. Therefore, hematopoiesis proceeds through a series of lineage commitment steps10. The immediate progeny of the most primitive HSCs are multipotent short-term reconstituting cells (STRCs, also called multipotent progenitor cells, MPPs) that retain full lineage potential yet have a relatively limited capacity for sustaining self-renewal divisions, thus actively producing cells for only a few weeks or months. STRCs give rise to oligopotent progenitors (i.e. common myeloid and common lymphoid progenitors (CMPs and CLPs)), which are restricted in their developmental potential, giving rise only to mature myeloid or lymphoid cells. The oligopotent progenitors in turn give rise to more lineagerestricted progenitors, with the final output being the mature functional circulating blood cells. The myeloid lineage includes those cells responsible for carbon dioxide and oxygen transport (erythrocytes), blood clotting (platelets) and those involved in mounting a phagocytic response to foreign organisms (neutrophilic, basophilic, and eosinophilic granulocytes and macrophages). The lymphoid  3  lineage includes cells involved in humoral (B-cells) and cellular immunity (T cells, Natural Killer cells) (Figure1.1)10. The lifespan of these various types of mature blood cells ranges from a few hours or days (neutrophils) to a few months (erythrocytes) to many years (memory T-cells). As a result, ~100 million mature blood cells need to be produced each day throughout the entire lifetime of the mouse. The process by which these cells are produced is called hematopoiesis. Our appreciation of the complexity and likely fluidity of the hematopoietic hierarchy continues to evolve with, for example, recent evidence of possible early branching of myelo-erythorid committed cells from pluripotent cells11. Most dramatically, the complexity and heterogeneiety of the HSC compartment is now becoming more apparent with respect both to their differentiation capacity and sustainability of self-renewal12-16.  1.2.2 The significance of HSCs Hematopoiesis involves the ordered production of more than 10 distinct mature cell types from HSCs which possess the ability of both multipotency (i.e. HSC can differentiate into all functional blood cells) and self-renewal (i.e. HSC can give rise to itself without differentiation). Therefore, during the lifetime of an organism, an adequate pool of HSCs is maintained through self-renewal divisions, while at the same time HSCs consistently meet the high demand for continuous replenishment of short-lived mature blood cells17. The ability of HSCs to self-renew is fundamental to hematopoietic homeostasis, development, following bm transplantation and/or in response to  4  different physiologic stresses. Self-renewal is the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter stem cells that retain the same ability to self-renew and generate differentiated cell lineages as the parental cell18. During homeostasis, a significant proportion of adult bm HSCs with long-term reconstitution potential is predominantly  quiescent  and  divides  infrequently19,20,  likely  undergoing  asymmetric self-renewing divisions to maintain the stem cell compartment and therefore continuously regenerate mature blood cells in a correctly balanced ratio. In contrast, during early development21 or following bm transplantation, HSCs likely undergo symmetric self-renewing divisions to expand or replenish the stem cell pool. For example, between embryonic day 12 (E12) day postcoitus (dpc) and E16 dpc the population of mouse fetal liver (FL) HSCs expands nearly 40-fold22. By adulthood, an additional 13-fold expansion brings the total HSC content of mouse bm up to 20,00023. HSC expansion also occurs during the initial phase of hematopoietic recovery following bm transplantation, when HSCs are stimulated to self-renew and replenish both mature and primitive hematopoietic compartments. When small numbers of HSCs are transplanted into myeloablated or pre-immune hosts, the increases in HSC numbers that follow may be even larger than those seen during development24-27. These findings document the high replicative potential of HSCs and illustrate HSC features fundamental for the development of powerful clinical therapies for leukemias and genetic blood disorders based on HSC transplantation (see section 1.2.4).  5  The importance of HSC self-renewal and differentiation is further underscored by the numerous examples where deregulation of these processes can have severe clinical consequences ranging from aplastic anemia to leukemia. Such concepts have been reviewed by Krivtsov and Armstrong28 and it suggests that similarly to normal hematopoiesis chronic and acute myeloid leukemias may originate and/or are maintained by a population of leukemic stem cells (LCSs) with the capacity to self-renew. Being a long-lived self-renewing cells, HSCs seem to be prone to accumulating substantial genetic or epigenetic abnormalities needed for overt leukemia. Thus the transformation of HSC into LSC may only require maintenance of self-renewal properties already in place in the HSC. Nevertheless, recent evidence has revealed that less primitive progenitor cells are also capable of giving rise to LSCs29. Such transformation likely occurs through reactivation and/or intensification of self renewal programs in a cell that does not originally display such properties. These key roles of HSCs further underline the importance of elucidating the unresolved mechanisms underlying HSC behaviour, which are relevant for the development of new tools both for clinical expansion of normal HSCs and conversely for disruption/inhibition of LSC growth.  1.2.3 The evidence for the existence of HSCs The concept and basis for bm transplantation emerged in response to the clinical need for cells capable of rescuing the hematopoietic system of individuals suffering from radiation accidents, followed by the experimental observation that  6  mice could be protected from radiation-induced death when they had their spleen shielded30 or by an infusion of spleen or bm cells31. Subsequently, it was shown that lethally irradiated mice protected by infused marrow had their hematopoietic tissue colonized by donor-derived cytogenetically distinguishable cells32. Three decades later, proviral marking studies provided definitive evidence that the lifelong reconstitution of the hematopoietic system in transplanted irradiated mice was attributable to the activity of a small number of long term repopulating cells (LTRCs) capable of self-renewal and multi-lineage differentiation33-35. One of the first assays of detecting and quantifying primitive hematopoietic cells in vivo was the colony forming unit – spleen (CFU-S) assay36. In this assay, thoroughly described by Szilvassy37, lethally irradiated syngeneic recipients are transplanted intravenously with bone marrow cells from donor mice. Within 2 weeks post-transplant a fraction of injected cells gives rise to macroscopic spleen colonies. These colonies were found to be clonogenic, derived from a single cell having CFU-S potential. Such colonies can be counted in order to measure the CFU-S frequency in the initial cell suspension. Moreover, transplantation of a spleen colony into a secondary recipient gives rise to more spleen colonies indicating that CFU-S can differentiate and self-renew. Therefore, initially this assay was thought to detect true HSCs. However, regardless of mapping out some major features of HSCs, it is no longer considered the defining assay for HSCs. Mature lymphoid cells were not detected in CFU-S colonies, indicating that the assay defines myeloid restricted early progenitors (i.e. erythriod, granulocytic, and megakaryocytic). Moreover, with the development of  7  transplantation-based HSC assays and cell purification strategies it was possible to physically separate CFU-S from true HSCs with the long-term repopulation ability38,39. Nevertheless, the results of the early studies of CFU-S led to recognition of the importance of quantitative assays specific for cells with longterm in vivo lympho-myeloid reconstitution ability (see section 1.3.2).  1.2.4 Potential applications of ex vivo expansion of HSCs in clinical and basic research settings The capacity for sustained self-renewal is fundamental for the increasing application of HSC-based therapies in a wide range of malignant and genetic disorders. From that perspective, the development of strategies to extensively expand HSCs ex vivo would be key to the success of such therapies. HSC transplantation following a myeloablative conditioning regimen is a key curative treatment for patients with many types of hematologic malignancies, bm failure, immunodeficiences, and hemoglobinopathies, as it results in a full or partial replacement of the diseased hematopoietic tissue with normal cells and/or provides additional immune effectors3,4. HSCs obtained directly from the patient (autologous HSCs) are mainly used for rescuing the patient with malignancy from the effects of high dose chemotherapy or may serve as the target for gene therapy vectors40. Standard clinical practice is to use mobilized pheripheral blood (mpb), collected from patients following cytoreductive cycles of chemotherapy to minimize the tumor burden41. However, autologous mpb product bears a risk of being contaminated with tumor cells. HSCs obtained from another person  8  (allogeneic HSCs) are used to treat hematological malignancies (i.e. leukemias and/or bm failure syndromes (i.e. Aplastic anemia)) where replacing with healthy donor cells is required. One of the major complications of an allogeneic transplantation is graft-versus-host (GVHD) disease, as allografts contain mature immune cells that can respond against host-specific antigens (reviewed by Shizuru et al.4). Thus amplification of HSC numbers would be immensely useful in conquering these existing limitations and possibly establishing improved transplantation approaches. Moreover, as HSC numbers directly control the efficacy of bm transplantation procedures42,43, successful HSC expansion could potentially allow the use of much smaller volumes of bm, thereby reducing the cost and the risk of HSC collection. It could also accelerate the levels of HSC recovery following bm transplantation, thus reducing morbidity and mortality associated with the procedure, and allow use of T-depleted donor grafts, which reduce the incidence of and severity of GVHD. Besides, a promising new source of HSC donor material is cord blood (CB), which gives reduced incidence of GVHD due to naïve immunological state of the cells44. However, there is also a higher rate of mortality because of failed or delayed engraftment. This latter issue relates to the small size of CB grafts and their consequently low stem cell content. Higher HSC doses have consistently been found to correlate with improved disease-free survival and reduced transplant-related mortality42,43. HSC-based gene therapy, involving collecting HSCs from the patient and genetically modifying them with a therapeutic transgene, is a growing treatment  9  option for patients with hematological defects2. Since these procedures require ex vivo culture of hematopoietic cells, often resulting in significant loss in HSC numbers and their engraftment potential45, ability to expand HSCs ex vivo would greatly improve the clinical outcomes of such therapies. In addition to these potential clinical applications, the strategies for obtaining amplified HSC populations ex vivo would provide invaluable tool and source for further studies of the mechanisms underlying HSC self-renewal process. As summarized above, much evidence has indicated that this process is controlled by a mechanism that involves a complex integration of extracellular cues and intracellular components. However, how these components are connected into a regulatory network and/or what are the unique molecular features that endow HSCs with the capacity to self-renew still remains unknown. Thus, as HSCs differ in their properties depending on their location (i.e. FL, bm) and the age of the organism, genome-wide comparative studies (i.e. chromatin immunprecipitation (ChIP)-sequencing in combination with expression and functional studies) may be the way to elucidate the subset of regulatory mechanisms that are absolutely necessary and sufficient for self-renewal, as well as conserved in a diverse HSC types. Moreover, it is thought that many leukemias are propagated by a distinct population of cells (LSCs) that may share some properties, including conserved self-renewal mechanisms,  with normal  HSCs46.  self-renewal  Therefore,  better  understanding  of  these  shared  mechanisms is of interest from the perspective of developing more effective therapies for leukemias.  10  1.3  HSCs  1.3.1 Ontological sources of HSCs Investigation of the initial origin and sites of production of HSCs during the genesis of the hematopoietic system have indicated that many features of these cells change throughout development. HSCs present at different times during development have been shown to differ in their average clonal output of daughter stem cells24,47, growth factor responsiveness48, cycling/activation status and phenotype21,49. All of these variations are proving to be relevant to providing important clues towards improving our understanding about the HSC behaviour and the mechanisms underlying self-renewal process. Following gastrulation, embryonic hematopoiesis in mice occurs in several mesodermal lineages as these precursor cells emigrate from the primitive streak to three distinct hemogenic tissues: the yolk sac, para-aortic splanchnopleure (PSp)/aorta-gonad-mesonephros  (AGM)  region,  and  the  placenta50.  Since  embryonic development is characterized by simultaneous hematopoietic activity in more than one anatomical site, blood progenitors rapidly move within embryo after the onset of circulation at embryonic day 8.5 (E8.5). In the stage referred to as “primitive hematopoiesis”, the first hematopoietic cells appear in the extra-embryonic yolk sac, at E7.5, concomitant with the developing vasculature. The murine yolk sac is composed of two tissue layers, extra-embryonic mesodermal cells, the origin of primitive nucleated erythrocytes and macrophages, and visceral endodermal cells, the origin of  11  endothelial cells. The simultaneous appearance of erythrocytes and endothelial cells has suggested that these two lineages arise from a common precursor termed the “hemangioblast”51,52. Soon after, also appearing in the yolk sac are erythro-myeloid progenitors 53 and neonatal repopulating HSCs54. In the stage referred to as “definitive hematopoiesis”, the AGM region50 and placenta55,56 are additional and independent sites of hematopoiesis in a mouse embryo, where multipotential progenitors and HSCs can be detected. Adult repopulating HSCs appear at E10.555-58. Soon after, multipotential progenitors and HSCs colonize the FL, the major site of hematopoiesis from E12 until birth. The frequency of HSCs reaches a peak in the FL on E14.5 with maintenance of their numbers until day 1622 after which these numbers decline, possibly due to an exodus of HSCs to the spleen and bm21. After birth, bm becomes the primary site of hematopoiesis1 where hematopoietic stem and progenitor cells mature, while the mature end cells are released into the peripheral blood (pb) circulation. Analysis of gene expression in HSCs from fetal versus adult sources have identified differential expression of cell surface receptors, adhesion molecules, signalling intermediates and transcription factors59, that are markers and candidate regulators of the differences in stem cell behaviour between different ontological sources. Indeed, cell cycle analysis has shown that HSCs found in E14.5 FL are actively in cycle60, whereas the vast majority of HSCs in the adult bm are generally in the G0 (quiescent) phase of cell cycle19,61. Two recent studies19,62 have exploited a histone 2B (H2B) - green fluorescent protein (GFP)  12  fusion protein-retaining assay for in vivo tracking of long-term HSC divisions and have revealed that the most primitive HSCs are dormant, with the division rate of ~150 days (or only 5 times during a lifetime of a mouse). Functional differences in the behaviour of HSCs from different stages of development have also been demonstrated. When compared with adult bm HSCs, FL HSCs exhibited faster regeneration rates of daughter HSCs47,59,63, spleen colony forming cells64, and more differentiated cells21,64, upon transplantion into irradiated recipients. Parallel alterations in differentiation properties have also been described, in which FL HSCs produce elevated proportions of myeloid59 cells compared with their adult bm counterparts. Interestingly, it has recently been suggested that an intrinsic switch from these fetal-like to adult-like HSC properties occurs rapidly, between 3 and 4 weeks post-birth59.  1.3.2 Functional characterization of HSCs Our current knowledge about normal hematopoiesis derives in large part from the availability of transplantation-based functional assays capable of detecting HSCs through their unique ability to give long-term lympho-myeloid reconstitution to myeloablated transplant recipients. The functional aspect of these assays is particularly important since most phenotypic identifiers can be altered under certain conditions (see section 1.3.3). Several such assays have been used for detection and quantification of mouse HSCs: repopulation unit assay based on competitive repopulation, competitive repopulation unit assay based on limit  13  dilution principles in combination with competitive repopulation, and purified single cell transplantation assay. In these assays, primitive cells are tested via injection into genetically distinct myeloablated (usually irradiated) recipients, along with some source of radioprotective cells. This is essential to ensure shortterm survival, since purified test cells may require several weeks to produce functional progeny38. Several months later, the differentiated hematopoietic progeny are analyzed to determine the presence or absence of myeloid and lymphoid cells generated from the test cells originally injected. The repopulation unit (RU) assay developed by Harrison65 measures the ability of a test cell population to repopulate irradiated hosts relative to another reference source of repopulating cells contained in the transplant innoculum. When co-injected with measured numbers of genotypically distinguishable cells with predefined hematopoietic activity, the relative competitive ability of the test cells can be measured based on the proportion of donor cells in their hematopoietic tissues, usually the blood. This assay thus compares the stem cell activity of a cell population relative to some reference population, but does not measure HSC frequency directly as measurements of competitive repopulation ability of populations of cells cannot distinguish between variations in LTRC number and variations in competitive ability per individual LTRC. This important information can only be obtained by utilizing the statistical power of limiting dilution, using the competitive repopulating unit assay, or by injecting single cells. However, additional statistical methods can be applied to the variance data obtained by repopulation unit assay to calculate the frequencies of the input  14  HSCs if it is assumed that the average population activity of each HSC in the test population is the same as in the reference population66. However, this can prove a possible limitation if such assumption is called into question (e.g. in case of genetically modified cells). For the purpose of studies carried out in this thesis, a competitive repopulation unit (CRU) assay based on limit dilution principles to detect and quantitate cells with lympho-myeloid repopulating ability was used67. The CRU assay provides the specificity required for the exclusive quantification of HSCs with life-long blood cell-producing activity23,68. This procedure uses the principles of limiting dilution analysis to measure the frequency of cells in a given suspension that have transplantable long-term repopulating ability and can individually generate both lymphoid and myeloid progeny. Most popular is the use of C57BL/6J (B6) mice, pre-treated with a lethal dose of radiation (myeloablative treatment), or C57BL/6J-KitW-41J (W41) mice, a B6 congenic strain with a partial loss of c-kit function through the acquisition of a point mutation69, pre-treated with sublethal dose70. The treatment of these hosts maximizes the sensitivity of the assay and reduces the competing endogenous stem cell population to a minimum, creating an environment in which the engrafting stem cells will be optimally stimulated. In order for a limiting dilution analysis of the stem cell content of the test cell suspension to be performed, the recipients must be able to survive regardless of whether they receive any stem cells in the test cells injected. Survival of normal recipients is assured by cotransplanting them with hematopoietic cells of the same genotype that contain  15  sufficient numbers of short-term repopulating cells but minimal numbers of longterm repopulating cells (usually 1-2 x 105 normal bm cells function as helper cells). Survival of c-kit mutant hosts is similarly assured by pre-treating them with a dose of radiation that allows significant numbers of endogenous cells to survive. The differentiated blood cell progeny of the test cells and the recipients must be genetically distinguishable and assessed at a time when they can be safely assumed to represent the exclusive output of cells with life-long stem cell potential. Strains of mice congenic with the C57BL/6 mouse are typically used to allow the blood cell progeny of the test cells to be uniquely identified by flow cytometric detection of a cell surface alloantigen CD45 (also known as leukocyte common antigen or Ly5), which is found on the cell surface of all hematopoietic cells except terminally differentiating erythrocytes71,72. Quantification of HSCs is achieved by application of Poisson statistical analysis on the proportion of animals that test negative for the test cell-derived repopulation at each cell dose transplanted, where the dose at which 37% of animals are negative is estimated to contain 1 HSC or 1 CRU. In practice, a threshold of ≥1% test cell-derived myeloid and lymphoid pb cells detected >4-6 months post-transplant has been shown to rigorously detect a long term lympho-myeloid repopulating cell. Phenotypic characterization and thus prospective isolation of HSCs (see section 1.3.3) further facilitated their functional characterization at the single cell level. Cell populations in which at least 40% of the cells can be detected as CRUs in single cell transplant experiments have been isolated by a variety of strategies demonstrating the robustness of this assay13-15,73-75. These studies  16  have shown that the transplantation of a single HSC into lethally irradiated mice can successfully rescue the entire hematopoietic system, through dual capacity of HSC to self-renew producing additional HSCs and differentiate to all blood cell lineages. In limiting dilution or purified single cell transplantation experiments, the precise definitions used to identify HSCs are of particular importance for the interpretation of results. In general, if long-term and multilineage donor-derived repopulation is seen, it is determined retrospectively that an HSC was present in the test cells. As technology has developed, this definition and the way it is measured have changed. Detailed examination of the functional characteristics of highly purified HSCs and refinements in purification strategies have yielded increased resolution of the complexity of the HSC population with respect both to differentiation capacity and sustainability of self-renewal. For example, single cell analyses of CD45+lin-RholowSP and/or CD45+EPCR+CD48-CD150+ (E-SLAM) population  revealed four patterns of heterogeneous differentiation behaviour  (referred to as α, β, γ, and δ HSC subtypes), among which two of the patterns (α and β HSC subtypes) were associated with durable self-renewal activity14,15. Other work has shown that the Rholowlin-c-kit+Sca-1+ fraction of bm cells can be further resolved into cells with sustained lympho-myeloid repopulating activity versus those that sustain it for only a limited time, although up to 6-8 month period, so-called intermediate-term HSCs16. However, the extent of self-renewal that must be observed before the HSC definition is met is still debated. It could be argued that the only true test for  17  self-renewal in vivo is to test the regenerated cells using the same assay (i.e. secondary transplantation).  1.3.3 Phenotypic characterization of HSCs Currently, HSC cannot yet be positively identified on the basis of any single unique morphological, physical or cell surface characteristic. Rather a number of different phenotypic markers are used in combination to enrich for HSCs. The most commonly analyzed markers are cell surface antigens against which specifically reactive monoclonal antibodies have been made. These antibodies can then be labeled either directly or indirectly (via a secondary antibody) with a unique fluorochrome and used to distinguish cells as positive or negative on the basis of their acquired fluorescence. Multi-parameter flow cytometry has the power to distinguish cells based on these molecularly-determined features, in an objective and quantitative way, with a high degree of specificity. In addition, this technology can be used not only for cell analysis but also for their separation into viable subsets defined by the analysis. These isolated cells can then be assayed for their functional attributes. In this way, the phenotype of different functionally defined cell populations can be identified. Initially, mouse bm subpopulations including primitive hematopoietic cells were prospectively isolated by selecting cells that lacked cell surface markers expressed on differentiated hematopoietic cells (so-called lineage or lin markers, expressed predominantly on terminally differentiated lymphocytes (CD45R/B220, CD3, CD4, CD8), myeloid (CD11b/Mac-1, Ly-6G/Gr-1), and erythroid (TER-119)  18  cells)37 and expressed stem cell antigen-1, or Sca-172 and c-kit76. Later experiments showed that the lin-Sca-1+c-kit+ (LSK) population could be further fractionated into functionally distinct subpopulations, based on differential expression of additional cell surface molecules, such as CD3477,78, CD2779, flk2/flt380-82, endoglin/CD10583, SLAM family receptors including CD150, CD48 and CD24484, EPCR/CD20185, and α-2 integrin/CD49b86, or by differences in staining with the vital dyes, Rhodamine (Rho)-12387 or Hoechst 33342 (Ho)88,89. Cells with the ability to efflux Ho are often visualized using two emission wavelengths, giving rise to the characteristic side population or SP phenotype90. Using a combination of some of these surface markers, for instance CD45+linRho-SP73,  or  CD45+EPCR+CD48-CD150+  (E-SLAM)15,  or  LSKCD34loFlt3-  RholoCD49blo16, quiescent cells with long-term multilineage reconstituting ability can now be routinely isolated to near homogeneity, at purities of >50%. Nevertheless, when examined at the clonal level, even highly purified HSCs produced significant variation in progeny output14. However, many stem cell markers are expressed differently in quiescent versus activated HSCs, including those present in developing hematopoietic tissues (FL), or mobilized in adults by cytokine administration or after myelosuppressive treatments, or when exposed to growth factors in culture. Thus, in spite of the identification of phenotypes of adult mouse bm cells that are almost exclusive for HSCs, the use of these phenotypes as surrogate indicators of HSCs can lead to false conclusions. Several examples of markers that show  19  such inconsistency in their expression on HSCs include CD3491, CD3892, Hoechst and Rhodamine efflux activity93, endoglin/CD10594, and Mac-121,95. Some latest reports hold the promise for establishing a purification strategy that robustly identifies activated HSCs by showing the existence of some markers that may be stable in both steady-state and activated HSCs, including SLAM family markers96,97, and EPCR15.  1.4  Regulation of HSC self-renewal process  Self-renewal division and preservation of HSC function is a complex process involving cell cycle stimulation in which competency for multi-lineage differentiation is maintained but not activated in one or both daughter cells, along with the maintenance of their genomic integrity. The decision of a HSC to selfrenew or become more restricted upon division is thought to reflect a combination of both cell autonomous (intrinsic) mechanisms and processes influenced by the surrounding (extrinsic) environment (Figure 1.2). Moreover, preserving genomic integrity of a HSC emerges as another important way of maintaining its homeostatic function, as being a long lived cell prone to accumulating DNA damage that can easily be propagated to daughter stem cells and downstream progeny through the process of self-renewal and differentiation.  20  1.4.1 Extrinsic regulation of HSC self-renewal Extrinsic control mechanisms refer to changes in cellular activity mediated by the interaction of the cell, often through specific cell surface receptors, with factors in the external environment (ie. cytokines, hormones, cell-cell contacts). Growing evidence strongly supports the concept that HSCs occupy a specific in vivo environment, known as the HSC niche. Two spatially and likely functionaly distinct HSC niches have been proposed, endosteal and perivascular. The molecular crosstalk between HSCs and cellular components and/or soluble or membrane-bound ligands of the niche has been shown to regulate the balance between HSC quiescence and self-renewal during hematopoietic homeostasis and/or injury98,99. The niche concept has provoked further research into the cells and molecules that extrinsically influence the self-renewal behaviour of HSCs. Such activity was demonstrated through in vitro studies primarily utilizing highly enriched HSCs cultured either on feeder cells (ie. stromal cells derived from AGM100, FL101, bm102, and osteoblasts103-105) or in serum- and feeder-free conditions, in the presence of recombinant cytokines. The latter studies have yielded some direct evidence of growth factor dependent modulation of HSC selfrenewal versus differentiation decisions. Some selected examples will be summarized in the section 1.4.1.2.  1.4.1.1  Niche signals  The term “niche”, describing HSC bm microenvironment, was first created in the 1970s106 based on an earlier observation that progenitors capable of forming  21  multilineage colonies in the spleens of irradiated mice (CFU-S) are more enriched near the endosteum than within the central bm107. More recently, it has been demonstrated that defective mutants108-112  resulted  in  bone development in several mouse  hematopoietic  deficiency.  Moreover,  genetic  manipulation that has increased osteoblast numbers in mice also increased the number of HSCs in the bm, providing direct evidence for the involvement of endosteum in HSC regulation and maintenance. In two independent studies, this has been achieved by osteoblast-specific expression of a constitutively active form of parathyriod hormone103, or inducible deletion of bone morphogenetic protein (BMP) receptor1A in bm stroma104. Finally, recently developed HSC purification strategies in combination with immunohistocytochemical localization techniques have confirmed that quiescent HSCs are often localized at the endosteal surface, associated with osteoblasts84,113,114. However, the latest studies using HSCs highly enriched using SLAM markers, have revealed the presence of HSCs at the surface of sinusoidal endothelium84, raising the possibility that during homeostasis, another HSC microenvironment, located more centrally in the bm cavities, may also exsist115. These perivascular niches are mainly essential for supporting injury-activated HSCs and their immediate progeny116, while clearly capable of harboring quiescent HSCs as well. Although the exact site of HSCs is still debatable, there appears to be consensus that in healthy adult mice most quiescent HSCs reside close to the endosteal lining within trabecular bm cavities. As reviewed in Trumpp et al.116, these endosteal (or osteoblastic) niches are composed of a unique combination  22  of osteoblasts, osteoclasts, stromal fibroblasts, vascular structures, extracellular matrix and sympathetic neurons. Quiescent HSCs are tightly anchored in their niche through interactions with a number of adhesion molecules expressed by both HSCs and niche stromal cells (i.e. N-cadherin, CD44 and numerous integrins),  together  with  secreted  extracellular  matrix  components  (i.e.  osteopontin and hyaluronic acid) and Ca++-mediated signalling. Once in close proximity to the niche, efficient receptor-ligand signalling occurs in HSCs, expressing KIT, TIE2, MPL, and CXCR4 receptors in response to their respective ligands, membrane-bound stem cell factor (SCF), angiopoietin 1 (ANG1), thrombopoietin (TPO), and CXCL12 secreted by osteoblasts. Functional studies indicate SCF maintains HSC function108,117, ANG1 and TPO promote HSC quiescence113,118,119, whereas CXCL12 regulates HSC migration and localization in the bm120. Mutant versions of any of these receptors induce HSC cycling, exhaustion and/or detachment from the niche116. Osteoblasts have also been implicated in HSC maintenance by activating Notch and Wnt signalling in HSCs and shown to be sufficient in promoting HSC self-renewal in culture121,122. However, in vivo, conditional deletion of the Notch receptor and ligand123 or Wnt signalling intermediates (i.e. β- and γ-catenin)124 does not affect adult HSC maintenance. Thus, it is possible that in vivo HSC self-renewal is maintained by redundant signals, rather than dependent upon any particular one. Since in the niche a complex array of extrinsic molecular signals along with cell-intrinsic regulatory networks control HSC function and balance their numbers in response to physiologic demands, mimicking such environment or at  23  least its essential components could refine in vitro conditions supportive of HSC self-renewal. Therefore, further resolution of the key regulators mediating HSC behaviour in the niche will likely provide major new insights into methods for achieving HSC expansion.  1.4.1.2  Control of HSC self-renewal by growth factors  Much effort has been directed at trying to identify growth factors that alone or in combination would support the proliferation, survival and ideally self-renewal and net expansion of HSCs. Using various combinations and concentrations of SCF, Flt3-ligand (Flt3L), interleukin (IL)-6 and/or IL-11, up to several fold net increases of mouse bm HSC have been documented125-127. Later studies revealed that while Flt3L or SCF alone stimulated proliferation, maintenance of HSC activity required activation of the gp130 pathway via IL-6 or IL-11 stimulation128. These findings were validated by culturing single highly purified HSCs (~50% pure) in 300 ng/ml SCF and 20 ng/ml IL-11 and showing maintenance of transplantable HSC activity in the first division progeny73,129. Experiments using TPO have demonstrated its positive effect on HSC expansion in serum- and feeder-free cultures130. Nevertheless, TPO alone sustains HSC numbers without inducing their proliferation, thus probably promoting HSC survival and maintenance, rather than affecting HSC mitogenesis. Taken together, these findings recognize the importance of high level activation of certain tyrosine kinase (KIT via SCF binding131,132) and cytokine receptors (gp130 via IL-11 or IL-6 binding133, or MPL via TPO binding134,135 and subsequent activation of several signalling pathways  24  (i.e. JAK/STAT136, MAPK, PI3/AKT137-139) in the regulation of HSC self-renewal process. In addition, recent gene expression profiling of day 15 murine fetal liver cells, supportive of HSC expansion in culture, has identified several new candidates (i.e. insulin-like growth factor 2 (IGF2) and angiopoietin-like proteins) subsequently shown to support 2-20-fold net expansion of HSCs in 10-day culture, together with SCF, TPO and fibroblast growth factor-1 (FGF-1)140,141. Finally, since several evolutionary conserved pathways have been implicated in having a role in HSC biology, their respective ligands have been shown to positively (Wnt3a122, Delta1142) or negatively (transforming growth factor beta (TGF-β143)) influence mouse HSC self-renewal, when added to a culture as growth factors. Overall many studies have been undertaken to elucidate the role of soluble extrinsic factors on HSC self-renewal and address the issue of how these factors may expand HSC population in vitro. However, the magnitude of HSC expansion they stimulate remains modest.  1.4.2 Intrinsic regulation of HSC self-renewal Intrinsic control mechanisms refer to those processes that take place within HSCs to confer their key properties and hence their behaviour. These encompass a variety of intracellular signalling molecules, cell cycle regulators, epigenetic modifiers, and complex transcriptional networks that determine cell potential and fate (reviewed in9,144,145). Some key intrinsic regulators, with the  25  focus on Hox transcription factors (section 1.4.2.5) particularly relevant to the body of work presented later in this thesis, are highlighted below.  1.4.2.1 Intracellular signalling molecules A handful of developmental cell signaling pathways, including Wnt/β-catenin, TGF-β/Smad, JAK/STAT, and Notch - control the vast majority of patterning and cell fate specification events during embryogenesis146. Some of these signaling pathways also appear to play important roles in later phases of fetal and adult hematopoiesis. For example, retroviral-mediated overexpression of β-catenin in HSCs resulted in expansion of HSC numbers in long-term cultures, accompanied by up-regulated expression of genes involved in stimulating HSC self-renewal, Notch1 and HoxB4121. However, later studies showed no increase in self-renewal when β-catenin was conditionally expressed in vivo147, and HSCs lacking βcatenin expression failed to result in an in vivo phenotype148. As  reviewed  in  Blank et al.9, recent reports have indicated that TGF-β/Smad signaling may have an important role in the regulation of HSC fate decisions, both as a positive and a negative regulator of HSC self-renewal in vivo. Also, enforced activation of Notch signalling or its downstream target Hes-1 has been shown to increase the selfrenewal capacity of in vivo repopulating HSCs. As mentioned in a previous section, many pathways activated by growth factors have been identified as having a role in HSC self-renewal. Thus, SCF, TPO, IGF2, and ligands that activate gp130 subsequently activate the JAK/STAT pathway. Expression of a dominant negative Stat3149 and deletion of Stat5A150 in  26  mouse HSCs have resulted in decreased bm and blood cellularity and a loss of competitive ability after transplantation, indicating the importance of both, Stat3 and Stat5A activation in hematopoiesis. Similarly, upregulation of Stat3151 and Stat5A152 by the expression of their constitutively activated form has shown enhanced self-renewal and regeneration activity of transplanted HSCs.  1.4.2.1  Cell cycle regulators  HSCs require complex cell cycle regulation mechanisms in order to confer the potential for repeated periods of quiescence and cell cycle re-entry. Retinoblastoma family proteins (Rb, p107, and p130) are major regulators of the G1 to S cell cycle transition by repressing of E2F transcription factors that otherwise induce genes required for DNA replication and S phase entry. Although the deletion of a single Rb gene in hematopoietic cells results in a mild phenotype, deletion of all three Rb family genes leads to proliferation of HSCs and ultimately myeloproliferative disease (reviewed by He et al.145). Expression of the D-type cyclins is dependent on mitogen activation allowing the formation of complexes with partner cyclin-dependant kinases (CDK). Cyclin D-CDK4/6 complex promotes cell cycle entry by phosphorylating and inactivating Rb proteins. In mice, deletion of all three D cyclins severely impairs the expansion of HSC numbers, leading to their depletion during fetal development (reviewed by He et al.145). The activity of the cyclin-CDK complexes is regulated by Ink4/ARF (p16, p15, p18, and p19) and Cip/Kip (p21, p27, and p57) family of CDK inhibitors with  27  important roles in regulating the progression of cells through specific phases of the cell cycle153. Several of these have been implicated as regulators of HSC self-renewal. p16154 and p18155 deficiencies each increase HSC frequency and enhance their longterm repopulating and self-renewal activity. p27 is also required to limit self-renewal of proliferating hematopoietic progenitors, but not stem cells156. Conversely, cells from adult p21-/- mice have reduced numbers of CFU-S and HSC activity possibly due to failure to respond to signals that induce HSC quiescence157,158.  1.4.2.2  Epigenetic factors  “Epigenetic regulation is defined as mechanisms that maintain cell-type specific gene expression patterns in daughter cells through cell division independently of the primary DNA sequence” (the definition taken from159). Epigenetic mechanisms involve DNA methylation, histone modifications, and regulation of chromatin structure and play important roles not only in normal stem cell homeostatsis but also in cancer development160. Polycomb and trithorax multiprotein complexes maintain proper gene expression of numerous developmental genes, including Hox genes, through histone modification159,161,162. In vertebrates, polycomb group proteins form two complexes: polycomb repressive complex 1 (PRC1), encompassing Bmi1, Phc1 (Rae28), Pcgf2 (Mel18), and Ring1 gene products and PRC2, comprised of Suz12, Ezh2, and Eed gene products. PRC2 proteins trimethylate histone H3 at lysine 27 (H3K27) and then assemble proteins of PRC1 complex that promote  28  chromatin compaction and repress transcription on this methylation mark163. In the mouse model, polycomb proteins have been implicated in regulating HSC self-renewal activity either positively (Bmi1164-166, Rae28167,168, and Ezh2169) or negatively (Mel18170). Trithorax proteins oppose polycomb repression by methylating H3K4 and activating transcription through recruiting nucleosome remodelling enzymes and histone acetylases171. It has been shown in the murine system that the Mll gene, a member of the trithorax group, plays an essential and nonredundant role in fetal and adult HSC self-renewal172 possibly through the maintenance of Hox gene expression, mainly for the Hoxb and Hoxc clusters173. Recent advances in the genome-wide mapping of DNA methylation revealed that methylated CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation. In mammals, methylation of CpG dinucleotides within the DNA is controlled by at least three different DNA methyltransferases (DNMTs): DNMT3a and DNMT3b for de novo methylation, and DNMT1 for methylation maintenance174-176. A conditional knockout study of DNMT3a and DNMT3b in adult HSCs demonstrated that DNA methylation by these enzymes is critical for self-renewal of HSCs but not for their differentiation to progenitors and mature cells177. Two recent studies also utilized an inducible, conditional knockout approach to demonstrate the role of DNMT1 in protecting essential HSC properties, by silencing differentiation programs that interfere with self-renewal and multipotency176,178.  29  1.4.2.3  Hematopoietic transcription factors  Gene knockout and overexpression studies have now provided extensive evidence of the important roles of multiple transcription factors in hematopoiesis and specifically in HSC function. Many transcription factors involved in regulation of HSC self-renewal have also been identified as a result of their involvement in fusion proteins created by translocations and associated with different types of leukemia in humans179. Transcription factors involved in specifying HSCs and their emergence during early embryogenesis include Scl/tal-1180-182 and its associated protein partner Lmo2183,184, both essential for primitive and definitive hematopoiesis as well as Aml1 (also known as Runx1), specifically required for definitive hematopoiesis185-187. Recently, Sox17 has emerged as a marker of fetal identity in HSCs and an important regulator of fetal and neonatal HSC self-renewal program188. In the absence of each of these transcription factors no blood cells are generated. However, none are required for the maintenance and self-renewal of adult HSCs. The long list of key transcription factors required for the self-renewal of both fetal and adult HSCs includes: Gata2189,190, Pu.1191,192, c-Myc, N-myc193,194, Myb195,196, CBP197, JunB198, Zfx199, Meis1200,201 and several Hox family members, described in more detail in the following section (1.4.2.5). Lastly, Gfi1202, Tel/Etv6203, and Tie2204 transcription factors are all required for the maintenance of adult but not fetal HSCs. Gfi1 and Tie2 promote the quiescence of adult HSCs, whereas TEL/ETV6 promotes their survival.  30  This body of information suggests that many self-renewal regulation mechanisms are conserved between fetal and adult HSCs, although some are clearly modified to account for developmental differences in HSC function.  1.4.2.4  HOX transcription factors  The Hox genes were first discovered in Drosophila melanogaster205. D. melanogaster has eight homeobox genes divided in two clusters, Antennapedia (Ant-C) and Bithorax (BX-C) complexes. Together the two groups of clustered genes make up the Drosophila homeotic complex (HOM-C). In mammals, there are two main groups of Hox genes, class I or the clustered Hox genes that have high homology to HOM-C and class II, non-clustered and divergent homeoboxcontaining genes206. This thesis focuses only on Class I Hox genes. Mammalian Class I Hox genes are an evolutionary preserved family, comprised of 39 members and organized in four clusters, A-D, each containing 911 genes on four different chromosomes207-209 (Figure 1.3). Based on homology, Hox genes in separate clusters can be aligned in groups, resulting in 13 paralogs (i.e. HoxA4, HoxB4, HoxC4 and HoxD4). Paralog groups 1-8 are more closely related to Ant-C genes, whereas paralog groups 9-13 are more closely related to the Abdominal-B (Abd-B) gene of BX-C210. The high homology within paralogs suggests that a quadruplication of a single gene cluster has occurred during evolution211-213. Hox proteins are DNA-binding transcription factors that specify the anterior-posterior axis and segment identity of metazoan organisms during early  31  embryonic development. They have domains for DNA binding and for proteinprotein interactions. The most prominent structure of all Hox proteins is their 60amino acid DNA binding domain, referred to as homeodomain214. Foot printing, electrophoretic mobility shift assays (EMSA) and trans-activation assays have shown that Hox proteins bind DNA as monomers to 5’-TAAT-3’ core motif215. As homeodomains themselves have limited target sequence recognition, additional specificity and affinity is provided by direct interaction of Hox proteins with Hox cofactors, the homeodomain-containing proteins Pbx and Meis1216,217. Interestingly, Hox genes of the A, B, and C but not D clusters are also transcribed during normal mouse and human hematopoiesis and their expression is largely confined to primitive subpopulations218-221, suggesting their functional role in early hematopoiesis. Thus, the role of Hox genes in normal hematopoiesis has been extensively studied through gain- or loss- of function studies in mouse models (Figure 1.3)6. Some examples of such studies, recently summarized by Argiropoulos and Humphries6, are outlined in the following segment. Several Hox genes (i.e. HoxA7222, HoxA9223, HoxA10224) have emerged from these studies to have a positive impact on HSC self-renewal when overexpressed in mouse bm cells. Another striking example of such impact is enforced expression of HOXB4 that has resulted in HSC expansion in vivo and in vitro, without causing leukemia225-227 . Efforts have been made to resolve the mechanisms underlying such effect, by identifying downstream targets of HOXB4228,229. However, little progress has been made due to the absence of overlapping targets. Interestingly, HOXB4 deficiency in mice did not show any disruption of definitive  32  hematopoiesis230,231, implying that HOXB4 is not required for normal HSC function and/or that its function might be carried out by other Hox genes. In contrast, mice lacking HOXA9 demonstrated defects in multiple hematopoietic lineages232,233, as well as HSCs234. Along with such severity of the knockout phenotype and representing one of the most highly expressed Hox gene in the HSC compartment, HOXA9 emerges as a potential key HOX regulator of HSC function. Moreover, overexpression studies clearly demonstrated its capacity to stimulate HSC self-renewal, thus enhancing their regeneration in vivo. However, mice engineered with bm expressing HOXA9, ultimately succumb to leukemia223. Recently, the involvement of Hox genes in human leukemia has been supported by their observed aberrant expression235-239 and by translocations involving their cofactor Pbx1240 and/or upstream regulators241. The discovery of chromosomal translocations involving Nup98 and several Hox genes has now provided direct evidence of the involvement of Hox genes in the pathobiology of human leukemia242. Further elucidation of the cellular and molecular processes that are involved in normal and/or leukemic hematopoiesis and controlled by the complex Hox-based regulatory network, holds promise for developing new tools to expand HSCs and for providing a deeper understanding of mechanisms underlying HSC self-renewal process. Indeed, several experimental models based on engineered overexpression of Hox genes in hematopoietic cells have provided evidence of the potent ability of certain Hox and variant Hox proteins to enhance HSC selfrenewal behaviour. These points will be further elaborated upon in section 1.5.1.  33  1.4.3  Maintenance of genomic integrity in HSCs  HSC DNA damage is imparted by a wide range of exogenous (i.e. genotoxic chemicals, UV and ionizing radiation) and endogenous (i.e. reactive oxygen species (ROS), telomere attrition, DNA replication errors and double strand breaks) sources243. HSCs exploit a number of tumor suppressor (i.e. Rb, p16 154,244,245  , p53246,247, FoxO transcription factors248,249) and/or DNA repair250-256  pathways that minimize and/or recognize and repair these different types of lesions. However, a certain extent of DNA damage evades repair and accumulates with age, potentially driving cells to senescence, apoptosis, or transformation. While there is a growing excitement about the prospect of ex vivo expansion of HSCs that could have significant utility for transplantation-based therapies257, major concerns remain unresolved regarding the consequences of such manipulation on telomere length and/or HSC function, particularly whether the prolonged symmetric self-renewal activity is associated with more rapid aging as evidenced by accelerated telomere loss. The relevant background will be elaborated upon further in the following sections (see 1.4.3.1 and 1.4.3.2).  1.4.3.1 Telomere, loss of telomeric DNA and telomerase The maintenance of telomeres by the enzyme telomerase represents a specialized form of genomic maintenance and is of well documented importance for sustaining controlled proliferative capacity of cells. Telomeres are a complex of guanine-rich repeat sequences (TTAGGG) and associated proteins, located at  34  the end of every eukaryotic chromosome. Its primary function is to enable the DNA repair machinery to distinguish between chromosome ends and double strand brakes and thus protect the chromosome ends against chromosomal fusion, recombination and terminal DNA degradation258. Over 40 years ago, Hayflick and Moorhead suggested that most normal cells are programmed for a given number of cell divisions and cannot divide indefinitely259. Ten years later Watson260 and Olovnikov261 identified the telomere end replication problem which is believed to be the primary cause of the limited replicative potential of most normal somatic cells. As a result, their telomeres continuously shorten with each cell division. When cells reach their replicative potential, at least some of their telomeres have become critically short leading to chromosomal instability, senescence and/or apoptosis. This continuous telomere shortening as cells proliferate, suggests that telomere length may serve as a mitotic clock, ticking off the passage of time with each cell division, thus providing a  measure  of  cell  replicative  history262  and  making  telomere  length  measurements an attractive tool for studying aging. In fact, studies have shown that humans, who normally have 5 - 20 kb of telomeric DNA, lose 50 to 200 base pairs with each successive round of cell division263.  However, several other  mechanisms have emerged that also cause telomere shortening including oxidative damage, the failure to unwind or correctly process higher-order structures of G-rich telomeric DNA and the deletion of telomeres by homologous recombination264-266. The relative importance of these different telomere erosion  35  pathways and their effect on the telomere shortening of diverse cell types has not yet been fully established. Although the replicative potential of most cells is believed to be limited due to telomere erosion, there are several cell types, including stem cells and many cancer cells, which surpass this limitation by expressing an enzyme known as telomerase. The telomerase enzyme is a large ribonucleoprotein consisting of catalytically active reverse transcriptase protein (Tert) and a RNA template (Terc or TR)267, both essential for normal enzymatic activity. Cells that express sufficient levels of the telomerase avoid the end replication problem by extending 3´chromosomal ends through the addition of single stranded TTAGGG repeats. In addition to telomerase expression some cells may generate telomeric DNA through recombination events, a mechanism known as alternative lengthening of telomeres (ALT)263.  1.4.3.2 Telomere maintenance in HSCs Although most healthy hematopoietic cells do not express telomerase and thus experience telomere shortening with each cell division, there are a few exceptions. These include some types of lymphocytes and HSCs, characterized by a need for a high proliferative capacity. Studies reported to date indicate that despite detectable levels of telomerase expression268, proliferation of HSCs is accompanied by telomere shortening during aging in humans269 and during serial transplantation in mice270,271, suggesting limited replicative potential of HSCs272274  . Moreover, although telomerase over-expression in mouse HSCs may  36  attenuate the telomere shortening, it seems to be insufficient to extend their serial transplant capacity275. Perhaps some of the most compelling evidence denoting the importance of telomerase expression, both in humans and mice, has emerged by studying the severe consequences of telomerase deficiency. Patients suffering from dyskeratosis congenita (DKC) or acquired aplastic anemia with the loss of function mutations in telomerase complex genes276,277 have short telomeres, frequently associated with decreased proliferative capacity of hematopoietic progenitors, bone marrow failure and possibly malignant progression due to genomic instability278. Similar to DKC patients, late generation telomerase deficient mice (as mice possess significantly longer telomeres than humans and it takes up to 4 generations until telomeres become critically short) generally suffer from genomic instability, defects in highly proliferative tissues279,280,  including  reduced  replicative  capacity271  and  repopulating  ability253,281 of HSCs, tumor formation282 and overall reduced lifespan. In addition, three recent manuscripts provide great insights into the importance of the Tert protein in hematopoiesis; during the development283, as a transcriptional modulator of the Wnt/β-catenin signalling pathway required for stem cell proliferation and self-renewal284, or as a facilitator of HSC proliferation and recovery of peripheral blood cell counts upon androgen therapy of bm failure syndromes285. Finally, an issue of particular interest would be to further elucidate the role of telomerase in telomere homeostasis and function of HSCs under conditions of prolonged self-renewal activity, required to effect substantial net expansion of HSC.  37  1.5  In vitro expansion of HSCs  1.5.1 Demonstrated strategies for mouse HSC expansion While a variety of culture conditions have now been described that allow enormous expansion of later clonogenic progenitors and substantial expansion of even more primitive cells, detected as long-term culture initiating cells, ex vivo expansion of rigorously defined HSCs has proven a greater challenge. Although may studies have demonstrated detectable expansion of HSCs in vitro in response to particular combination of growth factors122,126,140,141,286 (see section 1.4.1.2), the proliferation of HSCs in vitro inevitably leads to hematopoietic differentiation or death. The limited expansion of HSCs obtained in these culture conditions is most likely attributable to failure to promote symmetrical selfrenewal divisions, rather than to induce cycling or survival, as demonstrated by dye tracking strategies287 and CRU assays of clones generated in single cell cultures5,73,129,288. Recent attention has focused on cell intrinsic mediators/determinants of HSC self-renewal, whose activation has in some cases resulted in expansion of HSCs ex vivo. From that perspective, forced expression of activated β-catenin121 or Bmi-1166 has shown to expand HSCs and/or multipotent progenitors in culture up to 80- or 35-fold respectively. A recent study by Deneault et al.289, has combined the power of expression profiling and functional studies revealing 18 nuclear factors that conferred a clear repopulation advantage to HSCs. Specific Hox (i.e. HoxB4) and variant Hox (i.e. Nup98-Hox) genes have also emerged as  38  potent stimulators of mouse HSC expansion in vitro and are particularly relevant to the studies conducted in this thesis.  1.5.1.1 HOXB4 as a stimulator of HSC self-renewal in vitro As mentioned earlier, a remarkable example of the potent ability of Hox to enhance HSC self-renewal is demonstrated through retroviral-engineered overexpression of HOXB4, which has stimulated expansion of HSC numbers in vitro 40 to 80-fold226. Significant enhancement of HSC self-renewal could also be elicited by repeated delivery of HoxB4 protein to the cell as an exogenously supplied TAT-HOXB4 fusion protein290. Moreover, manipulation of other pathways has yielded significant increases in HOXB4-mediated HSC expansion and has added to our current understanding of HSC self-renewal. For example, the increase in HSC self-renewal generated by overexpression of HOXB4 was further enhanced in p21-deficient HSCs291 and suppression of Pbx1 expression enhanced HOXB4-mediated HSC expansion in vitro to a remarkable 100,000fold292.  1.5.1.2  NUP98-HOX fusions are powerful stimulators of HSC self-renewal  in vitro Results from our laboratory have shown that HOXB4 is not unique in its ability to stimulate HSC self-renewal and that significant expansion of HSCs in culture may extend to other Hox genes, in particular, engineered NUP98-HOX fusion genes7,8. All naturally occurring NUP98-HOX fusions reported to date include the  39  N-terminus of NUP98 which contains a region of multiple phenylalanine-glycine repeats which has been reported to act as a transcriptional co-activator through binding to CBP/p300293 and the C-terminus of the Hox gene product, including the intact homeodomain and a variable portion of the flanking amino acids294. The engineered NUP98-HOX fusion genes that have been studied include HOXB4 a member of the Antennapedia class and HOXA10, which belongs to the Abd-B-class of Hox genes. Both fusion genes were found to stimulate a marked expansion in vitro of multipotent spleen colony-forming cells. overexpression  of  the  NUP98-HOXA10  fusion  gene  blocked  Moreover, terminal  differentiation in vitro, leading to a sustained output cells with a “primitive” phenotype7. Subsequent studies aimed to explore if overexpression of these NUP98HOX fusion genes might have an impact on HSC self-renewal, resulting in even more potent ability to stimulate HSC expansion in short term in vitro culture than HoxB4. Indeed, studies initiated by Dr. Ohta in our lab, and then pursued in a collaborative effort, were able to demonstrate the profound impact on HSC expansion of novel NUP98-HOX fusion genes with over 1,000-fold absolute increases in HSC numbers occurring in short-term cultures initiated with large or limiting numbers of transduced cells8. Moreover, this effect was preserved when sequences flanking the homeobox (encoding the homeodomain) of the HOXA10 portion of the fusion were removed, thus identifying the homeobox as the essential Hox gene sequence required in this fusion gene. The >1,000-fold expansion of HSCs obtained using the NUP98-HOXA10hd fusion gene comes  40  close to the theoretical limit in a maximum period of 7-8 days of gene expression, assuming previously reported 12-14 hour doubling time for these cells73,295. Moreover, extension of the culture period to 17 days has showed that the transduced HSC numbers continued to increase up to a total of >10,000-fold. Proviral integration analysis of genomic DNA from recipients of NUP98HOXA10hd-transduced cells has revealed a highly polyclonal reconstitution consistent with in vitro expansion of a large number of HSCs. Expanded NUP98HOXA10hd-transduced HSCs have successfully repopulated myeloid and lymphoid lineages in vivo, were non-leukemogenic and showed no sign of proliferative senescence while remaining responsive to factors that control the HSC pool size. In addition, measurements of the number of progeny CRUs regenerated in the primary mice suggest that these reach normal CRU levels in the bm without becoming excessive8. However, it still remains unclear if NUP98-HOXA10hd-induced HSC expansion results from enhanced self-renewal activity of long-term repopulating cells or HSCs, as being target cells responsive to the self-renewal promoting activity of NUP98-HOXA10hd. Also, dramatic differential behaviour of NUP98HOX fusion genes on HSC function in vitro versus in vivo reinforces the importance of both intrinsic and extrinsic regulators that control self-renewal. In particular, such different behaviours drive interest in dissecting out the possible roles of growth factors and/or other microenvironmental factors (e.g. stromaniche) that enable unrestrained self-renewal behaviour in vitro in contrast to  41  normal regenerative and differentiation behaviour in vivo. Some of these issues have been addressed later in the thesis.  1.5.2 Demonstrated strategies for human HSC expansion Like in a mouse system, currently developing protocols for expansion of human HSCs ex vivo include the use of cytokine cocktails, stromal coculture, as well as cell culture in bioreactors. These ex vivo culture systems are designed for cell populations enriched for human HSCs296-302, in conjunction with in vivo functional assay for human HSC quantification, known as the SCID (severe combined immunodeficiency) repopulating cell (SRC) assay303. Common components used in ex vivo human CB expansion protocols and demonstrated to support up to 4-fold HSC expansion include SCF, IL-3, IL-6, granulocyte colony stimulating factor (G-CSF) and Flt3-L  304,305  , and SCF, Flt3-L,  IL-6, TPO and the soluble IL-6 receptor306. Latter has been shown to expand HSCs from bm and mobilized peripheral blood as well307. Moreover, several developmental regulators of cell fate determination including Notch308 and Sonic hedgehog309, stromal co-culture with mesenchymal stem cells as feeder cells310, and/or using bioreactor systems  311  have all been shown to modestly augment  these cytokine-induced effects on HSCs. In recent years, the dramatic effect of two chromatin-modifying agents (i.e. histone deacetylases), 5-aza-2'-deoxyctidine and trichostatin A, on the fate of human bm cells have been shown312 and later the addition of 5-aza-2'deoxyctidine/trichostatin A to human CB cell culture has demonstrated up to 10-  42  fold SRC expansion313 accompanied by increased expression of HSC selfrenewal stimulator HoxB4314. Similar effects on cultured human HSCs were seen in the presence of histone deacetylase inhibitor, valproic acid315. HoxB4 also has the potency to expand human HSCs, which has been demonstrated by its overexpression in human CB cells, showing up to 5-fold competitive growth advantage in vivo316,317. Similar competitive advantage of HoxB4-tarnsduced cells has been validated in dog and nonhuman primate models and even more striking  effect  seen  in  cells  that  were  ex  vivo  expanded  prior  to  transplantation318,319. A major stride toward the goal of ex vivo expansion of human HSCs has been made with the recent discovery of a small molecule called StemRegenin1 (SR1), the antagonist of the aryl hydrocarbon receptor (AHR)257. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice320.  1.6  Thesis objectives  As reviewed above, the self-renewal function of normal HSCs is essential to their ability to sustain lifelong hematopoiesis and forms the basis for an ever widening range of transplant based therapies for bone marrow failure, malignant and genetic disorders. At the same time there is increasing evidence that for a wide spectrum of leukemias the disease is driven by a rare subpopulation of cells (LSCs), with “stem-cell” like characteristics46. Current knowledge regarding the  43  precise regulatory network underlying HSC self-renewal or unique set of molecular markers that define HSC multipotent state remains incomplete. Accordingly, the overall goal of my PhD thesis research was to better characterize mouse HSCs with increased and prolonged self-renewal activity in order to improve understanding of the processes involved in the regulation of HSC self-renewal programs, relevant to the development of new tools both for clinical expansion of normal HSCs and ultimately for disruption of self-renewal pathways to inhibit LSC growth. This research builds on evidence that some naturally occurring and novel homeobox transcription factors that are expressed in primitive hematopoietic cells are involved in the regulation of normal and/or leukemic hematopoiesis. Specifically, my focus was to investigate the potency and mechanisms of action of one such factor, a fusion of the nucleoporin gene, NUP98, and the homeobox (encoding the homeodomain) of HOXA10 (NUP98HOXA10hd). This was pursued through several following aims. The first aim of my thesis was to characterize the efficiency of NUP98HOXA10hd to enhance the self-renewal program of HSCs (from both bm and FL sources) at the clonal level. This would provide the direct evidence that highly purified functionally defined long-term repopulating cells or HSCs are the target cells responsive to the self-renewal promoting activity of NUP98-HOXA10hd, as well as the direct measure of NUP98-HOXA10hd potency to expand HSCs ex vivo. The second aim was to identify the phenotype of NUP98-HOXA10hdexpanded HSCs in order to develop strategies for prospective isolation of HSCs with enhanced self-renewal capacity. Additionally, in attempt to develop a short-  44  term surrogate assay for detecting the presence of HSCs in culture, the correlation between the phenotype and in vivo functional readout of NUP98HOXA10hd-transduced HSCs was monitored following their exposure to multifactorial growth factor conditions. The final aim of this thesis was to explore the effect of enhanced HSC self-renewal in vivo and in vitro on their premature senescence (i.e. telomere shortening) and possibly impairment of their functional capacity. To this end the impact of expansion on HSC quantitative and qualitative exhaustion was examined under conditions of severe stress, including the absence of telomerase (Tert-/- background) and conditions of prolonged selfrenewal stimulation in vivo and in vitro.  45  Erythrocytes Platelets  Granulocytes Macrophages Dendritic cells  T-cells  NK-cells  B-cells  Figure 1.1 Hierarchical model of hematopoiesis (adapted from Weissman and Shizuru321). HSCs are placed at the top of the hematopoietic hierarchy and have the ability to give rise to all of the downstream lineages of the hematopoietic system with both myeloid and lymphoid elements. Arrow indicates the HSC as the only cell in the hierarchy with long-term self-renewal activity. BLP, B lymphocyte progenitor; ProT, T-cell progenitor; GMP, granulocyte/macrophage progenitor; MEP, megakaryocyte/erythroid progenitor; Mk-P, megakaryocyte progenitor; EP, erythroid progenitor.  46  Figure 1.2 Key regulators of HSCs (adapted from Rizo et al.322). A graphical representation of some key extrinsic and intrinsic regulators of HSCs and signalling pathways involved in the HSC fate in the niche. Extrinsic regulators appear on the outside of the cell and intrinsic regulators are in the inside of the cell.  47  Figure 1.3 Clustered Hox gene organization (adapted from Argiropoulos et al.6). The four Hox clusters are located on four different chromosomes. Based on the sequence homology, individual genes in different clusters are aligned into paralagous groups (identical colours). Boxes above genes or brackets under genes summarize resulting hematopoietic phenotypes of engineered overexpression or knockout of a particular Hox gene(s), respectively. BFU-E, erythroid burstforming unit; CFU-GM, granulocyte/macrophage colony-forming unit; MPD, myeloproliferative disorder; LL AML, long latency acute myeloid leukemia.  48  CHAPTER 2 Ontogeny stage-independent and large magnitude clonal expansion in vitro of HSCs stimulated by an engineered NUP98-HOX fusion transcription factor  2.1 Introduction  HSCs are rare cells within the hematopoietic hierarchy responsible for the permanent establishment of hematopoiesis10. The ability of HSCs to self-renew is essential  for  their  expansion  throughout  hematopoietic  development,  homeostasis, following bm transplantation and/or in response to different physiological stresses19-25. Elucidating the critical elements underlying this process is thus of critical importance for clinical purposes42,43. Numerous molecular elements have been shown to be essential to the self-renewal machinery of HSCs, including intracellular signalling molecules121,139,151,152,323-325, cell  cycle  regulators155,158,326,  chromatin  modifiers164-167,169,170,176-178,  and  transcription factors189-199,201-204. In addition, various growth factor receptors have been identified as mediators of environmental cues that can modulate the selfrenewal process122,126,140,141,286. Forced manipulation of intrinsic cellular elements has also been used with success as an alternative and potentially complementary track to obtain amplified HSC populations ex vivo8,226,289,291,292. The latter approach includes the engineered expression of HOXB4226,227, and various potent NUP98-HOX fusion constructs8,294,7. These studies also showed the homeobox domain, but not its flanking sequences to be an essential element  49  in the NUP98-HOXA10hd fusion construct. Greatly increasing the potential clinical implications of this work, in spite of its ability to rapidly stimulate very large expansions of adult bm HSC numbers, no evidence of perturbed HSC function or regulation in vivo has been identified8. Outstanding are questions about the cellular targets and biological mechanisms that lead to the production of expanded HSC numbers from NUP98HOXA10hd-treated bm cells. To address these issues, we designed a series of experiments in which highly purified suspensions of fetal and adult bm HSCs were used as targets in both bulk and single cell expansion culture protocols. The results demonstrate an ability of NUP98-HOXA10hd to reproducibly stimulate multi-log expansions from individual HSCs, under multiple growth factor stimulatory conditons and without effects on the growth kinetics or phenotype associated with proliferating normal HSCs. These findings support an ability of NUP98-HOXA10hd to directly potentiate the self-renewal machinery operative in HSCs throughout ontogeny and introduce the reality of large scale recovery of functionally normal HSCs at high purity.  2.2 Materials and methods  2.2.1 Mice Mice were bred and maintained at the British Columbia Cancer Agency Research Centre animal facility according to the guidelines of the Canadian Council on Animal Care. Transplant donors were C57Bl/6J (B6) mice that express CD45.2  50  or C57Bl/6Ly-Pep3b (Pep3b) mice that express CD45.1. Recipients of B6 cells were either C57Bl/6-W41/W41 (W41) mice that express CD45.2 or Pep3b mice; recipients of Pep3b cells were B6 mice. B6 heterozygous mice expressing both CD45.1 and CD45.2 were used as recipients in certain competition assays.  2.2.2 Flow cytometry Populations of cells with a lin-Sca1+kit+ (LSK), lin-Sca-1+CD43+(c-kit+)Mac1dim, CD45+lin-Rho-SP or CD45+EPCR+CD48-CD150+ (E-SLAM) phenotype were isolated using a FACSVantage, FACSDiVa or AriaII cell sorter (Becton Dickinson (BD), San Jose, CA, USA) as previously described14,15,59. For transduced cells, anti CD11b (Mac1) was left out of the lin cocktail. Data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA).  2.2.3 Viral transduction and cell culture GP+E86 cells producing high titer helper virus-free MSCV-IRES-GFP (GFP) and MSCV-NUP98-HOXA10hd-IRES-GFP  (NUP98-HOXA10hd)  virus7,67  were  seeded into the wells of round-bottom tissue culture treated 96-well plates at 4x104 irradiated (40 Gy of X-rays) cells per well and then single or aliquots of 2050 highly purified HSCs added. Cells were co-cultivated for 48 hours in 100 µl of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% fetal bovine serum (FBS), 5 µg/mL protamine sulfate (Sigma, Oakville, Canada), and either 10 ng/ml human (IL-6, 6 ng/ml murine IL-3 and 100 ng/ml murine SCF in the case of adult bm cell transductions or 50 ng/ml murine SCF in the case of  51  fetal liver cell transductions327. Media, serum and growth factors were all from STEMCELL Technologies (Vancouver, BC, Canada). The cells from each well were then recovered, transferred to non-tissue culture-treated 96-well plates and further cultured in 200 µl of the same medium without protamine sulfate until confluency (around day 7 for clonal or day 4 or 5 for bulk cultures). Thereafter, on every 2-3 days, cells were expanded in increasing volumes of up to 2ml of media by day 14 for clonal cultures and day 10 for bulk cultures, except for the factorial design experiments where a different experimental design was used (see below).  2.2.4 Microfluidic cultures A microfluidic cell culture array containing 1,600 chambers (4.1 nl each) and an integrated iso-osmotic reservoir was fabricated as previously reported328. Two days prior to loading cells into the array, the iso-osmotic bath was filled with DMEM and the microfluidic array primed with culture medium (DMEM supplemented with 15% FBS, 10 ng/ml IL-6, 6 ng/ml IL-3 and 100 ng/ml SCF). The environmental chamber (Live Cell Instrument, Chamlide) was set to 37oC, 5% CO2 and humidity levels were maintained close to saturation. Just before loading the cells, the bath content was replaced and medium inside the device was exchanged. Two days after the end of infection, cells were harvested, centrifuged at 250 g for 5 minutes and resuspended in 5-10 µL. Using a Teflon tube connected to a stainless steel pin, control GFP-transduced cells were first introduced into the device at a rate of 1uL/min until the first cells reached the end  52  of the array. The pump was then stopped to allow these cells to settle down by gravity to the bottom of the chambers. This process was repeated until the entire inoculum volume was loaded. Medium was then pumped through the array to flush out any unsettled cells. A picture set of the entire array was taken to identify wells inoculated with single control GFP-transduced cells. NUP98-HOXA10hdtransduced cells were then introduced into the device in a similar manner, and a second picture set was taken to identify wells inoculated with single cells (either control GFP- or NUP98-HOXA10hd-transduced). Bright field pictures of wells identified as containing single cells at the start were then acquired every 5 minutes using a microscope (Axiovert 200, Carl Zeiss) outfitted with an automated stage (ProScan II, Prior Scientific), a 20x objective and a CCD camera (Orca ER, Hamamatsu). A media bottle was connected to the device and medium was exchanged automatically every 3 hours for 72 hours. At the end of the experiment, the clones were imaged using bright field and green fluorescence to identify transduced cells.  2.2.5 Transplantation and analysis of transplanted mice HSCs numbers were quantified as described67 by performing limiting dilution (LD) CRU assays either in sublethally irradiated W41 mice (360 cGy of X-rays) or in lethally irradiated B6 or Pep3b mice (810 cGy of X-rays, given a life-sparing injection of 105 normal B6 or Pep3b bm cells). For the competitive transplant experiments, mice were injected with cell numbers from each source that were estimated to contain 30 HSCs based on previously documented HSC  53  frequencies in fresh fetal liver21, fresh adult bm23 and day-10 NUP98-HOXA10hdtransduced adult bm cells (Table 2.3). Mice were bled 2, 4 and 5-6 months post transplantation and analyzed for the presence of GFP+ T and B lymphoid and granulocyte/monocyte (GM) white blood cells (WBCs) after ammonium chloride lysis of the erythrocytes as previously described8. Recipients with ≥1% donorderived (GFP+) lympho-myeloid cells at 20 and/or 24 weeks post-transplant were considered to be repopulated by HSCs.  2.2.6 Factorial design experiments E-SLAM cells were transduced and after 7 days in standard bulk expansion cultures, lin-Sca-1+CD43+(c-kit+)Mac1dim cells were isolated and subdivided into 20 equal aliquots that were then further cultured for 7 additional days in all combinations of 4 growth factors. A total of 20 conditions were analyzed in 24 full factorial combinations plus 4 centre points. The high-level growth factor concentrations were 6 ng/ml for IL-3, 10 ng/ml for IL-6, 100 ng/ml for SCF and 100 ng/ml for human thrombopoietin (TPO, Genentech, Inc., South San Francisco, CA, USA). The centre point levels were 1:10 dilutions of the high concentrations and the low levels were 0 ng/ml of each growth factor. The full array of conditions tested is presented in Appendix E. Two types of endpoints were analyzed: 1) the number of lin-Sca-1+CD43+(c-kit+)Mac1dim cells present at the end of the cultures and 2) the fraction of GFP+ cells found in the circulating WBCs of transplanted mice after 6 weeks or 4 months. When no reconstitution was achieved, the fraction of reconstitution was defined as 0%. For full factorial  54  modeling, the growth factor concentrations were converted into scaled estimates via Equation 1, approximatng the low level absence of factors as 1:100 dilutions of the high growth factor levels. (Equation 1) The full factorial model analyzed using JMP® 8.0.2 software was then reduced to include only main and interaction effects that were significant (P<0.05) for at least one of the 3 responses measured. After model reduction, the prediction equation obtained was of the following form:  (Equation 2)  where Y is the modeled response, the βi values represent main factor effects (e.g., βIL3 is the main effect of IL-3), βij values represent second order interaction effects (e.g., βIL6,SF is the interaction effect of IL-6 and SCF), while βIL6,SF,TPO represents the third order interaction of IL-6, SCF and TPO. Each response was modelled separately and the model intercept β0 represents the predicted response Y obtained using the centre point levels of all 4 growth factors in combination.  2.2.7 Viral Integration analysis Southern blot analyses were performed as previously described8 on genomic DNA isolated using DNAzol reagent (Invitrogen, Carlsbad, CA, USA) and digested with EcoR1 which cleaves the integrated provirus at a single site.  55  Digested DNA was then separated in a 0.8% agarose gel by electrophoresis and transferred to zeta-probe membranes (Bio-Rad, Mississauga, ON, Canada). Membranes were probed with a [32P]dCTP-labelled GFP sequence.  2.3 Results  2.3.1 Efficient high-level expansion of HSCs in single cell cultures of NUP98-HOXA10hd-transduced CD45+lin-Rho-SP or E-SLAM cells Single CD45+lin-Rho-SP or E-SLAM cells containing HSCs at purities of 20% to 50%15,73 were isolated by fluorescence-activated cell sorting (FACS), individually transduced with a NUP98-HOXA10hd or GFP control retroviral vector and then further cultured with IL-3, IL-6 and SCF for a total of 14 days prior to measuring the total and CRU content of each individual culture as shown schematically in Figure 2.1A. Large and equivalent (~106–fold) clonal expansions of total nucleated cells were obtained from both control and NUP98-HOXA10hdtransduced input cells (Figure 2.1B). However, whereas none of the control GFPtransduced clones (n=16) contained detectable HSCs, based on the largest fractions tested (1/5th), HSCs were present at high frequencies in 25 of 28 (89%) clones initiated with NUP98-HOXA10hd-transduced CD45+lin-Rho-SP cells and in 15 of 17 (88%) clones initiated with NUP98-HOXA10hd-transduced E-SLAM cells. For many of these, 1/1000th or 1/2000th of the cells in each clone were not yet  at  limiting  dilution  and  showed  robust  long-term  lympho-myeloid  reconstitution of all recipients injected (Figure 2.1C). The average HSC  56  expansion in vitro achieved for all HSC clones tested was >1000-fold, as shown in figure 2.1E and further summarized in table 2.1. The effect of NUP98HOXA10hd overexpression on each HSC clone tested is presented in appendices A-D. Analysis of vector integration sites in Southern blots of bm DNA from representative recipients confirmed the clonal origin of the expanded HSCs in each well (Figure 2.1D).  2.3.2 The in vitro growth kinetics of NUP98-HOXA10hd-transduced HSCs is not perturbed The NUP98-HOXA10hd-induced expansion of highly purified HSCs in vitro could be mediated by an increased frequency of symmetric self-renewal divisions, a decrease in HSC cell cycle transit times and/or an increase in HSC survival. The generation of similar outputs of total nucleated cells by control and NUP98HOXA10hd-transduced HSC clones suggested that the cell cycle transit times and survival (>90% of transduced clones survived) of the NUP98-HOXA10hdtransduced cells throughout the culture period were not markedly altered. To investigate this question with greater resolution, we imaged the initial division kinetics of the transduced cells in a microfluidic array system328 (Figure 2.2A). The results show that highly purified (E-SLAM) HSCs transduced either with NUP98-HOXA10hd or a GFP control vector divide with the same (on average, 15 hour) cell cycle transit time (Figure 2.2B), suggesting that NUP98-HOXA10hd stimulates self-renewal fate decision without impact on cell division kinetics.  57  2.3.3 Proliferation phenotype of NUP98HOXA10hd-expanded HSCs allows their isolation at high purity We next asked whether the NUP98-HOXA10hd-expanded HSCs would also show a phenotype expected of HSCs that could allow their isolation in large numbers at high purity. To address this question, we initiated cultures with 20 30 adult bm E-SLAM cells (estimated to contain ~10-15 HSCs), transduced them with NUP98-HOXA10hd or GFP and then cultured them for a total of 10 days. The cells were then harvested and the HSC content measured by CRU assays of either total or various FACS-purified subpopulations (Figure 2.3A). Initial immunophenotyping confirmed a greatly reduced content of lin+ cells in the cultures of NUP98-HOXA10hd-transduced cells8,289,292, indicative of the differentiation block they experience (Figure 2.3B and 2.4A). The expected HSC expansions during the 10-day culture period of ~2000-fold were confirmed by limiting dilution assay (LDA) transplantation studies (Figure 2.3C and 2.4B; Table 2.2 and 2.3). Phenotypic analysis of the lin- subset in the cells harvested from the 10day cultures revealed that up to 10% of the NUP98-HOXA10hd-transduced cells were CD150+CD48-, while this subset was undetectable in the lin- cells derived from control vector-transduced cells (Figure 2.3B). However, only 37% of the recovered HSCs in the cultures of NUP98-HOXA10hd-transduced cells were present in the lin-CD150+CD48- subset and the remaining HSCs were equally distributed between the corresponding CD150+CD48+ and CD150- subsets (Figure 2.3C and 2.3D; Table 2.2).  58  To identify a phenotype that might give a greater enrichment and recovery of in vitro expanded NUP98-HOXA10hd-transduced HSCs, we examined a different phenotype previously described as suitable for purifying HSCs from E14.5 fetal livers21,59,95.  FACS analysis showed that cells with this lin-Sca-  1+CD43+(c-kit+)Mac1dim phenotype represented ~4% of the lin- population in the cultures of NUP98-HOXA10hd-transduced cells (Figure 2.4A). In contrast, they were barely detectable in the control cell cultures. Isolation and LDA transplantation assays of this fraction showed that it contained HSCs at a frequency of 1 in 4 and contained 87% of the total in vitro expanded HSCs (Figure 2.4B and 2.4C; Table 2.3). High level enrichment of HSCs in the Sca1+CD43+(c-kit+)Mac1dim fraction was further confirmed by transplanting 5 cells with this phenotype (1.25 HSCs) into multiple irradiated recipients. All 10 longterm chimeras obtained in the 12 mice injected with such transplants showed a balanced lympho-myeloid contribution to the circulating pb cells up to 6 months post-transplant (Figure 2.4D). Thus, isolation of GFP+lin-Sca-1+CD43+(ckit+)Mac1dim cells from cultures of NUP98-HOXA10hd-transduced cells offers a simple method for obtaining HSCs with increased self-renewal activity in vitro in large numbers and at very high purities. Indeed, we have subsequently established the feasibility of scaling up this expansion and purification strategy to obtain an estimated 1-2x105 highly purified HSCs at 25% purity from as few as 1,000 starting E-SLAM cells.  59  2.3.4 Factorial analysis of the effect of different conditions on NUP98HOXA10hd-transduced HSC expansion in vitro Given evidence that external cues can directly and rapidly modulate HSC selfrenewal responses in vitro73,129, we next asked whether this would also be true for NUP98-HOXA10hd-transduced HSCs. To this end, we performed a 2-level full factorial experiment to assess the effects of IL-3, IL-6, and SCF and TPO on NUP98-HOXA10hd-transduced HSC expansion in 7-day cultures initiated with lin-Sca-1+CD43+(c-kit+)Mac1dim cells isolated from previous 7-day expansion cultures. Aliquots containing 100 of these cells (estimated to contain 20-25 HSCs, see above) were exposed to various combinations of IL-3, IL-6, SCF and TPO (as shown in Appendix E) and 7 days later the cells were harvested, immunophenotyped and 1/10,000th of the cells were then transplanted into 2 irradiated recipients, for each condition tested. The total number of viable lin-Sca1+CD43+(c-kit+)Mac1dim cells recovered from the cultures and the level of chimerism obtained in the recipients 6 weeks and 4 months post-transplant were analyzed.  The results where at least one of these readouts was significant  (Figure 2.5) revealed remarkably consistent positive or negative trends. Both endpoints indicated positive effects of IL-6 and SCF (P < 0.01) on the expansion process. In addition, these endpoints both revealed a positive interaction between IL-6 and SCF (P = 0.0001). A phenotypic readout suggested that an activation of MPL receptor (via addition of TPO) has an inhibitory effect (P = 0.02) on NUP98-HOXA10hd-induced HSC expansion in vitro, seemingly to some extent through negative interactions with SCF (P = 0.02) and IL-6 (P = 0.03). The  60  ability of the lin-Sca-1+CD43+(c-kit+)Mac1dim phenotype to predict the results of functional assays of HSC responses (Figure 2.5) was further supported by the correlation of the phenotypic and 4 month endpoints (R2 = 0.80, p< 0.0001, Figure 2.5).  2.3.5 NUP98-HOXA10hd induces the in vitro expansion of fetal HSCs To determine if the self-renewal promoting properties of NUP98-HOXA10hd extended to HSCs from other stages of ontogeny, we transduced aliquots of 50 or single lin-Sca-1+CD43+(c-kit+)Mac1dim E14.5 fetal liver cells (estimated to be ~10% pure HSCs59) and then cultured them for 8 or 12 days, respectively, in the presence of hematopoietic growth factors prior to performing LDA transplant assays to determine the number of HSCs present. The results from 2 such experiments indicate a net expansion in HSCs numbers of ~900-fold (Figure 2.6A). Similarly, results from the clonal analyses showed that 3 of 5 clones analyzed (60%) produced ~450- to >1000 daughter HSCs detectable 14 days later (Figure 2.6A). Given that fetal liver HSCs are known to be able to outcompete adult bm in co-transplantation repopulation assays59,63, we next asked whether this property would be retained by their respective NUP98-HOXA10hd-transduced progeny HSCs. To examine this question, we transplanted groups of irradiated mice with equal numbers of genetically distinguishable fresh HSCs from both sources (~30 of each) or equal numbers of fresh fetal and transduced in vitro expanded adult HSCs (Figure 2.6B). As shown in Figure 2.6C, fetal liver HSCs  61  contributed to long-term reconstitution ~15-times more effectively than fresh or in vitro expanded adult HSCs. This is consistent with previous observations that the effects of NUP98-HOXA10hd on the self-renewal activity of HSCs are manifest in vitro but are constrained by the regulatory conditions operative in vivo.  2.3.6  NUP98-HOXA10hd-transduced  HSCs  that  undergo  high  level  expansion in vitro continue to expand in vivo We next investigated the possible consequences of high level in vitro expansion of HSCs on their subsequent in vivo self-renewal and regenerative activities. To address this question, the HSC content of 2 representative primary recipients was determined by flow cytometry and secondary LDA of the LSK cells produced (Figure 2.7A; Δ2). Each of these mice had been initially transplanted with ~30 HSCs derived from a single HSC, transduced and expanded in vitro (Appendix A, clone #7 and clone #8), resulting in high level (~60% and ~80%, respectively) long-term (6-7 months) lympho-myeloid reconstitution of the recipient. The proportion of transduced LSK bm cells in the 2 reconstituted mice was comparable to their counterparts in normal, unmanipulated mice. Secondary LDA of bm cells obtained from each of these 2 primary recipients show that the ~30 transduced HSCs injected into the primary recipients had, on average, expanded a further ~300-fold (Figure 2.7B; Δ2) which enabled the HSC compartment of each primary recipient to be reconstituted to up to 70% of its expected level. Moreover, the progeny of NUP98-HOXA10hd-transduced HSCs continued to  62  provide significant and balanced reconstitution of the myeloid and lymphoid lineages (Figure 2.7C - FACS plots in the middle). We next asked whether the ability of the original NUP98-HOXA10hdtransduced HSCs to expand in vitro would be retained by their progeny HSCs regenerated in vivo. Accordingly, LSK bm cells were isolated from the same primary recipients investigated above (Figure 2.7A; Δ3) and cultured in vitro for 6 days prior to being assessed by LDA transplants for the number of HSC present. Significant cell expansion was obtained with a complete absence of lin+ cells and the secondary limiting dilution transplants revealed that a further 100-fold expansion of the NUP98-HOXA10hd-transduced HSCs had occurred (Figure 2.7B; Δ3), with retention of a balanced lympho-myeloid differentiation potential (2.7C - FACS plots on the right).  2.4 Discussion  In this study, we describe the clonal expansion of highly purified preparations of both adult and fetal HSCs following their transduction with a NUP98-HOXA10hd vector and a strategy for repurifying the derived HSCs at purities of at least 25%. These findings demonstrate the consistency of the high (>1,000-fold) HSC expansions achievable from these cells within a 10-14 day culture period. The expanded HSCs also showed sustained lympho-myeloid repopulating ability without evidence of any lineage preference, or any deviation of their in vivo regeneration of a normal sized HSC compartment. Nevertheless, these HSCs  63  retain an ability to amplify their numbers in vitro under conditions that are inadequate to support normal HSC self-renewal divisions. These properties suggest an ability of NUP98-HOXA10hd to bypass the dependence of normal HSCs on unknown factors required to sustain their ability to self-renew. These findings are intriguing given the recent evidence from clonal analysis of adult bm E-SLAM cells has shown these to display variable stable differentiation and self-renewal phenotypes. Specifically, these studies have revealed 4 patterns of differentiation behaviour of which only 2, (those associated with myeloid potential, with or without lymphoid potential) are associated with durable self-renewal activity14,15. Our data suggest that all of these HSCs can be converted to display a singular differentiation and self-renewal phenotype. Thus, forced overexpression of NUP98-HOXA10hd in HSCs should provide a valuable tool for probing the controls that are responsible for this heterogeneity in the nontransduced starting cells. The finding that ~90% of transduced and in vitro expanded adult bm HSCs are lin-Sca-1+CD43+(c-kit+)Mac1dim cells and that this allows their prospective isolation at purities of at least 25% is also significant. This phenotype is consistent with the phenotype of proliferating HSC populations21,59,329, reflecting the likelihood of their status in the cultures from which they were isolated. Interestingly, it is known that normal proliferating HSCs cannot be detected at full efficiency due to their failure to engraft when transiting the S/G2/M phases of the cell cycle45,60,295,330,331. Thus, the purities determined here likely represent ~2-fold underestimates raising the values to ~50%. Moreover, the  64  demonstrated ability to apply this approach to larger cultures established the feasibility of using these cells for many genetic and chemical screening applications, as well as for additional molecular and biologic characterization studies. Finally, we found that in general a similar pattern of growth factor effects influenced the output of cultured non-manipulated128 and NUP98-HOXA10hdtransduced transplantable adult HSCs. Factorial analysis has shown that coincident activation of c-kit (via addition of SCF) and gp130 (via addition of IL-6) significantly sustained NUP98-HOXA10hd-induced HSC expansion in vitro. Moreover, a significant correlation was revealed between distinct methods (number of lin-Sca-1+CD43+(c-kit+)Mac1dim transduced cells obtained in vitro vs. level of chimerism achieved in vivo) used to detect the HSC activity obtained in 7-day culture under such multifactorial conditions. These findings illustrate a potential use of phenotype as an initial screening assay that would reduce the experimental burden compared to long-term transplantation. This phenotypebased assay should allow higher throughput screening and optimization of in vitro conditions affecting HSC self-renewal/expansion. This work provides a strategy for the prospective isolation of large numbers of HSCs with durable self-renewal activity and homogeneously balanced lympho-myeloid differentiation ability. Nevertheless, the enhanced selfrenewal ability they can display in vitro relative to their normal counterparts remains subject to the extrinsic factors to which they are exposed both in vitro and in vivo. Thus, this approach should facilitate future elucidation of how  65  intrinsic and extrinsic regulatory elements interact to determine HSC fate decisions by allowing more precise molecular interrogation of their responses to discrete stimuli. Our findings also emphasize the robustness of NUP98HOXA10hd to enhance the self-renewal program of HSCs from diverse sources (i.e. fetal and adult), thereby offering new opportunities to contrast the molecular features that distinguish and unite these HSCs. Further exploitation of these observations should not only provide novel methods for obtaining clinically relevant HSC numbers ex vivo, but likely also help to develop targeted therapies for cancers arising from inappropriately activated self-renewal pathways.  66  A  B  C  67  D  E  Figure 2.1 NUP98-HOXA10hd promotes multi-log expansion in vitro of individually purified adult bm HSCs. (A) Schematic of the experimental design followed to directly investigate the capacity of NUP98-HOXA10hd to enhance clonal HSC expansion in vitro. TNCs; total nucleated cells. (B) Average number of viable nucleated GFP- (grey) or NUP98-HOXA10hd-transduced (blue) cells at the end of 14-day culture. Values shown are the mean ± SD of 16 GFP- or 40 NUP98-HOXA10hd-transduced cell counts. (C) Proportion of peripheral blood leukocytes produced by a fraction (indicated as a transplantation dose) of NUP98-HOXA10hd-transduced and expanded CD45+lin-Rho-SP (top panel) and E-SLAM (bottom panel) clones transplanted into irradiated recipients 20 weeks previously. Each triangle represents an individual recipient/mouse. Solid triangles indicate recipients of the smallest fraction(s) tested of each clone, positive for the presence of transplanted cells. Open triangles indicate recipients of the highest fraction(s) tested of each  68  clone, negative for the presence of transplanted cells. Symbol (†) indicates a dead mouse. (D) DNA extracted from NUP98-HOXA10hd-transduced bone marrow cells of representative long-term reconstituted (24 weeks) recipients (as shown in 2.1C, top panel) was analyzed for proviral integrants by Southern blotting. Arrows indicate common integration pattern(s) among recipients of the cells from the same HSC expanded clone (NUP98-HOXA10hd-transduced clone #16, #17, #18, #20 and #21). Transplantation dose is expressed as fraction of the culture. (E) Grey bar indicates the maximal number of HSCs generated by day 14 in cultures of GFP-transduced cells, estimated by complete lack of reconstitution in recipients of 1/5th of 16 such cultures. Blue bar indicates the number of HSCs generated by day 14 in cultures of NUP98-HOXA10hdtransduced cells, as determined by limiting dilution CRU assay. Value shown is the mean ± SEM of results from 40 NUP98-HOXA10hd-transduced clones tested. Presented result is the minimal number of HSCs estimated since the limiting dilution was not reached for the majority of NUP98-HOXA10hdtransduced HSC clones tested.  69  Table 2.1 Summary of all highly purified HSC clones tested and average HSC expansion in vitro achieved in response to forced expression of NUP98HOXA10hd. Phenotype tested # of NUP98-HOXA10hd HSC clones tested # of expanded HSC clones Minimum cell dose (fraction of culture) transplanted Average minimum HSC expansion in vitro estimated (LD not reached) Range based on ± SEM Average HSC expansion in vitro documented (LD reached) Range based on ± SEM  CD45+lin-Rho-SP  E-SLAM  28 25 th 1/500 or 1/1000th or 1/2000th or 1/5000th  17 15 1/500th or 1/1000th  >806 - fold 381 ± 1730  >942 - fold 425 ± 2110  2520 - fold 1140 ± 5720  70  A  B  Figure 2.2 The in vitro cell division kinetics of highly purified HSCs transduced either with control or NUP98-HOA10hd vector. (A) Time-lapse imaging of NUP98-HOXA10hd-transduced HSC divisions in a chamber. (B) E-SLAM cells were transduced with NUP98-HOXA10hd or GFP control retroviral vector and cultured under conditions shown in 2.3A. 2 days following retroviral infection in the conventional culture, single transduced cells (42 of NUP98-HOXA10hd+ and 78 of GFP+) were deposited into individual chambers of the microfluidic cell culture system. Time-lapse imaging and  71  automated image analysis was used to score 1st, 2nd and 3rd division of each single cell over a period of 72 hours. Each symbol shows the proportion of single viable cells initially deposited into individual chambers of the microfluidic cell culture system that had divided by the time point shown.  72  A  B  73  C  D  Figure 2.3 The CD150+CD48- subset of day 10 NUP98-HOXA10hd-transduced and expanded population contains less than half of the total HSCs generated in vitro. (A) Schematic of the experimental design where following 10-day NUP98HOXA10hd-induced HSC expansion in vitro, bulk or purified cells were isolated based on the phenotype indicated in 2.3B and 2.4A and assayed for HSC content by LDA. Since depleted of HSCs by day 10, cultures containing GFPtransduced control cells were only used for immunophenotyping and comparison and not for further transplantation assays. (B) Representative profile of viable, linNUP98-HOXA10hd- and GFP-transduced day-10 cells, assessed for CD150 and CD48 expression. (C) Empty bars indicate the average number (expressed as the mean ± SD) of bulk or phenotypically defined NUP98-HOXA10hd-transduced cells generated at the end of 10-day culture. TNCs; total nucleated cells. Filled bars indicate the number of HSCs (expressed as the mean ± SEM) generated by day 10 in cultures of NUP98-HOXA10hd-transduced cells and the HSC content (±SEM) of phenotypically defined subsets (CD150+CD48-; CD150+CD48+; CD150-(CD48+CD48-)), as determined by the limiting dilution CRU assay. (D) The distribution of the total NUP98-HOXA10hd-transduced HSCs generated in 10-day culture according to indicated phenotypes (CD150+CD48-; CD150+CD48+; CD150-(CD48+CD48-)). Each subset was isolated by FACS and the HSC content was determined by limiting dilution. Black area represents the HSC content (<10% of the total) undetected by the indicated phenotype(s).  74  Table 2.2 Calculated day 10 HSC frequencies and HSC content estimates of total or various FACS-purified subpopulations of NUP98-HOXA10hd-transduced and in vitro cultured bm cells (as shown in figure 2.3C). +  Cell population analyzed Day 10 total nucleated cells ± SD Day 10 HSC frequency in vitro (range defined by ± SEM) Day 10 estimated HSC content 10-day HSC expansion in vitro  -  +  +  -  +/-  Bulk 2500000 ± 424264.1  CD150 CD48 subset 240000 ± 40729.3506  CD150 CD48 subset 360000 ± 61094.02589  CD150 CD48 subset 1620000 ± 274923.1165  1 in 116 (+) 1 in 68 (-) 1 in 198  1 in 30 (+) 1 in 16 (-) 1 in 57  1 in 58 (+) 1 in 28 (-) 1 in 121  1 in 259 (+) 1 in 123 (-) 1 in 545  ~21552  ~8000  ~6207  ~6255  ~2155-fold  75  A  B  C  D  76  Figure 2.4 Proliferation phenotype of NUP98HOXA10hd-expanded HSCs. (A) Representative profile of viable, lin- NUP98-HOXA10hd- and GFP-transduced day 10 cells assessed for Sca-1 and CD43 expression and further analyzed for the level of surface Mac1 protein expression. Viable, lin-, transduced cells expressing both Sca-1 and CD43 were also confirmed to express high levels of c-kit protein327. (B) Empty bars indicate the average number (expressed as the mean ± SD) of bulk or phenotypically defined NUP98-HOXA10hd-transduced cells at the end of 10-day culture. TNCs; total nucleated cells. Filled bars indicate the total number of HSCs generated by day 10 in cultures of NUP98-HOXA10hdtransduced cells and the HSC content of phenotypically defined subsets (lin-Sca1+c-kit+CD43+Mac1dim; lin-Sca-1+c-kit+CD43+Mac1-; lin-Sca-1+c-kit+CD43bright Mac1 ), as determined by the limiting dilution CRU assay. Values shown are the mean ± SEM. (C) The distribution of the total NUP98-HOXA10hd-transduced HSCs generated in 10-day culture according to indicated phenotypes: lin-Sca1+c-kit+CD43+Mac1dim; lin-Sca-1+c-kit+CD43+Mac1-; lin-Sca-1+c-kit+CD43bright Mac1 . Each subset was isolated by FACS and assayed for the HSC content by limiting dilution CRU assay. Black area represents the HSCs (<10% of the total) undetected by indicated phenotype(s). (D) On the left, proportion of pb leukocytes produced after >20 weeks post-transplant by 5 lin-Sca-1+ckit+CD43+Mac1dim NUP98-HOXA10hd-transduced and expanded cells. Each triangle represents an individual recipient/mouse. On the right, bars indicate the mean % lineage (Ly6G and Mac1 (red); B220 (blue); CD4 and CD8 (grey)) contribution to WBCs of mice reconstituted with the limited number (5 cells / mouse) of NUP98-HOXA10hd-transduced and expanded cells, re-purified for linSca-1+c-kit+CD43+Mac1dim phenotype, shown on the left. Error bars indicate the SD of the mean.  77  Table 2.3 Calculated day 10 HSC frequencies and HSC content estimates of total or various FACS-purified subpopulations of NUP98-HOXA10hd-transduced and in vitro cultured bm cells (as shown in figure 2.4B). -  Cell population analyzed Day 10 total nucleated cells ± SD Day 10 HSC frequency in vitro (range defined by ± SEM) Day 10 estimated HSC content 10-day HSC expansion in vitro  -  -  Bulk 3900000 ± 264575.1  lin + + Sca1 CD43 + (dim) c-kit Mac1 subset 128700 ± 8730.9790327  lin + + Sca1 CD43 + c-kit Mac1 subset 167700 ± 11376.73064  lin + Sca1 CD43 + ++ c-kit Mac1 subset 31200 ± 2116.601049  1 in 105 (+) 1 in 71 (-) 1 in 154  1 in 4 (+) 1 in 3 (-) 1 in 5  1 in 83 (+) 1 in 58 (-) 1 in 119  1 in 388 (+) 1 in 143 (-) 1 in 1051  ~37143  ~32175  ~2020  ~80  ~2476-fold  78  Figure 2.5 Growth factor effects on NUP98-HOXA10hd-induced HSC expansion in vitro. Graphic representation of the value of factor effects (βi) divided by the model intercept (β0) based on Equation 2. Three responses (Y) were modeled: (1) the number of transduced cells of lin-Sca-1+CD43+(c-kit+)Mac1dim phenotype obtained in 7-day multifactorial expansion cultures, (2) the % of transduced cells detected 6 weeks and (3) 4 months posttransplantation (PT) in pb of recipients of 1/10,000th of each 7-day multifactorial expansion culture. Inter-action effects are identified by the multiplication sign “x”. The factor effects are plotted with respect to growth factor concentrations scaled between -1 (low values) and 1 (high values). The growth factor levels tested are presented in Appendix E. Statistical significance is shown as *P < 0.05.  79  A  B  C  80  Figure 2.6 NUP98-HOXA10hd induces the in vitro expansion of fetal HSCs. (A) For bulk cultures, hatched bars indicate the number of total nucleated cells (TNCs) at the beginning (day 0) and at the end (day 10) of culture, following NUP98-HOXA10hd infection and in vitro expansion. Value shown for day 10 is the mean ± SD of results from 2 experiments. Filled bars indicate the HSC content in culture on day 0 and day 10, estimated based on HSC frequency calculated by limiting dilution CRU assay. Values shown are the mean ± SEM of results from two experiments. For clonal cultures, hatched bars indicate the number of TNCs, following NUP98-HOXA10hd infection and in vitro expansion in 14-day culture, for 5 FL HSC clones tested. Filled bars indicate the number of HSCs generated by day 14 in 5 clonally expanded cultures of NUP98HOXA10hd-transduced cells, as determined by limiting dilution CRU assay. Error bars represent ± SE. The limiting dilution was not reached for clone#1 and #2 and clones #4 and #5 did not contain HSCs. (B) Schematic of the experimental design followed to investigate in vivo competition capacity of fetal liver and adult bm hematopoietic cells. (C) Proportion of pb leukocyte contribution by fresh adult and fresh fetal (blue and red bar on the left) or day 10 NUP98-HOXA10hdtransduced and expanded adult and fresh fetal (blue and red bar on the right) HSCs transplanted (16 weeks earlier) to irradiated mice in competition. Values shown are the mean ± SD of results from eight transplanted recipients that received cells from both sources.  81  A  B  C  82  Figure 2.7 The progeny of in vitro expanded NUP98-HOXA10hd-transduced HSC clones continue to expand in vivo and re-expand in vitro. (A) Schematic of the experimental design followed to measure the level of NUP98-HOXA10hd-induced HSCs expansion during 14-day culture in vitro (Δ1), 7-month regeneration in vivo (Δ2), and 6-day re-expansion culture in vitro (Δ3). (B) Bars represent HSC content at the beginning (light blue) and at the end (dark blue) of each expansion period, measured by 10 or 20 LDA CRU assay. Δ1, Δ2, Δ3 are estimated based on changes in HSC input vs. output numbers. (C) Representative pb FACS plots of primary and secondary recipients transplanted with the progeny of NUP98-HOXA10hd-expanded HSCs, as indicated in A.  83  CHAPTER 3 Prolonged self-renewal activity unmasks telomerase control of telomere homeostasis and function of mouse HSCs  3.1 Introduction  The existence of HSCs with a capacity for sustained self-renewal is essential for lifelong blood cell production. Expansion of the stem cell pool requires the stimulation of symmetric self-renewal divisions and is critical both during early development22 and in later life332,333, as well as after transplantation or following hematopoietic injury. When small numbers of HSCs are transplanted into myeloablated or pre-immune hosts, the increases in HSC numbers that follow may be even larger than those seen during development24-26. These findings document the high replicative potential of HSCs. In mice, retroviral marking studies and, more recently, reconstitution studies starting from a single transplanted cell have shown that a single HSC can re-establish the hematopoiesis of a mouse, by continual creation of new HSCs capable of regenerating the system 74,334. The self-renewal function of HSCs and their ability to re-establish hematopoiesis permanently in a myelosuppressed host is the basis of an increasing range of therapies for bone bm failure, malignant and genetic disorders4,335. Broader use (e.g., from CB sources) and improved safety (e.g., by accelerating recovery) of such transplant therapies would be greatly facilitated by the development of methods for achieving significant prior expansion of HSC  84  numbers ex vivo8,141,226. However, it remains unclear, whether HSC self-renewal activity, provoked by either extrinsically or intrinsically induced mechanisms would at some point have deleterious consequences (e.g., by inducing HSC senescence or an impairment of some critical aspect of HSC function). Studies reported to date indicate that despite detectable levels of telomerase expression268, the telomeres of leukocytes present in the blood of increasingly older people are increasingly shorter. This finding suggests that some HSC proliferation is constantly occurring in humans269,336. Similar results have been reported for hematopoietic cells produced in murine recipients of serially transplanted hematopoietic cells270,271. The  severe  consequences  of  genetically  determined  telomerase  deficiencies provide additional compelling evidence of the importance of telomerase in both humans and mice. Patients suffering from DKC or acquired aplastic anemia with loss of function mutations in telomerase complex genes277,337 have short telomeres, frequently associated with a decreased proliferative capacity of their hematopoietic progenitors, bm failure and sometimes evidence of malignant progression due to genomic instability338,339. Similar to DKC patients, late generation telomerase-deficient mice (as mice possess significantly longer telomeres than humans and it takes up to 4 generations until the telomeres become critically short) generally suffer from genomic instability, defects in highly proliferative tissues279, including reduced replicative capacity271 and repopulating ability253 of their HSCs, tumor formation282 and overall reduced lifespan. In addition, 3 recent papers provide  85  further insights into the importance of the TERT protein in hematopoiesis as a transcriptional modulator of the Wnt/β-catenin signalling pathway required for HSC proliferation284 and self-renewal during development283 or as a facilitator of HSC proliferation and recovery of peripheral blood cell counts upon androgen therapy of bm failure syndromes285. The purpose of this study was to examine the extent of HSC telomere loss and possible impairment of HSC function under conditions of HSC self-renewal stimulation in vitro and in vivo and the effect of absent telomerase (Tert) on the responses obtained. The average length of telomere repeats in HSCs progeny was measured by a highly sensitive flow-fluorescent in situ hybridization (FISH) method340, here adapted for application to murine cells. This method uses labelled peptide nucleic acid probes specific for telomere repeats in combination with fluorescence measurements by flow cytometry thereby enabling the rapid analysis of large numbers of different cell types, discriminated by differences in light scattering and immunophenotypic properties. The conditions used to stimulate HSC self-renewal involved serially transplanting freshly isolated bm samples (self-renewal stimulation in vivo) or after their transduction with a NUP98-HOXA10hd (NA10hd) transgene and expansion in vitro prior to transplant (self-renewal stimulation in vitro and in vivo). This in vitro treatment was based on our previous finding that expression of this variant Hox transcription factor (NUP98-HOXA10hd = NUP98 fused to the homeodomain of HOXA10) reproducibly expands HSCs >1,000-fold in 10-day cultures with retention of normal HSC differentiation and increased self-renewal activity post-transplant8.  86  The results of the present studies confirm the importance of intact telomerase activity in maintaining the integrity of telomere length in HSCs that have undergone many self-renewal divisions and demonstrate its necessity in preserving the genomic integrity of their progeny.  3.2 Materials and methods  3.2.1 Mice Mice were bred and maintained at the Biomedical Research Centre (BRC) and the British Columbia Cancer Research Centre animal facilities according to the guidelines of the Canadian Council on Animal Care. Transplant donor and recipient pairs were the following: C57Bl6 that express CD45.2 and either C57Bl6/JSJL or C57Bl/6Ly-Pep3b that express CD45.1; or TERT-KOxC57Bl6 that express CD45.2 and either C57Bl6/JSJL or C57Bl/6Ly-Pep3b that express CD45.1. The Tert-/- strain which lacks telomerase expression was received as a gift from Dr. Lea Harrington where it was backcrossed over 10 times onto a C57Bl6 background341. Tert-/- mice were maintained by heterozygous breeding at the BRC (UBC, Vancouver, Canada).  3.2.2 Transduction of mouse bone marrow cells Generation of the MSCV-IRES-GFP (GFP) and the MSCV-NUP98-HOXA10hdIRES-GFP (NUP98-HOXA10hd or NA10hd) viral vectors was previously described67 . Bm cells were obtained from mice injected 4 days previously with  87  150 mg/kg 5-fluorouracil (5-FU) (Mayne, Pharma, Montreal, QC, Canada) and transduced as previously described8,67. Loosely adherent and nonadherent cells were recovered and either immediately transplanted into irradiated recipients or further cultured for an additional 6 days. For generating transduced bm cells to be transplanted immediately after retroviral infection, cultures were initiated with 106 cells in a 10 cm dish. Bulk expansion cultures were initiated with 1.5 x 105 cells in a 6 cm dish and then transferred to a 10 cm dish 2-3 days at the end of infection. Expansion cultures initiated with 5,000 unseparated bm cells containing limiting numbers of HSCs (1-2)8 were seeded in a 96-well plate and then transferred into a 24-well plate 2-3 days after the end of infection. By the end of infection, more than 30% of the total cells in all cultures were GFP+ and by the end of additional, 6-day expansion culture, more than 65% of the total cells in all cultures were GFP+.  3.2.3 Transplantation Cells were harvested by flushing the femora of 21-22 week-old C57Bl6 and C57Bl6-Tert-/- mice. Red blood cells were lysed and the remaining white blood cells (wbc) counted. Cells were either immediately transplanted into recipients or retrovirally transduced prior to transplantation. For longitudinal studies, one million, and for serial transplantation studies, one or ten million unmanipulated wild-type or Tert-/- bm cells were transplanted into each of 4-5 lethally irradiated (1.1 Gy of X-rays) C57Bl6/JSJL recipient mice. After 6 weeks, and 3 and 9 months, bm cells were aspirated from recipient mice  88  for telomere length measurements. Donor-derived bm cells were isolated on a FACSVantage (Becton Dickinson (BD), San Jose, CA), 3 months post-transplant from the 3 primary recipients showing the highest level of reconstitution within each group. Secondary recipients were lethally irradiated (1.1 Gy of X-rays) and transplanted with 5 x 106 donor-derived bm cells from the primary mice by i.v. injection. Tertiary transplantations were performed in a similar fashion with 5x106 donor-derived cells isolated from secondary recipients 3 months post transplantation. Pb was analysed for donor-derived granulocytes and the reconstitution level evaluated one week prior to each transplantation. 2x105 or 102-103 starting cell equivalents of NUP98-HOXA10hdtransduced wild-type and Tert-/- bm cells were transplanted into each of 3-4 lethally irradiated (0.81 Gy X-rays) C57Bl/6Ly-Pep3b recipient mice, either immediately after the retroviral infection or following 6-days in vitro, respectively. Cells recovered at the end of the 6-days in culture and also from the bm aspirates obtained from mice 3 and 6 months post transplant were subjected to telomere length measurements (see following section). At 6 months posttransplant 1-2 primary recipients showing the highest level of donor-derived reconstitution were selected and donor-derived bm cells were isolated on a FACSDiVa (BD) for transplantation into secondary mice (3x106 cells/lethally irradiated secondary recipient). Peripheral blood was analysed for donor-derived white blood cell reconstitution 3 and 6 months post-transplantation.  89  3.2.4 Assessment of HSC frequencies HSCs were detected and quantified using the LD CRU assay, as previously described67. Briefly, lethally irradiated C57Bl/6Ly-Pep3b mice (0.81 Gy X-rays) were transplanted by i.v. injection with variable numbers of fresh or NUP98HOXA10hd-transduced and in vitro expanded cells from WT or TERT-KO mice, along with a life-sparing dose of 105 normal C57Bl/6Ly-Pep3b bm cells. The proportion of mice in each group that showed multi-lineage repopulation with donor-derived (CD45.2+ or GFP+) cells was determined by flow cytometric analysis of pb wbc a minimum of 16 weeks post-transplantation. Only mice whose pb contained >1% donor-derived myeloid cells (Ly6G+ and Mac-1+), B cells (B220+), and T cells (CD4+ and CD8+) were considered to be positive. CRU frequencies were calculated using L-Calc software (STEMCELL Technologies).  3.2.5 Peripheral blood analysis Blood samples (100 µl) were obtained from the tail vain, erythrocytes lysed with ammonium chloride (STEMCELL Technologies) and the leukocytes suspended in Hanks’ Balanced Salt Solution with 2% fetal bovine serum (FBS) (STEMCELL Technologies). Thereafter, the remaining leukocytes where incubated on ice for 20 minutes with the following antibodies: CD45.1-fluorescein isothiocyanate (FITC), CD45.2-allophycocyanine (APC) and Gr1-phycoerythrin (PE) for analysis of recipients transplanted with unmanipulated bm cells. For the analysis of recipients transplanted with NA10-hd-transduced bm cells, all lysed blood samples were split into 3 fractions, each incubated on ice for 20 minutes with  90  following antibody combinations: biotinylated anti-Ly5.2 in combination with either PE-labeled antibody to B220; or a combination of PE-labeled antibodies to Ly6G and Mac-1; or a combination of PE-labeled antibodies to CD4 and CD8, followed by a 20 minute incubation with APC-labeled streptavidin. Finally, all samples were washed with Hanks’ Balanced Salt Solution with 2% FBS and 1µg/ml propidium iodide (PI, Sigma Chemicals, St.Louis, MO, USA) prior to analysis on a FACSCalibur (BD).  3.2.6 Colony forming cell (CFC) assays Clonogenic hematopoietic progenitor cells were assayed by plating 1,500 cells per ml of methyl-cellulose culture medium (Methocult M3234, STEMCELL Technologies), containing 3 U/ml human erythropoietin, 10 ng/ml mouse IL-3, 10 ng/mL human IL-6, and 50 ng/ml mouse SCF (all recombinant from STEMCELL Technologies). After 7 days of culture, colonies were counted using standard scoring criteria, the cells pooled and the viable cells counted also. Equal cell numbers were replated in duplicates for a total of 3 times at weekly intervals to assess serial replating capacity.  3.2.7 Flow-FISH After lysis of the red blood cells with ammonium chloride, FACS-isolated donor granulocytes (CD45.2+) from unmanipulated or donor (GFP+) wbcs from transduced bm samples were frozen and stored either in liquid nitrogen or at 1200C until analysis. The majority of transduced bm samples analyzed were  91  more than 90% GFP+ and analysed without further enrichment. Samples less than 90% GFP+ (lowest positive being 30%) were FACS-sorted for the transduced population prior to telomere length measurements.  To measure  telomere lengths with flow-FISH, each sample (2x105–1x106 cells) was split in half and mixed with 2x105 fixed cow thymocytes of known telomere length. One sample was stained, leaving the second unmarked to account for autofluorescence. DNA was then denatured for 15 minutes in 75% formamide at 87°C, followed by hybridization for 90 minutes at room temperature with a 0.3 µg/ml FITC-labelled (CCCTAA)3 PNA specific for the telomere sequence. Excess probe was removed by several washes in a Hydra robotic washing station. The first 4 washes were performed with 75% formamide while the 5th contained only PBS. Next, the DNA was counterstained with LDS 751 (Exciton, Dayton, OH) at 0.01 µg/ml for at least 20 minutes to visually separate the granulocytes from the cow thymocytes. Finally, the cells were analyzed on a FACS Calibur (BD) with telomere lengths assessed on gated granulocytes (for wbc samples) and calculated in Microsoft Excel.  3.2.8 Flow cytometry for γ-H2AX Single cell suspensions of bm harvested from either wild-type or Tert-/- mice, and from recipients reconstituted with either unmanipulated or NUP98-HOXA10hdtransduced cells, were fixed, as described342, with a mixture of paraformaldehyde and saponin (BD Cytofix/CytopermTM Buffer) after being incubated with antibodies for immunofluorescent staining of cell surface antigens for 30 min (Ly-  92  6A/E,  E13-161.7;  CD117,  2B8).  After  treatment  with  a  secondary  permeabilization reagent (BD CytopermTM Plus Buffer), cells were resuspended in 200 µl mouse monoclonal antiphosphohistone γ-H2AX antibody (Sigma), which was diluted 1:500 in 1X Perm/Wash Buffer (BD). Tubes were placed on a shaker platform at 225 rpm and incubated for 2 hours at room temperature. After secondary antibody labeling with 1:200 diluted Alexa-647 IgG (H+L)F(ab)2 fragment conjugate (Molecular Probe - Invitrogen), cells were returned to the shaker platform for 1 hour incubation at room temperature. Cells were then rinsed and resuspended in 1 µg/ml 4’,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI; Sigma) for DNA staining and analysed using an Aria II FACS equipped with 3 lasers (633 nm, 488 nm, and 405 nm, BD). For each transplanted recipient, selecting gate was determined based on comparison of the target population within donor (transplanted) and recipient (endogenous) progenitor cells.  3.3 Results  3.3.1 Telomerase deficiency affects telomere homeostasis under conditions of prolonged self-renewal stimulation In an initial series of experiments, primary recipients were transplanted with bm cells from either wild-type or Tert-/- mice which were not found to be different based on flow cytometric analyses of cells with a lin-Sca-1+c-kit+ (LSK) phenotype and assessment of HSC frequency by limiting dilution transplant  93  assays of cells with long-term lympho-myeloid repopulating activity (data not shown). Six weeks, 3 and 9 months after transplantation of primary mice the donor-derived bm cells regenerated were sampled by bm aspiration and their telomere lengths measured using flow FISH (Figure 3.1A). To examine the effects of telomerase deficiency on HSC function, the cells were subjected to a serial transplant protocol. Secondary and tertiary transplants were performed by transplanting donor-derived bm cells from the primary and the secondary recipients, respectively, and at the time of each transplantation, telomere length measurements were again performed on donor-derived bm cells (Figure 3.1C). Bm cells from early generation Tert-/- mice had significantly shorter telomere repeats (~24kb) than those of wild-type mice (~34kb) (Figure 3.1B, 3.1D, 3.2B, 3.2E), most likely as a consequence of the telomerase deficiency in the parental germline combined with the proliferative stress that occurs during early development22. Surprisingly, evidence of further erosion of telomeres related to the serial transplantation protocol, even in cells unable to produce telomerase, was not observed over 9 months post transplantation within individual mice (Figure 3.1B, 3.1D). Consistent with this, both wild-type and Tert-/- HSCs showed a similar reconstitution capacity in primary, secondary and tertiary transplants (Figure 3.1E). However, in the third round of transplantation, we observed a decrease in pb chimerism in recipients of either wild-type or Tert-/- HSCs (Figure 3.1E), that was not correlated with evident telomere attrition. Subsequent experiments were designed to determine if stimulating even higher levels of HSC expansion in vivo would lead to telomere attrition and/or  94  further effect on HSC function. Therefore, in addition to proliferative stress provoked by serial transplantation, we used forced expression of NUP98HOXA10hd to enhance the self-renewal of wild-type and Tert-/- bm cells regenerating in vivo8. Accordingly, we transduced bm cells with GFP (control) or NUP98-HOXA10hd retroviral vector and then transplanted them into primary recipients either immediately after infection or following an additional 6-day period in culture sufficient to achieve >1,000-fold net expansion of the input HSCs8. Expansion cultures were initiated with either large or reduced numbers of bm cells, estimated to contain ~30 or 1-2 HSCs, respectively. The latter approach allowed monitoring the possibility of clone to clone variation. Secondary transplants were performed 6 months later and telomere length measurements of donor-derived (NUP98-HOXA10hd-transduced) bm cells were performed after 3 and 6 months in primary hosts and 3 months post transplantation of secondary recipients (Figure 3.2A, 3.2D). The growth factor cocktail employed for gene transduction and in vitro expansion was chosen to induce HSC cycling required for high level gene transfer343, rather than to promote HSC self-renewal and maintain HSC numbers in culture. Consequently, recipients of control GFPtransduced bm cells showed low chimerism for transduced cells (for recipients of wild-type, <20%, or for Tert-/-, <4%,) or essentially no chimerism for transduced cells (for recipients of wild-type or Tert-/- transduced and additionally cultured cells), thus impeding telomere length analysis in these samples. However, no significant difference in the telomere length was observed throughout the expansion culture period (Appendix F) when assessed in the progeny of GFP-  95  and NUP98-HOXA10hd-transduced wild-type or Tert-/- bm cells cultured for up to 20-day period. A  recipient  of  GFP-transduced  wild-type  bm  cells  transplanted  immediately after transduction was followed for 9 months with no detectable telomere attrition (data not shown). There was also no significant change in the telomere length in the progeny of NUP98-HOXA10hd-transduced wild-type bm cells initially transplanted either immediately after transduction or following 6-day in vitro expansion into primary and then secondary recipients (Figure 3.2B, 3.2E), regardless of the number of input HSCs (large or reduced to 1-2 clones). These results indicate that NUP98-HOXA10hd-stimulated symmetric HSC self-renewal divisions required to explain the expansion measured in vivo and/or in vitro does not affect telomere homeostasis. A similar result was seen for the progeny of NUP98-HOXA10hd-transduced Tert-/- bm cells initially transplanted immediately after transduction into primary and then secondary recipients which showed only a slight but still non-significant decrease in telomere length (Figure 3.2B). In sharp contrast, the progeny of NUP98-HOXA10hd-transduced Tert-/HSCs and/or clones that had undergone the 6-day in vitro expansion exhibited a significant (~5kb) telomere loss during the 6 months of regeneration in primary transplant recipients and an additional ~5kb telomere loss after another 3 months of regeneration in the secondary transplant recipients (Figure 3.2E). This result highlights the significance of telomerase in maintaining HSC telomere homeostasis when sufficient symmetric self-renewal divisions are stimulated to occur. Importantly, the level of chimerism obtained in recipients of wild-type or  96  Tert-/- HSCs, stimulated to self-renew by NUP98-HOXA10hd either in vivo (Figure 3.2A) or in vitro (Figure 3.2D) also revealed a significantly decreased reconstituting ability of Tert-/- bm cells in primary and secondary transplants (Figure 3.2C, 3.2F). Interestingly, such a deficit in reconstituting ability of Tert-/bm cells (Figure 3.2F) was accompanied by skew to myeloid cells (data not shown) and a significant telomere loss (Figure 3.2E) only following NUP98HOXA10hd-enhanced HSC expansion in vitro (Figure 3.2D).  However, the  phase of NUP98-HOXA10hd-enhanced HSC expansion in vivo (Figure 3.2A, 3.2C), appears to be independent of detectable telomere attrition (Figure 3.2B). We also performed a limiting dilution analysis of the HSC frequency in the in vitro expanded cell suspensions initiated with wild-type and Tert-/- bm cells overexpressing  NUP98-HOXA10hd.  As  expected,  results  revealed  very  significant levels of net HSC expansion in both wild-type and Tert-/- cultures (~1800- and ~260-fold, respectively) although the net expansion was ~7-fold less for NUP98-HOXA10hd-transduced Tert-/- HSCs relative to wild-type HSCs (Figure 3.3B). To determine whether a more rapid indicator of the role of telomerase in maintaining the proliferative potential of primitive hematopoietic cells could be developed, we examined the response of NUP98-HOXA10hd-transduced clonogenic bm cells to being serially replated in methylcellulose medium. NUP98HOXA10hd-transduced  wild-type  progenitors  consistently  formed  large  granulocyte-macrophage colonies over at least 4 passages. In contrast, NUP98HOXA10hd-transduced Tert-/- progenitors were essentially exhausted by a third  97  replating (Figure 3.3A). Taken together, these results demonstrate that the absence of telomerase activity blunts the ability of NUP98-HOXA10hd to maintain the self-renewal capability of primitive hematopoietic cells, including HSCs, thereby implicating telomerase in the regulation of this property.  3.3.2 Primitive hematopoietic cells lacking telomerase activity exhibit signs of enhanced DNA damage Recent studies performed in mouse models of accelerated aging, including mice lacking telomerase activity, and/or defective in DNA repair pathways have shown that accumulation of DNA damage can deleteriously impact HSC function252,253. In fact, competitive transplantation assays have revealed reduced long-term reconstituting and self-renewal activity of HSCs from aged mutants relative to wild-type HSCs, ultimately leading to premature exhaustion of their numbers. These studies thus suggest that impaired HSC function may be a general consequence of DNA damage accumulation. Because our findings demonstrated a decreased ability of primitive Tert-/hematopoietic cells to respond to prolonged NUP98-HOXA10hd-enhanced selfrenewal stimulation in vivo and/or in vitro (Figure 3.2C, 3.2F and 3.3), we designed experiments to investigate whether this might be associated with a corrupted genomic integrity of these cells. To investigate this possibility, we used flow cytometry to analyze the LSK subset of unmanipulated, second generation Tert-/- bm cells for expression of phosphorylated histone H2AX (γ-H2AX), an indicator of DNA damage. This analysis showed an average of a 4-fold increase  98  in γ-H2AX expression within the telomerase deficient LSK subset compared to their wild-type counterparts (Figure 3.4A). A similar trend was observed when donor-derived bm cells were obtained from recipients reconstituted with unmanipulated wild-type and Tert-/- cells (Figure 3.4B), as well as donor-derived bm cells in recipients reconstituted with NUP98-HOXA10hd-transduced and in vitro expanded wild-type or Tert-/- cells (Figure 3.4C). This trend was further reinforced by direct comparison of γ-H2AX expression in recipient- and donorderived LSK compartment within each analyzed reconstituted recipient shown in 3.4B and 3.4C. Taken together, these results show that γ-H2AX expression was not increased simply as a result of the stimulus for HSC regeneration in transplanted mice and/or NUP98-HOXA10hd-enhanced expansion in vitro or in vivo, and that the evidence of DNA damage accumulation seen in primitive (LSK) Tert-/- hematopoietic cells may represent an early indicator of the reduced functional integrity of these cells when stimulated to proliferate.  3.4 Discussion  Our findings confirm and extend accumulating evidence for 2 functions of telomerase in HSC regulation. First, is a postulated role of telomerase in telomere maintenance.  Second, is a role of telomerase in preserving HSC  function. These activities were revealed in experiments in which Tert-/- HSCs were stimulated to execute increased self-renewal divisions by serial transplantation, as previously examined in studies by others270,271 in combination  99  with forced expression of NUP98-HOXA10hd and exposure to growth factors in vitro8. Importantly, under the same conditions, telomere homeostasis was not affected when NUP98-HOXA10hd was used to induce symmetric self-renewal divisions of wild-type HSCs either in vivo or in vitro. Contrary to previous findings270,271, we did not detect significant telomere shortening in the progeny of serially transplanted wild-type or Tert-/- HSCs in the absence of an additional stimulation of HSC self-renewal divisions. The absence of telomere loss by serially transplanted Tert-/- HSCs may be explained by the possible existence of telomere-length-independent barriers, alternative mechanisms for lengthening telomeres in dividing HSCs, and insufficient HSC turnover following whole bm transplantation. HSC replicative potential may be limited by more than just the length of their telomeres, as the expression of the catalytic component of telomerase was eventually able to prevent telomere shortening in HSCs subjected to more proliferation than that stimulated by 3 serial transplant cycles, but was not sufficient to sustain their transplant capacity271. Thus, it cannot be excluded that HSC telomeres may stay long because of a telomeraseindependent mechanism active in HSCs. Telomeric DNA may also be generated through recombination events, a mechanism known as ALT344 and shown to occur in embryonic stem cells and during early development345. Recombinationbased telomere elongation has also been identified in human tumors, immortalized human cell lines, telomerase-null mouse cell lines and late generation telomerase-null mice346-348, and possibly occurs in other stem cells of Mus musculus (with, on average, very long telomeres) as well. Finally, it was  100  recently shown that HSCs with the highest self-renewal capacity are maintained in a dormant state with their stem cell potential being subject to reversible activation upon injury19. Accordingly, it might be anticipated that the regeneration of hematopoiesis that is stimulated to occur in transplanted irradiated mice would involve a rapid induction and short-lived induction of HSC turnover, insufficient to affect their telomere length. Another explanation for the absence of detectable telomere shortening in wild-type HSCs in spite of their extensively stimulatation to self-renew is a potential upregulation of endogenous telomerase levels by genes active in HSCs, in line with recent reports demonstrating telomere maintenance and elongation in induced pluripotent stem (iPS) cells349. In the current study, we detected no significant difference in the telomere length in the progeny of GFP- and NUP98-HOXA10hd-transduced wild-type bm cells cultured for up to 16-days following transduction (total of 20 days in culture) or transplanted immediately after transduction into primary recipients. Moreover, preliminary affymetirx expression analysis showed no difference in telomerase expression of non-transduced (fresh) and NUP98-HOXA10hd-transduced highly purified HSCs15 (data not shown). Thus our data argue that NUP98-HOXA10hd does not trigger upregulation of endogenous telomerase levels in HSCs. The fact that our findings are in disagreement with previous studies may also be explained in part by differences in the study designs and methodologies used for telomere length measurements. Allsopp et al.270 used Q-FISH to measure telomere lengths in LSK cells and Southern blot analysis for whole bm cells; whereas, we inferred effects on HSCs from measurements applied to their  101  granulocyte progeny and used Flow-FISH for average telomere length measurements at the single cell level. Q-FISH requires cells to be stimulated in culture, arrested in metaphase and mounted onto slides350 to enable telomere lengths of individual chromosomes to be made on a limited number of cells. Accordingly, it is less quantitative than Flow-FISH. The more precise measurements possible with Flow-FISH indicate that self-renewal stress imposed by serial transplantation even in the presence of stimulation by NUP98HOXA10hd and in the absence of Tert does not result in significant telomere length erosion. Nevertheless, highly significant telomere length reduction (~10kb) was apparent in the absence of Tert after the forced stimulation of prolonged self-renewal divisions in vitro and in vivo. Consistent with the previous data, we did not observe any difference in the frequency or competitive regenerative activity of wild-type and early (first) generation Tert-/- HSCs. However, we noted levels of γ-H2AX expression in the LSK population of later (second) generation Tert-/- bm cells, as well as after extensive self-renewal induced by stimulating primitive NUP98-HOXA10hdtransduced Tert-/- hematopoietic cell proliferation. Interestingly, this occurred in cells that also showed an acquired deficiency in reconstituting ability post transplantation and self-renewal ability in vitro. Indeed, the appearance of this evidence of accumulating DNA damage in primitive Tert-/- hematopoietic cells paralleled their reduced functional activity. Similar consequences of DNA damage accumulating in late generation aging Tert-/- as well as wild-type HSCs has also been reported 253.  102  Recent evidence showing that TERT protein can have a physiological role independent of its canonical function in maintaining telomere homeostasis, suggests a broader involvement in the control of cellular transformation, proliferation, stem cell biology, survival and chromatin regulation283-285,351-355. Furthermore, along with recent findings of others283-285, we showed that telomerase may function as a regulator of the HSC self-renewal process, as our results revealed significant (~7-fold) decrease in capacity of Tert-/- HSCs to expand in vitro in response to NUP98-HOXA10hd stimulation, relative to their wild-type counterparts. However, it is important to note the lack of deleterious effects seen on the greatly expanded wild-type HSC populations generated in the presence of intracellular NUP98-HOXA10hd in relatively short term (6-day) cultures. This included lack of effects on both telomere homeostasis and overall genomic integrity. Although the current findings cannot be safely extrapolated to human HSCs, as inbred mice possess significantly longer telomeres than humans356, they do serve to underscore the importance of elucidating the mechanisms linking telomere shortening to impaired HSC function, which are likely to be relevant to the rational development of strategies for the effective treatment of many patients with bm failure syndromes (i.e. dyskeratosis congenita and/or aplastic anemia) or conversely with leukemia where this link may be uncoupled.  103  A  B  C  D  104  E  Figure 3.1 Mouse HSCs have a reservoir of telomeres sufficient to sustain their self-renewal during several cycles of serial transplantation. (A) Experimental protocol in which wbm cells from either wild-type or Tert-/- mice were transplanted into lethally irradiated primary recipients and their telomere length monitored at various time points post transplantation (PT). (B) The average telomere length of donor-derived wild-type (blue bars) or Tert-/- (red bars) bm cells 6 weeks, 3 and 9 months PT within primary recipients. Dotted lines indicate the telomere length of donor wild-type (blue) or Tert-/- (red) bm cells on the day of the primary transplantation. Error bars denote standard deviation (SD). Nwt = 5 and Ntert-/- = 5 at 6-week, 3- and 9-month time point analysis. (C) Serial transplantation protocol of either wild-type or Tert-/- wbm cells. (D) The average telomere length of donor-derived wild-type (blue bars) or Tert-/- (red bars) bm cells measured at the time of each transplantation. Dotted lines indicate the telomere length of donor wild-type (blue) or Tert-/- (red) bm cells on the day of the primary transplantation. Error bars denote SD. Nwt = 6; 6; 4 and Ntert-/- = 6; 4; 2 at 3-month PT time point analysis of 10, 20, and 30 recipients, respectively. (E) Percent donor reconstitution in pb of primary, secondary and tertiary recipients (generated as described in Figure 1C) transplanted with unmanipulated wild-type (blue triangles) and Tert-/- (red triangles) bm cells. Each triangle represents an individual recipient. Horizontal lines indicate mean values.  105  A  B  C  106  D  E  F  107  Figure 3.2 NUP98-HOXA10hd effect on telomere reconstitution activity of wild-type and Tert-/- HSCs.  maintenance  and  (A) Experimental protocol in which wbm cells from 5-FU pre-treated wild-type or Tert-/- mice were transduced with NUP98-HOXA10hd and transplanted into lethally irradiated primary recipients. Six months later, donor-derived wbm cells from the primary recipients were transplanted into secondary recipients. (B) Average telomere lengths of donor-derived wild-type (blue bars) or Tert-/- (red bars) bm cells obtained 3 and 6 months PT from the primary recipients and 3 months PT from the secondary recipients. Dotted lines indicate the telomere length of donor wild-type (blue) or Tert-/- (red) bm cells prior to infection and primary transplantation. Error bars denote SD. Nwt = 2; 2; 2 and Ntert-/- = 2; 2; 3 at 3- and 6-month PT time point analysis of 10 and 3-month PT time point analysis of 20 recipients, respectively. (C) Percent donor reconstitution in the pb of primary and secondary recipients (generated as described in Figure 3.2A) transplanted with NUP98-HOXA10hd-transduced wild-type (blue triangles) and Tert-/- (red triangles) bm cells. Statistical significance was determined by application of the paired student t-test and is shown as *P<0.05 or **P<0.01. Each triangle represents an individual recipient. (D) Experimental protocol in which wbm cells from 5-FU pre-treated wild-type or Tert-/- mice were transduced with NUP98-HOXA10hd, expanded for 6 days in vitro and transplanted into lethally irradiated primary recipients. Six months later, donor-derived wbm cells from the primary recipients were transplanted into secondary recipients. (E) Average telomere lengths of donor-derived wild-type (blue bars) or Tert-/- (red bars) bm cells were obtained at the end of expansion in vitro, 3 and 6 months PT from the primary recipients and 3 months PT from the secondary recipients. Dotted lines indicate telomere lengths of donor wild-type (blue) or Tert-/- (red) bm cells prior to infection, in vitro expansion and primary transplantation. Error bars denote SD. Nwt = 2; 4; 4; 4 and Ntert-/- = 2; 4; 4; 5 at 10-day time point analysis of cultured cells, 3- and 6-month PT time point analysis of 10 and 3-month PT time point analysis of 20 recipients, respectively. Statistical significance was determined by application of the paired student t-test and is shown as **P<0.01 or ***P<0.005. (F) Percent donor reconstitution in the pb of primary and secondary recipients (generated as described in Figure 3.2D) transplanted with NUP98-HOXA10hd-transduced and in vitro expanded wild-type (blue triangles) and Tert-/- (red triangles) bm cells. Statistical significance was determined by the application of the paired student t-test and is shown as *P<0.05 or ***P<0.005. Each triangle represents an individual recipient. Horizontal lines indicate mean values.  108  A  B  Figure 3.3 Absence of telomerase activity blunts NUP98-HOXA10hd-induced self-renewal of myeloid progenitors and HSCs in vitro. Cultures were initiated with wild-type or Tert-/- bm cells from 5-FU pre-treated mice. Cells were transduced with NUP98-HOXA10hd, expanded for 6 days in vitro and either plated in methylcellulose medium (A) or transplanted into lethally irradiated recipients (B). (A) Each 7 days of methylcellulose culture, generated colonies were counted, cells harvested, pooled and equal cell numbers replated for a total of 3 times in order to calculate the yield of granulocyte-macrophage colonies formed. Statistical significance was determined by application of the paired student t-test and is shown as *P<0.05. (B) Mice were transplanted with limiting dilutions of cells used to initiate the cultures and with cells harvested at the end of expansion in vitro. Proportions of circulating B, T, and myeloid donorderived (CD45.2+ or GFP+) WBCs were determined 4-6 months later. Fold in vitro expansions of wild-type and Tert-/- HSCs stimulated by NUP98-HOXA10hd were estimated by determining the frequency, and hence HSC content in suspensions used to initiate and harvested from in vitro cultures. Results are expressed as the mean ± SEM of 2 independent experiments.  109  A  B  C  110  Figure 3.4 Primitive Tert-/- hematopoietic cells express elevated levels of γH2AX. Expression of γ-H2AX and DNA content were analysed by flow-cytometry, within LSK subset of (A) Non-manipulated wild-type and Tert-/- bm. (B) Recipient(CD45.2-) and donor-derived (CD45.2+) bm from recipients reconstituted with non-manipulated wild-type or Tert-/- bm cells; (C) Recipient- (GFP-) and donorderived (GFP+) bm from recipients reconstituted with NUP98-HOXA10hdtransduced and in vitro expanded wild-type or Tert-/- bm cells.  111  CHAPTER 4 Conclusion and recommendation for future work  The results presented in this thesis clearly show that overexpression of NUP98HOXA10hd fusion gene significantly enhances the self-renewal capacity of adult mouse bm HSCs at the clonal level, thus promoting their multi-log expansion both in vitro and in vivo. In this thesis, I further describe key findings pertaining to the functional, phenotypic and division kinetic properties of self-renewing HSCs, and examine the impact of prolonged self-renewal on their senescence. Moreover, I show that NUP98-HOXA10hd effect extends to other functionally distinct HSC types (i.e. fetal and telomerase deficient HSCs). Such robust strategy to control HSC population size will benefit treatments for many haematological diseases including leukemia, since HSC high absolute numbers are required and critical for the steady-state hematopoiesis and in bm transplant settings. Besides, these findings set the stage for more detailed analyses of the mechanisms underlying NUP98-HOXA10hd effect, which may offer new clues to the mechanisms that regulate HSC self-renewal process.  4.1  Potential cellular mechanisms of NUP98-HOXA10hd-mediated HSC  expansion  The earliest description of a highly purified HSC population showed that 1 out of 4 Lin-Sca1+Kit+CD34- adult mouse bm cells was capable of repopulating an irradiated recipient for 3 months77. Since then, many refinements of mouse HSC  112  purification strategies have been described73,81,84 in order to enhance the purity of HSCs with long-term repopulating activity. Particularly relevant to the work presented here are ones that have exploited the high Ho90 and/or Rho73,357 dye exclusion activity and/or the more recently discovered EPCR85 and/or SLAM (CD150+CD48-) phenotypes15,84 that are also selective for HSCs from adult mouse bm. The most significant contributions of these advances have been to enable both direct and clonal functional analyses of large numbers of individual mouse HSCs. Initial studies presented in Chapter 2 exploited such approach and demonstrated the power of NUP98-HOXA10hd fusion gene to stimulate >1000fold in vitro expansion of HSCs at the clonal level, indicating highly purified functionally defined long-term repopulating cells (i.e. HSCs) as target cells responsive to NUP98-HOXA10hd effect. Moreover, transduced and in vitro expanded HSC clones were consistently found to sustain lifelong self-renewal potential as determined by secondary transplant assay and showed frequent stable propagation of the robust lympho-myeloid differentiation program in vivo. Further investigations, based on the evidence of numerous HSC clonal subtypes that differ with respect to their downstream lineage potential and/or self-renewal capacity12-15 along with recently developed strategies for their precise isolation15,16, may reveal more specific target and/or the spectrum of target cells responsive to self-renewal promoting activity of NUP98-HOXA10hd. Subsequent experiments presented in Chapter 2 confirmed the lin-Sca1+CD43+Mac1dim59 phenotype to be highly selective of HSCs from multiple activated settings (i.e. FL, cultured cells), as it enabled reisolation of almost  113  entire NUP98-HOXA10hd-transduced and in vitro expanded HSC population, at purities of >25%. Moving forward, these prospectively isolated populations should prove extremely useful for studying the molecular properties of HSCs that correlate with enhanced self-renewal activity. In addition, Chapter 2 provided evidence that the number of NUP98-HOXA10hd-transduced cells of lin-Sca1+CD43+Mac1dim phenotype, obtained in an array of extrinsic conditions, significantly correlates with the corresponding HSC activity determined by functional in vivo transplantation assay. This direct measurement of lin-Sca1+CD43+Mac1dim phenotype in order to replace functional endpoint for detecting HSC activity would be an important accomplishment in establishing a surrogate assay, rather than long-term transplantation assay, for assessing conditions supportive of HSC expansion. Additional support for establishing such strategy could  emerge  from  examining  the  correlation  between  the  lin-Sca-  1+CD43+Mac1dim phenotype and corresponding functionally defined HSC activity obtained under influence of variety of nuclear/intrinsic factors, recently implicated in HSC self-renewal by displaying HoxB4-like effect289. HSC population size is controlled in vivo by a number of extrinsic and intrinsic factors involved in rigorous regulation of HSC survival, proliferation, selfrenewal, and (lineage-specific) differentiation. While many aspects of these processes are intertwined, available evidence suggests that they are often regulated by distinct mechanisms. Accordingly, NUP98-HOXA10hd exerts its effect either by increasing HSC cycling rate or probability of self-renewal, or decreasing apoptosis. Although both processes depend on cell division,  114  proliferation encompasses all types of stem and progenitor cell divisions whereas self-renewal requires that at least one of the daughter cells has a developmental potential as the original cell, thus determining its fate. An in vitro study using cytokine combination similar to that used here found that essentially all long-term repopulating cells were induced into cycle358. Two recent studies have suggested that Hox transcription factors may influence stem cell-fate decisions rather than alter their proliferation rate and/or death. By examining DNA synthesis, cell-cycle phase distributions, and tracking cell divisions, Cellot et al.292 have showed that HOXB4hiPbx1lo Sca-1+Lin- cells do divide, but are not highly proliferative. Moreover, by using Notch reporter mouse with GFP acting as a sensor for differentiation, Wu et al.359 have demonstrated that NUP98-HOXA9 has no effect on the proliferation rate, but promotes symmetric divisions of LSK cells. Indeed, highly purified HSCs with and without NUP98-HOXA10hd were assessed individually for their proliferative response to early-acting cytokines ex vivo, showing identical cell cycle transit time, as presented in Chapter 2. This finding strongly suggests that the dramatic increase (compared to control) in HSC content obtained ex vivo in cultures containing NUP98-HOXA10hd-transduced cells is determined by their positive self-renewal response to the fusion gene. NUP98-HOXA10hd-transduced cells also showed enhanced level of HSC regeneration in vivo, in order to repopulate the post-irradiation hematopoietic system. Similarly to existing model of HSC regeneration following normal adult bm transplantation24,25, NUP98-HOXA10hd overexperssion could be increasing the rate of self-renewing divisions during early recovery phase post-  115  transplantation, thereby increasing the maximal HSC level (higher than seen in control setting) achieved in vivo. Intriguingly, once progenitor and mature cell populations recovered, system returned to steady state where HSC numbers were maintained rather than expanded and the mice did not develop leukemia. This is furthermore suggestive of NUP98-HOXA10hd having effect on HSC fate rather than proliferation rate, given that NUP98-HOXA10hd-transduced HSCs respond to a negative extrinsic stimuli, most likely forcing them back to quiescent state. To test this possibility, several known niche factors implicated in negative regulation of HSC numbers (i.e. TGFβ143,360, TNFα361, osteopontin362) could be examined for their effect on NUP98-HOXA10hd-induced HSC expansion. Another interesting candidate emerging from results of the multifactorial analysis of cytokine effects on NUP98-HOXA10hd-induced HSC expansion presented in Chapter 2 is TPO. Also as implicated earlier in HSC survival363, proliferation and quiescence118,119 one can envisage that inhibition of TPO/MPL signalling (via AMM2 (anti-MPL antibody) administration) in mice reconstituted with NUP98HOXA10hd-transduced HSCs would force them into cell cycle, leading to excessive HSC numbers in vivo and ultimately leukemia. Besides exerting its normal function, by producing all blood cell lineages throughout life and reconstituting a complete hematopoietic system in transplanted recipients, NUP98-HOXA10hd-transduced HSCs do not exhibit any signs of replicative senescence. In fact, findings presented in Chapter 3 clearly showed telomere maintenance in NUP98-HOXA10hd-transduced HSCs under conditions of prolonged self-renewal stimulation both in vivo and in vitro. Only in  116  the absence of telomerase NUP98-HOXA10hd-transduced HSCs exhibited a significant telomere loss and functional impairment. These findings go in line with recent reports demonstrating telomere maintenance and elongation in iPS cells349,364, suggesting that telomerase upregulation is a feature of the pluripotent state. Therefore, it would be interesting to directly examine whether NUP98HOXA10hd upregulates the endogenous telomerase levels in HSCs and whether functional impairment (e.g. diminished self-renewal activity) of telomerasedeficient NUP98-HOXA10hd-transduced HSCs would be restored by telomerase reintroduction. Results of such experiments could directly reinforce the role of telomerase in regulation of HSC self-renewal process.  4.2  Potential molecular mechanisms of NUP98-HOXA10hd-mediated HSC  expansion  It is now clear that HSCs are not functionally homogeneous. In fact, even in identical genetic backgrounds, intrinsic differences in HSC properties have been described when comparing HSCs from mice of the same12-15 or different ages333,365,366 or different developmental stages47,59,63. Interestingly, findings presented in this thesis reveal the power of NUP98-HOXA10hd to enhance the existing self-renewal program of a spectrum of adult bm HSC subtypes14, as well as HSCs from different stages of development, including FL HSCs (Chapter 2) and telomerase deficient HSCs sharing some features of aged bm HSCs (Chapter 3). This is an exciting realization, suggesting that among diverse self-  117  renewal machineries a subset of the genes might be uniquely regulated in HSCs with different functional properties and the mechanisms by which they are regulated, would offer insights into the fundamental control of the HSC properties (i.e. self-renewal and lineage output). If the pathways affecting these decisions could then be modulated, it would have far-reaching implications for many aspects of hematopoietic stem cell biology. With this mindset, comparative highthroughput gene expression analysis84,367-371 can begin to define the fundamental and/or cell type- and developmental stage-specific self-renewal program(s) and facilitate the understanding of the mechanism(s) underlying such fate option throughout development, homeostasis and disease. Furthermore, transcriptional networks are now being developed that allow interconnection and crosscomparisons of generated datasets372. Recently, substantial evidence has emphasized the importance of epigenetic regulation of transcript availability on stem cell properties. As primitive hematopoietic cells express low levels of many genes normally associated with cell differentiation373, it has thus been hypothesized that open chromatin structure is maintained in primitive cells, with progressive epigenetic changes occurring during differentiation374. Changes in transcription factor levels coincide with these epigenetic changes, resulting in the up-regulation and down-regulation of appropriate lineage-specific genes in a stepwise fashion375,376. It would thus be of particular interest to attempt to modify the epigenome of HSCs and determine whether they remain responsive to NUP98-HOXA10hd effect. One strategy would be to alter the expression in HSCs of one or more of the Polycomb group  118  genes, with well-known roles in epigenetic regulation as discussed in Chapter 1. Another approach would be to remodel the chromatin in a more random fashion by treatment with drugs (i.e. 5-azo2’deoxycytidine312,313, valproic acid377, trichostatin A312,313,378, trapoxin378, and chlamydocin378) that interfere with histone deacetylation and/or DNA methylation. Finally, throughout a collaborative effort in the lab, a comprehensive, genome-wide analysis of changes in the epigenome is now in progress. For these studies, we have taken advantage of NUP98HOXA10hd that as shown here allows for >1,000-fold expansion of HSCs in short-term liquid culture and for efficient generation of transplantable AML following co-transduction with Meis1379, a Hox transcription factor co-factor, overexpressed in AML and potent collaborating gene in numerous murine leukemia models. We have exploited Illumina platform sequencing to perform genome-wide analysis of Meis1-induced changes in key histone marks associated with activation (H3K4me3), priming (H3K4me3 and H3K27me3) or silencing (H3K9 and/or H3K27me3) chromatin states that should provide novel insights into the subsets of important genes and pathways linked to leukemic transformation by Meis1. Another approach that may also offer new clues to the mechanisms underlying NUP98-HOXA10hd effect involves delineating the key regions and functions of the fusion protein, contributing to its HSC self-renewal activity.  119  4.2.1 The role of NUP98 fusion partner The importance of the NUP98 moiety in the NUP98-fusions has been well established380,381. However, the mechanistic contribution of the NUP98 moiety to the fusion proteins remains unclear. Molecular-genetic analyses of multiple patient samples containing translocations of NUP98 suggest a minimal NUP98domain encompassing roughly half of the NUP98 protein382. This N terminal truncation of NUP98 contains a number of protein motifs, including a GLEBS motif, required for interaction with other nucleoporin proteins383, a Rae-1 interaction domain, important in both mediating mRNA transport function384 and interaction with the anaphase promoting complex (APC)385, and a high density of FG/GLFG repeats, implicated the in the recruitment of transcriptional activators (CBP/p300)293 and in some cases repressors (HDAC1) as well386. Initiated by Dr. Eric Yung and later pursued in a collaborative effort, we have conducted a structure-function analysis of NUP98 in the context of NUP98-HOXA10hd. Results clearly demonstrate that the NUP98 fusion partner does not function in a manner that requires interactions with either the nuclear pore complex (NPC) or the Rae1/anaphase promoting complex (APC), both of which would have been consistent with its role as a nucleoporin protein. Instead, NUP98 seems to function in a transactivation manner in part by recruitment of CBP/p300 via its FG/GLFG repeats. However, additional NUP98-mediated interactions that have impact on HSC self-renewal are yet to be defined.  120  4.2.2 The role as a transcription factor The potent effect of NUP98-HOXA10hd fusion on HSC self-renewal is possibly tightly linked to its role as a transcription factor. However, the target genes and regulatory networks that NUP98-HOXA10hd controls remain unknown. Robust methods for isolating nearly pure murine HSCs, the ability to influence them to almost exclusively self-renew in vitro and finally the ability to re-purify large numbers of these actively self-renewing HSCs (as described in Chapter 2) would make it possible to analyze the identity of the genes controlled by NUP98HOXA10hd with increased precision. Such analysis would combine chromatin immunoprecipitation (ChIP) with sequencing technology to identify in vivo protein-DNA interactions on a genome wide scale and could be complemented with global analysis of gene expression profile, in order to establish the relationship between key genes affected by NUP98-HOXA10hd and their expression  status.  Identification  of  NUP98-HOXA10hd  target  genes  in  combination with expression and perhaps functional studies would provide the opportunity to infer pathways that might be involved in regulation of self-renewal and also examine associated chromatin changes (e.g., histone acetylation and methylation), which may provide important clues to the mechanism of NUP98HOXA10hd-induced effect .  121  4.3  Potential therapeutic applications  The results presented in this thesis show great promise for therapeutic applications. Ex vivo HSC expansion mediated by NUP98-HOXA10hd can potentially be used for gene therapy and bm transplantation applications. As outlined in Chapter 1, HSC numbers currently limit the ability to use CB grafts in adult patients. Also, procedures which require ex vivo culture of hematopoietic cells, including gene therapy and leukemic cell purging of autologus grafts, often result in significant stem cell losses. As HSC number directly controls the efficacy of bm transplantation procedures42,43, the ability to expand these cells ex vivo will be enormously useful. In addition, gene therapy models are often hampered by poor gene transfer and engraftment efficiencies, thus NUP98-HOXA10hd might help to generate complete reconstitution of a hematopoietic system by genecorrected cells. However, further studies into the safety and efficacy of using this factor in the human setting are warranted. Early results indicate that NUP98-HOXA10hd overexpression does have a positive effect on human HSC expansion ex vivo (personal communications with Dr. Suzan Imren). Studies into the safety of using NUP98-HOXA10hd on human cells are also essential. Although NUP98HOXA10hd overexpression has never led to leukemias in mice, other Hox gene alterations have been associated with leukemogenesis (e.g. HOXA9223,387). Transient transfer of NUP98-HOXA10hd could be achieved either through an inducible system (e.g. tetracycline-inducible388), or by transfer of the protein (e.g.  122  as an HIV TAT fusion389). Modifications such as these might allow for more effective and/or safer utilization of the NUP98-HOXA10hd advantages.  123  REFERENCES  1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.  12.  13. 14. 15.  Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. Feb 22 2008;132(4):631-644. Cavazzana-Calvo M, Hacein-Bey-Abina S. Correction of genetic blood defects by gene transfer. Curr Opin Hematol. Nov 2001;8(6):360-367. Anasetti C, Petersdorf EW, Martin PJ, Woolfrey A, Hansen JA. Trends in transplantation of hematopoietic stem cells from unrelated donors. Curr Opin Hematol. Nov 2001;8(6):337-341. Shizuru JA, Negrin RS, Weissman IL. Hematopoietic stem and progenitor cells: clinical and preclinical regeneration of the hematolymphoid system. Annu Rev Med. 2005;56:509-538. Ema H, Takano H, Sudo K, Nakauchi H. In vitro self-renewal division of hematopoietic stem cells. J Exp Med. Nov 6 2000;192(9):1281-1288. Argiropoulos B, Humphries RK. Hox genes in hematopoiesis and leukemogenesis. Oncogene. Oct 15 2007;26(47):6766-6776. Pineault N, Abramovich C, Ohta H, Humphries RK. Differential and common leukemogenic potentials of multiple NUP98-Hox fusion proteins alone or with Meis1. Mol Cell Biol. Mar 2004;24(5):1907-1917. Ohta H, Sekulovic S, Bakovic S, et al. Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions. Exp Hematol. May 2007;35(5):817-830. Blank U, Karlsson G, Karlsson S. Signaling pathways governing stem-cell fate. Blood. Jan 15 2008;111(2):492-503. Bryder D, Rossi DJ, Weissman IL. Hematopoietic stem cells: the paradigmatic tissue-specific stem cell. Am J Pathol. Aug 2006;169(2):338346. Adolfsson J, Mansson R, Buza-Vidas N, et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell. Apr 22 2005;121(2):295-306. Muller-Sieburg CE, Cho RH, Karlsson L, Huang JF, Sieburg HB. Myeloidbiased hematopoietic stem cells have extensive self-renewal capacity but generate diminished lymphoid progeny with impaired IL-7 responsiveness. Blood. Jun 1 2004;103(11):4111-4118. Sieburg HB, Cho RH, Dykstra B, Uchida N, Eaves CJ, Muller-Sieburg CE. The hematopoietic stem compartment consists of a limited number of discrete stem cell subsets. Blood. Mar 15 2006;107(6):2311-2316. Dykstra B, Kent D, Bowie M, et al. Long-term propagation of distinct hematopoietic differentiation programs in vivo. Cell Stem Cell. Aug 16 2007;1(2):218-229. Kent DG, Copley MR, Benz C, et al. Prospective isolation and molecular characterization of hematopoietic stem cells with durable self-renewal potential. Blood. Jun 18 2009;113(25):6342-6350.  124  16. 17. 18. 19. 20. 21. 22. 23.  24. 25. 26. 27. 28. 29. 30. 31.  Benveniste P, Frelin C, Janmohamed S, et al. Intermediate-term hematopoietic stem cells with extended but time-limited reconstitution potential. Cell Stem Cell. Jan 8 2010;6(1):48-58. Seita J, Weissman IL. Hematopoietic stem cell: self-renewal versus differentiation. Wiley Interdiscip Rev Syst Biol Med. Nov-Dec 2010;2(6):640-653. Molofsky AV, Pardal R, Morrison SJ. Diverse mechanisms regulate stem cell self-renewal. Curr Opin Cell Biol. Dec 2004;16(6):700-707. Wilson A, Laurenti E, Oser G, et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell. Dec 12 2008;135(6):1118-1129. Essers MA, Offner S, Blanco-Bose WE, et al. IFNalpha activates dormant haematopoietic stem cells in vivo. Nature. Apr 16 2009;458(7240):904908. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci U S A. Oct 24 1995;92(22):10302-10306. Ema H, Nakauchi H. Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood. Apr 1 2000;95(7):2284-2288. Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ. Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc Natl Acad Sci U S A. Nov 1990;87(22):8736-8740. Pawliuk R, Eaves C, Humphries RK. Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood. Oct 15 1996;88(8):2852-2858. Iscove NN, Nawa K. Hematopoietic stem cells expand during serial transplantation in vivo without apparent exhaustion. Curr Biol. Oct 1 1997;7(10):805-808. Thorsteinsdottir U, Sauvageau G, Humphries RK. Enhanced in vivo regenerative potential of HOXB4-transduced hematopoietic stem cells with regulation of their pool size. Blood. Oct 15 1999;94(8):2605-2612. Lansdorp PM. Self-renewal of stem cells. Biol Blood Marrow Transplant. Oct 1997;3(4):171-178. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nat Rev Cancer. Nov 2007;7(11):823833. Krivtsov AV, Twomey D, Feng Z, et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature. Aug 17 2006;442(7104):818-822. Jacobson LO, Marks EK, Lorenz E. The hematological effects of ionizing radiations. Radiology. Mar 1949;52(3):371-395. Lorenz E, Uphoff D, Reid TR, Shelton E. Modification of irradiation injury in mice and guinea pigs by bone marrow injections. J Natl Cancer Inst. Aug 1951;12(1):197-201.  125  32. 33. 34.  35. 36. 37. 38. 39.  40. 41. 42.  43. 44. 45. 46. 47.  Ford CE, Hamerton JL, Barnes DW, Loutit JF. Cytological identification of radiation-chimaeras. Nature. Mar 10 1956;177(4506):452-454. Lemischka IR, Raulet DH, Mulligan RC. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell. Jun 20 1986;45(6):917-927. Dick JE, Magli MC, Huszar D, Phillips RA, Bernstein A. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell. Aug 1985;42(1):71-79. Keller G, Paige C, Gilboa E, Wagner EF. Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors. Nature. Nov 14-20 1985;318(6042):149-154. Till JE, Mc CE. A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res. Feb 1961;14:213-222. Szilvassy SJ. The biology of hematopoietic stem cells. Arch Med Res. Nov-Dec 2003;34(6):446-460. Jones RJ, Wagner JE, Celano P, Zicha MS, Sharkis SJ. Separation of pluripotent haematopoietic stem cells from spleen colony-forming cells. Nature. Sep 13 1990;347(6289):188-189. Ploemacher RE, Brons RH. Separation of CFU-S from primitive cells responsible for reconstitution of the bone marrow hemopoietic stem cell compartment following irradiation: evidence for a pre-CFU-S cell. Exp Hematol. Mar 1989;17(3):263-266. Sorrentino BP. Clinical strategies for expansion of haematopoietic stem cells. Nat Rev Immunol. Nov 2004;4(11):878-888. Kondo M, Wagers AJ, Manz MG, et al. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu Rev Immunol. 2003;21:759-806. Mavroudis D, Read E, Cottler-Fox M, et al. CD34+ cell dose predicts survival, posttransplant morbidity, and rate of hematologic recovery after allogeneic marrow transplants for hematologic malignancies. Blood. Oct 15 1996;88(8):3223-3229. Sierra J, Bjerke J, Hansen J, et al. Marrow transplants from unrelated donors as treatment for acute leukemia. Leuk Lymphoma. Nov 2000;39(56):495-507. Rocha V, Cornish J, Sievers EL, et al. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood. May 15 2001;97(10):2962-2971. Glimm H, Oh IH, Eaves CJ. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G(2)/M transit and do not reenter G(0). Blood. Dec 15 2000;96(13):4185-4193. Rosen JM, Jordan CT. The increasing complexity of the cancer stem cell paradigm. Science. Jun 26 2009;324(5935):1670-1673. Rebel VI, Miller CL, Eaves CJ, Lansdorp PM. The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts. Blood. Apr 15 1996;87(8):3500-3507.  126  48.  49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.  61. 62.  Miller CL, Rebel VI, Helgason CD, Lansdorp PM, Eaves CJ. Impaired steel factor responsiveness differentially affects the detection and longterm maintenance of fetal liver hematopoietic stem cells in vivo. Blood. Feb 15 1997;89(4):1214-1223. Huang H, Auerbach R. Identification and characterization of hematopoietic stem cells from the yolk sac of the early mouse embryo. Proc Natl Acad Sci U S A. Nov 1 1993;90(21):10110-10114. Dzierzak E, Speck NA. Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat Immunol. Feb 2008;9(2):129136. Keller G, Lacaud G, Robertson S. Development of the hematopoietic system in the mouse. Exp Hematol. May 1999;27(5):777-787. Lacaud G, Robertson S, Palis J, Kennedy M, Keller G. Regulation of hemangioblast development. Ann N Y Acad Sci. Jun 2001;938:96-107; discussion 108. Palis J, Robertson S, Kennedy M, Wall C, Keller G. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development. Nov 1999;126(22):5073-5084. Yoder MC, Hiatt K, Dutt P, Mukherjee P, Bodine DM, Orlic D. Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity. Sep 1997;7(3):335-344. Gekas C, Dieterlen-Lievre F, Orkin SH, Mikkola HK. The placenta is a niche for hematopoietic stem cells. Dev Cell. Mar 2005;8(3):365-375. Ottersbach K, Dzierzak E. The murine placenta contains hematopoietic stem cells within the vascular labyrinth region. Dev Cell. Mar 2005;8(3):377-387. Muller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E. Development of hematopoietic stem cell activity in the mouse embryo. Immunity. Jul 1994;1(4):291-301. de Bruijn MF, Speck NA, Peeters MC, Dzierzak E. Definitive hematopoietic stem cells first develop within the major arterial regions of the mouse embryo. EMBO J. Jun 1 2000;19(11):2465-2474. Bowie MB, Kent DG, Dykstra B, et al. Identification of a new intrinsically timed developmental checkpoint that reprograms key hematopoietic stem cell properties. Proc Natl Acad Sci U S A. Apr 3 2007;104(14):5878-5882. Bowie MB, McKnight KD, Kent DG, McCaffrey L, Hoodless PA, Eaves CJ. Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect. J Clin Invest. Oct 2006;116(10):28082816. Cheshier SH, Morrison SJ, Liao X, Weissman IL. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci U S A. Mar 16 1999;96(6):3120-3125. Foudi A, Hochedlinger K, Van Buren D, et al. Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol. Jan 2009;27(1):84-90.  127  63. 64. 65. 66.  67. 68.  69. 70.  71. 72. 73.  74. 75. 76.  Harrison DE, Zhong RK, Jordan CT, Lemischka IR, Astle CM. Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term. Exp Hematol. Apr 1997;25(4):293-297. Jordan CT, McKearn JP, Lemischka IR. Cellular and developmental properties of fetal hematopoietic stem cells. Cell. Jun 15 1990;61(6):953963. Harrison DE. Competitive repopulation: a new assay for long-term stem cell functional capacity. Blood. Jan 1980;55(1):77-81. Harrison DE, Jordan CT, Zhong RK, Astle CM. Primitive hemopoietic stem cells: direct assay of most productive populations by competitive repopulation with simple binomial, correlation and covariance calculations. Exp Hematol. Feb 1993;21(2):206-219. Sekulovic S, Imren S, Humphries K. High level in vitro expansion of murine hematopoietic stem cells. Curr Protoc Stem Cell Biol. Jan 2008;Chapter 2:Unit 2A 7. Szilvassy SJ, Cory S. Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil-treated murine marrow. Blood. May 1 1993;81(9):23102320. Nocka K, Tan JC, Chiu E, et al. Molecular bases of dominant negative and loss of function mutations at the murine c-kit/white spotting locus: W37, Wv, W41 and W. EMBO J. Jun 1990;9(6):1805-1813. Miller CL, Rebel VI, Lemieux ME, Helgason CD, Lansdorp PM, Eaves CJ. Studies of W mutant mice provide evidence for alternate mechanisms capable of activating hematopoietic stem cells. Exp Hematol. Feb 1996;24(2):185-194. Thomas ML. The leukocyte common antigen family. Annu Rev Immunol. 1989;7:339-369. Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse hematopoietic stem cells. Science. Jul 1 1988;241(4861):58-62. Uchida N, Dykstra B, Lyons KJ, Leung FY, Eaves CJ. Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics. Exp Hematol. Dec 2003;31(12):1338-1347. Benveniste P, Cantin C, Hyam D, Iscove NN. Hematopoietic stem cells engraft in mice with absolute efficiency. Nat Immunol. Jul 2003;4(7):708713. Ema H, Sudo K, Seita J, et al. Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice. Dev Cell. Jun 2005;8(6):907-914. Okada S, Nakauchi H, Nagayoshi K, Nishikawa S, Miura Y, Suda T. In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells. Blood. Dec 15 1992;80(12):3044-3050.  128  77. 78. 79. 80.  81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91.  Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. Jul 12 1996;273(5272):242-245. Zhao Y, Lin Y, Zhan Y, et al. Murine hematopoietic stem cell characterization and its regulation in BM transplantation. Blood. Nov 1 2000;96(9):3016-3022. Wiesmann A, Phillips RL, Mojica M, et al. Expression of CD27 on murine hematopoietic stem and progenitor cells. Immunity. Feb 2000;12(2):193199. Adolfsson J, Borge OJ, Bryder D, et al. Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity. Oct 2001;15(4):659-669. Christensen JL, Weissman IL. Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells. Proc Natl Acad Sci U S A. Dec 4 2001;98(25):14541-14546. Forsberg EC, Serwold T, Kogan S, Weissman IL, Passegue E. New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic progenitors. Cell. Jul 28 2006;126(2):415-426. Chen CZ, Li M, de Graaf D, et al. Identification of endoglin as a functional marker that defines long-term repopulating hematopoietic stem cells. Proc Natl Acad Sci U S A. Nov 26 2002;99(24):15468-15473. Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C, Morrison SJ. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. Jul 1 2005;121(7):1109-1121. Balazs AB, Fabian AJ, Esmon CT, Mulligan RC. Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow. Blood. Mar 15 2006;107(6):2317-2321. Wagers AJ, Weissman IL. Differential expression of alpha2 integrin separates long-term and short-term reconstituting Lin-/loThy1.1(lo)c-kit+ Sca-1+ hematopoietic stem cells. Stem Cells. Apr 2006;24(4):1087-1094. Li CL, Johnson GR. Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization. Blood. Mar 15 1995;85(6):14721479. Matsuzaki Y, Kinjo K, Mulligan RC, Okano H. Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity. Jan 2004;20(1):87-93. Pearce DJ, Ridler CM, Simpson C, Bonnet D. Multiparameter analysis of murine bone marrow side population cells. Blood. Apr 1 2004;103(7):2541-2546. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med. Apr 1 1996;183(4):1797-1806. Sato T, Laver JH, Ogawa M. Reversible expression of CD34 by murine hematopoietic stem cells. Blood. Oct 15 1999;94(8):2548-2554.  129  92. 93.  94. 95.  96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107.  Tajima F, Deguchi T, Laver JH, Zeng H, Ogawa M. Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells. Blood. May 1 2001;97(9):2618-2624. Uchida N, Dykstra B, Lyons K, Leung F, Kristiansen M, Eaves C. ABC transporter activities of murine hematopoietic stem cells vary according to their developmental and activation status. Blood. Jun 15 2004;103(12):4487-4495. Zhang CC, Lodish HF. Murine hematopoietic stem cells change their surface phenotype during ex vivo expansion. Blood. Jun 1 2005;105(11):4314-4320. Rebel VI, Miller CL, Thornbury GR, Dragowska WH, Eaves CJ, Lansdorp PM. A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse. Exp Hematol. Apr 1996;24(5):638-648. Yilmaz OH, Kiel MJ, Morrison SJ. SLAM family markers are conserved among hematopoietic stem cells from old and reconstituted mice and markedly increase their purity. Blood. Feb 1 2006;107(3):924-930. Kim I, He S, Yilmaz OH, Kiel MJ, Morrison SJ. Enhanced purification of fetal liver hematopoietic stem cells using SLAM family receptors. Blood. Jul 15 2006;108(2):737-744. Wilson A, Trumpp A. Bone-marrow haematopoietic-stem-cell niches. Nat Rev Immunol. Feb 2006;6(2):93-106. Morrison SJ, Spradling AC. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell. Feb 22 2008;132(4):598-611. Taoudi S, Gonneau C, Moore K, et al. Extensive hematopoietic stem cell generation in the AGM region via maturation of VE-cadherin+CD45+ predefinitive HSCs. Cell Stem Cell. Jul 3 2008;3(1):99-108. Oostendorp RA, Robin C, Steinhoff C, et al. Long-term maintenance of hematopoietic stem cells does not require contact with embryo-derived stromal cells in cocultures. Stem Cells. Jun-Jul 2005;23(6):842-851. Moore KA, Ema H, Lemischka IR. In vitro maintenance of highly purified, transplantable hematopoietic stem cells. Blood. Jun 15 1997;89(12):43374347. Calvi LM, Adams GB, Weibrecht KW, et al. Osteoblastic cells regulate the haematopoietic stem cell niche. Nature. Oct 23 2003;425(6960):841-846. Zhang J, Niu C, Ye L, et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature. Oct 23 2003;425(6960):836841. Mayack SR, Wagers AJ. Osteolineage niche cells initiate hematopoietic stem cell mobilization. Blood. Aug 1 2008;112(3):519-531. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells. 1978;4(1-2):7-25. Lord BI, Testa NG, Hendry JH. The relative spatial distributions of CFUs and CFUc in the normal mouse femur. Blood. Jul 1975;46(1):65-72.  130  108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123.  Barker JE. Sl/Sld hematopoietic progenitors are deficient in situ. Exp Hematol. Feb 1994;22(2):174-177. Barker JE. Early transplantation to a normal microenvironment prevents the development of Steel hematopoietic stem cell defects. Exp Hematol. Jun 1997;25(6):542-547. Komori T, Yagi H, Nomura S, et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. May 30 1997;89(5):755-764. Visnjic D, Kalajzic I, Gronowicz G, et al. Conditional ablation of the osteoblast lineage in Col2.3deltatk transgenic mice. J Bone Miner Res. Dec 2001;16(12):2222-2231. Visnjic D, Kalajzic Z, Rowe DW, Katavic V, Lorenzo J, Aguila HL. Hematopoiesis is severely altered in mice with an induced osteoblast deficiency. Blood. May 1 2004;103(9):3258-3264. Arai F, Hirao A, Ohmura M, et al. Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell. Jul 23 2004;118(2):149-161. Nilsson SK, Johnston HM, Coverdale JA. Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches. Blood. Apr 15 2001;97(8):2293-2299. Kiel MJ, Morrison SJ. Uncertainty in the niches that maintain haematopoietic stem cells. Nat Rev Immunol. Apr 2008;8(4):290-301. Trumpp A, Essers M, Wilson A. Awakening dormant haematopoietic stem cells. Nat Rev Immunol. Mar 2010;10(3):201-209. McCarthy KF, Ledney GD, Mitchell R. A deficiency of hematopoietic stem cells in steel mice. Cell Tissue Kinet. Mar 1977;10(2):121-126. Yoshihara H, Arai F, Hosokawa K, et al. Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche. Cell Stem Cell. Dec 13 2007;1(6):685-697. Qian H, Buza-Vidas N, Hyland CD, et al. Critical role of thrombopoietin in maintaining adult quiescent hematopoietic stem cells. Cell Stem Cell. Dec 13 2007;1(6):671-684. Petit I, Szyper-Kravitz M, Nagler A, et al. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR4. Nat Immunol. Jul 2002;3(7):687-694. Reya T, Duncan AW, Ailles L, et al. A role for Wnt signalling in selfrenewal of haematopoietic stem cells. Nature. May 22 2003;423(6938):409-414. Willert K, Brown JD, Danenberg E, et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature. May 22 2003;423(6938):448-452. Mancini SJ, Mantei N, Dumortier A, Suter U, MacDonald HR, Radtke F. Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation. Blood. Mar 15 2005;105(6):2340-2342.  131  124. 125.  126. 127.  128.  129. 130.  131. 132. 133.  134.  135. 136.  Koch U, Wilson A, Cobas M, Kemler R, Macdonald HR, Radtke F. Simultaneous loss of beta- and gamma-catenin does not perturb hematopoiesis or lymphopoiesis. Blood. Jan 1 2008;111(1):160-164. Holyoake TL, Freshney MG, McNair L, et al. Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow. Blood. Jun 1 1996;87(11):4589-4595. Miller CL, Eaves CJ. Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci U S A. Dec 9 1997;94(25):13648-13653. Audet J, Miller CL, Rose-John S, Piret JM, Eaves CJ. Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic stem cells. Proc Natl Acad Sci U S A. Feb 13 2001;98(4):1757-1762. Audet J, Miller CL, Eaves CJ, Piret JM. Common and distinct features of cytokine effects on hematopoietic stem and progenitor cells revealed by dose-response surface analysis. Biotechnol Bioeng. Nov 20 2002;80(4):393-404. Kent DG, Dykstra BJ, Cheyne J, Ma E, Eaves CJ. Steel factor coordinately regulates the molecular signature and biologic function of hematopoietic stem cells. Blood. Aug 1 2008;112(3):560-567. Matsunaga T, Kato T, Miyazaki H, Ogawa M. Thrombopoietin promotes the survival of murine hematopoietic long-term reconstituting cells: comparison with the effects of FLT3/FLK-2 ligand and interleukin-6. Blood. Jul 15 1998;92(2):452-461. Huang E, Nocka K, Beier DR, et al. The hematopoietic growth factor KL is encoded by the Sl locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell. Oct 5 1990;63(1):225-233. Ashman LK. The biology of stem cell factor and its receptor C-kit. Int J Biochem Cell Biol. Oct 1999;31(10):1037-1051. Yoshida K, Taga T, Saito M, et al. Targeted disruption of gp130, a common signal transducer for the interleukin 6 family of cytokines, leads to myocardial and hematological disorders. Proc Natl Acad Sci U S A. Jan 9 1996;93(1):407-411. Alexander WS, Roberts AW, Nicola NA, Li R, Metcalf D. Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl. Blood. Mar 15 1996;87(6):2162-2170. Carver-Moore K, Broxmeyer HE, Luoh SM, et al. Low levels of erythroid and myeloid progenitors in thrombopoietin-and c-mpl-deficient mice. Blood. Aug 1 1996;88(3):803-808. Kiger AA, Jones DL, Schulz C, Rogers MB, Fuller MT. Stem cell selfrenewal specified by JAK-STAT activation in response to a support cell cue. Science. Dec 21 2001;294(5551):2542-2545.  132  137. 138. 139. 140. 141. 142.  143.  144. 145. 146. 147.  148. 149. 150.  151.  Wandzioch E, Edling CE, Palmer RH, Carlsson L, Hallberg B. Activation of the MAP kinase pathway by c-Kit is PI-3 kinase dependent in hematopoietic progenitor/stem cell lines. Blood. Jul 1 2004;104(1):51-57. Zhang J, Grindley JC, Yin T, et al. PTEN maintains haematopoietic stem cells and acts in lineage choice and leukaemia prevention. Nature. May 25 2006;441(7092):518-522. Yilmaz OH, Valdez R, Theisen BK, et al. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. May 25 2006;441(7092):475-482. Zhang CC, Lodish HF. Insulin-like growth factor 2 expressed in a novel fetal liver cell population is a growth factor for hematopoietic stem cells. Blood. Apr 1 2004;103(7):2513-2521. Zhang CC, Kaba M, Ge G, et al. Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nat Med. Feb 2006;12(2):240245. Varnum-Finney B, Brashem-Stein C, Bernstein ID. Combined effects of Notch signaling and cytokines induce a multiple log increase in precursors with lymphoid and myeloid reconstituting ability. Blood. Mar 1 2003;101(5):1784-1789. Sitnicka E, Ruscetti FW, Priestley GV, Wolf NS, Bartelmez SH. Transforming growth factor beta 1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells. Blood. Jul 1 1996;88(1):82-88. Zon LI. Intrinsic and extrinsic control of haematopoietic stem-cell selfrenewal. Nature. May 15 2008;453(7193):306-313. He S, Nakada D, Morrison SJ. Mechanisms of stem cell self-renewal. Annu Rev Cell Dev Biol. 2009;25:377-406. Gerhart J. 1998 Warkany lecture: signaling pathways in development. Teratology. Oct 1999;60(4):226-239. Kirstetter P, Anderson K, Porse BT, Jacobsen SE, Nerlov C. Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block. Nat Immunol. Oct 2006;7(10):1048-1056. Cobas M, Wilson A, Ernst B, et al. Beta-catenin is dispensable for hematopoiesis and lymphopoiesis. J Exp Med. Jan 19 2004;199(2):221229. Oh IH, Eaves CJ. Overexpression of a dominant negative form of STAT3 selectively impairs hematopoietic stem cell activity. Oncogene. Jul 18 2002;21(31):4778-4787. Bunting KD, Bradley HL, Hawley TS, Moriggl R, Sorrentino BP, Ihle JN. Reduced lymphomyeloid repopulating activity from adult bone marrow and fetal liver of mice lacking expression of STAT5. Blood. Jan 15 2002;99(2):479-487. Chung YJ, Park BB, Kang YJ, Kim TM, Eaves CJ, Oh IH. Unique effects of Stat3 on the early phase of hematopoietic stem cell regeneration. Blood. Aug 15 2006;108(4):1208-1215.  133  152. 153. 154. 155.  156. 157.  158. 159. 160. 161. 162. 163. 164. 165. 166.  Kato Y, Iwama A, Tadokoro Y, et al. Selective activation of STAT5 unveils its role in stem cell self-renewal in normal and leukemic hematopoiesis. J Exp Med. Jul 4 2005;202(1):169-179. Sherr CJ, Roberts JM. Living with or without cyclins and cyclin-dependent kinases. Genes Dev. Nov 15 2004;18(22):2699-2711. Janzen V, Forkert R, Fleming HE, et al. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature. Sep 28 2006;443(7110):421-426. Yuan Y, Shen H, Franklin DS, Scadden DT, Cheng T. In vivo selfrenewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C. Nat Cell Biol. May 2004;6(5):436-442. Cheng T, Rodrigues N, Dombkowski D, Stier S, Scadden DT. Stem cell repopulation efficiency but not pool size is governed by p27(kip1). Nat Med. Nov 2000;6(11):1235-1240. Mantel C, Luo Z, Canfield J, Braun S, Deng C, Broxmeyer HE. Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo. Blood. Nov 15 1996;88(10):3710-3719. Cheng T, Rodrigues N, Shen H, et al. Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science. Mar 10 2000;287(5459):1804-1808. Konuma T, Oguro H, Iwama A. Role of the polycomb group proteins in hematopoietic stem cells. Dev Growth Differ. Aug 2010;52(6):505-516. Lunyak VV, Rosenfeld MG. Epigenetic regulation of stem cell fate. Hum Mol Genet. Apr 15 2008;17(R1):R28-36. Schuettengruber B, Chourrout D, Vervoort M, Leblanc B, Cavalli G. Genome regulation by polycomb and trithorax proteins. Cell. Feb 23 2007;128(4):735-745. Sauvageau M, Sauvageau G. Polycomb group proteins: multi-faceted regulators of somatic stem cells and cancer. Cell Stem Cell. Sep 3 2010;7(3):299-313. Ringrose L, Paro R. Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet. 2004;38:413443. Park IK, Qian D, Kiel M, et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. May 15 2003;423(6937):302-305. Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature. May 15 2003;423(6937):255260. Iwama A, Oguro H, Negishi M, et al. Enhanced self-renewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1. Immunity. Dec 2004;21(6):843-851.  134  167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181.  Ohta H, Sawada A, Kim JY, et al. Polycomb group gene rae28 is required for sustaining activity of hematopoietic stem cells. J Exp Med. Mar 18 2002;195(6):759-770. Kim JY, Sawada A, Tokimasa S, et al. Defective long-term repopulating ability in hematopoietic stem cells lacking the Polycomb-group gene rae28. Eur J Haematol. Aug 2004;73(2):75-84. Kamminga LM, Bystrykh LV, de Boer A, et al. The Polycomb group gene Ezh2 prevents hematopoietic stem cell exhaustion. Blood. Mar 1 2006;107(5):2170-2179. Kajiume T, Ninomiya Y, Ishihara H, Kanno R, Kanno M. Polycomb group gene mel-18 modulates the self-renewal activity and cell cycle status of hematopoietic stem cells. Exp Hematol. Jun 2004;32(6):571-578. Mikkelsen TS, Ku M, Jaffe DB, et al. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature. Aug 2 2007;448(7153):553-560. McMahon KA, Hiew SY, Hadjur S, et al. Mll has a critical role in fetal and adult hematopoietic stem cell self-renewal. Cell Stem Cell. Sep 13 2007;1(3):338-345. Ernst P, Mabon M, Davidson AJ, Zon LI, Korsmeyer SJ. An Mll-dependent Hox program drives hematopoietic progenitor expansion. Curr Biol. Nov 23 2004;14(22):2063-2069. Meissner A, Mikkelsen TS, Gu H, et al. Genome-scale DNA methylation maps of pluripotent and differentiated cells. Nature. Aug 7 2008;454(7205):766-770. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet. Mar 2003;33 Suppl:245-254. Broske AM, Vockentanz L, Kharazi S, et al. DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction. Nat Genet. Nov 2009;41(11):1207-1215. Tadokoro Y, Ema H, Okano M, Li E, Nakauchi H. De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells. J Exp Med. Apr 16 2007;204(4):715-722. Trowbridge JJ, Snow JW, Kim J, Orkin SH. DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells. Cell Stem Cell. Oct 2 2009;5(4):442-449. Look AT. Oncogenic transcription factors in the human acute leukemias. Science. Nov 7 1997;278(5340):1059-1064. Shivdasani RA, Mayer EL, Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature. Feb 2 1995;373(6513):432-434. Mikkola HK, Klintman J, Yang H, et al. Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stemcell leukaemia SCL/tal-1 gene. Nature. Jan 30 2003;421(6922):547-551.  135  182. 183. 184. 185.  186. 187.  188. 189. 190. 191. 192. 193. 194. 195. 196.  D'Souza SL, Elefanty AG, Keller G. SCL/Tal-1 is essential for hematopoietic commitment of the hemangioblast but not for its development. Blood. May 15 2005;105(10):3862-3870. Warren AJ, Colledge WH, Carlton MB, Evans MJ, Smith AJ, Rabbitts TH. The oncogenic cysteine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell. Jul 15 1994;78(1):45-57. Kim SI, Bresnick EH. Transcriptional control of erythropoiesis: emerging mechanisms and principles. Oncogene. Oct 15 2007;26(47):6777-6794. Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell. Jan 26 1996;84(2):321330. Lacaud G, Gore L, Kennedy M, et al. Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro. Blood. Jul 15 2002;100(2):458-466. Ichikawa M, Asai T, Saito T, et al. AML-1 is required for megakaryocytic maturation and lymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adult hematopoiesis. Nat Med. Mar 2004;10(3):299-304. Kim I, Saunders TL, Morrison SJ. Sox17 dependence distinguishes the transcriptional regulation of fetal from adult hematopoietic stem cells. Cell. Aug 10 2007;130(3):470-483. Tsai FY, Keller G, Kuo FC, et al. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature. Sep 15 1994;371(6494):221-226. Rodrigues NP, Janzen V, Forkert R, et al. Haploinsufficiency of GATA-2 perturbs adult hematopoietic stem-cell homeostasis. Blood. Jul 15 2005;106(2):477-484. Kim HG, de Guzman CG, Swindle CS, et al. The ETS family transcription factor PU.1 is necessary for the maintenance of fetal liver hematopoietic stem cells. Blood. Dec 15 2004;104(13):3894-3900. Iwasaki H, Somoza C, Shigematsu H, et al. Distinctive and indispensable roles of PU.1 in maintenance of hematopoietic stem cells and their differentiation. Blood. Sep 1 2005;106(5):1590-1600. Wilson A, Murphy MJ, Oskarsson T, et al. c-Myc controls the balance between hematopoietic stem cell self-renewal and differentiation. Genes Dev. Nov 15 2004;18(22):2747-2763. Laurenti E, Varnum-Finney B, Wilson A, et al. Hematopoietic stem cell function and survival depend on c-Myc and N-Myc activity. Cell Stem Cell. Dec 4 2008;3(6):611-624. Mucenski ML, McLain K, Kier AB, et al. A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell. May 17 1991;65(4):677-689. Sandberg ML, Sutton SE, Pletcher MT, et al. c-Myb and p300 regulate hematopoietic stem cell proliferation and differentiation. Dev Cell. Feb 2005;8(2):153-166.  136  197.  198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213.  Rebel VI, Kung AL, Tanner EA, Yang H, Bronson RT, Livingston DM. Distinct roles for CREB-binding protein and p300 in hematopoietic stem cell self-renewal. Proc Natl Acad Sci U S A. Nov 12 2002;99(23):1478914794. Passegue E, Wagner EF, Weissman IL. JunB deficiency leads to a myeloproliferative disorder arising from hematopoietic stem cells. Cell. Oct 29 2004;119(3):431-443. Galan-Caridad JM, Harel S, Arenzana TL, et al. Zfx controls the selfrenewal of embryonic and hematopoietic stem cells. Cell. Apr 20 2007;129(2):345-357. Hisa T, Spence SE, Rachel RA, et al. Hematopoietic, angiogenic and eye defects in Meis1 mutant animals. EMBO J. Jan 28 2004;23(2):450-459. Azcoitia V, Aracil M, Martinez AC, Torres M. The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo. Dev Biol. Apr 15 2005;280(2):307-320. Hock H, Hamblen MJ, Rooke HM, et al. Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells. Nature. Oct 21 2004;431(7011):1002-1007. Hock H, Meade E, Medeiros S, et al. Tel/Etv6 is an essential and selective regulator of adult hematopoietic stem cell survival. Genes Dev. Oct 1 2004;18(19):2336-2341. Puri MC, Bernstein A. Requirement for the TIE family of receptor tyrosine kinases in adult but not fetal hematopoiesis. Proc Natl Acad Sci U S A. Oct 28 2003;100(22):12753-12758. Lewis EB. A gene complex controlling segmentation in Drosophila. Nature. Dec 7 1978;276(5688):565-570. Krumlauf R. Hox genes in vertebrate development. Cell. Jul 29 1994;78(2):191-201. Boncinelli E, Acampora D, Pannese M, et al. Organization of human class I homeobox genes. Genome. 1989;31(2):745-756. Scott MP. Vertebrate homeobox gene nomenclature. Cell. Nov 13 1992;71(4):551-553. Zeltser L, Desplan C, Heintz N. Hoxb-13: a new Hox gene in a distant region of the HOXB cluster maintains colinearity. Development. Aug 1996;122(8):2475-2484. Duboule D, Dolle P. The structural and functional organization of the murine HOX gene family resembles that of Drosophila homeotic genes. EMBO J. May 1989;8(5):1497-1505. Holland PW, Garcia-Fernandez J, Williams NA, Sidow A. Gene duplications and the origins of vertebrate development. Dev Suppl. 1994:125-133. Kappen C, Ruddle FH. Evolution of a regulatory gene family: HOM/HOX genes. Curr Opin Genet Dev. Dec 1993;3(6):931-938. Schughart K, Kappen C, Ruddle FH. Duplication of large genomic regions during the evolution of vertebrate homeobox genes. Proc Natl Acad Sci U S A. Sep 1989;86(18):7067-7071.  137  214. 215. 216. 217. 218. 219.  220. 221. 222. 223. 224. 225. 226. 227.  228. 229.  Gehring WJ, Affolter M, Burglin T. Homeodomain proteins. Annu Rev Biochem. 1994;63:487-526. Kalionis B, O'Farrell PH. A universal target sequence is bound in vitro by diverse homeodomains. Mech Dev. Sep 1993;43(1):57-70. Mann RS. The specificity of homeotic gene function. Bioessays. Oct 1995;17(10):855-863. Shen WF, Montgomery JC, Rozenfeld S, et al. AbdB-like Hox proteins stabilize DNA binding by the Meis1 homeodomain proteins. Mol Cell Biol. Nov 1997;17(11):6448-6458. Pineault N, Helgason CD, Lawrence HJ, Humphries RK. Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp Hematol. Jan 2002;30(1):49-57. Sauvageau G, Lansdorp PM, Eaves CJ, et al. Differential expression of homeobox genes in functionally distinct CD34+ subpopulations of human bone marrow cells. Proc Natl Acad Sci U S A. Dec 6 1994;91(25):1222312227. Giampaolo A, Pelosi E, Valtieri M, et al. HOXB gene expression and function in differentiating purified hematopoietic progenitors. Stem Cells. May 1995;13 Suppl 1:90-105. Moretti P, Simmons P, Thomas P, et al. Identification of homeobox genes expressed in human haemopoietic progenitor cells. Gene. Jul 8 1994;144(2):213-219. Nakamura T, Largaespada DA, Shaughnessy JD, Jr., Jenkins NA, Copeland NG. Cooperative activation of Hoxa and Pbx1-related genes in murine myeloid leukaemias. Nat Genet. Feb 1996;12(2):149-153. Thorsteinsdottir U, Mamo A, Kroon E, et al. Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion. Blood. Jan 1 2002;99(1):121-129. Magnusson M, Brun AC, Miyake N, et al. HOXA10 is a critical regulator for hematopoietic stem cells and erythroid/megakaryocyte development. Blood. May 1 2007;109(9):3687-3696. Antonchuk J, Sauvageau G, Humphries RK. HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation. Exp Hematol. Sep 2001;29(9):1125-1134. Antonchuk J, Sauvageau G, Humphries RK. HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell. Apr 5 2002;109(1):39-45. Sauvageau G, Thorsteinsdottir U, Eaves CJ, et al. Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev. Jul 15 1995;9(14):1753-1765. Schiedlmeier B, Santos AC, Ribeiro A, et al. HOXB4's road map to stem cell expansion. Proc Natl Acad Sci U S A. Oct 23 2007;104(43):1695216957. Jiang J, Yu H, Shou Y, et al. Hemgn is a direct transcriptional target of HOXB4 and induces expansion of murine myeloid progenitor cells. Blood. Aug 5 2010;116(5):711-719.  138  230. 231. 232.  233.  234. 235. 236. 237.  238. 239. 240. 241. 242. 243. 244.  Bijl J, Thompson A, Ramirez-Solis R, et al. Analysis of HSC activity and compensatory Hox gene expression profile in Hoxb cluster mutant fetal liver cells. Blood. Jul 1 2006;108(1):116-122. Brun AC, Bjornsson JM, Magnusson M, et al. Hoxb4-deficient mice undergo normal hematopoietic development but exhibit a mild proliferation defect in hematopoietic stem cells. Blood. Jun 1 2004;103(11):4126-4133. Izon DJ, Rozenfeld S, Fong ST, Komuves L, Largman C, Lawrence HJ. Loss of function of the homeobox gene Hoxa-9 perturbs early T-cell development and induces apoptosis in primitive thymocytes. Blood. Jul 15 1998;92(2):383-393. Lawrence HJ, Helgason CD, Sauvageau G, et al. Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood. Mar 15 1997;89(6):19221930. Lawrence HJ, Christensen J, Fong S, et al. Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells. Blood. Dec 1 2005;106(12):3988-3994. Lawrence HJ, Rozenfeld S, Cruz C, et al. Frequent co-expression of the HOXA9 and MEIS1 homeobox genes in human myeloid leukemias. Leukemia. Dec 1999;13(12):1993-1999. Rozovskaia T, Feinstein E, Mor O, et al. Upregulation of Meis1 and HoxA9 in acute lymphocytic leukemias with the t(4 : 11) abnormality. Oncogene. Feb 15 2001;20(7):874-878. Kawagoe H, Humphries RK, Blair A, Sutherland HJ, Hogge DE. Expression of HOX genes, HOX cofactors, and MLL in phenotypically and functionally defined subpopulations of leukemic and normal human hematopoietic cells. Leukemia. May 1999;13(5):687-698. Afonja O, Smith JE, Jr., Cheng DM, et al. MEIS1 and HOXA7 genes in human acute myeloid leukemia. Leuk Res. Oct 2000;24(10):849-855. Imamura T, Morimoto A, Takanashi M, et al. Frequent co-expression of HoxA9 and Meis1 genes in infant acute lymphoblastic leukaemia with MLL rearrangement. Br J Haematol. Oct 2002;119(1):119-121. Kamps MP, Baltimore D. E2A-Pbx1, the t(1;19) translocation protein of human pre-B-cell acute lymphocytic leukemia, causes acute myeloid leukemia in mice. Mol Cell Biol. Jan 1993;13(1):351-357. Ayton PM, Cleary ML. Molecular mechanisms of leukemogenesis mediated by MLL fusion proteins. Oncogene. Sep 10 2001;20(40):56955707. Slape C, Aplan PD. The role of NUP98 gene fusions in hematologic malignancy. Leuk Lymphoma. Jul 2004;45(7):1341-1350. Rossi DJ, Jamieson CH, Weissman IL. Stems cells and the pathways to aging and cancer. Cell. Feb 22 2008;132(4):681-696. Krishnamurthy J, Torrice C, Ramsey MR, et al. Ink4a/Arf expression is a biomarker of aging. J Clin Invest. Nov 2004;114(9):1299-1307.  139  245. 246. 247. 248. 249. 250. 251. 252. 253. 254. 255. 256. 257. 258. 259. 260. 261.  Zindy F, Quelle DE, Roussel MF, Sherr CJ. Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging. Oncogene. Jul 10 1997;15(2):203-211. TeKippe M, Harrison DE, Chen J. Expansion of hematopoietic stem cell phenotype and activity in Trp53-null mice. Exp Hematol. Jun 2003;31(6):521-527. Dumble M, Moore L, Chambers SM, et al. The impact of altered p53 dosage on hematopoietic stem cell dynamics during aging. Blood. Feb 15 2007;109(4):1736-1742. Tothova Z, Gilliland DG. FoxO transcription factors and stem cell homeostasis: insights from the hematopoietic system. Cell Stem Cell. Aug 16 2007;1(2):140-152. Miyamoto K, Araki KY, Naka K, et al. Foxo3a is essential for maintenance of the hematopoietic stem cell pool. Cell Stem Cell. Jun 7 2007;1(1):101112. Ruzankina Y, Pinzon-Guzman C, Asare A, et al. Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss. Cell Stem Cell. Jun 7 2007;1(1):113-126. Navarro S, Meza NW, Quintana-Bustamante O, et al. Hematopoietic dysfunction in a mouse model for Fanconi anemia group D1. Mol Ther. Oct 2006;14(4):525-535. Nijnik A, Woodbine L, Marchetti C, et al. DNA repair is limiting for haematopoietic stem cells during ageing. Nature. Jun 7 2007;447(7145):686-690. Rossi DJ, Bryder D, Seita J, Nussenzweig A, Hoeijmakers J, Weissman IL. Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature. Jun 7 2007;447(7145):725-729. Reese JS, Liu L, Gerson SL. Repopulating defect of mismatch repairdeficient hematopoietic stem cells. Blood. Sep 1 2003;102(5):1626-1633. Bender CF, Sikes ML, Sullivan R, et al. Cancer predisposition and hematopoietic failure in Rad50(S/S) mice. Genes Dev. Sep 1 2002;16(17):2237-2251. Ito K, Hirao A, Arai F, et al. Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells. Nature. Oct 21 2004;431(7011):997-1002. Sauvageau G, Humphries RK. Medicine. The blood stem cell Holy Grail? Science. Sep 10 2010;329(5997):1291-1292. Blackburn EH. Switching and signaling at the telomere. Cell. 2001;106:661-673. Hayflick L MP. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961;25:585-621. Watson JD. Origin of concatameric T4 DNA. Nat New Biol. 1972;239:197201. Olovnikov AM. A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol. 1973;41:181-190.  140  262. 263. 264. 265. 266. 267. 268. 269.  270. 271. 272. 273. 274. 275. 276. 277. 278.  Rando TA. Prognostic Value of telomere length: The long and short of it. American Neurological Association. 2006;60:155-157. Greenwood MJ L, P. Telomeres, telomerase, and hematopoietic stem cell biology. Achives of Medical Research. 2003;34:489-495. Ding H SM, Wu X, et al. Regulation of murine telomere length by Rtel: An essential gene encoding a helicase-like protein. Cell. 2004;117:873-886. Crabbe L VR, Haggblom CI, et al. Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. Science. 2004;306:19511953. Wang RC SA, De Lange T. Homologous recombination generates T-loopsized deletions at human telomeres. Cell. 2004;119:355-368. Yu GL BJ, Attardi LD, Blackburn EH. In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature. 1990;344:126-132. Chiu CP, Dragowska W, Kim NW, et al. Differential expression of telomerase activity in hematopoietic progenitors from adult human bone marrow. Stem Cells. Mar 1996;14(2):239-248. Vaziri H, Dragowska W, Allsopp RC, Thomas TE, Harley CB, Lansdorp PM. Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci U S A. Oct 11 1994;91(21):9857-9860. Allsopp RC, Cheshier S, Weissman IL. Telomere shortening accompanies increased cell cycle activity during serial transplantation of hematopoietic stem cells. J Exp Med. Apr 16 2001;193(8):917-924. Allsopp RC, Morin GB, DePinho R, Harley CB, Weissman IL. Telomerase is required to slow telomere shortening and extend replicative lifespan of HSCs during serial transplantation. Blood. Jul 15 2003;102(2):517-520. Harrison DE, Astle CM. Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure. J Exp Med. Dec 1 1982;156(6):1767-1779. Elwood N. Telomere biology of human hematopoietic stem cells. Cancer Control. Mar-Apr 2004;11(2):77-85. Lansdorp PM. Role of telomerase in hematopoietic stem cells. Ann N Y Acad Sci. Jun 2005;1044:220-227. Allsopp RC, Morin GB, Horner JW, DePinho R, Harley CB, Weissman IL. Effect of TERT over-expression on the long-term transplantation capacity of hematopoietic stem cells. Nat Med. Apr 2003;9(4):369-371. Vulliamy T, Marrone A, Dokal I, Mason PJ. Association between aplastic anaemia and mutations in telomerase RNA. Lancet. Jun 22 2002;359(9324):2168-2170. Yamaguchi H, Calado RT, Ly H, et al. Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia. N Engl J Med. Apr 7 2005;352(14):1413-1424. Calado RT, Regal JA, Hills M, et al. Constitutional hypomorphic telomerase mutations in patients with acute myeloid leukemia. Proc Natl Acad Sci U S A. Jan 27 2009;106(4):1187-1192.  141  279. 280. 281. 282. 283. 284. 285. 286. 287. 288. 289. 290. 291. 292. 293.  Lee HW, Blasco MA, Gottlieb GJ, Horner JW, 2nd, Greider CW, DePinho RA. Essential role of mouse telomerase in highly proliferative organs. Nature. Apr 9 1998;392(6676):569-574. Herrera E, Samper E, Blasco MA. Telomere shortening in mTR-/- embryos is associated with failure to close the neural tube. EMBO J. Mar 1 1999;18(5):1172-1181. Samper E, Fernandez P, Eguia R, et al. Long-term repopulating ability of telomerase-deficient murine hematopoietic stem cells. Blood. Apr 15 2002;99(8):2767-2775. Blasco MA, Lee HW, Hande MP, et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. Oct 3 1997;91(1):25-34. Imamura S, Uchiyama J, Koshimizu E, et al. A non-canonical function of zebrafish telomerase reverse transcriptase is required for developmental hematopoiesis. PLoS One. 2008;3(10):e3364. Park JI, Venteicher AS, Hong JY, et al. Telomerase modulates Wnt signalling by association with target gene chromatin. Nature. Jul 2 2009;460(7251):66-72. Calado RT, Yewdell WT, Wilkerson KL, et al. Sex hormones, acting on the TERT gene, increase telomerase activity in human primary hematopoietic cells. Blood. Sep 10 2009;114(11):2236-2243. Huynh H, Iizuka S, Kaba M, et al. Insulin-like growth factor-binding protein 2 secreted by a tumorigenic cell line supports ex vivo expansion of mouse hematopoietic stem cells. Stem Cells. Jun 2008;26(6):1628-1635. Oostendorp RA, Audet J, Eaves CJ. High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo. Blood. Feb 1 2000;95(3):855-862. Nakauchi H, Sudo K, Ema H. Quantitative assessment of the stem cell self-renewal capacity. Ann N Y Acad Sci. Jun 2001;938:18-24; discussion 24-15. Deneault E, Cellot S, Faubert A, et al. A functional screen to identify novel effectors of hematopoietic stem cell activity. Cell. Apr 17 2009;137(2):369379. Krosl J, Austin P, Beslu N, Kroon E, Humphries RK, Sauvageau G. In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein. Nat Med. Nov 2003;9(11):1428-1432. Miyake N, Brun AC, Magnusson M, Miyake K, Scadden DT, Karlsson S. HOXB4-induced self-renewal of hematopoietic stem cells is significantly enhanced by p21 deficiency. Stem Cells. Mar 2006;24(3):653-661. Cellot S, Krosl J, Chagraoui J, Meloche S, Humphries RK, Sauvageau G. Sustained in vitro trigger of self-renewal divisions in Hoxb4hiPbx1(10) hematopoietic stem cells. Exp Hematol. May 2007;35(5):802-816. Kasper LH, Brindle PK, Schnabel CA, Pritchard CE, Cleary ML, van Deursen JM. CREB binding protein interacts with nucleoporin-specific FG repeats that activate transcription and mediate NUP98-HOXA9 oncogenicity. Mol Cell Biol. Jan 1999;19(1):764-776.  142  294. 295. 296.  297.  298.  299.  300.  301.  302. 303.  304.  305.  Lam DH, Aplan PD. NUP98 gene fusions in hematologic malignancies. Leukemia. Nov 2001;15(11):1689-1695. Habibian HK, Peters SO, Hsieh CC, et al. The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit. J Exp Med. Jul 20 1998;188(2):393-398. Glimm H, Eisterer W, Lee K, et al. Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice. J Clin Invest. Jan 2001;107(2):199-206. Hogan CJ, Shpall EJ, Keller G. Differential long-term and multilineage engraftment potential from subfractions of human CD34+ cord blood cells transplanted into NOD/SCID mice. Proc Natl Acad Sci U S A. Jan 8 2002;99(1):413-418. Kerre TC, De Smet G, De Smedt M, et al. Both CD34+38+ and CD34+38cells home specifically to the bone marrow of NOD/LtSZ scid/scid mice but show different kinetics in expansion. J Immunol. Oct 1 2001;167(7):36923698. Ratajczak MZ, Pletcher CH, Marlicz W, et al. CD34+, kit+, rhodamine123(low) phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells. Leukemia. Jun 1998;12(6):942-950. Uchida N, Combs J, Chen S, Zanjani E, Hoffman R, Tsukamoto A. Primitive human hematopoietic cells displaying differential efflux of the rhodamine 123 dye have distinct biological activities. Blood. Aug 15 1996;88(4):1297-1305. Hess DA, Meyerrose TE, Wirthlin L, et al. Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity. Blood. Sep 15 2004;104(6):16481655. Storms RW, Green PD, Safford KM, et al. Distinct hematopoietic progenitor compartments are delineated by the expression of aldehyde dehydrogenase and CD34. Blood. Jul 1 2005;106(1):95-102. Larochelle A, Vormoor J, Hanenberg H, et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med. Dec 1996;2(12):1329-1337. Conneally E, Cashman J, Petzer A, Eaves C. Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci U S A. Sep 2 1997;94(18):9836-9841. Glimm H, Eaves CJ. Direct evidence for multiple self-renewal divisions of human in vivo repopulating hematopoietic cells in short-term culture. Blood. Oct 1 1999;94(7):2161-2168.  143  306. 307. 308. 309. 310. 311.  312. 313. 314.  315. 316. 317.  318. 319.  Ueda T, Tsuji K, Yoshino H, et al. Expansion of human NOD/SCIDrepopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL6, and soluble IL-6 receptor. J Clin Invest. Apr 2000;105(7):1013-1021. Gammaitoni L, Bruno S, Sanavio F, et al. Ex vivo expansion of human adult stem cells capable of primary and secondary hemopoietic reconstitution. Exp Hematol. Mar 2003;31(3):261-270. Karanu FN, Murdoch B, Gallacher L, et al. The notch ligand jagged-1 represents a novel growth factor of human hematopoietic stem cells. J Exp Med. Nov 6 2000;192(9):1365-1372. Bhardwaj G, Murdoch B, Wu D, et al. Sonic hedgehog induces the proliferation of primitive human hematopoietic cells via BMP regulation. Nat Immunol. Feb 2001;2(2):172-180. McNiece I, Harrington J, Turney J, Kellner J, Shpall EJ. Ex vivo expansion of cord blood mononuclear cells on mesenchymal stem cells. Cytotherapy. 2004;6(4):311-317. Madlambayan GJ, Rogers I, Kirouac DC, et al. Dynamic changes in cellular and microenvironmental composition can be controlled to elicit in vitro human hematopoietic stem cell expansion. Exp Hematol. Oct 2005;33(10):1229-1239. Milhem M, Mahmud N, Lavelle D, et al. Modification of hematopoietic stem cell fate by 5aza 2'deoxycytidine and trichostatin A. Blood. Jun 1 2004;103(11):4102-4110. Araki H, Mahmud N, Milhem M, et al. Expansion of human umbilical cord blood SCID-repopulating cells using chromatin-modifying agents. Exp Hematol. Feb 2006;34(2):140-149. Araki H, Yoshinaga K, Boccuni P, Zhao Y, Hoffman R, Mahmud N. Chromatin-modifying agents permit human hematopoietic stem cells to undergo multiple cell divisions while retaining their repopulating potential. Blood. Apr 15 2007;109(8):3570-3578. De Felice L, Tatarelli C, Mascolo MG, et al. Histone deacetylase inhibitor valproic acid enhances the cytokine-induced expansion of human hematopoietic stem cells. Cancer Res. Feb 15 2005;65(4):1505-1513. Buske C, Feuring-Buske M, Abramovich C, et al. Deregulated expression of HOXB4 enhances the primitive growth activity of human hematopoietic cells. Blood. Aug 1 2002;100(3):862-868. Schiedlmeier B, Klump H, Will E, et al. High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation. Blood. Mar 1 2003;101(5):1759-1768. Zhang XB, Beard BC, Beebe K, Storer B, Humphries RK, Kiem HP. Differential effects of HOXB4 on nonhuman primate short- and long-term repopulating cells. PLoS Med. May 2006;3(5):e173. Zhang XB, Schwartz JL, Humphries RK, Kiem HP. Effects of HOXB4 overexpression on ex vivo expansion and immortalization of hematopoietic cells from different species. Stem Cells. Aug 2007;25(8):2074-2081.  144  320. 321.  322. 323. 324.  325. 326. 327. 328. 329. 330. 331.  332. 333. 334.  Boitano AE, Wang J, Romeo R, et al. Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells. Science. Sep 10 2010;329(5997):1345-1348. Weissman IL, Shizuru JA. The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases. Blood. Nov 1 2008;112(9):3543-3553. Rizo A, Vellenga E, de Haan G, Schuringa JJ. Signaling pathways in selfrenewing hematopoietic and leukemic stem cells: do all stem cells need a niche? Hum Mol Genet. Oct 15 2006;15 Spec No 2:R210-219. Varnum-Finney B, Purton LE, Yu M, et al. The Notch ligand, Jagged-1, influences the development of primitive hematopoietic precursor cells. Blood. Jun 1 1998;91(11):4084-4091. Stier S, Cheng T, Dombkowski D, Carlesso N, Scadden DT. Notch1 activation increases hematopoietic stem cell self-renewal in vivo and favors lymphoid over myeloid lineage outcome. Blood. Apr 1 2002;99(7):2369-2378. Karlsson G, Blank U, Moody JL, et al. Smad4 is critical for self-renewal of hematopoietic stem cells. J Exp Med. Mar 19 2007;204(3):467-474. Walkley CR, Fero ML, Chien WM, Purton LE, McArthur GA. Negative cellcycle regulators cooperatively control self-renewal and differentiation of haematopoietic stem cells. Nat Cell Biol. Feb 2005;7(2):172-178. Bowie MB, Kent DG, Copley MR, Eaves CJ. Steel factor responsiveness regulates the high self-renewal phenotype of fetal hematopoietic stem cells. Blood. Jun 1 2007;109(11):5043-5048. Lecault V, Vaninsberghe M, Sekulovic S, et al. High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays. Nat Methods. 2011;8(7):581-586. Randall TD, Weissman IL. Phenotypic and functional changes induced at the clonal level in hematopoietic stem cells after 5-fluorouracil treatment. Blood. May 15 1997;89(10):3596-3606. Fleming WH, Alpern EJ, Uchida N, Ikuta K, Spangrude GJ, Weissman IL. Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells. J Cell Biol. Aug 1993;122(4):897-902. Orschell-Traycoff CM, Hiatt K, Dagher RN, Rice S, Yoder MC, Srour EF. Homing and engraftment potential of Sca-1(+)lin(-) cells fractionated on the basis of adhesion molecule expression and position in cell cycle. Blood. Aug 15 2000;96(4):1380-1387. Morrison SJ, Wandycz AM, Akashi K, Globerson A, Weissman IL. The aging of hematopoietic stem cells. Nat Med. Sep 1996;2(9):1011-1016. Rossi DJ, Bryder D, Zahn JM, et al. Cell intrinsic alterations underlie hematopoietic stem cell aging. Proc Natl Acad Sci U S A. Jun 28 2005;102(26):9194-9199. Krause DS, Theise ND, Collector MI, et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell. May 4 2001;105(3):369-377.  145  335. 336. 337. 338. 339. 340. 341. 342.  343.  344. 345. 346. 347.  348. 349. 350.  Verma IM, Weitzman MD. Gene therapy: twenty-first century medicine. Annu Rev Biochem. 2005;74:711-738. Hills M, Lucke K, Chavez EA, Eaves CJ, Lansdorp PM. Probing the mitotic history and developmental stage of hematopoietic cells using single telomere length analysis (STELA). Blood. Jun 4 2009;113(23):5765-5775. Fogarty PF, Yamaguchi H, Wiestner A, et al. Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA. Lancet. Nov 15 2003;362(9396):1628-1630. Calado RT, Young NS. Telomere diseases. N Engl J Med. Dec 10 2009;361(24):2353-2365. Walne AJ, Dokal I. Advances in the understanding of dyskeratosis congenita. Br J Haematol. Apr 2009;145(2):164-172. Baerlocher GM, Vulto I, de Jong G, Lansdorp PM. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc. 2006;1(5):2365-2376. Erdmann N, Liu Y, Harrington L. Distinct dosage requirements for the maintenance of long and short telomeres in mTert heterozygous mice. Proc Natl Acad Sci U S A. Apr 20 2004;101(16):6080-6085. Banuelos CA, Banath JP, MacPhail SH, et al. Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks. DNA Repair (Amst). Sep 1 2008;7(9):14711483. Bodine DM, Karlsson S, Nienhuis AW. Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells. Proc Natl Acad Sci U S A. Nov 1989;86(22):8897-8901. Henson JD NA, Yeager TR, Reddel RR. Alternative lengthening of telomeres in mammalian cells. Oncogene. 2002;21:598-610. Zalzman M, Falco G, Sharova LV, et al. Zscan4 regulates telomere elongation and genomic stability in ES cells. Nature. Apr 8 2010;464(7290):858-863. Bryan TM EA, Gupta J, Bacchetti S, Reddel RR. Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J. 1995;14:4240-4248. Niida H SY, Hande MP, Matsumoto T, Takehara S, Tachibana M, Oshimura M, Lansdorp PM, Furuichi Y. Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA. Molecular Cell Biology. 2000;20:4115-4127. Muntoni A, Reddel RR. The first molecular details of ALT in human tumor cells. Hum Mol Genet. Oct 15 2005;14 Spec No. 2:R191-196. Marion RM, Strati K, Li H, et al. Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell. Feb 6 2009;4(2):141-154. Poon SS, Lansdorp PM. Quantitative fluorescence in situ hybridization (QFISH). Curr Protoc Cell Biol. Nov 2001;Chapter 18:Unit 18 14.  146  351. 352. 353.  354. 355. 356. 357. 358. 359. 360. 361.  362. 363.  364. 365.  Stewart SA, Hahn WC, O'Connor BF, et al. Telomerase contributes to tumorigenesis by a telomere length-independent mechanism. Proc Natl Acad Sci U S A. Oct 1 2002;99(20):12606-12611. Smith LL, Coller HA, Roberts JM. Telomerase modulates expression of growth-controlling genes and enhances cell proliferation. Nat Cell Biol. May 2003;5(5):474-479. Armstrong L, Saretzki G, Peters H, et al. Overexpression of telomerase confers growth advantage, stress resistance, and enhanced differentiation of ESCs toward the hematopoietic lineage. Stem Cells. Apr 2005;23(4):516-529. Masutomi K, Possemato R, Wong JM, et al. The telomerase reverse transcriptase regulates chromatin state and DNA damage responses. Proc Natl Acad Sci U S A. Jun 7 2005;102(23):8222-8227. Lee J, Sung YH, Cheong C, et al. TERT promotes cellular and organismal survival independently of telomerase activity. Oncogene. Jun 12 2008;27(26):3754-3760. Lansdorp PM. Telomeres and disease. EMBO J. Sep 2 2009;28(17):25322540. Li CL, Johnson GR. Rhodamine123 reveals heterogeneity within murine Lin-, Sca-1+ hemopoietic stem cells. J Exp Med. Jun 1 1992;175(6):14431447. Trevisan M, Yan XQ, Iscove NN. Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo. Blood. Dec 1 1996;88(11):4149-4158. Wu M, Kwon HY, Rattis F, et al. Imaging hematopoietic precursor division in real time. Cell Stem Cell. Nov 2007;1(5):541-554. Fortunel N, Hatzfeld J, Aoustin L, et al. Specific dose-response effects of TGF-beta1 on developmentally distinct hematopoietic stem/progenitor cells from human umbilical cord blood. Hematol J. 2000;1(2):126-135. Bryder D, Ramsfjell V, Dybedal I, et al. Self-renewal of multipotent longterm repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation. J Exp Med. Oct 1 2001;194(7):941-952. Stier S, Ko Y, Forkert R, et al. Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size. J Exp Med. Jun 6 2005;201(11):1781-1791. Borge OJ, Ramsfjell V, Veiby OP, Murphy MJ, Jr., Lok S, Jacobsen SE. Thrombopoietin, but not erythropoietin promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro. Blood. Oct 15 1996;88(8):2859-2870. Agarwal S, Loh YH, McLoughlin EM, et al. Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients. Nature. Mar 11 2010;464(7286):292-296. Sudo K, Ema H, Morita Y, Nakauchi H. Age-associated characteristics of murine hematopoietic stem cells. J Exp Med. Nov 6 2000;192(9):12731280.  147  366.  367. 368. 369. 370. 371.  372. 373. 374. 375. 376. 377. 378. 379.  380.  Beerman I, Bhattacharya D, Zandi S, et al. Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. Proc Natl Acad Sci U S A. Mar 23 2010;107(12):5465-5470. Ivanova NB, Dimos JT, Schaniel C, Hackney JA, Moore KA, Lemischka IR. A stem cell molecular signature. Science. Oct 18 2002;298(5593):601604. Phillips RL, Ernst RE, Brunk B, et al. The genetic program of hematopoietic stem cells. Science. Jun 2 2000;288(5471):1635-1640. Ramalho-Santos M, Yoon S, Matsuzaki Y, Mulligan RC, Melton DA. "Stemness": transcriptional profiling of embryonic and adult stem cells. Science. Oct 18 2002;298(5593):597-600. Venezia TA, Merchant AA, Ramos CA, et al. Molecular signatures of proliferation and quiescence in hematopoietic stem cells. PLoS Biol. Oct 2004;2(10):e301. Iscove NN, Barbara M, Gu M, Gibson M, Modi C, Winegarden N. Representation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA. Nat Biotechnol. Sep 2002;20(9):940-943. Miranda-Saavedra D, De S, Trotter MW, Teichmann SA, Gottgens B. BloodExpress: a database of gene expression in mouse haematopoiesis. Nucleic Acids Res. Jan 2009;37(Database issue):D873-879. Akashi K. Lineage promiscuity and plasticity in hematopoietic development. Ann N Y Acad Sci. Jun 2005;1044:125-131. Cross MA, Enver T. The lineage commitment of haemopoietic progenitor cells. Curr Opin Genet Dev. Oct 1997;7(5):609-613. Bonifer C. Epigenetic plasticity of hematopoietic cells. Cell Cycle. Feb 2005;4(2):211-214. Bonifer C, Hoogenkamp M, Krysinska H, Tagoh H. How transcription factors program chromatin--lessons from studies of the regulation of myeloid-specific genes. Semin Immunol. Aug 2008;20(4):257-263. Bug G, Gul H, Schwarz K, et al. Valproic acid stimulates proliferation and self-renewal of hematopoietic stem cells. Cancer Res. Apr 1 2005;65(7):2537-2541. Young JC, Wu S, Hansteen G, et al. Inhibitors of histone deacetylases promote hematopoietic stem cell self-renewal. Cytotherapy. 2004;6(4):328-336. Pineault N, Abramovich C, Humphries RK. Transplantable cell lines generated with NUP98-Hox fusion genes undergo leukemic progression by Meis1 independent of its binding to DNA. Leukemia. Apr 2005;19(4):636-643. Hirose K, Abramovich C, Argiropoulos B, Humphries RK. Leukemogenic properties of NUP98-PMX1 are linked to NUP98 and homeodomain sequence functions but not to binding properties of PMX1 to serum response factor. Oncogene. Oct 9 2008;27(46):6056-6067.  148  381. 382. 383.  384. 385. 386. 387.  388. 389.  Yassin ER, Sarma NJ, Abdul-Nabi AM, et al. Dissection of the transformation of primary human hematopoietic cells by the oncogene NUP98-HOXA9. PLoS One. 2009;4(8):e6719. Moore MA, Chung KY, Plasilova M, et al. NUP98 dysregulation in myeloid leukemogenesis. Ann N Y Acad Sci. Jun 2007;1106:114-142. Wang X, Babu JR, Harden JM, et al. The mitotic checkpoint protein hBUB3 and the mRNA export factor hRAE1 interact with GLE2p-binding sequence (GLEBS)-containing proteins. J Biol Chem. Jul 13 2001;276(28):26559-26567. Blevins MB, Smith AM, Phillips EM, Powers MA. Complex formation among the RNA export proteins Nup98, Rae1/Gle2, and TAP. J Biol Chem. Jun 6 2003;278(23):20979-20988. Baker DJ, Dawlaty MM, Galardy P, van Deursen JM. Mitotic regulation of the anaphase-promoting complex. Cell Mol Life Sci. Mar 2007;64(5):589600. Bai XT, Gu BW, Yin T, et al. Trans-repressive effect of NUP98-PMX1 on PMX1-regulated c-FOS gene through recruitment of histone deacetylase 1 by FG repeats. Cancer Res. May 1 2006;66(9):4584-4590. Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM, Sauvageau G. Hoxa9 transforms primary bone marrow cells through specific collaboration with Meis1a but not Pbx1b. EMBO J. Jul 1 1998;17(13):3714-3725. Kyba M, Perlingeiro RC, Daley GQ. HoxB4 confers definitive lymphoidmyeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell. Apr 5 2002;109(1):29-37. Nagahara H, Vocero-Akbani AM, Snyder EL, et al. Transduction of fulllength TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat Med. Dec 1998;4(12):1449-1452.  149  APPENDICES Appendix A: Effect of NUP98-HOXA10hd on individual HSC clones of CD45+linRho-SP phenotype. BA; before analysis, NA; not available.  (+) recipients / total # of M transplanted  LD reached (yes/no)  Estimated (min.) HSC expansion  # of HSCs generated in vitro  1/1 - 2/2 - 2/2  no  >351.072  1/1 - 2/2 - 2/2  no  >351.098  1/1 - 2/2 - 2/2  no  >351.096  0/1 - 0/2 - 0/2  no  not detected  1/1 - 2/2 - 1/1  no  >152.224  1/1 - 2/2 - died BA  no  >34.884  1/1 - 2/2 - 2/2  no  >351.074  1/1 - 2/2 - 2/2  no  >351.1  1/1 - 2/2 - 2/2 - 2/2  no  >1098.608  1/1 - 2/2 - 2/2  no  >351.1  1/1 - 2/2 - 2/2 - 1/1  no  >804.72  1/50th/1M - 1/500th/2M  1/1 - 2/2  no  >348.9  1100000  1/50th/1M - 1/500th/2M  1/1 - 2/2  no  >348.92  #14  2200000  1/50th/1M - 1/500th/2M  1/1 - 2/2  no  >348.92  #15  100000  1/50th/1M - 1/500th/2M  1/1 - 2/2  no  >348.92  #16  1500000  1/100th/2M - 1/1000th/3M  2/2 -3/3  no  >1098.9  #17  1600000  1/100th/2M - 1/1000th/3M  2/2 -3/3  no  >1098.88  #18  1600000  2/2 -3/3  no  >1098.88  #19  2900000  1/1 -2/2 - 3/3  no  >2309.56  #20  3900000  1/1 -2/2 - 3/3  no  >2309.58  #21  2500000  1/1 -2/2 - 3/3  no  >2309.5  #22  2000000  1/1 -2/2 - 1/2  yes  ~1718.8  #23  400000  1/100th/2M - 1/1000th/3M 1/50th/1M - 1/500th/2M 1/2000th/3M 1/50th/1M - 1/500th/2M 1/2000th/3M 1/50th/1M - 1/500th/2M 1/2000th/3M 1/50th/1M - 1/500th/2M 1/2000th/2M 1/100th/2M - 1/1000th/3M 1/5000th/2M  0/2 - 0/3 - 0/2  no  not detected  #24  650000  (3/3)  no  >1098.63  #25  2100000  0/2 - 0/3 - 0/2  no  not detected  #26  800000  1/1 - 3/3 - 0/2  yes  ~2140.08  #27  500000  1/1 - 3/3 - 0/1  yes  ~2772.55  #28  2500000  1/2 - 0/2  yes  ~3465.75  NUP98HOXA10hdtransduced clones  Total nucleated cells  #1  1060000  #2  220000  #3  60000  #4  40000  #5  40000  #6  380000  #7  580000  #8  100000  #9  2380000  #10  50000  #11  20000  1/5th/1M - 1/50th/2M 1/500th/2M 1/5th/1M - 1/50th/2M 1/500th/2M 1/5th/1M - 1/50th/2M 1/500th/2M 1/5th/1M - 1/50th/2M 1/500th/2M 1/5th/1M - 1/50th/2M 1/500th/1M 1/5th/1M - 1/50th/2M 1/500th/2M 1/5th/1M - 1/50th/2M 1/500th/2M 1/5th/1M - 1/50th/2M 1/500th/2M 1/5th/1M - 1/50th/2M 1/500th/2M - 1/1000th/2M 1/5th/1M - 1/50th/2M 1/500th/2M 1/5th/1M - 1/50th/2M 1/500th/2M - 1/1000th/1M  #12  750000  #13  Fraction transplanted / each mouse (M)  1/1000th/3M 1/100th/2M - 1/1000th/3M 1/5000th/2M 1/100th/1M - 1/1000th/3M 1/5000th/2M 1/100th/1M - 1/1000th/3M 1/5000th/1M 1/5000th/2M 1/10000th/2M  Range defined by ± 1 S.E. 132.712 ± 928.772 132.726 ± 928.818 132.72 ± 928.818 NA 60.804 ± 381.104 12.92 ± 94.354 132.704 ± 928.812 132.72 ± 928.82 577.15 ± 2091.068 132.715 ± 928.82 383.392 ± 1689.062 129 ± 943.65 129.03± 943.58 128.92 ± 943.58 129.02 ± 943.63 523.05 ± 2308.5 523.04 ± 2308.48 523.04 ± 2308.48 1188.42 ± 4487.75 1188.33 ± 4487.73 1188.5 ± 4487.75 827.2 ± 3571.6 NA 522.47 ± 2310.035 NA 1139.52 ± 4019.04 1355.4 ± 5671.5 1249.5 ± 9612.5  150  Appendix B: Average peripheral blood lineage contribution to donor-derived compartment of representative recipients shown in figure 2.1C (top panel). NA; not available. NUP98HOXA10hdtransduced clones  % myeloid cells ± SD  % B cells ± SD  % T cells ± SD  (% Ly6G/Mac1)  (% B220)  (% CD4/CD8)  15 ± 7  64 ± 22  21 ± 15  #2  8±1  71 ± 4  21 ± 3  #3  15 ± 10  33 ± 1  52 ± 11  #4  NA  NA  NA  #5  11  47  42  #6  24 ± 17  51 ± 21  25 ± 4  #7  12 ± 2  54 ± 4  34 ± 6  #8  15 ± 2  39 ± 2  46 ± 4  #9  34 ± 6  24 ± 8  42 ± 6  #10  19 ± 5  42 ± 4  39 ± 7  #11  17 ± 7  19 ± 20  64 ± 27  #12  24 ± 18  18 ± 4  58 ± 22  #13  9±1  38 ± 16  53 ± 17  #14  17 ± 2  57 ± 3  26 ± 1  #15  30 ± 6  40 ± 8  30 ± 2  #16  20 ± 3  20 ± 18  60 ± 20  #17  22 ± 2  20 ± 5  58 ± 7  #18  14 ± 3  51 ± 9  35 ± 9  #19  12 ± 3  63 ± 13  25 ± 10  #20  15 ± 5  62 ± 6  22 ± 2  #21  18 ± 5  63 ± 19  19 ± 7  #22  34  19  47  #23  NA  NA  NA  #24  34 ± 4  36 ± 5  30 ± 7  #25  NA  NA  NA  #26  33 ± 6  22 ± 4  45 ± 10  #27  13 ± 11  11 ± 7  76 ± 18  #28  23  17  60  #1  151  Appendix C: Effect of NUP98-HOXA10hd on individual HSC clones of E-SLAM phenotype. NA; not available.  NUP98HOXA10hdtransduced clones  Total nucleated cells  7  2500000  #2  1250000  #3  2500000  #4  2000000  #5  1500000  #6  1250000  #7  1600000  #8  900000  #9  1650000  #10  1400000  #11  1900000  #12  700000  #13  800000  #14  900000  #15  500000  #16  1500000  1/100th/2M 1/1000th/3M 1/100th/2M 1/1000th/3M 1/100th/2M 1/1000th/3M 1/100th/2M 1/1000th/3M 1/100th/2M 1/1000th/3M 1/100th/2M 1/1000th/3M 1/100th/1M 1/1000th/3M 1/100th/1M 1/1000th/3M 1/100th/1M 1/1000th/3M 1/100th/1M 1/1000th/2M 1/100th/1M 1/1000th/3M 1/50th/1M 1/500th/2M 1/50th/1M 1/500th/2M 1/50th/1M 1/500th/2M 1/100th/1M 1/1000th/2M 1/100th/1M 1/1000th/2M  #17  600000  1/1000th/2M  Fraction transplanted / each mouse (M)  (+) recipients / total # of M transplanted  LD reached (yes/no)  Estimated (min.) HSC expansion  # of HSCs generated in vitro  2/2 -3/3  no  >1098.75  2/2 -3/3  no  >1098.875  2/2 -3/3  no  >1098.75  2/2 -3/3  no  >1098.6  2/2 -3/3  no  >1098.9  2/2 -3/3  no  >1098.625  1/1 -3/3  no  >1098.72  1/1 -3/3  no  >1098.72  1/1 -3/3  no  >1098.735  1/1 -2/2  no  >697.9  1/1 -3/3  no  >1098.77  522.75 ± 2309.25 523 ± 2308.5 523 ± 2308.5 522.4 ± 2310 523.05 ± 2308.5 522.5 ± 2310 522.72 ± 2309.28 522.72 ± 2309.22 522.72 ± 2309.175 258.02 ± 1887.2 522.69 ± 2309.26  0/1 - 0/2  no  not detected  NA  0/1 - 0/2  no  not detected  1/1 - 2/2  no  >348.93  1/1 - 2/2  no  >697.85  1/1 - 2/2  no  >697.8  (2/2)  no  >693.12  NA 128.7 ± 943.65 258.05 ± 1887.25 258 ± 1887.3 249.9 ± 1922.52  Range defined by ± 1 S.E.  152  Appendix D: Average peripheral blood lineage contribution to donor-derived compartment of representative recipients shown in figure 2.1C (bottom panel). NA; not available. NUP98HOXA10hdtransduced clones  % myeloid cells ± SD  % B cells ± SD  % T cells ± SD  (% Ly6G/Mac1)  (% B220)  (% CD4/CD8)  #1  9±5  17 ± 6  74 ± 11  #2  14 ± 4  19 ± 4  67 ± 6  #3  19 ± 11  30 ± 5  51 ± 6  #4  8±2  29 ± 10  63 ± 10  #5  11 ± 4  8±5  81 ± 9  #6  7±3  18 ± 7  75 ± 8  #7  24 ± 7  16 ± 2  60 ± 6  #8  14 ± 2  17 ± 4  69 ± 5  #9  25 ± 2  23 ± 6  52 ± 6  #10  34 ± 1  13 ± 1  53 ± 1  #11  NA  NA  NA  #12  NA  NA  NA  #13  NA  NA  NA  #14  28 ± 7  33 ± 12  39 ± 6  #15  17 ± 3  22 ± 3  61 ± 1  #16  29 ± 10  28 ± 1  43 ± 10  #17  19 ± 3  23 ± 5  58 ± 3  153  Appendix E: Design matrix for the two-level factorial experiment. IL-3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20  IL-6 -1 -1 0 1 0 0 1 1 -1 1 -1 0 -1 1 -1 1 -1 -1 1 1  SCF -1 -1 0 -1 0 0 1 -1 1 1 1 0 -1 -1 -1 1 1 1 -1 1  TPO -1 1 0 -1 0 0 -1 1 -1 1 1 0 1 1 -1 1 -1 1 -1 -1  -1 1 0 1 0 0 -1 -1 1 1 -1 0 -1 1 1 -1 -1 1 -1 1  The concentrations for each of the 4 factors were as follows: -1 = low value; 0 = mid value (10-fold lower concentrations than high value); 1 = high value. For IL-3 -1 = 0 ng/ml, 0 = 0.6 ng/ml, 1 = 6 ng/ml; for IL-6 -1 = 0 ng/ml, 0 = 1 ng/ml, 1 = 10 ng/ml, for SF -1 = 0 ng/ml, 0 = 10 ng/ml, 1 = 100 ng/ml; for TPO -1 = 0 ng/ml, 0 = 10 ng/ml, 1 = 100 ng/ml.  154  Appendix F: NUP98-HOXA10hd effect on telomere length of wild-type and Tert-/bone marrow cells during in vitro culture. Presented are telomere lengths of fresh (Day 0) wild-type and Tert-/- bm cells (granulocytes), or following GFP and NA10hd infection and additional 6- (Day 10) or 20-day (Day 24) culture.  155  

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