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Effect of c-kit and flt-3 overexpression on primitive hematopoietic cells Chu, Pak-Yan Pat 2003

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EFFECT OF C-KIT AND  FLT-3 OVEREXPRESSION ON  PRIMITIVE HEMATOPOIETIC CELLS  by Pak-Yan Pat Chu B.Sc.(Honours Biological Science), University of Windsor, 1996  A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Medical Genetics, Faculty of Medicine  We accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A July 2003 © Pak-Yan Pat Chu, 2003  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head, of my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d without my w r i t t e n p e r m i s s i o n .  Department of  AW/crt,/  GieneflcS  The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada  ABSTRACT Recent studies have demonstrated that hematopoietic stem cell (HSC) self-renewal divisions in vitro are favoured by exposure of the cells to elevated levels of Steel factor (SF) or flt-3 ligand (FL). This suggested that enhanced HSC self-renewal by these cytokines might be limited by the level of expression of the corresponding c-kit or flt-3 receptors on HSCs. In this thesis, I investigated this hypothesis by examining how retroviral-mediated overexpression of either c-kit or flt-3 affects the responsiveness of hematopoietic cells to SF and FL, respectively, first in a cell line model and then in primary mouse bone marrow cells,  c-kit and flt3-  transduced BaF3 cells were able to proliferate in lower concentrations of SF and F L , respectively, than control-transduced cells, although evidence of high dose inhibition was also noted. In primitive mouse bone marrow cells transduced with the same c-kit ory7f-3-encoding vectors, a similar effect on short term total cell expansion in vitro was seen. This included a cytokine-specific dose sensitization of progenitor and HSC expansion without detectable effects on their subsequent commitment or differentiation. Depressed responsiveness to very high cytokine concentrations was also seen in primary transduced cells such that no significant further net amplification of the transduced HSCs could be achieved in vitro.  In vivo  competitive reconstitution assays were performed to determine whether the enhanced cytokine sensitivity of receptor-overexpressing HSCs would give them a growth advantage in vivo. Although continued expression of functional receptors on the in vivo generated progeny of the transduced HSCs could be clearly documented and their self-renewal shown to be intact, their behaviour in vivo could not be distinguished from control-transduced cells, even when the corresponding ligand was administered exogenously or induced endogenously. It thus appears that neither c-kit nor flt-3 receptor levels on HSCs are limiting to their capacity to self-amplify in vivo. These results suggest that other molecular interactions of HSCs with their environment may be more potent regulators of HSC self-renewal responses in vivo and need to be identified to improve HSC amplification in vitro.  ii  TABLE OF CONTENTS ABSTRACT  ii  LIST OF TABLES  vi  LIST OF FIGURES  ;  .........vii  LIST OF ABBREVIATIONS  ix  ACKNOWLEDGMENTS Chapter 1: 1.1.  xii  Introduction  1  Proj ect overview  1  1.2. Hematopoiesis 1.2.1. Hierarchical model of hematopoiesis Pluripotent HSCs Progenitor cells Terminal maturation of blood cells 1.3. Characterization of HSCs 1.3.1. Definition of HSCs 1.3.2. Quantitation of HSCs 1.3.3. Phenotyping and purification of HSCs 1.4. Properties of HSCs 1.4.1. Self-renewal potential of HSCs 1.4.2. Commitment of HSCs 1.4.3. Cycling status of HSCs in vivo 1.4.4. Homing and mobilization of HSCs 1.5. Regulation of HSCs 1.5.1. Intrinsic factors / transcription factors 1.5.2. Environmental factors / cytokines 1.6. Effect of environmental factors on HSC self-renewal 1.6.1. Effect of growth-promoting cytokines Instructive vs selective actions of cytokines Kinetics of HSC stimulation 1.6.2. Effect of growth-inhibiting cytokines 1.6.3. Effect of morphogens 1.7. Sub-class III tyrosine kinase receptors and their corresponding ligands 1.7.1. c-kitandSF Molecular structure Expression of c-kit in primitive hematopoietic cells 1.7.2. Flt-3 and F L Molecular structure Expression of flt-3 in primitive hematopoietic cells 1.7.3. Roles of c-kit and flt-3 in primitive hematopoietic cell In vitro studies In vivo studies 1.7.4. Involvement of c-kit and flt-3 in hematopoietic diseases 1.8. Genetic manipulation of hematopoietic cells using recombinant retroviruses iii  1 2 5 6 6 7 7 7 9 11 11 12 12 13 14 14 16 18 18 19 21 21 22 23 25 25 26 27 27 28 29 29 33 34 34  1.8.1. Retrovirus biology 1.8.2. Production of recombinant retroviruses 1.8.3. Recombinant retroviral vectors 1.8.4. Murine HSCs as target for retroviral-mediated gene transfer 1.9. Present studies and thesis objective.  Chapter 2:  Materials and methods  40  2.1. Reagents 2.1.1. Cell lines 2.1.2. Cytokines 2.1.3. Mice 2.1.4. Isolation of mouse B M cells 2.2. Retoviral vectors construction and molecular analysis 2.2.1. Vector construction 2.2.2. Vector sequence validation 2.3. Transduction protocols 2.3.1. Production of retroviral supernatant 2.3.2. Transduction protocols for cell lines 2.3.3. Transduction protocol for murine B M cells 2.3.4. Assessment of gene transfer efficiency in bulk cell populations 2.3.5. Viral titer and helper virus assays 2.4. In vitro assays 2.4.1. BaF3 cell proliferation assays 2.4.2. Primary murine B M hematopoietic cell culture 2.5. Progenitor and stem cell assays 2.5.1. C F C assays 2.5.2. C R U assay for HSCs Transplantation procedure Assessment of HSCs in engrafted mice Secondary B M transplantations 2.6. Flow cytometry and cell sorting 2.7. Statistical methods  Chapter 3:  3.1. Introduction 3.2. Results 3.2.1. Construction and validation of c-kit and flt-3 retroviral vectors 3.2.2. Generation of polyclonal and derivative monoclonal BaF3cell populations expressing different levels of c-kit and flt-3 3.2.3. Altered mitogenic responses of M-KIT and M-FLT-transduced BaF3 cells 3.3. Discussion  49 49 50 50 52 57 70  Effect of overexpressing c-kit or flt-3 on the in vitro self-renewal and  differentiation responses of primitive hematopoietic cells 4.1. 4.2.  40 40 40 41 41 41 41 42 43 43 43 44 44 45 45 45 45 46 46 46 46 46 47 47 48  The effect of variable levels of c-kit and flt-3 receptor expression on  responsiveness to the cognate ligand in a cell line model  Chapter 4:  35 36 36 37 38  Introduction Results  iv  73  4.2.1. Optimization of a protocol for retroviral transduction of primitive murine B M cells 74 4.2.2. Effect of forced overexpression of c-kit or flt-3 in primary B M cells on total cell expansion in vitro in response to SF and F L stimulation 81 4.2.3. Effect of forced overexpression of c-kit or flt-3 in primary B M cells on progenitor cell expansion in vitro in response to SF and F L stimulation 87 4.2.4. Effect of forced overexpression of c-kit or flt-3 in primary B M cells on the expansion of HSCs in vitro in response to SF or F L 89 4.3. Discussion 100  Chapter 5:  The role of c-kit and flt-3 in HSC expansion in vivo  5.1. Introduction 5.2. Results 5.2.1. In vivo competitive reconstitution experiments 5.2.2. Effect of in vivo injection of stimulatory factors 5.3. Discussion  Chapter 6:  General discussion  103 103 105 105 115 119  121  REFERENCES  125  v  LIST OF TABLES Table 4.1.  Titers of different c-kit retroviruses and the relative level of expression of c-kit in transduced BaF3 cells 76  Table 4.2.  Improved retrovirus production and transduction of primitive murine B M cells...... 79  Table 4.3.  Calculated repopulating activity of HSCs (CRUs) present in retrovirally transduced populations 90  Table 4.4.  Data used to calculate the expansion in vitro of M-KIT-transduced HSCs cultured with different concentrations of SF 97  Table 4.5.  Data used to calculate the expansion in vitro of M-FLT-IG-transduced HSCs cultured with different concentrations of F L  98  Table 5.1.  Calculated net expansion of transduced HSCs in primary mice  112  Table 5.2.  Level of engraftment in recipients of mixtures of receptor and MIG-transduced cells before and after injection of SF or LPS  116  vi  LIST OF FIGURES Figure 1.1.  The current model of the hematopoietic hierarchy  4  Figure 1.2.  The general molecular structure of the subclass III receptor T K family and their corresponding ligands 24  Figure 1.3.  Clone formation and progenitor expansion from single cell cultures of CD34 CD38" adult human B M cells  32  Figure 3.1.  Retroviral constructs  51  Figure 3.2.  FACS analysis and sorting of transduced BaF3 cells  53  Figure 3.3.  Fluorescence profiles of various polyclonal populations of transduced BaF3 cells  54  Mean and range of fluorescence intensity of individual clones of transduced BaF3 cells  56  +  Figure 3.4.  Figure 3.5.  Mitogenie responses of M-KIT and M-FLT-transduced BaF3 populations to IL3 58  Figure 3.6.  Mitogenic responses of M-KIT and M-FLT-transduced BaF3 populations to SF andFL 61  Figure 3.7.  Variation in the mitogenic responses of BaF3 cells to SF and F L according to their relative levels of expression of c-kit or flt-3  65  Figure 4.1.  Structures of the retroviral constructs  75  Figure 4.2.  Protocol for producing high titer retroviral supernatants  78  Figure 4.3.  Detection of transduced receptors on murine B M cells  80  Figure 4.4.  Effect of overexpression of c-kit and flt3 on total cell expansion in vitro  82  Figure 4.5.  Ligand-specific cell-autonomous expansion of receptor-transduced cells  86  Figure 4.6.  Effect of overexpression of c-kit and flt3 on C F C production in vitro  88  Figure 4.7.  Gene transfer efficiencies to HSCs  92  Figure 4.8.  Comparison of the lineage distribution of WBCs produced by receptor and control-transduced HSCs after their transplantation into mice  94  Figure 4.9.  Method used to calculate the extent of HSC expansion achieved in vitro  96  Figure 4.10.  Effect of overexpression of c-kit and flt3 on the maintenance of HSCs in vitro ..99  vii  Figure 5.1.  In vivo competitive reconstitution experimental design  Figure 5.2.  FACS analysis of WBCs from recipients of mixed populations of control and receptor-transduced B M cells 107  Figure 5.3.  Time course study of the competitive reconstituting activity of receptortransduced HSCs in primary recipients  109  Competitive HSC-reconstituting activity of receptor-transduced HSCs in primary recipients assessed by transplantation into secondary recipients  Ill  Figure 5.4.  106  Figure 5.5.  Cytokine sensitivities of B M cells from mice engrafted with receptor-transduced HSCs 114  Figure 5.6.  Structure, expression and function of a retroviral vector encoding a c-fms-flt3 chimeric receptor 117  viii  LIST OF ABBREVIATIONS 5-FU AGM  5-fluorouracil Aorta-gonad-mesonephros  ALL  Acute lymphoblastic leukemia  AML  Acute myelogenous leukemia  APC  Allophycocyanin  BFU-E  Burst-forming unit erythroid  BIT  Bovine serum albumin, insulin, transferrin,  BM  Bone marrow  BMP  Bone morphogenetic proteins  BrdU  Bromodeoxyuridine  CD  Cluster differentiation  CFC  Colony-forming cell  CFC-E  Colony-forming cell - erythroid  CFC-Eo  Colony-forming cell - eosinophil  CFC-G  Colony-forming cell - granulocyte  CFU-GM  Colony-forming cell - granulocyte macrophage  CFC-M  Colony-forming cell - macrophage  CFC-Ma  Colony-forming cell - mast cell  CFC-Mk  Colony-forming cell - megakaryocyte  CFU-S  Colony-forming unit-spleen  CML  Chronic myelogenous leukemia  CRU  Competitive repopulation unit  CSF  Colony stimulating factors  CXCR4  Chemokine CXC receptor 4  Cy-5  Cyanine 5-succinimidylester  DMEM  Dulbecco's modified Eagle medium  ECM  Extracellular matrix  EGFR  Epidermal growth factor receptor  EPO  Erythropoietin ix  E C cells  Embryonic carcinoma cells  ES cells  Embryonic stem cells  EST  Expressed sequence tag  FACS  Fluorescent activated cell sorting  FCS  Fetal calf serum  FITC  Fluorescein isothiocyanate  FL  Flt-3 ligand  G-CSF  Granulocyte - colony stimulating factors  GFP  Green fluorescent protein  GM-CSF  Granulocyte/macrophage - colony stimulating factors  HF  Hank's Balanced Salt Solution plus 2% fetal calf seru  HSC  Hematopoietic stem cell  ICAM-1  Intercellular adhesion molecule-1  Ig  Immunoglobulin  IMDM  Iscove's modified Dulbecco's medium  IRES  Internal ribosomal entry site  IL  Interleukins  JAK  Janus Kinase  LIF  Leukemia inhibitory factor  Lin  Lineage  LIST  Ligand-receptor signaling-threshold  LPS  Lipopolysaccharide (bacterial)  LTC  Long-term culture  LTC-IC  Long-term culture-initiating cell  LTR  Long-term reconstituting or long terminal repeat  2-ME  2-mercaptoefhanol  MCP-1  Monocyte chemoattractant protein-1  M-CSF  Macrophage - colony stimulating factors  MFI  Mean fluorescence intensities  MlP-la  Macrophage inflammatory protein-1 alpha  MMuLV  Moloney murine leukemia virus  MSCV  Murine Stem Cell Virus X  NOD/SCID  Non-obese diabetic-severe combined immunodeficiency  PAS  Para-aortic splanchnopleure  PE  Phycoerythrin  PI  Propidium iodide  Rh-123  Rhodamine-123  RTK  Receptor tyrosine kinase  SI  Steel  SDF-1  Stroma-derived factor-1  SEM  Standard error of mean  SF  Steel factor  Shh  Sonic hedgehog  STAT  Signal transducer and activation of transcription  STR  Short-term reconstituting  TGF-P  Transforming growth factor-beta  TK  Tyrosine kinase  TNF  Tumor necrosis factor  TPO  Thrombopoietin  VLA-4  Very late antigen-4  W  White-spotting  WGA  Wheat germ agglutinin  Wnt  Wingless  xi  ACKNOWLEDGMENTS This thesis project is the most challenging task I have ever encountered. Throught out this project, I have gained, besides knowledge, a chance to explore the complexity of nature. It is truly one of the most valuable experiences.  I want to thank all the people in Terry Fox  Laboratory from whom I have gained a lot of help andfriendshipsover these years. There are several people whom I would like to especially thank here, because I think they have given me the most significant help: Dr. Connie Eaves, my supervisor, for giving me great help and support throughout this project. Her incredible energy, knowledge, enthusiasm and professionalism have a profound impact not just on this project but also on my personal development. My parents and grandparents, for their unconditional support.  Their support has  provided me with a rare opportunity of complete freedom and a safety net to pursue the path I have chosen. Gigi, for all the happy moments she has spent with me. Because of them, I feel very fortunate. Finally, I would also like to thank Roman M . Babicki for his generous financial support in the form of studentships for 2 years of my graduate program.  xii  CHAPTER 1: INTRODUCTION 1.1.  Project overview A stem cell is a cell that has the ability both to self-replicate and to give rise to  differentiated mature cells.  Hematopoietic stem cells (HSCs) are the cells ultimately  responsible for the life long production of all blood cell types. HSCs have been postulated to exist for more than 50 years, and their transplantation is routinely used to treat patients with cancers and other inherited disorders of the blood and immune systems. However, in spite of the intensive research in this field for the last several decades, many aspects of H S C biology remain unresolved. In particular, we still know very little about the molecular mechanisms that determine how HSCs maintain their long-term multilineage differentiation and extensive proliferative potential through many cell cycles. In this thesis, I have used a genetic approach to investigate whether the maintenance of these properties may be altered by changes in the levels of receptors that allow HSCs to respond to growth factors that can alter H S C selfrenewal in a concentration-dependent fashion. I believe that the theoretical model being tested in this thesis is important because further understanding of the mechanisms of H S C maintenance in vitro and in vivo is relevant to the future successful manipulation of HSCs for a variety of clinical applications.  1.2.  Hematopoiesis The hematopoietic system contains at least 10 different mature blood cell types, each  with a set of specialized properties crucial for life-supporting functions. These include oxygen transport, killing and removal of microorganisms, resolution of inflammatory responses and blood clotting. The mature blood cells are distributed throughout the body by a network of arteries, veins and lymphatics whereby the cellular and plasma components can be brought into contact with all of the other tissues. Most mature blood cells have a relatively short normal lifespan (ranging in humans from approximately 48 hours for neutrophils to 120 days for erythrocytes and in mice, from approximately 18 hours to 40 days for the same cell types). Most are also incapable of further division (Beulter et al. 1995). As a result, large numbers of blood cells are lost from the body each day (estimated as ~ 10 Abkowitz et al. 1995).  1  /day in humans) (Ogawa 1993;  The fact that the number of mature blood cells is maintained at relatively constant levels throughout adult life indicates the existence of a process whereby newly differentiated blood cells must be continuously produced from more primitive precursors.  This process,  which is referred to as hematopoiesis, is complex and dynamic, both in terms of the different types and numbers of blood cells produced and in the mechanisms that regulate their outputs in response to changing needs. Studies in mice have shown that the para-aortic splanchnopleure (PAS) and aortagonad-mesonephros (AGM) region are the earliest intra-embryonic sites of hematopoiesis (Godin et al. 1993; Medvinsky et al. 1993; Dzierzak et al. 1998; Nishikawa et al. 2001). It is believed that many HSCs first arise in the P A S / A G M region, from which they enter the embryonic circulation and then colonize the developing liver. However, some HSCs may also arise independently in the yolk sac blood islands where the first mature blood cells are formed (Dzierzak and Medvinsky 1995).  The liver then becomes the major site of hematopoiesis  throughout the rest of fetal life. After birth, hematopoiesis shifts to the bone marrow (BM).  1.2.1. Hierarchical model of hematopoiesis A major advance of understanding of hematopoiesis was prompted by early studies that demonstrated the rescue of lethally irradiated mice by the intravenous injection of histocompatible B M cells and the ability of a small subset of the injected B M cells to generate macroscopic colonies in the spleen of the irradiated recipients (Till and McCulloch 1961). Further analysis indicated that by 12 days post-transplant, these spleen colonies contain different lineages of hematopoietic cells, all of which are the progeny of a single rare cell that was called a colony-forming unit-spleen (CFU-S).  These studies also showed that spleen  colonies generated in this way contain daughter CFU-S, indicating that the original CFU-S were capable of self-generation (Till et al. 1964). These observations gave rise to the concept that CFU-S are HSCs.  Subsequent studies of the effect of 5-fluorouracil (5-FU) provided  evidence of pre-CFU-S cells (Hodgson and Bradley 1979).  Later, improved methods for  fractionating B M cells on the basis of a variety of different parameters showed that >90% of the CFU-S in normal adult mouse B M do not have long term repopulating ability (Hodgson and Bradley 1979; Spooncer et al. 1985; Ploemacher and Brons 1989; Ploemacher et al. 1993; Szilvassy and Cory 1993). Thus, while the CFU-S assay is useful for quantitating a primitive  2  multi-potent myeloid progenitor, it does not reliably or specifically measure the most primitive HSCs with long term repopulating activity (Jones et al. 1990). The use of semi-solid media for B M cell cultures allowed another major advance in the understanding of hematopoiesis (Bradley and Metcalf 1966; Ichikawa et al. 1966; Metcalf and Nicola 1984).  Such media allow the formation of individual colonies of specific or mixed  lineages of hematopoietic cells in vitro to be visualized.  These colonies were shown to  originate from single cells, and the initiating cells were termed (in vitro) colony-forming cells (CFCs).  CFCs are more numerous than CFU-S and their presence in spleen colonies  demonstrated that they were produced by CFU-S (Wu et al. 1967). Most of the colonies that develop from unseparated suspensions of hematopoietic cells consist of cells of only a single lineage, regardless of the mixture of cytokines/growth factors present in the culture medium. This suggests that the progenitors of these colonies are "committed" to a particular lineage differentiation pathway before being placed in the culture. Most CFCs also appear to lack selfrenewal capacity. They are therefore thought to represent a type of intermediate progenitor. More recently, strategies to separate progenitors with particular differentiation potentialities, based on differences in their physical and surface marker characteristics have also been developed (Ploemacher and Brons 1989; Jones et al. 1990; Wolf et al. 1993; Lemieux et al. 1995). Taken together, these observations support a model of hematopoiesis in which the hematopoietic differentiation process spans multiple steps leading to the generation of a hierarchy of progenitor subsets of generally increasing frequency. For simplicity, these are conventionally categorized as: stem cells, progenitor cells and terminally differentiating cells - the cells in each compartment representing the amplified progeny of cells from the preceding compartment (Zon 2001) (Figure 1.1a).  3  a)  b)  Dividing and terminally differentiating cells . . •  ....  Figure 1.1. The current model of the hematopoietic hierarchy (a) A simplified model of the compartments in the hematopoietic hierarchy, illustrating the increasing size of the compartments, unidirectional progress of differentiation (vertical white arrows) and the accompanying decrease of cellular proliferative potential (inverted triangle). Commitment occurs in the stem cells (black) with no transdifferentiation between lineages (compartments separated by vertical dashed lines), (b) Additional subpopulations in the hierarchy defined by different in vivo or in vitro functional assays. In the stem cell (black) compartment, competitive repopulation units (CRUs) define the stem cells, whereas the colony-forming unitspleen (CFU-S) and long-term culture-initiating cells (LTC-ICs) include/comprise mainly shortterm repopulating cells. The extent of overlap between cell populations is not well defined. CFC, colony-forming cell (Figure adapted from Zon 2 0 0 1 ) .  According to this model, the production of mature blood cells from HSCs is unidirectional and achieved through the processes of cell amplification and differentiation along the myeloid and lymphoid lineages. This model also assumes that the blood cells in the later (more mature) levels of the hierarchy have less proliferative and differentiation potential, and that no dedifferentiation or transdifferentiation occurs between lineages. More recently, many additional experimental studies have suggested further sub-populations within each compartment (see below) leading to a more complex hierarchical model (Figure 1.1b). However, the basic pattern and assumptions remain the same. Pluripotent HSCs The continued development of increasingly sophisticated cell separation technologies has allowed pluripotent HSC populations to be isolated in highly enriched form based on their unique constellations of physical and cell-surface properties (Spangrude et al. 1988; Morrison and Weissman 1994) (see also section 1.3.3).  Several experiments have shown that some  single HSCs can be sufficient to produce most of the blood cells generated in a mouse for many months (Jordan and Lemischka 1990; Lemischka 1992; Osawa et al. 1996a). However, other studies have also documented the functional heterogeneity of HSCs (Dick et al. 1985; Lemischka et al. 1986; Jordan and Lemischka 1990; Morrison and Weissman 1995; Goodell et al. 1996). Heterogeneity is revealed by the different repopulation abilities of phenotypically indistinguishable HSC-containing cell populations after their transplantation into irradiated mice  or by  the  demonstration  of  sustained  multi-lineage  repopulating abilities  by  phenotypically distinct cells (see also section 1.3.2). In general, adult mouse B M cells with multi-lineage repopulating ability have been found to comprise 2 separable populations: 1) long-term reconstituting (LTR) cells that can sustain hematopoiesis for more than 4 months in irradiated recipients, and 2) short-term reconstituting (STR) cells that can initially regenerate all of the blood elements but only for a few weeks (Jones et al. 1989; Jordan and Lemischka 1990; Keller and Snodgrass 1990; Harrison and Zhong 1992; Harrison et al. 1993; Zhong et al. 1996) (Figure 1.1b). Differences in the surface antigen profile of these 2 populations suggest that they may represent different stages of differentiation (Uchida et al. 1996; Zhao et al. 2000).  While the L T R cells are  assumed to be the more primitive, they may be unable to protect animals from lethal irradiation when injected in low numbers (Zhao et al. 2000). STR cells are thought to have less self5  renewal capacity, but may play an important role in the early recovery of mature blood cells in the immediate post-transplant period (Jordan and Lemischka 1990; Morrison and Weissman 1994; Uchida et al. 1996; Zhao et al. 2000). Recently, subsets of STR cells that are either myeloid or lymphoid-restricted have also been identified (Kondo et al. 1997; Akashi et al. 2000; Glimm et al. 2001). Progenitor cells Within the intermediate class of progenitor cells identified as CFCs, a clear substratification is evident, based on the developmental potentialities these cells display.  In  general, 3 types of colonies can be seen in short term cultures. These are thought to reflect 3 hierarchically ordered sub-classes of CFCs as follows: 1) immature "blast CFCs" that form colonies in which a high proportion of the cells at the end of 2 weeks are, themselves, CFCs and thus produce secondary colonies of multiple lineages when replated into secondary cultures (Keller and Phillips 1982; Nakahata and Ogawa 1982); 2) multi-lineage CFCs, such as CFC-GEMM  (CFC-granulocyte, erythrocyte, megakaryocyte and macrophage) that form  colonies containing mature cells of all of these lineages (Johnson and Metcalf 1977; Fauser and Messner 1979; Humphries et al. 1981) as well as some CFCs; and 3) more lineage-restricted CFCs that form colonies containing mature cells of one or 2 lineages. The latter include C F C E (CFU-E) (erythroid), CFC-Eo (eosinophil), C F C - G (granulocyte), C F C - M (macrophage), CFC-Mast (mast cell), CFC-Mk (megakaryocyte), etc. In addition, single lineage CFCs can be stratified according to their proliferative potential, the more primitive CFCs giving rise to larger colonies in which mature elements appear later. Terminal maturation of blood cells Morphologically recognizable blood cell precursors can be observed and followed for the last 3 to 5 consecutive cell cycles of their development. The changes that allow these cells to be distinguished and ordered in a sequence can be readily visualized by light microscopy of stained cell preparations. In summary, the features of the hematopoietic hierarchy are: 1) an initial loss of self-renewal potential; 2) a progressive distribution of restricted differentiation potentialities among increasingly larger populations of progenitor cells, and 3) an assumed irreversibility in the establishment of progenitor subpopulations (Zon 2001).  1.3.  Characterization of HSCs  1.3.1. Definition of HSCs HSCs are defined in this thesis as cells that are able to reconstitute all of the lymphoid and myeloid lineages for the lifetime of the individual (Lemischka et al. 1986). The existence of such cells was first demonstrated by studies that utilized radiation-induced chromosomal markers in primitive murine B M cells to track the common origin of lymphoid and myeloid cells regenerated in engrafted recipients (Wu et al. 1968; Abramson et al. 1977).  These  observations were subsequently confirmed using the unique integration site of retrovirallytransduced mouse B M or fetal liver cells to identify multi-lineage clones regenerated in vivo (Mintz et al. 1984; Dick et al. 1985; Keller et al. 1985; Lemischka et al. 1986; Capel and Mintz 1989; Jordan and Lemischka 1990; Keller and Snodgrass 1990).  Such experiments clearly  demonstrated that a small number of HSCs can be sufficient for the long-term restoration of normal multi-lineage hematopoiesis in a myeloablated mouse. In humans, the first evidence for HSCs came from the recognized involvement of multiple lineages in patients with myeloproliferative diseases (Dameshek 1951) that were subsequently shown to represent clonal disorders (Fialkow 1979). The existence of normal lympho-myeloid repopulating human HSCs was first formally demonstrated in 1989 by D N A analysis of recipients of allogeneic B M transplants (Turhan et al. 1989) and later demonstrated experimentally by limiting dilution xenografting experiments (Bhatia et al. 1997a; Conneally et al. 1997).  1.3.2. Quantitation of HSCs The detection and quantitation of HSCs relies on the use of functional assays that allow their rigorous discrimination from closely related primitive hematopoietic cells with limited reconstituting activity (e.g., short term repopulating cells) and/or more restricted differentiation potentialities (see also section The reason for this is that a unique set of molecular correlates of the functional attributes of HSCs are not yet known. Accordingly, HSCs cannot be identified or measured directly (see below). assays are used to quantify HSCs.  In the murine system, 2 types of functional  Both rely on the transplantation of the test cells into  recipients whose endogenous hematopoiesis has been greatly suppressed, either by irradiation and/or because the recipients carry a mutation that results in deficient HSC activity. This is done to optimize the sensitivity of the assay by maximally stimulating hematological recovery  and H S C activation. Both assays use a prolonged (>4 months) post-transplant end-point of engraftment by the test cells.  Demonstration of such prolonged engraftment is essential to  ensure specificity of the read-out for long term repopulating cell activity since the progeny of other types of more restricted transplantable cells can also be detected for periods of up to 4 months (Jordan and Lemischka 1990; Morrison and Weissman 1994; Zhong et al. 1996; Miller etal. 1997). One functional assay for HSCs is based solely on the principle of competitive repopulation. This method compares the long-term repopulating ability of 2 sources of cotransplanted hematopoietic  cells whose progeny can be distinguished by  genetically  determined D N A , phenotypic or biochemical markers. The first of these populations is termed the competitor population, and usually consists of a pre-determined number of normal adult mouse B M cells that, by themselves, will readily repopulate and radioprotect the recipients. Various numbers of B M cells from a second population, termed the test population, are simultaneously injected with high numbers of the competitor population (generally 10 normal 6  adult B M cells).  The relative contributions to hematopoiesis by both cell populations are  measured 4 months post-transplantation (or later).  The frequency of HSCs in the test  population is then estimated based on the assumed existence of an inverse relationship between the variance and the input HSC number as predicted by the binomial equation, assuming a similar output potential of the HSCs in the test and competitor populations (Harrison et al. 1993). The other functional assay measures the frequency of HSCs directly using a limiting dilution analysis procedure (Szilvassy et al. 1990). This assay also incorporates the principle of competitive repopulation in that all recipients must contain (or receive) a minimal number of HSCs sufficient to ensure their survival. Survival of all recipients is necessary to avoid data bias, since the frequency of HSCs in the test suspension is determined from a statistical analysis of the proportion of mice transplanted with various doses of test cells that fail to show long-term (>4 months) lympho-myeloid engraftment by the test cells (above a defined detection threshold). Survival of the recipients is achieved either by co-transplanting a small number of HSCs of the same genotype as the recipient, or by using a recipient with a mutation that results in deficient endogenous HSC activity, such as the C57BL/6-W /W ' 4!  4  (W41) mouse.  In the latter instance, a lower (sublethal) dose of irradiation can then be used (Miller and Eaves  8  1997). However, the number of competitor or endogenous HSCs present in the recipient needs to be kept to a minimum in order to maximize the sensitivity of the assay (Szilvassy et al. 1990; Trevisan and Iscove 1995). Genetic markers, such as the Y chromosome for tracking the progeny of grafts of male cells into female recipients (Szilvassy et al. 1990), or a cell surface allo-antigen expressed on both lymphoid and myeloid cells (e.g., CD45/Ly5.1 or Ly5.2) (Spangrude and Scollay 1990; Szilvassy and Cory 1993; Rebel et al. 1994) can be used to identify the test cell origin of the blood cells produced post-transplant. Only recipients that are significantly (i.e., >1%) repopulated by both myeloid and lymphoid elements derived from the test cells for at least 4 months are considered positive (Miller and Eaves 1997). The input cell quantified by this assay is termed a competitive repopulation unit (CRU). Murine CRUs are present in the B M of normal adult mice at a frequency of approximately one per 10 cells 4  (Szilvassy et al. 1990; Miller and Eaves 1997). Because the quantitation of HSCs from the in vivo C R U assay is dependent on their reaching the B M microenvironment of the recipient and being stimulated to proliferate there, the type of treatment used to condition the recipients can influence C R U detection. Recent evidence also suggests that the cycling status of HSCs may affect their ability to be detected in a transplant assay (see section 1.4.4).  1.3.3. Phenotyping and purification of HSCs Functional assays for HSCs require them to proliferate and to differentiate for extended periods of time. Therefore, more immediate methods for the direct identification of HSCs are highly desirable. In the murine system, numerous strategies to obtain enriched populations of HSCs have been developed. These include bulk immunomagnetic and rosetting procedures, as well as multi-parameter fluorescent activated cell sorting (FACS) to remove or positively select for subpopulations according to their different physical characteristics (such as buoyant density and/or size), immunophenotypes (detected by antibody staining of variably expressed cell-surface markers) and abilities to efflux certain fluorescent dyes. Comparative studies of these properties exhibited by different types of B M cells have allowed many characteristics of HSCs in adult B M to be delineated, as follows: i) HSCs are relatively small cells with a lowdensity and a blast morphology (Orlic et al. 1993); ii) they express high levels of the Ly-6A/E (Sca-1) antigen (Spangrude et al. 1988; Spangrude and Brooks 1992), class I major histocompatibility antigens (H-2K) (Szilvassy et al. 1989; Spangrude and Scollay 1990), the CD38 antigen (Randall et al. 1996; Zhao et al. 2000) and c-kit (Ogawa et al. 1991; Ikuta and  9  Weissman 1992; Orlic et al. 1993) (see also section; iii) HSCs in normal adult mouse B M also express no or low levels of the sialomucin known as CD34 (a ligand for a cell adhesion molecule, L-selectin) (Osawa et al. 1996a; Sato et al. 1999; Zhao et al. 2000) nor many antigens present on mature blood cells (Muller-Sieburg et al. 1988; Spangrude et al. 1988; Spangrude and Brooks 1992; Orlic et al. 1993).  Such lineage-restricted cell surface  markers include CD3, CD4, CD8 CD45R (B220), CD90 (Thy-1), IgM (lymphoid), Ly-6G (Gr1), (myeloid) and Terll9 (erythroid); iv) HSCs are also largely separable from most other hematopoietic cells by their ability to efflux rhodamine-123 (Rh 123) (Mulder and Visser 1987; Ploemacher and Brons 1989; Orlic et al. 1993; Schinkel et al. 1997) and Hoechst 33342 (Baines and Visser 1983; Goodell et al. 1996; Zhou et al. 2001). Recent studies have demonstrated that HSCs can be enriched > 1000-fold by isolating subpopulations of adult mouse B M using various combinations of the above strategies to achieve HSC frequencies of 1 in 5 test cells (Wolf et al. 1993; Morrison and Weissman 1994; Spangrude et al. 1995; Osawa et al. 1996a; Wagers et al. 2002). However, these values may be underestimates because not every HSC thus isolated would be expected to home to the hematopoietic tissue and be activated following injection in vivo although this concept has recently been challenged (see section 1.4.4).  Moreover, it is important to note that  heterogeneity in repopulating behaviour has been observed even among the most enriched HSC populations. It is also important to note that as yet no known phenotypes can be used for the direct and definitive identification of HSCs. Numerous examples of dissociation in the regulation of surface and physiological marker expression and maintenance of HSC functions have been described (Rebel et al. 1994; Spangrude et al. 1995; Randall and Weissman 1997; Sato et al. 1999). In some cases, these changes appear to be related to HSC activation (Srour et al. 1992; Sato et al. 1999) that, in turn, may be variably related to cell cycle progression (Oh et al. 2000). Based on studies of human cells, it was believed that most of the primitive hematopoietic cells in the mouse would be likewise found to express high levels of the CD34 surface antigen (Krause et al. 1996; Bhatia et al. 1997b; Conneally et al. 1997). However, recent studies have shown that CD34 expression on murine HSCs appears only when they are activated (Osawa et al. 1996a; Goodell et al. 1997; Sato et al. 1999; Tajima et al. 2001; Ogawa 2002). In adult  10  humans, some primitive progenitors have now been found to be CD34" and capable of generating CD34 progeny (Bhatia et al. 1998; Zanjani et al. 1998; Nakamura et al. 1999). +  1.4. Properties of HSCs 1.4.1. Self-renewal potential of HSCs At the single cell level, a HSC self-renewal division can be operationally defined as proliferation without differentiation (i.e., maintenance of the undifferentiated state with all lineage options open).  Experimentally, evidence of such HSC self-renewal divisions can be  demonstrated and even quantitated by the analysis of the HSC progeny of primary HSCs regenerated in primary recipients.  This requires transplantating the cells from the primary  recipients into secondary recipients to demonstrate that progeny HSCs with longterm multilineage repopulating activity had been produced in the primary recipients. Several studies have clearly demonstrated the ability of HSCs to execute self-renewal divisions both in vivo (Lemischka et al. 1986; Jordan and Lemischka 1990; Keller and Snodgrass 1990) and in vitro (Fraser et al. 1990; Miller and Eaves 1997; Ema et al. 2000; Oostendorp et al. 2000). In some of these, it could be shown either directly (Ema et al. 2000; Oostendorp et al. 2000) or by integration site analysis of retrovirally marked clones (Lemischka et al. 1986; Fraser et al. 1990; Jordan and Lemischka 1990; Keller and Snodgrass 1990) that numerous recipients could be reconstituted by the progeny of a single original HSC. This provided a rigorous proof of self-renewal at the level of single HSCs. However, the numbers of self-renewal divisions that individual HSCs can execute and the extent to which this function can be altered by external factors (such as cytokines) have remained controversial issues (see section 1.6.1). Following B M transplantation, the donor HSC population is able to expand in vivo up to 100-fold in the following 8 to 12 month period (Osawa et al. 1996b; Pawliuk et al. 1996). Nevertheless, normally HSCs regenerate to levels that are <10% of the original (pre-transplant) B M value, despite full reconstitution of B M cellularity and numbers of later progenitor cell types (Harrison et al. 1978; Ross et al. 1982; Pawliuk et al. 1996; Iscove and Nawa 1997; Thorsteinsdottir et al. 1999). Originally, it was thought that HSCs possess an inherent limit for self-renewal that explained their decreasing regenerative activity after serial transplantation. However, it now seems more likely that serially transplanted HSCs retain their ability to reconstitute all lineages of recipients and can be shown to do so if they are retransplanted in adequate numbers (Keller and Snodgrass 1990; Iscove and Nawa 1997). 11  It has thus been  suggested that negative regulatory feedback mechanisms imposed in vivo by more mature cells may prematurely suppress HSC recoveries in the post-transplant period (Pawliuk et al. 1996).  1.4.2. Commitment of HSCs Lineage commitment of HSCs can be defined as the decision a HSC makes to restrict its differentiation potential, eventually to produce cells of a single lineage.  Commitment  usually requires not only the activation of expression of a particular set of genes (the lineage program) but also the shutdown of alternative program(s) (Hu et al. 1997).  A number of  studies have shown that changes in the expression patterns of certain transcription factors appear important to the commitment process in HSCs and their immediate progeny (Ness and Engel 1994; Shivdasani and Orkin 1996; Tenen et al. 1997).  Commitment events are not  necessarily associated with overt morphological changes, although accompanying changes in the expression of various membrane proteins and receptors can often be documented (MullerSieburg et al. 1988; Mayani et al. 1993a). Some studies of HSCs support a deterministic theory where H S C commitment is envisaged to be a fixed, stepwise process (Metcalf 1998). However, other studies suggest that the pattern of commitment in populations of HSC can also display random, stochastic features (Ogawa 1993)  (see  also section  1.6.1).  In addition, some studies suggest that the  differentiation process may not necessarily be coordinately regulated with other cellular processes.  For example, one study reported that the differentiation of a pluripotent  hematopoietic cell could proceed to completion in the complete absence of cell division (Fairbairn et al. 1993).  1.4.3. Cycling status of HSCs in vivo Much evidence now indicates that most of the HSCs in the B M of the normal adult are deeply quiescent. Based on early studies, an HSC clonal succession model (Kay 1965) and an HSC dormancy model (Lajtha 1979) were proposed. The first of these 2 models suggested that hematopoiesis is maintained by the sequential activation of dormant HSCs that proliferate, differentiate and eventually become exhausted. The second model suggested that the dormant (Go) state of most HSCs is reversible and HSCs are randomly recruited to enter and exit the cell cycle. The concept that most HSCs in normal adults are quiescent at any given moment was initially indicated by studies showing that the treatment of mice in vivo with cycle-active drugs (e.g., 5-FU) reduced cycling progenitor cell numbers to a much greater extent than pre12  CFU-S (Hodgson and Bradley 1979). This finding was subsequently confirmed with more HSC-specific assays (Lerner and Harrison 1990; Lemieux et al. 1995). Recent experiments that measured the accumulation  in vivo of cells with a phenotype associated with HSCs that  had incorporated bromodeoxyuridine (BrdU) over a period of continuous exposure revealed that approximately 99% of these cells had entered S phase within 6 months. (Bradford et al. 1997; Cheshier et al. 1999). These experiments indicated that the HSC population is in a slow, but continuous, state of turnover, with the caveat that the populations monitored were not functionally defined nor they were pure HSCs.  1.4.4. Homing and mobilization of HSCs Although  in vivo hematopoiesis normally occurs in specific tissues such as the fetal  liver, neonatal spleen and adult B M , it has been known for many years that HSCs also circulate in the blood (Brecher 1951; Hanks 1964; Fujioka 1967). The number of HSCs in the blood also dramatically increases, albeit transiently after a variety of  in vivo treatments, including  sublethal or localized irradiation, (Barnes and Loutit 1967) or myelotoxic chemotherapy (Pettengell et al. 1993; Sutherland et al. 1994). HSCs appear to be released from the B M into the circulation in response to signals that are activated when blood or B M cell numbers are significantly reduced. HSCs also proliferate and are mobilized into the bloodstream in response to  in vivo injections of growth factors, such as granulocyte colony-stimulating factor (G-CSF)  (Socinski et al. 1988; Sheridan et al. 1992; Pettengell et al. 1993; Molineux et al. 1997). These mobilized HSCs then seed secondary hematopoietic tissues, such as the spleen (Morrison et al. 1997b). Their ability to be mobilized is probably the result of cell cycle-related changes in their interactions with extracellular matrix (ECM) or cells within the B M .  The efficiency of  HSC homing to the B M has been quantified by transplanting B M cells from primary into secondary recipients early after transplantation of the primary recipients, e.g., after 24 hours. Such studies have yielded marrow homing values of ~ 10% (Szilvassy et al. 2003). However, very recently it has been reported that B M subpopulations can be isolated in which every cell can engraft a mouse and 50% with longterm repopulating activity (Benveniste et al. 2003). This would mean that although all HSCs injected are rapidly cleared from the blood stream only a fraction of them actually go to the B M initially. It has also recently become apparent that proliferating HSCs acquire a marked, reversible inability to engraft intravenously transplanted recipients as they traverse the S/G2/M 13  phases of the cell cycle (Srour et al. 1992; Habibian et al. 1998; Glimm et al. 2000; Szilvassy et al. 2000; Cashman et al. 2002). Although the mechanisms that regulate HSC cycling statusrelated engraftment deficiencies are not known, it has been suggested that changes in the expression of cell adhesion molecules may be involved (Orschell-Traycoff et al. 2000). In addition, in vitro stromal cell culture experiments (Miyake et al. 1991) and other in vivo intervention experiments (Papayannopoulou and Nakamoto 1993; Craddock et al. 1997; Zanjani et al. 1999) have suggested that the molecular mechanisms regulating mobilization and homing in general may involve the activities of very late antigen-4 (VLA-4) and the chemokine C X C receptor 4 (CXCR4) (Peled et al. 1999).  Involvement of other adhesion  molecules, such as intercellular adhesion molecule-1 (ICAM-1) (Arkin et al. 1991), CD44 (Lewinsohn et al. 1990) and V L A - 5 (van der Loo et al. 1998) has also been suggested but the roles of these molecules are not yet clear.  1.5.  Regulation of HSCs The normal regulation of HSCs must achieve an appropriate balance between self-  renewal and differentiation, commitment to different lineages, proliferation and quiescence, and maintenance of cell viability versus cell death. Evidence exists to suggest that all of these HSC  properties  are subject  to  regulation,  either by  cell-autonomous  (intrinsic)  or  environmental (extrinsic) factors (Watt and Hogan 2000).  1.5.1. Intrinsic factors / transcription factors Intrinsic mechanisms that are critical for the functional integrity of HSCs include: i) those that regulate telomere length (Lee et al. 1998); ii) those that control intracellular signaling regulating cell cycle progression and apoptosis; and iii) downstream transcription factors that control gene expression. Most of the information regarding the functions of such intrinsic factors in HSCs has come from genetic approaches, such as gene targeting (loss-offunction) or transgenic (gain-of function) strategies, particularly in the investigation of various transcription factors. From loss-of-function studies, a number of transcription factors have been identified as critical to the control of hematopoietic potential. Examples of such transcription factors are cmyb, A M L 1 (CBF-a2) (Castilla et al. 1996; Okuda et al. 1996), SCL (tal-1) (Shivdasani et al. 1995; Porcher et al. 1996; Begley and Green 1999), T E L / E T V 6  (Wang et al. 1998), L M 0 2  (Rbtn2) (Warren et al. 1994) and GATA2 (Orkin 1996; Shivdasani and Orkin 1996). Mice 14  lacking any of these genes have a complete absence of primitive hematopoietic cells. Thus, the role of these transcription factors is interpreted as specification of hematopoietic competence and/or the establishment of HSC populations during development. Transcription factors have also been identified through the study of cis-binding factors of lineage-specific genes and genes activated in leukemias.  In such studies, the role of the  transcription factor is interpreted as acting at different levels in the  differentiation  hematopoietic hierarchy; for example at the branching of the lymphoid and myeloid lineages or later (Shivdasani and Orkin 1996; Sieweke and Graf 1998; Orkin 2000). Such transcription factors are believed to play a role in lineage specification. For example, specification of the myeloid lineages appears to be regulated by a mutually antagonistic mechanism involving GATA-1 and P U . l , in which these 2 proteins can directly interact and inhibit the actions of each other (Rekhtman et al. 1999; Nerlov et al. 2000).  GATA-1 promotes erythroid and  megakaryocyte differentiation (Kulessa et al. 1995), whereas P U . l promotes the lymphoid and myeloid lineages (Nerlov and Graf 1998). Mice lacking GATA-1 or its co-factor have shown blocked erythropoiesis at the proerythroblast stage (Pevny et al. 1991; Simon et al. 1992; Pevny et al. 1995; Fujiwara et al. 1996)  and defects in megakaryocyte  development  (Shivdasani et al. 1997; Tsang et al. 1998; Vyas et al. 1999). Mice lacking PU.l have reduced or absent monocytes, granulocytes, T-lymphocytes and B-lymphocytes (Scott et al. 1994; McKercher et al. 1996; Scott et al. 1997; Anderson et al. 1998). Thus, the lineage choice is decided by the relative levels of these 2 transcription factors.  On the other hand, flcaros  promotes specification to the lymphoid lineage. Mice lacking Ikaros do not have lymphocytes or their precursors. Further specification within the lymphoid lineage comes from factors such as Pax5, which directs cells toward the B-lymphoid lineage by repressing differentiation along alternate lineages (Urbanek et al. 1994; Rolink et al. 1999). Gain-of-function approaches using retroviral transduction of transcription factors have also provided key insights into the regulation of HSCs.  A notable example is the forced  overexpression of the HOXB-4 homeobox gene product in HSCs.  This confers on the  transduced HSCs a selective ability to amplify their numbers both in vitro and in vivo (Sauvageau et al. 1995; Antonchuk et al. 2001; Antonchuk et al. 2002). HoxB-4-transduced HSCs retain normal multi-lineage differentiation ability and rapidly regenerate HSCs numbers to normal levels in vivo but do not become transformed. One of the interpretations of the  15  function of Hox-B4 in HSCs is that it may quantitatively shift the probability of HSC selfrenewal.  A very recent study further demonstrated that mice deficient in both Hoxb3 and  Hoxb4 have defects in endogenous hematopoiesis  and decreased numbers of primitive  hematopoietic cells (Bjornsson et al. 2003). The primitive hematopoietic cells in mice lacking Hoxb3 and Hoxb4 showed impaired proliferative and self-renewal capacity. indicates that Hoxb4 normally plays a role in regulating HSC self-renewal.  This study  Other homeobox  gene products, such as Hox-A9 and Hox-Al 1 have also been transduced into HSCs, although none have yielded results exactly like Hox-B4 in regard to HSC regulation (Lawrence et al. 1996; Hawley et al. 1997; Thorsteinsdottir et al. 2002).  1.5.2. Environmental factors / cytokines The non-hematopoietic stroma of the B M contains many different cell types, including fibroblasts, endothelial cells (Dexter et al. 1977; Zon 2001). These cells, in combination with ECM  components  and the basement membrane of blood vessels, form the complex  microenvironment believed to be responsible for regulating HSC survival and proliferation in vivo (Wineman et al. 1996; Thiemann et al. 1998; Punzel et al. 1999b). Analyses of murine and human stromal cells isolated from B M , as well as of stromal cell lines have shown that these cells can produce many different kinds of molecules, including many types of growth factors/cytokines, chemokines (see section 1.6.2), morphogens such as the bone morphogenetic proteins (BMP), sonic hedgehog (Shh), Wnt and Notch ligands (Morrison et al. 1997a; Metcalf 1998)  (see  section  1.6.3), E C M components  such as fibronectin, heparan-sulfate and  thrombospondin (Verfaillie et al. 1991; Long et al. 1992; Siczkowski et al. 1992), and certain peptide hormones (Zon 2001). The hematopoietic cytokines are a sub-group of molecules that function as regulators of proliferation (both mitogens and growth inhibitors), differentiation, cell migration (chemotactic factors), and survival of hematopoietic cells (Metcalf 1991a; Ogawa 1993). There is a network of more than 30 hematopoietic cytokines that are believed to play important roles in both paracrine (within the BM) and hormonal (via the circulation) regulation of hematopoiesis in vivo (Metcalf 1993). Hematopoietic cytokines include the various colony stimulating factors (CSF), such as G-CSF, granulocyte/macrophage-CSF (GM-CSF), macrophage-CSF (M-CSF), more than 20 so-called Interleukins (ILs) and other growth factors such as erythropoietin (EPO), thrombopoietin (TPO), flt-3 ligand (FL), Steel factor (SF), leukemia inhibitory factor 16  (LIF), transforming growth factor-beta (TGF-P), macrophage inflammatory protein-1 alpha (MlP-la) and tumor necrosis factor (TNF) (Zon 2001; Eaves 2002). Many of these cytokines were detected initially in functional tests of their ability to stimulate lineage-restricted progenitor growth and differentiation in vitro. However, subsequent studies have demonstrated that many of the cytokines initially thought to be lineage-restricted in their target specificity are actually pleiotropic, i.e., have multiple effects on different types of target cells at different levels in the hematopoietic hierarchy. For example, G-CSF, that stimulates the proliferation of monopotent neutrophil progenitors, can also activate the respiratory burst function of mature human neutrophils (Avalos 1996) as well as stimulating the proliferation of primitive hematopoietic cells in concert with other cytokines (Ogawa 1993). Some cytokines may have either a stimulatory or an inhibitory effect on different hematopoietic cell targets (Broxmeyer et al. 1990; Keller et al. 1992; Hirayama et al. 1994).  For example, the proliferation of  primitive human hematopoietic cells is inhibited by TGF-P (Cashman et al. 1990; Keller et al. 1992) with acceleration of differentiation (Krystal et al. 1994); however, proliferation of later granulopoietic progenitor types can be enhanced by TGF-P (Keller et al. 1991). Cytokines exert their effects by first binding to specific receptors expressed on the surface of hematopoietic cells. This causes the formation of ligand-receptor complexes (often dimers) that then lead to the phosphorylation of tyrosine residues in the intracellular portion of the signal-transducing chain of the receptor complex (Stahl and Yancopoulos 1993; Kondo et al. 2000). Cytokine receptors have been classified into different families based on a number of shared characteristics suggestive of a common origin during evolution.  One family of  receptors is the tyrosine kinase (TK) receptor gene family (Schlessinger 2000). This family includes c-kit and flt-3, the receptors for SF and F L , respectively. Both of these receptors can have important effects in the stimulation of primitive hematopoietic cells (Lyman and Williams 1995; Lyman and Jacobsen 1998; Miller et al. 1999; Audet et al. 2002) (see section 1.7). A second receptor family is the hematopoietic growth factor receptor family. These receptors are often molecular complexes of more than one protein.  However, all lack  endogenous T K activity, but upon activation, recruit and activate nearby non-receptor TKs and thereby activate the Jak/Stat and Ras/MAPK pathways (Stahl and Yancopoulos 1993; Brizzi et al. 1996; Dorsch et al. 1997; Burdach et al. 1998). Ligands for these receptors include G-CSF, GM-CSF, IL-3, EPO and TPO, both of which are active on primitive progenitors. IL-2, IL-4 17  and IL-7 receptors all share a common y chain and are active on lymphoid cells (Taniguchi and Minami 1993; Taniguchi 1995). A third receptor family is the gpl30 family, which includes receptors for IL-6, IL-11, and leukemia inhibitory factor (LIF).  The gpl30 receptors are all receptor complexes that  include a ligand-specific binding protein but share the gpl30 signaling molecule.  Like the  hematopoietic growth factor receptors, they too lack intrinsic T K activity (Kishimoto 1994; Sui et al. 1995; Ebihara et al. 1997; Hirano et al. 1997). These shared features of certain receptors may help to explain some of the overlap in activities of different cytokines. For example, G-CSF, GM-CSF and IL-3 can all support the proliferation and the complete differentiation of granulopoietic progenitor cells (Metcalf 1993), that is probably due to the overlapping intracellular signaling pathways activated by these receptors in the same cells (Miyajima et al. 1993; Hirayama et al. 1994). In vivo, cytokines are believed to form networks of complex feedback systems involving many interactions of cells with different cytokines (Metcalf 1993; Ogawa 1993; Sachs 1996; Lyman and Jacobsen 1998; Roeder et al. 1998). variety of target hematopoietic  Cytokines can also act on a  cells at different stages of differentiation and some  cytokines/chemokines may bind to more than one receptor. Many cells produce a number of different cytokines and many cytokines are produced by more than one cell type. Indeed the production of cytokines and expression of cytokine receptors are both regulated by cytokine feedback mechanisms, themselves (Callard et al. 1999). There are thus a tremendous number of ways cytokines can influence the effects of one another on hematopoietic target cells.  1.6.  Effect of environmental factors on HSC self-renewal  1.6.1. Effect of growth-promoting cytokines A characteristic of primitive hematopoietic cells is their requirement for stimulation by more than a single cytokine to allow rapid activation of cell cycling and preservation of their proliferative potential (Broxmeyer et al. 1991; de Vries et al. 1991; Metcalf and Nicola 1991; Migliaccio et al. 1991; Tsuji et al. 1992; Metcalf 1993; Ogawa 1993; Jacobsen et al. 1995; Keller et al. 1996). Success in promoting the growth of various primitive progenitors using multiple growth factors led many investigators to test whether similar growth factor combinations would also stimulate HSC expansion in vitro. Eventually, using enriched H S C 18  populations and serum-free medium, maintenance of HSC numbers was achieved (Rebel et al. 1994; Holyoake et al. 1996; Ramsfjell et al. 1996; YOnemura et al. 1997; Matsunaga et al. 1998b; Ramsfjell et al. 1999). Subsequently net expansions of murine HSCs of ~4-fold in 10 days were shown to be reproducibly achievable (Miller and Eaves 1997; Audet et al. 2001; Audet et al. 2002).  Several groups have shown that human H S C numbers can also be  minimally amplified in vitro (Bhatia et al. 1997a; Conneally et al. 1997; Nakahata 2001). Instructive vs selective actions of cytokines Till et al (1964) first proposed that HSC self-renewal behaviour is stochastically controlled, based on the observation that the generation of daughter CFU-S in individual spleen colonies fitted a probabilistic model. Many studies have subsequently supported this model including in vitro observations of CFU-S generation (and committed progenitor generation) in multi-lineage colonies (Humphries et al. 1981; Nakahata et al. 1982; Nakahata and Ogawa 1982; Leary et al. 1984; Mayani et al. 1993b; Petzer et al. 1996a). Recent studies still show little evidence that cytokines are "instructive" in the sense that they Can influence the ability of HSC to differentiate along a particular pathway (Metcalf 1991b; Metcalf 1998). For example, cytokine or cytokine receptor knock-out experiments argue against cytokines playing an instructive role in the differentiation process (Wu et al. 1995; Lin et al. 1996). In these studies, disruption of either the EPO or EPO receptor gene did not result in disruption of erythroid progenitor development.  These knock-out experiments  show that  lineage-commitment  decisions can be made in the absence of particular cytokines, although they do not rule out the possibility that compensatory pathways may obscure authentic functions. Studies of ectopically expressed cytokine receptors have also not provided support for an instructive model of hematopoietic cell lineage commitment based on cytokine activation. For example, overexpression of the EPO receptor in macrophage progenitor cells did not induce erythroid differentiation upon stimulation with EPO (McArthur et al. 1995). Conversely, forced expression of the M-CSF receptor by erythroid progenitors allowed M-CSF to stimulate erythroid colony formation (McArthur et al. 1994). Additional results supporting a permissive model of hematopoietic cytokine action in the regulation of commitment were obtained from experiments in which the TPO receptor gene (c-mpl) was replaced with a chimeric construct encoding the extracellular domain of c-mpl and the cytoplasmic signaling domain of the G-CSF receptor (Stoffel et al. 1999). Despite the absence of a functional c-mpl 19  signaling domain, the mice had a normal platelet count, indicating that in vivo the cytoplasmic domain  of  G-CSFR  can  functionally replace  M P L signaling  to  support  normal  megakaryopoiesis and platelet formation. On the other hand, evidence of instructive effects of cytokines on hematopoietic cell differentiation has been obtained in some experimental settings.  Studies of bipotential G M -  C F C differentiation demonstrated that these cells generate granulocytes preferentially when cultured in the presence of GM-CSF, and macrophages if cultured in M-CSF (Metcalf 1980; Metcalf and Burgess 1982). Evidence that adult mouse B M cells can be reprogrammed to generate embryonic hematopoietic progeny after being introduced into the developing blastocyst (Geiger et al. 1998) has also indicated that the environment can play an instructive role. However, these observations relate to the process of lineage selection and may not be informative about mechanisms that affect HSC self-renewal. The complexity of parameters involved in HSC regulation also suggests that instructive and stochastic effects may not be mutually exclusive. It is now clear that the mechanisms regulating HSC survival, mitogenesis and self-renewal can be, to some extent, modulated by exposure to different types and/or concentration of external cues (Jacobsen et al. 1995; Ramsfjell et al. 1999; Zandstra et al. 2000; Audet et al. 2001). In vitro HSC expansion studies have also suggested that IL-3 may be detrimental to the ability of primitive murine progenitors and HSCs to maintain their pluripotentiality (Yonemura et al. 1996; Matsunaga et al. 1998a) and, when present at too high a concentration, IL-3 also induces the differentiation of primitive human hematopoietic cells (Zandstra et al. 1997b). Regardless of the biological response elicited (self-renewal, proliferation, or survival), TPO, F L , SF, and IL-11 have been identified as the most potent cytokines active on HSCs (Miller and Eaves 1997; Audet et al. 2002).  SF and F L appear to have unique and non-  redundant activities on HSCs (Lyman and Jacobsen 1998; Audet et al. 2002). A recent study has shown that F L and SF alone will stimulate >85% of highly enriched murine B M HSCs to proliferate in single-cell serum-free cultures (Nakauchi et al. 2001).  However, optimal  retention of HSC activity requires additional exposure to a ligand that will adequately activate gpl30 (Audet et al. 2001). Investigation of the effects of both relative and absolute differences in SF and F L cytokine concentrations on HSC responses has indicated that their viability, 20  proliferation, and self-renewal behave differently.  Promotion of HSC self-renewal requires  exposure to the highest cytokine concentrations (Zandstra et al. 1997b) (see section 1.7.3). Kinetics of HSC stimulation The kinetics and/or intensity of stimulation (Marshall 1995) (see section, and other factors to which a cell is exposed (Sachs 1996; Callard et al. 1999; Schlessinger 2000) may have a considerable influence on how it responds. Variant forms of cytokines, such as membrane-bound, matrix-associated or membrane-anchored forms, may also elicit different biological outcomes (Gordon et al. 1987; Anderson et al. 1990; Otsuka et al. 1991; Lisovsky et al. 1996) (see section Many stromal cell types can support some HSC maintenance in vitro (Toksoz et al. 1992; Koller et al. 1997; Brandt et al. 1998; Bennaceur-Griscelli et al. 1999), although the concentration of cytokines found in stromal cultures is well below what is commonly added to stroma-free cultures (Burroughs et al. 1994; Punzel et al. 1999a). Moreover, membrane forms of SF have been found to be indispensable for normal hematopoietic development, with soluble forms being unable to substitute for them (Witte 1990). These observations suggest that the membrane-bound form of at least some cytokines is likely to be important to HSC regulation in vivo. In addition to potentially altering signaling events when the ligand cannot be internalized, the effective local concentration of a cytokine may be increased when it is bound on the cell membrane or to E C M components, such as glycosaminoglycans (Gordon et al. 1987; Long et al. 1992), thus providing a more potent stimulus for enhancing HSC self-renewal.  1.6.2. Effect of growth-inhibiting cytokines In addition to growth-promoting cytokines, a number of cytokines may selectively inhibit the self-renewal of HSCs, or may even induce cell death. Examples are the TGF-P proteins (Eaves et al. 1991) and members of the chemokine family, such as macrophage inhibitory M l P - l a (Graham et al. 1990; Dunlop et al. 1992; Broxmeyer et al. 1993; Eaves et al. 1993), monocyte chemoattractant protein-1 (MCP-1) (Cashman et al. 1998) and stromaderived factor 1 (SDF-1) (Cashman et al. 2002).  It has been shown that in unperturbed  stromal-cell containing long-term cultures (LTCs) of human cells, the primitive cells contained within the adherent cell layer become quiescent after a week, but can be transiently activated by media changes. In contrast, the more mature CFCs proliferate continuously (Cashman et al. 1985). Later it was discovered that the molecular mechanism responsible for the endogenously 21  induced quiescence involved the co-operative activity of TGF-p (Cashman et al. 1990) and chemokines such as MCP-1 (Cashman et al. 1998) and SDF-1 (Cashman et al. 2002). In immunodeficient non-obese diabetic-scid (NOD/SCID) mice engrafted with human hematopoietic cells, the function of these inhibitory cytokines in vivo was also examined. TGF-P, MCP-1, M l P - l a or SDF-1 when injected into engrafted mice were able to arrest the cycling of different types of human progenitor cells. However, only the injection of TGF-P and SDF-1 could inhibit the proliferation of human HSCs in this in vivo model (Cashman et al. 1999; Cashman et al. 2002).  1.6.3. Effect of morphogens Recent evidence has suggested that Notch, BMP, Shh and Wnt signaling pathways also play a role in controlling the self-renewal activities of HSCs (Bhatia et al. 1999; VarnumFinney et al. 2000; Bhardwaj et al. 2001; Reya et al. 2003). Characterization of the roles of these factors in non-hematopoietic systems have indicated that considerable cross talk can exist between these signaling pathways (Polakis 2000) and the Wnt signaling pathway has been shown to synergize with Notch, BMP, and Shh functions (Kuhl et al. 2000). The Wnt proteins belong to a major family of developmentally important signaling molecules, first identified for their instructive roles during embryonic development in Drosophila  melanogaster (Sharma 1973).  The Wnt gene family encodes  secreted  glycoproteins that act as paracrine or autocrine factors and are highly conserved in vertebrates. Members of the Wnt family confer distinct cellular functions depending on the nature of the target cell and expression of the corresponding Frizzled (Frz) receptors (Huelsken and Birchmeier 2001). Evidence for a role of Wnt signaling in regulating mesodermal cell fate, from which hematopoiesis is initiated (Mead and Zon 1998), combined with the expression of Wnt in primitive hematopoietic cells (Van Den Berg et al. 1998), has suggested a potential role for Wnt in the regulation of primitive hematopoietic cells.  Moreover, hematopoietic  progenitors from mouse and humans have shown increased self-renewal activities in response to conditioned media from Wnt-expressing cells or purified Wnt protein (Austin et al. 1997; Van Den Berg et al. 1998; Murdoch et al. 2003; Willert et al. 2003) A very recent study has further shown that increased HSC self-renewal could be achieved by modulating p-catenin, a major component of the Wnt signaling pathway (Reya et 22  al. 2003).  In this study, the investigators found that over-expression of P-catenin in HSCs  resulted in increased proliferation of purified HSCs candidates while significantly inhibiting their differentiation in vitro for a period of several weeks. They also obtained evidence that Wnt signaling may be required for the growth response of normal HSCs to other cytokines in vitro and may be important to HSC expansion in transplanted mice. Finally, they have shown that both HoxB4 and Notchl are upregulated in response to Wnt signaling in HSCs.  This  raises the possibility that the effects of Wnt signaling on HSCs are mediated through HoxB4 and/or Notchl.  1.7.  Sub-class III tyrosine kinase receptors and their corresponding ligands The sub-class III receptor tyrosine kinase (RTK) family includes c-fms, c-kit,flt-3and  the PDGF A and B receptors (Gronwald et al. 1988; Matsui et al. 1989).  Homologies in  receptor chain composition, structure, and sub-molecular motif characteristic of this R T K family have been recognized (Figure 1.2).  Moreover, the genes that encode this family  demonstrate overall conservation in exon size, number, sequence, and intro/exon boundaries, suggesting that they arose from duplications of an ancestral gene (Agnes et al. 1994). Although most of these family members and their corresponding ligands are known to have important roles in hematopoiesis, as mentioned above, the c-kit and flt-3 ligand-receptor pairs appear to have unique activities in more primitive hematopoietic cells.  23  Receptor tyrosine kinase Ig domians TM JM TK1 IK  SP  i-  i  /  Size (amino acid number)  -540  -20  -100  TK2  -170  Ligand of RTK SP  Size (amino acid number)  H1-4  -190  TM CT  -27  Figure 1.2. The general molecular structure of the subclass III receptor T K family and their corresponding ligands The approximate size of Ig, transmembrane (TM) and TK1 and 2 domains of a typical subclass III RTK family member are indicated (upper panel). The approximate size of the extracellular and T M domains of the type 1 transmembrane isoforms of the RTK ligand are also shown (lower panel). The cleavage site for the formation of soluble ligand isoforms is in the tether region, represented by the arrow. SP, signal peptide (removed in the post-translational process); JM, juxtamembrane domain; IK, interkinase domain; CT, carboxy-terminal domain; H 1-4, helix region 1 to 4 (adapted from Lyman and Jacobsen 1998).  24  1.7.1. c-kit and SF Molecular structure Initial knowledge of the physiological function of c-kit was developed from analyses of mice with naturally occurring mutations at the white-spotting (W) locus.  Such mice exhibit  defects in the development of multiple tissues including hematopoietic cells as well as melanocytes (Paulson and Bernstein 1995).  Reciprocal B M transplantation experiments  demonstrated that wild-type B M cells rescue the hematopoietic defect in W mutants, even without irradiation of the recipient. However, B M cells from mice with more pronounced W mutations do not generate macroscopic spleen colonies or rescue irradiated recipients (Bernstein 1959; McCulloch 1964). These findings suggested that a gene at the W locus was essential for HSC function. Later it was shown that the relevant gene at the W locus is c-kit (Chabot et al. 1988; Geissler et al. 1988), a gene first identified as a component of an oncogenic retrovirus (Chabot et al. 1988) and subsequently identified structurally as belonging to the R T K family of growth factor receptors. Mice with mutations at the Steel (SJ) locus have a phenotype virtually identical to W mutant mice (severe anemia, pigmentation defect, and sterility) (Russell 1979). However, in the case of SI mutant mice, these defects were found to be due to alterations in the environment in which the affected cells develop, rather than in the affected cells, themselves. Thus, B M cells from SI mutant mice can repopulate lethally irradiated wild-type or W animals, but the macrocytic anemia in SI mutant mice is not cured by the transplantation of wild-type B M cells (McCulloch et al. 1965). Later it was shown that the SI gene encodes a growth factor (SF) that is the ligand for c-kit (Copeland et al. 1990; Nocka et al. 1990; Williams et al. 1990; Zsebo et al. 1990). The murine and human c-kit receptors are both 976 amino acids in length, have 9 potential sites for N-linked glycosylation in their extracellular domains (Yarden et al. 1987; Qiu et al. 1988), and are glycosylated at one or more of these sites (Majumder et al. 1988). The c-kit receptor contains 5 immunoglobulin (Ig)-like domains in the extracellular region that are involved in ligand binding and receptor dimerization. The cytoplasmic region contains a split T K domain containing an ATP-binding region and a phosphotransferase domain (Figure 1.2).  The predicted size of the protein backbone alone is approximately 108 kD. However,  25  immunoprecipitation shows 2 proteins of approximately 140 kD and 155 kD, (Yarden et al. 1987), presumably due to glycosylation differences. The murine and human SF proteins are each 273 amino acids in length, with a 25 amino acid leader, a 185 amino acid extracellular domain, a 27 amino acid transmembrane domain, and a 36 amino acid cytoplasmic tail (Figure 1.2). The primary translation product of the SF gene is a type 1 transmembrane protein, i.e., the N-terminus of the protein is located outside of the cell. There are 4 cysteine residues that are conserved between SF and F L (see also section These cysteine residues form 2 intramolecular disulfide bonds that establish the 3 dimensional structure of the protein (Lu et al. 1991).  Although SF forms homodimers in  solution, they are not covalently linked (Arakawa et al. 1991). The mature mouse and human SF proteins undergo proteolytic cleavage to generate a soluble, biologically active, 164-165 amino acid protein (Anderson et al. 1990; Huang et al. 1990; Martin et al. 1990; Zsebo et al. 1990) with a primary cleavage site encoded within exon 6 (Martin et al. 1990) and a secondary cleavage site within exon 7 that could be targeted if the primary site were absent due to alternative splicing of the transcript (Anderson et al. 1990; Flanagan et al. 1991; Toksoz et al. 1992). Both high (kd, 16 to 310 pmol/L) and low (kd, 11 to 65 nmol/L) affinity binding of SF to its receptor have been reported (Broudy et al. 1992; Turner et al. 1992; Broudy et al. 1994). Some primary cells and cell lines have only high affinity sites, whereas others have both (Broudy et al. 1992; Turner et al. 1992). However, another study suggested that neither the number of c-kit receptors per cell nor the types of receptors present correlated with the ability of cells to proliferate in response to SF (Broudy et al. 1994). Expression of c-kit in primitive hematopoietic cells c-kit is expressed on cells of a number of lineages and from a number of tissues. During embryogenesis, c-kit is first expressed in the ectoderm (Orr-Urtreger et al. 1990; Motro et al. 1991). By Embryonic day 8.5 (E8.5), c-kit is expressed in the yolk-sac blood islands. Later in development, it is expressed by the hematopoietic cells that appear in the A G M , and then c-kit cells parallel the subsequent progression of hematopoiesis at different sites - i.e., +  first in the fetal liver, then in the spleen, and finally the B M (Orr-Urtreger et al. 1990; Motro et al. 1991).  The close correlation between c-kit expression, de novo development of  hematopoietic cells, and their presumed migration to new sites of hematopoiesis support the 26  belief that c-kit activation is involved in the survival and/or migration of hematopoietic cells during early fetal development. In the adult B M , c-kit is expressed by approximately 8% of hematopoietic cells (Ogawa et al. 1991).  Half of these c-kit murine B M cells co-express lineage-specific cell surface +  antigens such as Gr-1 and Mac-1 (lin ) that are characteristic of maturing myeloid cells. The +  other half of c-kit B M cells are lin". These cells express higher levels of c-kit suggesting that +  terminal myeloid differentiation is accompanied by a down-regulation of c-kit expression (Ogawa et al. 1991). Many studies have suggested that most, if not all, HSCs (purified by various methods from B M , fetal liver, or the AGM) are in this c-kit lin~ population (Okada et +  al. 1991; fkuta and Weissman 1992; Orlic et al. 1993; Okuda et al. 1996; Osawa et al. 1996a; Sanchez etal. 1996).  1.7.2. Flt-3 and FL Molecular structure The flt-3 gene was cloned independently by 2 different groups. One group exploited the sequence homology offlt-3 to other members of the R T K gene family (Rosnet et al. 1991). A second group used degenerate oligonucleotides (based on conserved regions within the kinase domain of RTKs) in a PCR-based strategy to isolate a novel receptor fragment from highly purified murine fetal liver stem cells (Matthews et al. 1991). The gene encoding the ligand for flt-3 (FL) was also cloned by different groups using different strategies.  One  screened a T-cell derived cDNA library to isolate a cell-surface protein that would bind to the soluble form of flt-3 (Lyman et al. 1993a).  The other used the purified protein to design  degenerate oligonucleotide primers to amplify a portion of the F L gene by PCR, which was then used to isolate a bacterial clone containing a full-length murine cDNA (Hannum et al. 1994). Once the murine F L cDNA had been isolated, it was used to isolate a human F L cDNA (Lyman etal. 1994). Murine and human flt-3 receptors contain 1000 and 993 amino acids, respectively, and have 9 and 10 potential extracellular N-linked glycosylation sites, respectively (Lyman et al. 1993b). Immunoprecipitation has shown 2 proteins of 130-143 kD and 155-160 kD (Lyman et al. 1993b; Rosnet et al. 1996).  The predicted size of the protein backbone alone is  approximately 110 kD. Pulse-chase analysis demonstrated that the larger protein arises from the smaller protein (likely as a result of glycosylation changes). 27  Only the larger protein is  found on the cell surface (Lyman et al. 1993b). One isoform of the murine flt-3 receptor is missing the fifth of the 5 Ig-like regions in the extracellular domain as a result of the skipping of 2 exons during transcription (Lavagna et al. 1995). The physiologic significance of this flt-3 receptor isoform is unknown. The primary translation product of the F L gene is a type 1 transmembrane protein (Figure 1.2).  The mouse and human proteins contain 231 and 235 amino acids, respectively.  The mouse and human F L proteins are 72% identical at the amino acid level, and homology is greater in the extracellular region (73%) than in the cytoplasmic domain (57%).  Like SF,  preliminary data suggest that F L also exists as a noncovalently linked homodimer, which contains 3 intramolecular disulfide bonds (Lyman and Jacobsen 1998). Multiple isoforms of both mouse and human F L have been identified. The predominant isoform of human F L is the transmembrane protein that is biologically active on the cell surface (Lyman et al. 1993a; Hannum et al. 1994; Lyman et al. 1994). This isoform is also found in the mouse, although it is not the most abundant isoform (Lyman and Jacobsen 1998). These transmembrane F L proteins can be proteolytically cleaved to generate a soluble form of the protein that is also biologically active (Lyman et al. 1993a). The most abundant isoform of murine F L is a 220 amino acid membrane-bound (Lyman and Jacobsen 1998). Soluble F L can also be synthesized from a relatively rare isoform in both mouse and human cells by alternative splicing of exon 6, which introduces a stop codon at the end of the extracellular domain (Lyman and Jacobsen 1998). The binding affinity of human F L for the flt-3 receptor on human myeloid leukemia cells has been estimated to be 200 to 500 pmol/L (Turner et al. 1996) and only high-affinity binding is seen. There is no evidence that SF or F L bind to any other protein other than the ckit and flt-3 receptors, respectively.  Similarly, no other ligands that bind the flt-3 and c-kit  receptors are known (Lyman and Jacobsen 1998). Expression of flt-3 in primitive hematopoietic cells The flt-3 gene is expressed in the placenta beginning at El3.5, and increases until birth (Rosnet et al. 1991). The flt-3 gene is also expressed in sites of hematopoiesis in the fetal liver. In the thymus, flt-3 begins to be expressed in the fetal thymus on El6.5. F L is also expressed in the yolk sac, fetal liver, and placenta (Hannum et al. 1994; Lyman et al. 1994). In the adult,  28  flt-3 and F L are co-expressed in spleen, B M , thymus, ovary, testis, liver, kidney, and intestine (Lyman and Jacobsen 1998). Flt-3 expression on hematopoietic cells appears restricted predominantly to primitive cells. Consistent with the cloning of flt-3 from fetal liver-enriched progenitors, 96% of those fetal liver cells and 88% of lin"Sca kit B M cells express flt-3 (Rasko et al. 1995). These cells +  +  contain distinct flt-3 and flt-3" subpopulations and the long-term repopulating activity appears +  to be predominantly but not exclusively found in the flt-3" fraction. A greater proportion of flt3  +  HSCs are in active cell cycle as compared to flt-3" HSCs and growth factor treatment  upregulates flt-3 expression in HSC-enriched populations (Zeigler et al. 1994). Therefore it is possible that originally flt-3" HSCs upregulate flt-3 in response to other growth factors, and acquire F L responsiveness in this fashion before their functions as HSCs are altered.  1.7.3. Roles of c-kit and flt-3 in primitive hematopoietic cell In vitro studies Investigation of the effects of incubating primitive hematopoietic cells in vitro to different relative and absolute concentrations of SF and F L has helped to delineate the different roles of c-kit and flt-3 in normal hematopoiesis. Several studies involving single-cell cloning and delayed addition of cytokines also demonstrated that SF and F L act directly on primitive hematopoietic cells, and not through secondary effects mediated by other cells (Lyman and Jacobsen 1998). In the absence of other cytokines, both SF (Bodine et al. 1992; Katayama et al. 1993; L i and Johnson 1994; Keller et al. 1995) and F L (Muench et al. 1995; Rasko et al. 1995; Takahira et al. 1996; Veiby et al. 1996) appear able to selectively promote the viability rather than the proliferation of primitive hematopoietic cells. Soluble SF can also promote the adhesion of hematopoietic cells to extracellular matrix proteins in vitro, such as fibronectin, (Levesque et al. 1996) or to adhesion molecules expressed by cells in the microenvironment, such as V C A M - 1 (Kovach et al. 1995), which may then have a later effect on H S C selfrenewal (see also section SF appears to be more efficient than F L at recruiting murine HSCs into the cell cycle, independent of which other cytokine is used as the synergistic factor (Lyman et al. 1993a; Hannum et al. 1994; Zeigler et al. 1994; Broxmeyer et al. 1995; Hudak et al. 1995; Jacobsen et al. 1995; Rasko et al. 1995; Ramsfjell et al. 1996; Ramsfjell et al. 1997; Audet et al. 2002). In contrast, several studies indicate that F L is more efficient than SF (or at least as efficient as SF) 29  in stimulating primitive human cells to proliferate (Gabbianelli et al. 1995; Petzer et al. 1996b; Rusten et al. 1996; Shah et al. 1996; Dao et al. 1997; Zandstra et al. 1997b). Studies of relative cytokine dose-responses and interaction parameters have indicated a possible quantitative way of how c-kit and flt-3 regulate different populations in the hematopoietic hierarchy (HSCs, CFCs and total cells) (Zandstra et al. 1997b; Audet et al. 2002).  These studies have  demonstrated that it is possible to enhance HSC amplification by increasing the extracellular concentration of cytokines.  Notably this required at least 10-fold higher cytokine  concentrations than were necessary to stimulate maximal expansion of CFCs or total cells from a common primitive starting population in the same cultures (Zandstra et al. 1997b; Audet et al. 2002). Moreover, from the study of these effects at the single cell level, it was possible to demonstrate that the effects on HSC expansion could not be explained by changes in H S C survival. (Figure 1.3).  This dose-dependent change in primitive cell responses suggests that  different response outcomes can be regulated by different intensities of activated intracellular signaling mechanisms (Zandstra et al. 2000).  An example of a similar threshold model is  found in T and B cell signaling, where a sufficient number of receptors must be triggered to yield a full cellular response (Valitutti et al. 1995; Chidgey and Boyd 1997; Valitutti and Lanzavecchia 1997; Bachmann et al. 1999; Benschop et al. 1999). According to such a model, when a relevant ligand-receptor interaction is kept above a required threshold level in HSCs, differentiation would continues to be suppressed, thus permitting a self-renewal division to occur. a A more detailed, large scale statistical analysis of the effects on murine HSCs of IL11, SF, and F L concentrations and their interactions showed that IL-11 has a maximal stimulatory effect on HSC expansion at 20 ng/mL with higher concentrations being inhibitory (Audet et al. 2002). In contrast, no saturation or decrease in HSC expansion was observed as the SF and F L concentrations were increased slightly beyond 300 ng/mL, suggesting that HSCs might be further expanded with even higher SF or F L concentrations. However, an unexpected negative interaction between SF and F L on HSCs was also observed that caused a significant decrease in H S C expansion when SF and F L were combined at high concentrations. reason for this negative interaction on HSC expansion is unclear.  The  However, it has been  speculated that, as the concentration of F L and SF increases, heterodimers of SF and F L form in solution and these in turn cause the formation of receptor heterodimers (Audet et al. 2002). It has been reported that formation of heterodimers between different members of the 30  epidermal growth factor receptor (EGFR) family is possible (Hackel et al. 1999) and crossactivations of c-kit and flt-3 have also been reported (Otto et al. 2001).  31  High cytokine Self-renewal  /\  / \ / \ /\  Low cytokine Differentiation  /\  / \ / \ /\  Figure 1. 3. Clone formation and progenitor expansion from single cell cultures of CD34 CD38 adult human BM cells +  In the study by Zandstra et. al., human B M HSC candidates (CD34 CD38") cells were isolated and cultured as single cells in serum-free medium containing either high (300 ng/mL SF, F L , and 60 ng/mL IL-3) or low (30 ng/mL SF, F L , and 6 ng/mL IL-3 ) cytokine concentrations for 10 days. Clone formation and C F C and LTC-IC expansion were determined at the end of a 10-day culture period. The frequency of input cells that proliferated was the same for both cytokine cocktails but the expansion of primitive cells (gray circles) was higher in the cocktail containing the higher concentrations of cytokines. These results suggest that self-renewal versus differentiation decisions can be modulated by the cytokine signaling intensity induced by the activated cytokine/receptor complex (Figure adapted from Zandstra et. al. 1997). +  32 In vivo studies As noted above, W and SI mutant mice exhibit defects in hematopoiesis.  Despite the  severe macrocytic anemia and the resulting embryonic lethality associated with some alleles of W and SI, some mutants are viable. In these, disruption of hematopoiesis appears restricted to erythropoiesis and mast cell generation. Specifically, in the B M of W41 mutant mice that have a partial c-kit signaling deficiency, the numbers of myeloid, pre-B, erythroid, multipotent progenitor cells, lin"Sca-l candidate stem cells and LTC-ICs are at near-normal levels (Miller +  et al. 1996). However, long-term repopulating HSC numbers are reduced about 20-fold (Miller et al. 1996). W41 fetal liver cells are qualitatively and quantitatively similar to normal mice in their short-term reconstituting ability but have less competitive long-term reconstituting ability than normal fetal liver HSCs (Miller et al. 1997). Sl/Sl mice lack functional SF and die at day 15 or 16 of gestation (Lyman and Jacobsen 1998). However, the number of cells in the HSC candidate populations (lin Sca-l Thy-l ) +  +  l0  and also CFU-S numbers in these mice increase normally in Sl/Sl mice from day 13 to 15 (Ikuta and Weissman 1992). Nevertheless, the enhanced production of SF seen in adults given myeloablative treatments (Hunt et al. 1992; Yan et al. 1994) and the ability of endogenously produced or exogenously administered SF to promote the survival of and recovery of hematopoiesis in myeloablated mice (Zsebo et al. 1992; Neta et al. 1993; Patchen et al. 1994; Yan et al. 1994) is consistent with the view that SF plays a role in promoting H S C reconstitution. Taken together, these observations suggest that SF might not be essential for HSC development in the mouse embryo but that, under certain circumstances, adult murine HSCs self-renewal might be SF-dependent. Whether flt-3 or F L is required for normal hematopoiesis has also been addressed by creating mice that carry a homozygous deletion of most of the gene encoding flt-3 (Mackarehtschian et al. 1995) or F L (McKenna et al. 2000). flt-3 null mice have normal levels of peripheral blood cells and are generally healthy and fertile, in contrast to the lethality observed in mice homozygous for the deletion of the genes encoding the c-kit receptor or its ligand (Bernstein et al. 1991). However, the loss of functional flt-3 receptors does result in a reduced number of early B-cell precursors and a defect in HSCs, as measured in a long-term competitive repopulation assay. This defect was demonstrable as a reduced ability of primitive cells from flt-3 null mice to compete with their wild-type counterparts in irradiated recipient 33  reconstitution assays (Mackarehtschian et al. 1995). flt-3 null mice crossed with mice carrying mutations in the c-kit receptor generated W/W-flt-3 null mice that died between 20 and 50 days after birth with more severely reduced numbers of hematopoietic cells than either of the parental strains (Mackarehtschian et al. 1995). These findings demonstrated that both flt-3 and c-kit receptors have distinct roles in ensuring the generation of a full complement of HSCs in the adult. F L null mice, like flt-3 receptor null mice, have a normal, healthy appearance, but also have reduced numbers in myeloid and B-lymphoid progenitor cells, dendritic cells, and natural killer cells (McKenna et al. 2000).  1.7.4. Involvement of c-kit and flt-3 in hematopoietic diseases c-kit is commonly expressed on human acute myelogenous leukemia (AML) blasts and SF is co-expressed with c-kit in 30% of such cases (Wang et al. 1989; Ikeda et al. 1991; Broudy et al. 1992).  c-kit receptor levels on human A M L blast cells are variable but, in  general, are similar to, or less than, c-kit levels on normal HSCs and progenitor cells (Cole et al. 1996). The c-kit receptor is also expressed on the blasts in a majority of samples from chronic myelogenous leukemia (CML) patients in blast crisis (Buhring et al. 1991). Flt-3 receptors are seen even more frequently on human A M L blasts than c-kit. Flt-3 is expressed in 93% of patients with A M L and approximately 20% of patients with A M L have an internal tandem duplication of the juxtamembrane domain of Flt-3 (Nakao et al. 1996; Kiyoi et al. 1999) that correlates with a poor prognosis.  Flt-3 receptors are present on the blasts of  100% of patients with B cell acute lymphoblastic leukemia (ALL) (McKenna et al. 1996) and 75% of patients with T cell A L L (Birg et al. 1992; DaSilva et al. 1994). Flt-3 cells were +  detected in 29% of C M L patients with accelerated phase disease and in 75% of C M L patients in blast crisis (Birg et al. 1992).  1.8.  Genetic manipulation of hematopoietic cells using recombinant retroviruses Until recently the most commonly used vehicles for exogenous gene transfer to primary  hematopoietic cells have been replication-incompetent recombinant retroviral vectors (Kohn 1997). The advantage of these vectors is their ability to transduce a variety of cell types, as well as to integrate stably into the genome of the transduced cells in the form of a D N A provirus. The use of such vectors has provided a powerful approach to track the proliferation and differentiation of individual HSC clones (Jordan and Lemischka 1990; Keller and Snodgrass 1990; Pawliuk et al. 1996) and to genetically manipulate HSCs and their progeny. 34  1.8.1. Retrovirus biology All retroviruses are basically similar in virion structure, genomic organization and mode of replication (Coffin 1996).  One of the most intensively studied retroviruses is the  Moloney murine leukemia virus (MMuLV) (Varmus 1988). The M M u L V virion is about 100 nm in diameter, and is enveloped by a glycoprotein-containing lipid bilayer.  The virion  contains a R N A genome that is reverse-transcribed into D N A upon entry of the virus into the host cell. The viral cDNA is then integrated into the host cell genome. The structure of an integrated wild type M M u L V viral (or proviral) D N A includes a transcriptional control sequence, a polyadenylation signal, and sequences required for integration in the long terminal repeat (LTR) regions at the 5' and 3' ends of the provirus.  The sequence required for  packaging the viral genome into a virus particle is designated psi (cp), and is located downstream of the 5' LTR. The proteins required for replication and packaging the retrovirus are encoded in 3 distinct open reading frames (ORFs) of the provirus; namely, the gag (group specific antigens), pol (polymerase/reverse transcriptase/integrase), and env (envelope), with gag-pol-env as the 5' to 3' order of these genes. The gag gene encodes a polypeptide that is cleaved into at least 3 proteins designated as the matrix, capsid and nucleocapsid protein. The pol gene encodes 2 proteins, reverse transcriptase, an RNA-dependent D N A polymerase, and the integrase protein necessary for integration of the viral cDNA into the D N A of the host cell. The env gene encodes the glycoprotein that surrounds the virion, and is a heterodimeric complex of both transmembrane and surface domains.  The larger surface domain is  responsible for binding to the cell surface receptor. The transmembrane domain anchors the complex to the virion envelope and contains domains responsible for the fusion of viral and cellular membranes (Varmus 1988). The retroviral replication cycle can be divided into 2 phases. The first phase includes binding of the virus particle to a specific receptor on the cell surface, entry of the virion core into the host cell and integration of the viral genetic material into the host genome. The second phase consists of synthesis and processing the viral mRNAs and protein from the provirus, assembly of the virion and finally release (non-lytic budding) of replication-competent mature virions (Varmus 1988).  35  1.8.2. Production of recombinant retroviruses To generate a recombinant replication-incompetent retrovirus, the viral structural genes (gag, pol, env) are replaced with a marker gene or the gene of interest and the viral structural genes required for virus production are provided in trans by retroviral packaging cell lines. Many of these cell lines have been derived from fibroblasts that have been engineered to produce retroviral structural proteins through the introduction of gag, pol and env genes by stable transfection, but are unable to package viral R N A because the cp sequence is missing. Thus when a retroviral vector plasmid is introduced into the packaging cells, the retroviral R N A subsequently produced can combine with the retroviral proteins produced by the packaging cell line to produce infectious recombinant viral particles.  These are capable of  transducing competent target cells but are incapable of directing further virus production (Varmus 1988). Recently, highly transferable packaging cell lines have been produced (Pear et al. 1993) and co-transfection of these cells with a gag/pol plasmid and env plasmid as well as the vector plasmid allows the generation of higher titers of recombinant retrovirus than were previously possible.  Recombinant virus production using these cells is transient, with virus  production peaking 48 to 72 hours after co-transfection of the various plasmids (Pear et al. 1993). These transient virus production methods are attractive because conventional stable retrovirus-producingfibroblastsroutinely require >4 weeks to generate and even longer periods to allow the isolation and expansion of selected stable, high titer clones. The titers obtained by transient transfection can also be similar to those produced by selected stable producer cell lines. In practice, replication competent-free virus titers rarely exceed 10 particles/mL.  1.8.3. Recombinant retroviral vectors Several factors can influence the performance of any particular retroviral construct including the regulatory elements used to drive expression, the number and size of transcriptional units, the viral backbone used, the direction of transcription, and the presence or absence of selectable markers.  Viral LTRs have consistently been shown to be strong  promoters, resulting in higher levels of gene expression as compared to a variety of internal promoters of viral or cellular origin (Correll et al. 1994). However, viral LTRs in the M M u L V are also highly susceptible to transcriptional silencing in a variety of primitive cell types including embryonic carcinoma (EC) cells (Kempler et al. 1993), embryonic stem (ES) cells 36  (Seliger et al. 1986) and various "primitive" hematopoietic cell lines (Baum et al. 1995). Several viral mutants that are able to express transferred genes at high levels in E C and ES cells have been isolated (Colicelli and Goff 1987; Hilberg et al. 1987). By combining the LTR, the 5' untranslated regions, and a number of convenient cloning sites, Hawley et al (Hawley et al. 1994) have produced a series of Murine Stem Cell Virus (MSCV) vectors that are relatively resistant to silencing. Further data have suggested that the provirus from the M S C V vector is able to transcribe transduced genes efficiently in HSCs and their progeny for extended periods of time in vivo (Sorrentino et al. 1995; Pawliuk et al. 1997).  1.8.4. Murine HSCs as target for retro viral-mediated gene transfer Murine B M marking and transplantation studies have shown that HSCs can be transduced without compromising their ability to repopulate the lymphoid and myeloid lineages (Dick et al. 1985; Lemischka et al. 1986; Jordan and Lemischka 1990).  The  efficiency of transduction of HSCs depends on a number of parameters including whether or not they are cycling and whether or not they have upregulated expression of the receptor for the retrovirus at the time of exposure. In addition, studies of more mature progenitors suggest that growth factors activate other, as yet uncharacterized mechanisms that enhance retroviral transduction (Hogge and Humphries 1987). Because the vast majority of HSCs in the normal adult are quiescent (see section 1.4.3.), a number of strategies are used to stimulate them to start to proliferate prior to exposure to retroviruses. One is to pretreat mice by injecting them with 5-FU to selectively kill more mature hematopoietic progenitor cells, that then causes a large proportion of HSCs to be induced into cycle (Harrison and Lerner 1991).  Additional stimulation of the cells with  cytokine cocktails including various combinations of IL-3, IL-6, SF, FL, IL-1 and LIF have been found to further enhance retrovirus-mediated gene transfer into murine, human and nonhuman primate HSCs (Bodine et al. 1989; Luskey et al. 1992; Einerhand et al. 1993; Peters et al. 1996; Conneally et al. 1997).  Therefore, most protocols include such a 48-hour  "prestimulation" of the target cells prior to exposure to retrovirus (Bodine et al. 1989). During transduction, another important parameter is the number of virus particles to which each target cell is exposed.  Since standard retroviruses are not stable, they cannot be  concentrated and additional strategies to increase their titers or effective titers have proven helpful. One of these is the use of fibronectin to concentrate virus particles on a molecular 37  surface that co-localizes to primitive hematopoietic cells (Moritz et al. 1994). Both of these activities of fibronectin were subsequently found to be attributable to a single 30/35 kD fragment of the fibronectin molecule (Hanenberg et al. 1996). Another consideration is the duration of the period of viral exposure. It has been shown that the absolute number of HSCs decreases dramatically during the transduction period (particularly between day 2 and day 4 of the transduction procedure) (Antonchuk et al. 2002). Therefore, although longer exposure of the target cells to virus may increase HSC transduction efficiency, the absolute number of transduced H S C that can ultimately be recovered may be less than the number present after a shorter 2-day transduction procedure. The retroviral receptor is the primary determinant of the type of cells that can be transduced by a given virus. Many of these receptors have multiple transmembrane domains and  function as transporter molecules.  These include the receptor for ectropic murine  retrovirus (MCAT) (Kim et al. 1991; Wang et al. 1991), amphotropic murine retrovirus (Ram1) (Miller et al. 1994) and the common receptor for gibbon ape leukemia virus (Kavanaugh et al.  1994).  M C A T and Ram-1 receptors are transport proteins that perform essential  housekeeping functions. M C A T serves as a cationic amino acid transporter (Wang et al. 1991) and Ram-1 is a sodium-dependent phosphate symporter (Kavanaugh et al. 1994). Transduction of murine HSCs using ecotropic retrovirus has been found to be more efficient than using amphotropic virus, presumably due to the lower levels of expression of Ram-1 on primitive murine hematopoietic cells as compared to human hematopoietic cells (Kavanaugh et al. 1994).  1.9.  Present studies and thesis objective B M transplantation studies have demonstrated that HSCs numbers can expand by  >8000 fold during the course of several serial B M transplants(Iscove and Nawa 1997). However, to date, no studies have been able to demonstrate >5-fold reproducible H S C expansion in vitro (Miller and Eaves 1997). Recent in vitro studies have now shown that HSC expansion is critically influenced by both the types and concentrations of the cytokines used in the culture (Zandstra et al. 1997b; Audet et al. 2002). In particular, these studies demonstrated that an elevated level of F L or SF in the cultures would preferentially increase the expansion of the more primitive cell populations, but without significant increase in the growth of the more mature progenitor populations.  A model of HSC self-renewal control has been proposed, 38  suggesting that the probability o f a self-renewal response o f H S C s is increased when the relevant ligand-receptor interaction is maintained above a threshold level (Zandstra et al.  2000). According to this model, the control o f H S C self-renewal is dependent o n at least 2 variables- the ligand concentration and the accessibility o f the corresponding receptors. In this thesis, I have focused on testing the second variable.  M y experimental approach was to  overexpress flt-3 or c-kit receptors by retroviral transduction o f H S C s and to then evaluate the effect o f these manipulations on the sensitivity o f H S C self renewal responses to F L and SF. The prediction was that an increased number o f receptors would sensitize the H S C s to attain a greater self-renewal response i n the presence o f a lower concentration o f ligand. M y specific objectives were therefore as follows: 1)  T o construct and validate retroviral vectors encoding functional c-kit or flt-3  cDNAs. 2)  T o compare the ligand-stimulated responses o f cloned lines o f c-kit or flt-3  transduced factor-dependent cells expressing different levels o f flt-3 or c-kit receptors. 3)  T o study the effect o f flt-3 or c-kit overexpression on H S C self-renewal and  differentiation in vitro. 4)  T o study the effect o f flt-3 and c-kit overexpression on H S C function in vivo  39  CHAPTER 2: MATERIALS AND METHODS 2.1. Reagents 2.1.1. Cell lines All cell lines were grown from periodically checked stocks that had been shown to be mycoplasma-free.  They were maintained at 37°C with 5% CO2 in medium containing 100  U/mL penicillin and 100 U/mL streptomycin obtained from StemCell Technologies Inc. (Vancouver, BC), unless specified otherwise. Phoenix-Eco cells are modified 293T cells, originally derived from human embryonic kidney tissue. They contain the SV40T antigen gene and the retroviral ecotropic env and gagpol genes to allow packaging of mRNA containing the retroviral packaging (\|/) sequence into ecotropic retrovirus (see section 1.8.1). Phoenix-Eco cells were obtained from Dr. G.P. Nolan (Stanford University, Palo Alto, CA) and were maintained in a growth medium consisting of Dulbecco's modified Eagle medium (DMEM), 2 m M L-glutamine and 10% fetal calf serum (FCS) that had been heat-inactivated (by incubation at 56 °C for 30 minutes). Cells were kept in continuous log phase growth by maintenance between 20% and 80% confluence. . NIH-3T3 murine fibroblasts were purchased from the American Type Tissue Collection (ATCC, Rockville, MD) and maintained in D M E M plus 2 m M L-glutamine and 10% bovine calf serum BaF3 cells are a murine IL-3-dependent murine cell line with features of pro-B cells (Perkins et al. 1996).  They were obtained from Dr. G. Krystal (Terry Fox Laboratory,  Vancouver, BC) and maintained in suspension cultures in RPMI 1640 plus 10% heatinactivated FCS, 10 M 2-mercaptoethanol (2-ME) and 10 ng/mL murine IL-3. 4  2.1.2. Cytokines Most of the cytokines used were purified recombinant murine (rm) or human (rh) proteins prepared in the Terry Fox Laboratory by transient expression of the appropriate cDNA in COS cells (rmIL-3 and rmSF). Rh F L was obtained from Immunex (Seattle, WA), rh IL-11 from Genetics Institute (Cambridge, M A ) , rh IL-6 from Cangene (Mississauga, ON), rh thrombopoietin (TPO) from Genenetech (San Francisco, CA) and rh erythropoietin (EPO) from StemCell Technologies.  40  2.1.3. Mice The  mice used i n these experiments were C 5 7 B L / 6 J (B6) mice and various B 6 -  congenic strains, i.e. Bd-W /]^ 41  41  (W41), and B6-Pep3b (Pep3b). In most experiments, Pep3B  mice were used as marrow donors and either B 6 or W41 mice as recipients, all at 8 to 14 weeks of age. The hematopoietic cells from the Pep3b strain are phenotypically distinguishable from those from B 6 or W41 mice on the basis o f allelic differences at the L y 5 (CD45) locus. Pep3b are homozygous Ly-5.1, whereas B 6 and W41 mice are homozygous for the Ly-5.2 allotype. A l l mice were bred and maintained i n the A n i m a l Facility i n the British Columbia Cancer Research Centre  from  parental strain breeders  Laboratories (Bar Harbor, M E ) .  originally obtained  from  The Jackson  They were housed i n micro-isolator cages with sterile water,  food, and bedding. Irradiated mice also received acidified water (pH 3.0) for 4 months after irradiation.  2.1.4. Isolation of mouse BM cells BM  cells were obtained  from  Pep3b (test) or B 6 (competitor) mice injected  intravenously 4 days previously with 150 mg/kg body weight o f 5 - F U (Faulding, Vaudreuil, PQ) i n phosphate-buffed saline, b y flushing dissected femurs and tibias using 22 and 26 gauge needles, respectively. Harvested B M cells were filtered through a cell strainer (100 u m N y l o n , Falcon, Becton Dickinson Labware, Franklin Lakes, NJ.) to remove clumps and resuspended i n D M E M supplemented with 2 % F C S at 10 to 10 cells/mL. 6  7  This cell suspension was then  placed carefully on top o f a 225 u M Ficoll solution (Type 400, Sigma) containing 257 m M sodium diatrizoate (Sigma) and centrifuged at 500g for 30 minutes at room temperature. The layer o f cells at the interface between the medium and Ficoll were recovered and washed twice. The cells were then finally re-suspended i n Iscove's modified Dulbecco's medium ( I M D M ) with 2 % F C S or other supplement as required for subsequent use i n direct assays or virus transduction studies.  2.2. Retoviral vectors construction and molecular analysis 2.2.1. Vector construction A l l viral vectors used i n this study were constructed from one o f 2 backbone vectors: M S C V - I R E S - G F P ( M I G , Figure 3.1) and a derivative o f this, referred to here as M S C V ( M ) and created b y removal o f the I R E S - G F P cassette as a Xho-Clal fragment from the M I G 41  vector. MIG contains the L T R sequences of the murine stem cell virus (MSCV) (Hawley et al. 1992), an internal ribosomal entry site (IRES) element (derived from the encephalomyocarditis virus) and the cDNA of the enhanced green fluorescent protein (GFP) (Antonchuk et al. 2001). It was obtained from Dr. K. Humphries (Terry Fox Laboratory, Vancouver, BC). The murine c-kit cDNA encompassing the complete coding sequence was isolated as an EcoRl-Hindlll  fragment (total 3.6 Kbp, with 2.9 Kbp coding sequence) from a plasmid  obtained from Dr. P. Besmer (Memorial Sloan-Kettering Cancer Center, New York, NY). It was inserted by cohesive-end ligation into the multiple cloning site upstream of the 3'-LTR of the M vector to create the MSCV-c-kit (M-KIT) vector (Figure 3.1). The murine flt-3 cDNA (total 3.2 Kbp, with 3.0 Kbp coding sequences) was cut into EcoRl-EcoRl and EcoRI-Xhol fragments, respectively, from a plasmid obtained from Dr. D. Birnbaum (INSERM, Marseille, France). The 2 fragments were then re-ligated and inserted simultaneously into the multiple cloning site (cleaved EcoRl and Xhol sites) of the MIG vector to create the MSCV-Flt-3-IRES-GFP (M-FLT-IG) vector.  The IRES-GFP fragment was  subsequently removed to create a M S C V - F L T (M-FLT) vector (Figure 3.1). A chimeric receptor cDNA composed of the M-CSF receptor (c-fms) extracellular domain and the flt-3 receptor intracellular domain (called fms-flt-3 [FF3]) was obtained from Dr. Lemischka (Princeton University, New Jersey). This cDNA was cut into EcoRl-EcoRl and EcoRl-Xhol fragments, respectively, and was re-ligated and inserted simultaneously into the multiple cloning site (cleaved EcoRl and Xhol sites) of the MIG vetor to create the MSCV-ff3IRES-GFP (M-FF3-IG)) vector (Figure 5.6a).  2.2.2. Vector sequence validation Highly purified (by QIAquickTM column, Qiagen, Missisauga, ON) M-KIT, M - F L T IG and M-FF3-IG vectors were diluted to 100 ng/uL in 10 m M Tris (pH=8.5) solution and used as templates for sequencing.  The Sanger method of sequencing, using fluorescence-  labelled dideoxy-dNTP-mediated chain termination was performed by the NAPS Unit at the University of British Columbia (Vancouver, BC). Complete c-kit, flt-3 and FF3 cDNAs were sequenced.  The results obtained for c-kit  and flt-3 were found to match published data (Qiu et al. 1988; Rosnet et al. 1991) and the data in the expressed sequence tag (EST) database in GenBank® (Boguski et al. 1993). However, a 42  single nucleotide sequence difference (C instead of A) was found in the FF3 cDNA at cDNA sequence #1565 (start count from the first A T G site, located in the transmembrane domain), that would result in a change in the encoded amino acid from Gin to His.  2.3.Transduction protocols 2.3.1. Production of retroviral supernatant Log-phase Phoenix-Eco cells were plated at 3.5 x 10 per 100 mm tissue culture dish in 6  12 mL growth medium and 22 hours later, when the cells were about 80% confluent, 6 mL was removed. At the same time, a transfection mixture was prepared by adding 30 p.g of retroviral vector D N A in 0.1X Tris-EDTA solution (TE, pH=8.0) plus 5 p.g of an ecotropic viral env gene-expressing plasmid (Env-1) and 5 p.g of a gag-pol gene-expressing plasmid (GP3, both obtained from Dr. R. Kay, Terry Fox Laboratory) with 250mM CaCh to give a final volume of 500 (J.L. Addition of the latter 2 plasmids increased the viral titer by approximately 5-fold (see section 4.2.1). This solution was then added dropwise (10 drops of ~ 50 uLper drop) to 500 uL of pre-warmed 2x HBS (pH= 7.05, 50 mM HEPES, 10 mM KC1, 12mM dextrose, 280 m M NaCl and 1.5 m M Na2HP04) with slow shaking to produce a fine D N A precipitate. The resultant mixture was then added immediately to the Phoenix-Eco cells.  These were then  incubated at 37 °C for 12 hours at which time the medium was removed and replaced with 12 mL of fresh growth medium. After a further 16 hours of incubation, the medium was again replaced and finally harvested another 20 hours. This virus-containing supernatant was then filtered through a 45 urn low-protein binding filter (Millex, Millipore Co. Bedford, M A ) and then kept on ice for use within a few hours or frozen and stored at -70 °C until required.  2.3.2. Transduction protocols for cell lines NIH-3T3 and BaF3 target cells were harvested and inoculated at 0.7 x 10 and 10 cells 5  5  per 35 mm well, respectively. NIH-3T3 cells were inoculated 24 hours prior to being exposed to virus-containing medium to allow adherence of the cells to the bottom of the well. BaF3 cells were inoculated immediately before being transduced. The target cells were covered by (in the case of NIH-3T3) or suspended in (in the case of BaF3) at least 0.4 mL of different dilutions of virus-containing supernatant to which 5 u.g/mL protamine sulphate was added and the cells incubated with virus for 4 hours. 4 mL of fresh growth medium was then added and the cells incubated for another 44 hours. 43  2.3.3. Transduction protocol for murine BM cells Isolated day 4 5-FU treated mouse B M cells (see section 2.1.4) were first cultured (prestimulated) for 48 hours without virus in Iscove's modified Dulbecco's medium (IMDM) containing 10 mg/mL bovine serum albumin, 10 mg/mL rh insulin, 0.2 mg/mL iron-saturated human transferrin, (BIT, StemCell), 40 p.g/mL low density lipoproteins (LDL, Sigma Chemicals, St. Louis, MO), 10" M 2-ME (Sigma) and the following cytokines: 300 ng/mL rm 4  SF, 1 ng/mL rh F L , and 20 ng/mL rh IL-11 (mHSC cytokine cocktail) to maximize the stimulation of the HSCs with optimal maintenance of their multilineage differentiation potential (Audet et al. 2002). At the end of 48 hours, the cells were then harvested, centrifuged and re-suspended at 2 to 5 x 10 cells per mL in viral supernatant diluted 1:1 (v/v) in D M E M 6  plus 15% heat-inactivated FCS, 5 |j.g/mL protamine sulphate and same growth factors as used in the pre-stimulation medium the HSC cytokine cocktail. The re-suspended cells were then placed in a volume of at least 0.7 mL in a 60 mm petri dish, that had been pre-coated with 3 p.g/cm fibronectin (Sigma) (overnight at 4 °C) and pre-loaded with viral supernatant (for 2 hour at 4 °C just before the transduction procedure). After 4 hours, 4 mL of fresh D M E M plus 15% FCS containing the mHSC cytokine cocktail were added to the culture and incubation continued for another 12 hours. The non-adherent cells in the transduction dishes were then harvested, centrifuged and re-suspended in a freshly prepared mixture of 1:1 diluted viral supernatant in D M E M plus serum and the mHSC cytokine cocktail. The cells were returned to the same dishes again for an another 4 hours of incubation prior to adding a further 4 ml of D M E M plus 15% FCS and the mHSC cytokine cocktail and incubated another 24 hours (Figure 4.2.). At the end of this time all cells were harvested, washed once in Hank's Balanced Salt Solution plus 2% FCS (HF) and re-suspended as required for in vitro and in vivo studies.  2.3.4. Assessment of gene transfer efficiency in bulk cell populations To assess the efficiency of NIH-3T3 and BaF3 cell transduction, these cells were harvested at the end of the transduction protocol and then analyzed by flow cytometry to determine the proportion of GFP or receptor-positive cells, according to the virus used. For primary B M cells, aliquots of cells were diluted to 5 x 10 cells/ml in D M E M plus 15% FCS 4  plus the mHSC cytokine cocktail and cultured for an additional 24 hours prior to being analyzed by flow cytometry for GFP or transduced receptor expression.  44  2.3.5. Viral titer and helper virus assays Viral titers were determined by measuring the highest dilution of viral supernatant that was still able to transfer expression of GFP (MIG-based virus) or c-kit (M-KIT based virus) to at least 5% of murine NIH-3T3 or BaF3 cells as measured by flow cytometry (see below). To test for the presence of helper virus, supernatant from a confluent virus-transduced NIH-3T3 cell culture (that had been incubated for at least 16 hours) was harvested, filtered and transferred to fresh NIH-3T3 cells for another 16 hours.  Flow cytometric analysis was  performed on the indicator NIH-3T3 cells 48 hours later.  Absence of helper virus was  indicated by failure to detect transfer of GFP fluorescence (or c-kit expression) from transduced target cells to the indicator cells (Cone and Mulligan 1984).  2.4 Jn vitro assays 2.4.1. BaF3 cell proliferation assays BaF3 cells were washed, resuspended in fresh growth medium, and aliquoted at 10  4  cells/well (in U-shaped 96-well plates). Various test growth factors were then added to each well such that the final volume was 0.1 mL/well. After 20 (in IL-3 containing medium) or 38 (in SF or F L containing medium) hours of incubation at 37 °C, the cells were pulsed with 1 uCi of H-thymidine (specific activity of 2 Ci/mmol, Mandel NEN) for 4 or 10 hours, 3  respectively. The D N A of the cells was then harvested onto filter mats using a cell harvester (LKB Wallac 1295-001, Turku, Finland), and the quantity of H-thymidine incorporated into 3  D N A measured in a liquid scintillation counter (LKB 1205 Betaplate).  2.4.2. Primary murine BM hematopoietic cell culture Primary B M expansion cultures were initiated with 5 to 10 x 10 transduced B M cells 3  per mL of culture medium (medium volume varying from 3 to 20 mL depending on the type of assay). The cells were cultured for 4 or 7 days at 37 °C in a humidified atmosphere of 5% C 0  2  in air. A l l expansion cultures were performed in D M E M supplemented with 15% FCS plus 20 ng/mL human IL-11 and test cytokines as indicated. At the end of the incubation period, the cells in suspension were removed and adherent cells were detached either by incubation in 0.5 mL typsin-EDTA for 4 minutes at 37 °C, or by using a cell scraper (Falcon), followed by 2 rinses with D M E M containing 15% FCS. The harvested cells were centrifuged at 350 g for 5  45  minutes, and cell counts performed followed by flow cytometry analysis.  C F C and H S C  assays were undertaken using appropriate aliquots.  2.5.Progenitor and stem cell assays 2.5.1. CFC assays Appropriate dilutions of B M cells were suspended in a solution of 1 % methylcellulose in Alpha medium supplemented with 15% FCS, 1% BSA, 10~ M 2-Me, 10 mg/mL rh insulin, 4  200mg/mL human transferrin (iron-saturated), 2mM L-glutamine (MethoCult, StemCell) supplemented with 50 ng/mL rm SF, 10 ng/mL rm IL-3, 10 ng/mL rh IL-6 and 3 units/mL rh EPO and l . l m L aliquots were then plated in 35 mm petri dishes (StemCell Technologies). Cultures were incubated at 37 °C for 10 to 14 days and the numbers and types of colonies present were then scored as granulocyte-macrophage (from CFU-GM), predominantly erythroid (from BFU-E) or obviously mixed (from C F U - G E M M ) according to standard criteria (Humphries etal. 1981).  2.5.2. CRU assay for HSCs Transplantation procedure B6 mice were given a lethal dose of irradiation (900 cGy, 110 cGy/min,  137  C s y-rays)  and W41 mice a sub-lethal dose of 400 cGy prior to being injected intravenously with test cells. In experiments in which B6 mice were injected with limiting numbers of CRUs (HSCs) in the test cell suspension (<10 CRUs/mouse), 10 freshly isolated normal B6 B M cells were 5  co-injected to ensure the survival of the recipients (Rebel et al., 1994). At defined intervals later, 100-200 uL samples of blood were obtained for analysis (see below) by tail vein puncture. The progeny cells of the injected donor cells were tracked by expression of Ly5.1 and/or GFP or the transduced receptor. Assessment of HSCs in engrafted mice One, 2 and 4 months post-transplantation, the level of engraftment by donor-derived cells (% of cell that are Ly-5.1 , or G F P Ly-5.2 cells) was determined from the assessment of +  +  +  the myeloid and lymphoid cells in the blood of the recipients. For the estimation of the initial transplanted donor HSC numbers, the average level of engraftment in each group of recipients transplanted with same number of donor cells was used to calculate initial donor HSC values. For these calculations, only data from groups with at least 6 mice engrafted with average 5% to 46  66% were used because the relationship between the number of C R U (HSC) transplanted and the output of WBCs per HSC deviates from linearity above the 66% engraftment level and becomes highly variable below 5% (Audet et al. 2001). Donor C R U (HSC) frequencies were determined assuming an average contribution of 8.3% of the circulating WBCs per H S C injected (Audet et al. 2001) (see section 4.2.3). BM transplantations BM  cells were aspirated 6 months after transplantation from the femurs of  anaesthetized primary recipients using a 22 gauge needle.  Approximately 10 aspirated B M 6  cells were transplanted into each sublethally irradiated secondary recipient (W41) mouse. The number of donor-derived CRUs regenerated in the primary recipients was determined by assessing the level of engraftment of the secondary mice with donor-derived WBCs, as described above.  2.6.Flow cytometry and cell sorting Fresh samples of blood, B M and spleen cells were first incubated at a dilution of 1:30 (v/v) in 0.165 M ammonium chloride (StemCell Technologies) for 15 minutes to lyze the erythrocytes.  The remaining nucleated cells were then suspended in HF and incubated  sequentially on ice with 6 \xg/mh of anti-Fc receptor Ab (produced in Terry Fox Laboratory from 2.4G2 hybridoma that had originally been obtained from A T C C ) (Unkeless, 1979) for 15 minutes and then with primary Abs in HF on ice for 30 minutes. The cells were then washed once and stained with secondary Abs (if necessary), washed once again in HF containing 1 p.g/mL propidium iodide (PI, Sigma) and finally re-suspended in 0.5 mL HF. Flow cytometric analysis was performed using a FACSCalibur flow cytometry machine (Becton Dickinson, San Jose, C A ) equipped with CellQuest software (Becton Dickinson). The following monoclonal primary Abs were used as required for the particular analysis to be performed: anti-mouse flt-3 and anti-mouse c-frns (Upstate Biotechnology, Lake Placid, NY), cyanine-5-succinimidylester (Cy5)-conjugated anti-Ly-5.1 (produced in Terry Fox Laboratory); phycoerythrin (PE), biotinylated or allophycocyanin (APC)-conjugated anti-c-KIT ; PE-conjugated anti-mouse flt3,  anti-Mac-1, anti-Gr-1,  anti-CD4,  anti-CD8,  anti-B200,  anti-CDllc,  anti-Sca-1  (Pharmingen, San Diego, CA). PE-conjugated donkey anti-rabbit IgG secondary Abs, control rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated streptavidin were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA).. The optimal working concentrations 47  of the Abs used were determined by prior titration. For each analysis, a minimum of 5,000 events was acquired. Normal (GFP"), receptor-negative, non-specific isotype Ab-stained and unstained cells were used to set up negative gates or quadrants containing over 99% of these cells.  Test cells appearing as events outside of these regions were then defined as positive  when >5 such events were seen. When more than 2 fluorochromes (plus GFP-staining) were used, cells labeled with single fluorochromes were used to set the compensation values. In some experiments, Ab stained and G F P  +  cells were sorted using a FACStar  +  (Becton  Dickinson) equipped with 5 W argon and 30 mW helium neon lasers.  2.7.Statistical methods All results are expressed as the mean + S E M except specified.  Significance of  differences between groups was determined using the two-tailed Student t-test (Sokal and Rohlf 1981) and a p value of 0.05.  The method of least squares and linear and polynomial  regressions were calculated using the Origin ™ version 4.1 computer software program (Micocal, MA).  48  CHAPTER 3: THE EFFECT OF VARIABLE LEVELS OF C-KTT AND FLT-3 RECEPTOR EXPRESSION ON RESPONSIVENESS TO THE COGNATE LIGAND EV A CELL LINE MODEL 3.1. Introduction The question of whether or not HSC self-renewal can be instructively regulated by cytokines has remained highly controversial (see section Results seen after exposure of highly enriched HSC populations to different cytokine cocktails have suggested that high concentrations of SF (in mice) and F L (in human) promote HSC self-renewal independent of their mitogenic activities (Zandstra et al. 1997b). Based on these observations, a ligand-receptor signaling-threshold (LIST) model of stem cell differentiation control has been proposed, which suggests that in addition to the quality (type) of receptor-ligand interaction induced, the quantitative and/or kinetic nature of this interaction may be important to determining the response outcome. This model suggests that the intensity of ligand-receptor signaling must be kept above a set threshold level in order to continuously suppress the differentiation process of HSCs and thereby increase the chance of a self-renewal division occurring (Zandstra et al. 2000). According to this model, if the SF or F L responsiveness of HSCs could be increased, more HSCs would execute self-renewal divisions at lower cytokine concentrations. As a first test of this principle, I have chosen the BaF3 cell line model for studies of cells engineered to express different levels of the c-kit or flt3 receptors. The hypothesis of these studies is that the biological responses induced by these ligands are limited by the numbers of receptors present on the cell surface and hence cells with higher c-kit or flt-3 expression levels will show increased SF or F L sensitivities, respectively.  BaF3 cells are IL-3-dependent  hematopoietic cell line and it had already been shown that they could proliferate in response to SF or F L when engineered to express c-kit or flt-3 ectopically (Dosil et al. 1993).  Such  proliferative responses can be readily quantitated by measuring the amount of H-thymidine the cells will incorporate into their D N A after a brief period of exposure to the growth factor and this was the endpoint I used to compare the SF and F L responsiveness of BaF3 cells engineered to express different levels of c-kit or flt-3 by retroviral transduction.  49  3.2. Results 3.2.1. Construction and validation of c-kit and flt-3 retroviral vectors To study the effect of c-kit or flt-3 expression level on the mitogenic response of BaF3 cells to SF and F L , respectively, 2 retroviral vectors containing the murine c-kit (M-KIT vector) or flt-3 (M-FLT vector) cDNA were first constructed using the MIG vector (Figure 3.1). The lengths of the c-kit and flt-3 cDNAs are 3.7 kb and 3.2 kb, respectively. The proteincoding region is 2.9 kb for c-kit and 3.0 kb for flt-3. Almost all of the non-coding sequence (0.8 kb for c-kit and 0.2 forflt-3)of both cDNAs are located at the 3' end region of the cDNAs. Both receptor cDNAs were placed under the control of the M S C V LTR enhancer and promoter regulatory elements. The molecular structures of M-KIT and M - F L T vector were validated by restriction enzyme digest mapping and complete D N A sequencing, and the sequences of c-kit and flt-3 were the same as those published (see section 2.2.2). Transient M-KIT and M - F L T retrovirus producer cells were generated by transfecting these vectors into the ecotropic Phoenix packaging cell line, and supernatants with a retroviral titer of ~ 2 to 5 x 10 Units/mL 5  routinely obtained. The biological activities of these vectors were confirmed by demonstration of their ability to confer on the transduced BaF3 cells a specific ligand-induced mitogenic activity as described below.  50  1.4 Kb  5'H  MIG (3.2 Kb)  N  1.4 Kb  —i—'•  H  0.4 Kb  1  3  E X AH V  LTR (MSCV)  I I  IRES  LTR (MSCV)  GFP  0.4 Kb  3.7 Kb  1.4 Kb  +  M-KIT (5.5 Kb)  3.2 Kb  1.4 Kb  0.4 Kb H  N  E  _L  _L  E B  N  LTR  c-kit cDNA  LTR  M-FLT (5.0 Kb)  flt3 cDNA  LTR  1  N  S  LTR  Figure 3.1. Retroviral constructs Structure and size of the 3 retroviral vectors used. Only the region between the murine stem cell virus (MSCV) retroviral regulatory L T R element is shown. The rest of the vector is the plasmid backbone, which is about 3.1 kb long and shared by all 3 vectors. The restriction sites that are important for vector identification and construction are as follows: Apal (A), Clal(C), EcoRl (E), Hindlll (H), Nhel (N), Smal (S) and Xhol (X). The structures of each of the vectors were confirmed by restriction enzyme digestion and D N A sequencing. The total length indicated fragment sizes are also shown. IRES, internal ribosome entry sites; EGFP, enhanced green fluorescence protein.  51  3.2.2. Generation of polyclonal and derivative monoclonal BaF3cell populations expressing different levels of c-kit and flt-3 Based on the relative fluorescence intensity exhibited by PE-conjugated receptorspecific Ab-stained cells, 4 polyclonal populations were isolated from both M-KIT and M - F L T transduced BaF3 cells 48 hours after termination of their exposure to virus (labeled A - D in order of increasing fluorescence). A G F P polyclonal population was also isolated from MIG+  transduced BaF3 cells to serve as a control for the transduced cells in each experiment (Figure 3.2). None of these MIG-transduced control cells expressed detectable levels of cell surfaceckit or flt-3 (Figure 3.3, MIG group). Parental BaF3 cells stained with control IgG-PE were used as a negative control to set the gates on the FACS to define positive cells. The relative level of expression of the ectopic receptor in the transduced polyclonal populations was inferred from the mean fluorescence intensity (MFI) of the transduced cells stained with PEconjugated specific Abs.  After maintenance of these isolated populations in vitro in the  presence of 10 ng/mL IL-3 for one week, re-assessment by FACS of anti-receptor Ab-stained cells confirmed the stability of expression of the ectopic receptors in the transduced cells, as shown by their stable MFI values (Figure 3.3).  52  n O N  O  (+)  M I G  •  o  • 11 11 i 11 11 11 11 i 11 11 i  -D (2000-8000) C (500-2000) B (120-500) A (30-120)  n O  LU  IN  M - K I T  •  o  11"11  i ii ii i r  II  II  D (600-1500) C (250-600) B (100-250) : h A (40-100) v  M - F L T  FSC-Height Figure 3.2. FACS analysis and sorting of transduced BaF3 cells Representative FACS profiles of MIG (top), M-KIT (middle) and M-FLT (bottom)-transduced BaF3 cells. Parental BaF3 cells stained with non-specific IgG Ab conjugated with PE were used as a negative control to set the negative region (lower-right quadrant). Anti-murine c-kit Ab and anti-murine flt3-Ab conjugated with PE were used to detect ectopic receptor expression on M KIT and M-FLT-transduced cells respectively. MIG-transduced BaF3 cells do not express detectable levels of c-kit or flt-3 (see Figure 3.3, MIG group). The squared areas in the upper right quadrants represent the gated regions used to define cells with different fluorescence intensities (either due to GFP or PE; actual values shown in brackets) and labeled as A, B, C, D. FCS = forward light scatter and provide an indicator of the relative size of the cells.  53  M - K I T  M - F L T  Ab-PE Figure 3.3.  Ab-PE  Fluorescence profiles of various polyclonal populations of  transduced BaF3 cells  FACS analysis of MIG (top panel) and each of the 4 polyclonal M-KIT or M - F L T transduced BaF3 populations (A to D, see Figure 3.2). Data for cells stained 1 week after isolation is shown. The cells were stained with either PE-conjugated anti-murine c-kit Ab (left panels) or anti-murine flt3 Ab (right panels) and then analyzed by FACS. The region marked by M l denotes the range of fluorescence exhibited by 95% of cells transduced with MIG. The MFI and the range of fluorescence exhibited by 95% of each population (indicated as M2) is shown for each population. 54  Aliquots of each of the 8 polyclonal populations generated were plated at a low density in mefhylcellulose medium (with 10 ng/mL IL-3) to allow the formation of well isolated colonies. These were plucked individually and then further expanded in liquid culture and then subsequently analyzed for their ability to bind anti-c-kit or anti-flt-3 Abs by FACS. Sixteen of these clones (8 expressing c-kit and 8 expressing flt-3) were then selected for further studies, to obtain a series with a >10-fold range of receptor levels (MFI values) and a narrow distribution of fluorescence values within the clone (Figure 3.4). FACS analysis was again performed on these selected clones after a further 2-week period of culture in IL-3-containing medium and the results confirmed that the level of receptor expression in each of these clones was also stable (see Figure 3.4, open symbols).  55  10000 1000 100 10 1000 100 10  H  y  "7  T°  *  #  0*  H  H MIG  •7  y y  tf  o  >  o  o o v> > v>  Figure 3.4. Mean and range offluorescenceintensity of individual clones of transduced BaF3 cells Each ofthe 8 M-KIT (a) and 8 M-FLT (b)-transduced BaF3 clonal populations selected for study were stained with PE-conjugated anti-receptor Abs 1 week (solid symbols) and 3 weeks (open symbols) after isolation and then analyzed by FACS. Each point shown represents the mean ± 2SD of the MFI. MIG control value [cross in the (b)] is also shown. Squares - subclones from the A polyclonal population; diamonds -subclones from B; triangles - subclones from C; circles - subclones from D.  56  3.2.3. Altered mitogenic responses of M-KIT and M-FLT-transduced BaF3 cells To first test whether the normal IL-3 responsiveness of the M-KIT or M - F L T transduced BaF3 cells remained the same, both polyclonal and monoclonal populations of transduced BaF3 cells expressing different levels of receptors were stimulated with IL-3 and their proliferative responses compared to control (MIG)-transduced BaF3 cells. As shown in Figure 3.5a, the 4 polyclonal M-FLT-transduced populations responded to IL-3 in a manner similar to the MIG controls. The same was true for most of the M - F L T clones, although 2 of these showed an unexplained reduced ability to respond to IL-3. Interesting, this behaviour was even more common and more pronounced in the M-KIT transduced cells (Figure 3.5a & b).  57  75  a) Polyclonal cells  MIG  5(H  4"Jfc*  y  M-FLT  'A  25%J*  ijt^—  *J?i  0  I  •  s  i  S  *  -*1  75  M-KIT  •  .  b) M-KIT clones MIG  *.  50  1.4  Q_ U  25  T • T • T  4  J'i T  T  A  M-KIT  75 c) M-FLT clones  f  50 H  { T  MIG  I' • /  25 H j  i - ' j / i  .  T  0 -I 0.001  0.01  0.1  1  IL-3 (ng/ml) 58  10  100  Figure 3.5.  Mitogenic responses of M-KIT and  BaF3 populations  to  M-FLT-transduced  IL-3  10 MIG, M-KIT and M-FLT-transduced polyclonal (a) and clonal (b and c) BaF3 populations were cultured in 0.1 mL of medium containing different concentrations of IL-3 for 24 hours. H -thymidine was added for the last 4 hours of incubation. For M KIT (black) and M - F L T (gray)-transduced cells, squares, diamonds, triangles, circles represent populations derived from populations A , B, C and D. Crosses represent the MIG control values. Populations within an individual transduction group were averaged and were fit with a sigmoidal model by the method of least squares shown by the solid line. Data for the polyclonal populations (a) are representative results from a single experiment (n=2); data from the clonal populations (b & c) represent the average results from 3 independent experiments ± SEM. 4  3  59  Next, these c-kit and flt-3 expressing BaF3 populations were assayed for their ability to respond to their corresponding ligands, SF and F L (Figure 3.6). For the polyclonal populations of both M-KIT and M-FLT-transduced cells, data from population A and B (the cells with the lowest levels of transduced receptor expression) were grouped as "low expresser" (Figure 3.6a, open symbols). Similarly, data from populations C and D (the cells with the highest level of receptor expression) were grouped as "high expresser" (Figure 3.6a, solid symbols). The data for the clonal populations of both M-KIT and M-FLT-transduced cells with different levels of receptor expression were similarly grouped into one that contained the 4 populations with the lowest levels of expression of the transduced receptor (Figure 3.6b, open symbols) and one that contained the 4 populations with the highest levels of expression of the transduced receptor (Figure 3.6b, solid symbols).  The populations expressing the higher levels of c-kit or flt-3  showed some ability to proliferate in response to SF or F L as a substitute for IL-3.  For those  expressing low levels of these receptors, responsiveness to SF or F L assessed in this way was reduced or absent. MIG-transduced control cells showed no response to either SF or F L over the full range of concentrations tested (to 300 ng/mL). Overall, the flt-3-expressing cells were more sensitive to stimulation with F L than the c-kit-expressing cells were to stimulation with SF. Thus, responsiveness to F L was seen when M-FLT-transduced cells were exposed to F L concentrations ranging from 1 to 100 ng/mL, whereas responses of M-KIT-transduced cells required their exposure to >10 ng/mL SF.  60  a) M-KIT polyclonal cells 20  H  c  10 H  D CO  B, A  oiO  b) M-KIT clones 20  B3 10 H  D1, C1 T  0.001  0.01  0.1  1  SF (ng/ml)  61  10  100  D2 B2, B1, A2, A1  Z  1000  c) M-FLT polyclonal 20 H  10  D B, A iX  X  X -X- X i-X  d) M-FLT clones 20  I  C1  A—-4  C2  A  A  10 H  / D1, D2, B1, A3  0 0.001  0.01  #^fa|^44*a*^ A2, A1 0.1  1  FL (ng/ml)  62  10  100  1000  Figure 3.6.  Mitogenic responses of M-KIT and  BaF3 populations to SF and  M-FLT-transduced  FL  10 MIG, M-KIT and M-FLT-transduced polyclonal (a & c) and clonal (b & d) BaF3 populations were cultured in 0.1 mL of medium containing different concentrations of SF (a & b) or F L (c & d) for 48 hours. H-fhymidine was added for the last 10 hours of incubation. For both M-KIT (a & b) and M - F L T (c & d)- transduced cells different symbols represent populations derived from populations with different levels of c-kit or flt-3 receptor expression (squares = A, diamonds = B, triangles = C, circles = D). Crosses represent the MIG control values. Populations within an individual transduction group were separated into high (solid symbols) and low (open symbols) receptor-expressing sub-groups (see text for detail). Data from the polyclonal populations (a & c) are representative results from a single experiment (n=2); data from the clonal populations (b & d) represent the average results from 3 independent experiments ± SEM. 4  3  63  Figure 3.7 shows a more detailed analysis of the mitogenic responsiveness of individual M-KIT and M-FLT-transduced BaF3 cells to specific concentrations of SF and F L , respectively, as a function of their levels of expression of c-kit and flt-3. Although a positive correlation in both cases was anticipated, such a simple relationship was borne out only in the case of the flt-3-expressing cells exposed to low concentrations of F L (Figure 3.7e, 0.2 ng/mL FL).  For the c-kit expressing cells, responses to a relatively low SF concentration appeared  independent of the level of receptor expression on the target cells (Figure 3.7a, 0.2 ng/mL SF). At higher levels of either SF or FL, the responses of the corresponding receptor-transduced cells were best fitted by a negative polynomial regression (Figure 3.7 c, d, f, g and h) indicative of an initial positive correlation with a reduced responsiveness above a certain level of receptor expression.  64  M-KIT  0.3 R=-0.04, p=0.84  a) 0.2 ng/mL SF T  0.2 -  II  0.1 CO  0.0 Q_ O  ?  1  1.5 R =0.19, p=0.09 2  b) 3 ng/mL SF  1.0 ii  0.5 1 1 J_  ^Eil 2^  0.0  1  T •  400  400 MFI  65  1*  800  1200  M-KIT r R =0.29 p=0.02* 2  c) 48 ng/mL SF  T  12-  1 •  CO  lt_dlJlt_j Q. O  18  T •  R =0.24, p=0.03<  d) 300 ng/mL SF  2  12  -400 MFI  66  M-FLT e) 0.2 ng/mL FL T  MFI  67  R=0.69 p<0.0001*  M-FLT 27  g) 48 ng/mL FL  R =0.65, p<0.0001 2  18  T:  o  27 y I  h) 300 ng/mL FL  R =0.73, p<0.0001 2  184  100  •100  MFI  68  200  300  Figure 3.7. Variation in the mitogenic responses of BaF3 cells to SF and FL according to their relative levels of expression of c-kit or flt-3 10 M - K I T ( a to d) and M - F L T (e to h)-transduced clonal populations were cultured in 0.1 m L o f medium containing different concentration of SF (a to d) or F L (e to h) for 48 hours. H-thymidine was added to the culture for the last 10 hours of incubation. The relative level o f expression of c-kit and flt-3 in each clone is indicated by the M F I after staining with anti-receptor specific Abs. The data for each transduced clone was analyzed at 0.20 (a & e), 3 (b & f), 48 (c & g) and 300 (d & h) ng/mL SF or F L concentrations. Data were analyzed by linear or polynomial regression and shown as the solid lines. The correlation coefficient (R) and probability (P) value o f the analysis were shown. M I G controls are shown as the groups with the lowest M F I . 4  3  69  3.3. Discussion The responsiveness of hematopoietic cells to SF or F L is likely to depend on many parameters, including those that determine ligand binding and initiation of signaling cascades as well as many downstream events that mediate the ultimate biological response elicited. These may include survival, proliferation, commitment and differentiation.  As a first step  towards examining the potential role of the level of expression of the receptors for these 2 growth factors on the cell surface, I created a series of BaF3 cell lines in which c-kit and flt-3 were expressed at different levels to allow this variable to be examined against a relatively "homogeneous" cell background. The acquired mitogenic response of these BaF3 cells to different concentrations of SF and F L was then determined. The results confirmed the ability of ectopic expression of c-kit or flt-3 to confer SF and F L responsiveness on these cells and clearly demonstrated that increased expression level of ckit or flt-3 enhances their sensitivity to the corresponding ligand at least at relatively low concentrations of SF and F L , respectively.  However, neither of these receptors was an  efficient substitute for the IL-3 receptor and their ability to replace the role of the IL-3 receptor required a threshold level of c-kit or flt-3 expression. This was demonstrated by the fact that BaF3 cells with the lowest levels of c-kit or flt-3 expression had little or no detectable ability to respond to SF or F L regardless of the concentration present. Interestingly, c-kit was much less effective in replacing the IL-3 receptor than flt-3, in spite of the Ab-staining data suggesting that c-kit was expressed at relatively higher levels on the transduced BaF3 cells. In addition, a negative effect of the highest levels of expression of these receptors on the responsiveness of the cells to high concentrations of SF or FL, respectively, was noted. Although in some cell systems, increased receptor expression has been found to correlate with increased sensitivity to ligand stimulation of mitogenesis (Roberts and Gullick 1989; Watanabe et al. 1989), other findings question the generality of this relationship. For example, neither c-kit nor flt-3 receptor levels on human acute myeloid leukemia (AML) samples correlate with the proliferative response obtained following SF or F L stimulation (Stacchini et al. 1996) and the number of receptors per human umbilical vein endothelial cell does not correlate with the ability of these cells to proliferate in response to SF (Broudy et al. 1994). Cellular expression of TGF-p or TNF receptors also do not necessarily correlate with responsiveness to the cognate ligand (Ruggiero et al. 1987; Hebert and Birnbaum 1989). On the 70  other hand, none of these findings preclude alternative explanations since the cells compared were not necessarily similar in other respects.  The present studies have the advantage that  effect of receptor density could be analyzed on the same cell background, thus circumventing the likely involvement of other differences contributing to the effects seen. The molecular mechanism underlying the reduced responsiveness of highly c-kit (or flt3)-expressing BaF3 cells to SF (or FL) was not further investigated here. However, it may be speculated that this was caused by limitations introduced in the availability of signaling intermediates required for a mitogenic response or an increased activation of pathways leading to apoptosis, e.g. by Ras, as shown in previous studies (Kauffmann-Zeh et al. 1997). It is known that activated Ras proteins can have either positive or negative effects on the regulation of apoptosis.  In part, this is due to the ability of Ras to control directly multiple effector  pathways, including those activated by PI3-K and Raf. PI3-K provides a universal survival signal (through activation of PKB/Akt or NF-kappa B) and Raf can inhibit survival (Downward 1998). Examination of the level of expression inferred from anti-receptor Ab-stained cells showed the MFI for the flt-3-expressing cells to be much lower than those for the c-kitexpressing cells, even though the vectors for flt-3 and c-kit were constructed in a similar manner to try to avoid disparities in expressions for similar integrated copy numbers. Nevertheless, the length of the non-translated region in the 3' end is different for c-kit and flt-3 (c-kit is 500 bp longer than flt-3) and this may have contributed to a difference in transcription rates. Potential differences in the binding efficiencies or PE-labeling of the 2 anti-receptor Abs used would also undermine cross-comparisons between the MFI values generated. Therefore, the strongest comparisons are between different clones expressing the same receptor at different levels. Other methods, such as RT-PCR or real-time PCR, would help to determine the extent of differences in expression of the 2 transduced receptors. In summary, the results obtained with the BaF3 cell model used here support the concept that optimal activation of a particular biological response occurs over a specific range of c-kit/SF or flt-3/FL interactions, above or below which a "suboptimal" response is achieved. These studies also suggest a requirement for the formation of a threshold number of cellsurface cytokine-receptor complexes to determine whether or not a cell can initiate and/or sustain a particular biological response.  In HSCs, such a strategy might serve to alter their 71  self-renewal under conditions of variable SF or F L exposure. The experiments in the following 2 chapters were undertaken to test this hypothesis directly.  72  CHAPTER 4: EFFECT OF OVEREXPRESSING C-KIT OR FLT-3 ON THE IN VITRO SELF-RENEWAL AND DIFFERENTIATION RESPONSES OF PRIMITIVE HEMATOPOIETIC CELLS 4.1. Introduction Previous studies have indicated that expansion of murine HSCs isolated from normal adult BM was maximal when the cells were exposed to very high concentrations of SF (>100 ng/mL) (Audet et al. 2002). This suggested that enhanced HSC self-renewal may be limited by the level of c-kit expressed. Similarly, the enhancing effect of FL (seen only when SF levels are relatively low) required very high concentrations of FL. The present studies were therefore designed to determine whether forced overexpression of either c-kit or flt-3 could be achieved in primitive primary murine BM cells and if so, whether this could affect their selfrenewal/expansion in vitro in response to specific SF or FL concentrations.  73  4.2. Results 4.2.1. Optimization of a protocol for retroviral transduction of primitive murine B M cells Construction of the retroviral vector M-KIT was described in Chapter 3. The retroviral vector M-FLT-IG was constructed by modifying the MIG vector by insertion of the flt-3 cDNA upstream of the IRES-GFP cassette in the MIG vector.  To determine whether the  arrangement and/or inclusion of different regulatory elements in the retroviral vector would allow for more efficient retroviral production and higher transgene expression level in target cells, several additional c-^Y-encoding vectors were constructed and tested (Figure 4.1). It had previously been reported that inclusion of the E F - l a enhancer in the L T R element of the vector would result in an increased retroviral titer and higher level of expression of the transferred gene in a lymphoid cell line system (personal communication, Dr. R. Kay). I therefore first compared the titers of virus-containing supernatants obtained with vectors that included this element using a standard virus production protocol, and then measured the relative levels of ckit-expression in BaF3 cells transduced with the various viruses obtained. The results of these initial experiments are summarized in Table 4.1. They showed that the highest titers and levels of expression of c-kit were obtained with the original M S C V construct. vector was chosen for all subsequent studies.  74  Accordingly, this  MSCV LTR  c-kit cDNA  MSCV LTR  M M E - K I T  MSCV / MMLV LTR  c-kit cDNA  MMLV / E F - 1 a / MSCV LTR  M M E - K I T - s  MMLV / E F - 1 a / MSCV LTR  MSCV / MMLV LTR  c-kit cDNA  SupF  MSCV / MMLV LTR  c-kit cDNA  IRES-GFP  M M E - K I T - I G  MMLV / E F - 1 a / MSCV LTR  Figure 4.1. Structures ofthe retroviral constructs Schematic representation ofthe MSCV-ckit (M-KIT), MSCV / MMLV / EF-la-ckit (MME-KIT) M S C V / M M L V / EF-la-ckit-supF (MME-KIT-s), MSCV / MMLV / E F - l a ckit-IRES-GFP (MME-KIT-IG) retroviral vector plasmids. 5' MSCV / MMLV LTR contains the MSCV enhancer and a hybrid MSCV / MMLV promoter. 3' MMLV / EF-la / MSCV LTR contain hybrid MMLV / E F - l a enhancer and a MSCV promoter. MMLV, Murine Molonary Leukemia Virus; EF-la, Elongation factor-la enhancer; IRES, Internal Ribosome Entry Site; SupF, suppressor tRNA for plasmid selection in E.coli The structure of each vector was confirmed by restriction enzyme digest (data not shown). Only the regions between the retroviral regulatory LTR elements are shown.  75  Table 4.1. Titers of different c-kit retroviruses and the relative level of expression of c-kit in transduced BaF3 cells Retrovirus  Titer (X10 U/mL) 5  MFI  _  a  M-KIT  5, 10  791  MME-KIT  2,4  519  MME-KIT-s  3,5  549  MME-KIT-IG  0.4,1  387  a  The values ranges shown werefrom2 independently prepared supernatants assayed separately on BaF3 cells.  b  Representative MFI values of the c-kit-expressing target (BaF3) cell transduced by each retrovirus are indicated. The MFI values was obtained by the same method as described in Figure 3.3.  76  The protocol used to transduce murine B M cells is shown schematically in Figure 4.2. This protocol involved co-transfecting Phoenix packaging cells with 2 additional vectors, pAX142 Eco-Env-1 and pGP3 (kindly provided by Dr. R. Kay), together with the test vector to enhance retroviral env and gal-pol protein production, respectively.  This modification  increased the titer of the virus obtained 5 to 10-fold when compared to conventional methods (Table 4.2). The transduction efficiency of murine B M cells using this protocol was typically 30 to 90% for the 3 viruses used (MIG, M-FLT-IG and M-KIT, Table 4.2). Evidence of expression of the transduced receptors was obtained from FACS analysis of anti-receptor Ab-stained cells obtained post-transduction (Figure 4.3).  A 2- to 10-fold  higher expression of flt-3 or c-kit could be seen in the transduced B M cells by comparison to the MIG control-transduced B M cells (Figure 4.3).  77  GP3  Env-1  Test Vector  48 h Phoenix-Eco cells  Day 4 5-FU Mouse BM cells  48 h •  * USUI  p_ V coated dish N  Serum free media ^  • virus-containing supernatant  • J  y  1  5  Experiments  %  v -  F  C  S  J  300 ng/mL SF 1 ng/mL FL 20 ng/mL IL-11  Figure 4.2. Protocol for murine B M cell transduction Briefly, high titer retroviral supernatant was produced by the Phoenix-Eco cells, and this supernatant was subsequently used for retroviral transduction of day 4 5 - F U treated mouse B M cells. Detailed descriptions of the high titer retroviral supernatant production and the B M cells retroviral transduction procedure are in section 2.3.1 and 2.3.3, respectively.  78  Table 4.2. Improved retrovirus production and transduction of primitive murine BM cells Transduction group  Titer range (XI0 U / m l ) 5  a  Transduction efficiency of B M cells ( % ) a  MIG  60 - 100  >90  M-FLT-IG  10-30  30-60  M-KIT  10 - 30  40 - 70  The range shown delimits the range of values from > 5 independent retroviral supernatant preparations, titering assays and gene transfer experiments to primary mouse B M cells.  79  Figure 4.3. Detection of transduced receptors on murine B M cells M I G (top), M - K I T (middle) and M - F L T - I G (bottom)-transduced murine B M cells were stained with either anti-c-kit (left panels) or anti-flt3 (right panels) Abs-conjugated with P E and analyzed by F A C S 48 hours post-transduction. Representative F A C S profiles are shown. The squared areas in each profile define the regions in which transduced receptors were detected. The percentage of positive (transduced) cells is shown. G F P cells are also shown on the Y-axis of the profile. +  80  4.2.2. Effect of forced overexpression of c-kit or flt-3 in primary BM cells on total cell expansion in vitro in response to SF and FL stimulation To examine the effect of overexpression of c-kit or flt-3 on the SF and F L responses of murine primary B M cells, cells from 5-FU pretreated mice were transduced and then, immediately post-transduction, 10 cells/mL (without selection of the transduced cells) were 4  cultured for. 4 or 7 days in IL-11 (20 ng/mL) plus various concentrations of either SF or FL. At the end of this time, the total number of viable B M cells was measured and the fold-expansion determined.  As shown in Figure 4.4, there was a sigmoidal relationship between the  concentration of F L or SF added and the change in cell numbers over time, confirming the acquisition of specific responsiveness to both SF (by M-KIT-transduced B M cells) and F L (by M-FLT-IG-transduced B M cells). As found for BaF3 cells transduced with these cDNAs, the M-FLT-IG-transduced cells became more sensitive to F L stimulation than the M-KITtransduced cells to SF stimulation, as shown by their ability to respond to 10-fold lower cytokine concentrations.  81  82  83  Figure 4.4. Effect of overexpression of c-kit and flt3 on total cell expansion in vitro Day 4 5-FU B M cells were transduced with MIG (crosses), M-KIT (triangles) or M FLT-IG (circles) and 24 hours after the second exposure to virus were resuspended in medium (initial cell density about 5 xlO cells/mL) containing 20 ng/mL IL-11 and different concentrations of murine SF (a, b) or human FL (c, d). Total viable cells were counted after 4 (a, c) or 7 days (b, d). Fold-expansion was calculated by dividing the final cell number in the culture by the corresponding initial cell number. Results are reported as mean ± SEM from 3 to 8 independent experiments and the lines shown werefittedwith a sigmoidal model by the method of least squares. * indicates a significant difference (p<0.05) from the MIG value. 3  84  To determine if the expansion of the primary B M cells is cell autonomous, M-KIT or M-FLT-IG-transduced L y 5 . T B M cells were mixed with MIG-transduced Ly5.1" cells at a ratio of 1:4 and then these cell mixtures were cultured as before in IL-11 plus various concentrations of SF or FL. The ratios of Ly5.1 /Ly5.1" cells were then monitored over a +  range of F L or SF concentrations. Figure 4.5.  The results from these experiments are summarized in  In the presence of IL-11 plus increasing concentrations of F L , the ratio of  Ly5.1 /Ly5.1" cells in the mixture of M-KIT and MIG-transduced cells remained relatively +  constant at the initial 1:4 value (i.e., ~20% M-KIT-transduced cells). However, under the same conditions, the ratio of Ly5.1 / Ly5.1~ cells in the mixture of M - F L T and MIG-transduced cells +  increased as a function of the F L concentration from 1:4 to 4:1 (Figure 4.5a). Similarly, when SF was substituted for FL, the ratio of Ly5.1 /Ly5.1" cells remained relatively constant in the +  cultures of M - F L T plus MIG-transduced cells, but increased from 1:4 to 4:1 in the cultures of M-KIT plus MIG-transduced cells (Figure 4.5b). These findings indicate that the responses of the M-KIT and M-FLT-transduced B M cells to proliferate were due to direct stimulation of these cells by SF and FL, respectively.  85  1.0  a)  a>  KIT / M I G  0.5 ]  o FLT/MIG LO  0.0 0.1  H-  o c o  rS  • m—m  1  100  10  SF (ng/ml)  1000  1.0  o a o  B  )  *  m aa  0  FLT / MIG  0.5 -  - A KIT/MIG  0.0 0.1  1  1  1  1  10  100  1000  FL (ng/ml) Figure 4.5. Ligand-dependent competitive expansion of receptortransduced cells M-KIT or M-FLT-IG-transduced Ly5.1 B M cells were mixed with MIG-transduced Ly5.1" in a 1 : 4 ratio to create a mixed population of KIT / MIG (triangles) and FLT / MIG (circles) cells. These populations were suspended in medium (initial cell density of ~ 10 cells/mL) containing 20 ng/mL IL-11 and different concentrations of murine SF (a) or human FL (b). Cells were harvested after 7 days and FACS analyses performed to determine the proportion of Ly5.1 cells in the culture. Results are reported as the averages of 2 independent experiments. +  4  +  86  4.2.3. Effect of forced overexpression of c-kit orflt-3in primary BM cells on progenitor cell expansion in vitro in response to SF and FL stimulation The effect of c-kit or flt-3 overexpression in murine hematopoietic cells on progenitor expansion was also studied in the same experiments used to evaluate effects on total cell expansion. As shown in Figure 4.6, maximum expansion of CFCs in the*M-KIT-transduced population occurred at a 3-fold lower dose of SF (<10 ng/mL) than in either the MIG or M FLT-transduced population (Figure 4.6a). Interestingly, when very high concentrations of SF were present (>100 ng/mL), C F C production in the M-KIT-transduced cells was inhibited, an effect not seen in the cultures of MIG or FLT-transduced cells. After 7 days of incubation, the results were more variable and difficult to interpret. As shown in Figure 4.6b, the production of CFCs by M-FLT-transduced cells was also enhanced at lower F L concentrations by comparison to MIG or M-KIT-transduced cells.  Thus, overexpression of c-kit or flt-3 in  primary B M cells also increased the sensitivity of very primitive cells to proliferate in response to the corresponding ligand.  87  30  .C  a) CFC expansion after 4 days in culture  0.1  1  10  FL (ng/ml)  100  1000  1000  Figure 4.6. Effect of overexpression of c-kit and flt3 on CFC production in vitro Day 4 5-FU B M cells were transduced with M I G (crosses), M - K I T (triangles) or M - F L T IG (circles) and 24 hours after the second exposure to virus, were resuspended in medium (initial cell density ~ 5 x l O cells/mL) containing 20 ng/mL IL-11 and different concentrations of murine SF (a) or human F L (b). The cells were then washed and assayed for CFCs. Fold-expansion in vitro was calculated by dividing the C F C number detected at the end of the expansion culture period by the number of CFCs present in the cells used to initiate the cultures. Results are reported as the mean ± S E M from 3 to 5 independent experiments and the lines shown were fitted with either a sigmoidal or polynomial model by the method of least squares. * indicates a significant difference (p<0.05) from the M I G value. 3  88  4.2.4. Effect of forced overexpression of c-kit or flt-3 in primary BM cells on the expansion of HSCs in vitro in response to SF or FL In a final series of experiments of similar design, I examined the effect of ectopic c-kit or flt-3 expression on HSC expansion in response to IL-11 plus varying concentrations of SF or FL. To quantitate HSC yields in these experiments, the modified method for C R U quantitation validated by Audet et al (Audet et al. 2001) was used. This involved measuring the percentage of donor-derived WBCs in transplanted mice >4 months later and then calculating the number of HSCs (CRUs) transplanted, assuming each contributes, on average, 8.3 ± 0.4 % of the total normalized W B C count. To test the validity of this assumption in the context of the present experiments, the proportion of donor-derived WBCs per HSC was calculated, assuming 1 H S C per 2000 day 4 5-FU B M cells (Szilvassy et al. 1990), and an ~5-fold decrease in HSC numbers by the end of the transduction protocol (Antonchuk et al. 2002). The results, shown in Table 4.3 are consistent with the previously published results for freshly isolated or cultured normal Scal lin" B M cells (Audet et al. 2001). +  89  Table 4.3. Calculated repopulating activity of HSCs present in retrovirally transduced populations Retroviral Vector  Experiment no. (no. of recipient mice)  Ave % donorderived WBC  Estimated HSCs injected per recipient  % donorderived W B C per HSC  a  b  MIG  1 (n=4) 2 (n=4) 3 (n=4) 4 (n=4) 5 (n=4) 6 (n=3)  45 39 41 28 32 22  4.5 4.6 5.0 4.1 4.2 3.0  8.1±1.1  M-KIT  1 (n=4) 2 (n=3) 5 (n=4) 6 (n=3)  43 33 36 21  5.0 4.1 4.2 3.0  8.1+0.7  3 4 5 6  53 42 25 14  4.5 4.6 4.2 3.0  7.9+3.2  M-FLT-IG  (n=4) (n=3) (n=4) (n-3)  The average % of Ly5.1 cells in recipients at 4 months post-transplant. The estimated number of HSCs injected per recipient was based on the assumption that 1 H S C would be present per 10 initial day 4 5-FU B M cell at the end of the transduction protocol (i.e., a 5-fold decrease from 1 per 2000 cells). Results are expressed as the mean ± S E M of values calculated from 3 independent experiments.  a  +  b  4  90  I next determined the proportion of HSCs that had actually been transduced to see if the gene transfer values obtained for the total B M cells (Table 4.2) would extrapolate to the H S C compartment.  Accordingly, some mice were transplanted with freshly transduced cells (24  hours after the last exposure to virus) and then assessed 4 months post-transplant to determine the percentage of G F P or c-kit cells within their Ly5.1 (donor-derived) WBCs. As shown in +  +  +  Figure 4.7a, these values ranged from 38% (M-FLT) to 56% (M-KIT) to 85% (MIG). These high levels of gene transfer to HSCs suggested that it might be unnecessary to sort for the transduced subset to see significant effects on cytokine enhanced HSC expansion in vitro.  91  a) After culture 1 0 0  7 5  5 0  2 5  0  b) Before culture 1 0 0  -  7 5  -  5 0  -  2 5  -  0  MIG  M-KIT  M-FLT-IG  Figure 4.7. Gene transfer efficiencies to HSCs Peripheral blood from mice transplanted with transduced Ly5.1 B M cells before (a) or after (b) being culture for 7 days in various conditions were assessed 4 to 6 months later by FACS to estimate the proportion of WBCs that were derived from transduced HSCs present in the input inocula. Percentages of MIG (white bars) and M-FLT-IG (gray bars) transduced HSCs were estimated by assessment of the % GFP cells in the Ly5.1 (donor-derived) WBCs. The percentage of M-KIT (black bars)-transduced HSCs was estimated by assessment of the % c-kit cells in the Ly5.1 WBCs. Results are reported as mean ± SEM from 3 independent experiments. +  +  +  +  +  92  To determine whether overexpression of c-kit or flt-3 in HSCs might alter the distribution of lineages represented amongst their W B C progeny generated in vivo, FACS analysis of blood samples stained with 5 commonly used markers of mature WBCs (B220, CD4, CD8, Mac-1 and Gr-1) was undertaken. The percentage of transduced donor-derived cells expressing each of the 5 markers is shown in Figure 4.8. These analyses showed no significant deviations between the differentiated cells produced in vivo by M-KIT or M - F L T transduced HSCs and the control HSCs either before (or after, see below) culturing the cells.  93  J2  100 -  8 75  CQ  Q_ 50 +  £ 25 0 O  "g 100 c  b) After culture M KF  re  is 75 5  c  ^  50 25 0 B220  CD4  CD8  Mac-1  Gr-1  Antibody used Figure 4.8. Comparison of the lineage distribution of WBCs produced by receptor and control-transduced HSCs after their transplantation into mice Peripheral bloodfrommice transplanted with transduced Ly5.1 BM cells before (a) or after (b) being cultured for 7 days in various conditions were analyzed 4 to 6 months later by FACS to evaluate the numbers and types of mature cells represented. Cells were stained with anti-B220, CD4, CDS, Mac-1 and Gr-1 Abs conjugated with PE and the proportion of positive cells co-stained with anti- Ly5.1 and anti-c-kit or that were GFP was determined. Result for MIG (M, white bars), M-FLT-IG (F, gray bars) and M-KIT (K, black bars)-transduced cells are reported as mean ± SEM from 3 independent experiments. +  +  +  94  Transduced (but unselected) B M cells were cultured for 7 days in medium containing IL-11 (20 ng/mL) and SF (at 10 or 300 ng/mL) or FL (at 1 or 10 ng/mL), and the extent of HSC expansion (relative to their numbers immediately post-transduction) was determined as described in Figure 4.9 and the details of calculation are shown in Table 4.4 and Table 4.5. In the presence of 10 ng/mL SF, HSC numbers in the MIG-transduced control cultures declined a further 7-fold similar to what has been reported in non-transduced HSCs (Audet et al. 2002) and with 300 ng/mL SF present, HSC numbers were preserved at close to those measured immediately post-transduction. In the parallel cultures containing the M-KIT-transduced cells, significantly higher (p <0.05) HSC numbers were obtained in both cases (by a factor of 2- to 4fold) (Figure 4.10a).  Since only -30% of the HSCs harvested from these cultures were  overexpressing c-kit (Figure 4.7a), the expansion of the M-KIT-transduced HSCs can be calculated to have been enhanced ~ 7-fold at 10 ng/mL SF and ~3-fold at 300 ng/mL SF. Similarly, in cultures containing 20 ng/mL IL-11 plus 1 ng/mL FL, control HSCs declined ~10-fold, and with 10 ng/mL FL, HSC numbers could be preserved; again consistent with previously published data (Audet et al. 2002). However, when M-FLT-transduced HSCs were present, the total number of CRU was ~6-fold higher (p <0.05) with 1 ng/mL FL present and ~1.5-fold higher with 10 ng/mL FL (Figure 4.10b). However, since the frequency of flt-3 CRU in these cultures was only -30% (Figure 4.7a), the corresponding enhanced expansions of the flt-3 HSCs would have been 20-fold and 2-fold, respectively. These findings suggest that +  enhanced sensitivity of HSCs to SF or FL can also be achieved by ectopic overexpression of either c-kit or flt-3, respectively.  95  +  5-FU BM cells Transduction Test culture fl  7 days Different proportions of pooled test cultures  Defined aliquots of transduced BM cells  Irradiated recipients (n>3)  Irradiated recipients (n>3)  > 4 months  > 4 months  Only recipient groups with average donor WBC engraftment levels between 5 and 70% used to calculate final HSC frequencies  Only recipient groups with average donor WBC engraftment levels between 5 and 70% used to calculate initial HSC frequencies  HSC expansion =  Final HSC frequency Initial HSC frequency  Figure 4 . 9 . Method used to calculate the extent of HSC expansion achieved in vitro See Table 4.4 and Figure 4.10 for detailed explanation. 96  Table 4.4. Data used to calculate the expansion in vitro of M-KIT-transduced HSCs cultured with different concentrations of SF Experimental group  Cytokine (ng/mL)  Experiment n o . (no. of recipient mice)  a  No. of starting equivalent cells injected per mouse (X 10 ) 5.0 4.1 3.0 4  3 (n=4) 4 (n=4) 6 (n=3)  MIG pre-culture  Estimated HSC frequency (per 10 cells)  c  3 (n=6) 4 (n=5) 5 (n=4)  12.6 20.6 7.1  9±6 18 ± 7 9±4  0.1 0.1 0.2  MIG post-culture  SF (300)  4 (n=6) 5 (n=6) 6 (n=6)  4.1 2.7 3.8  21 ± 6 17±6 19 ± 5  0.6 0.8 0.6  (n=4) (n=4) (n=4) (n=3)  5.0 4.1 4.2 3.0  43 ± 8 33 ± 7 36 ± 6 21 ± 12  1.0 1.0 1.0 0.9  3 4 5 6  M-KIT post-culture  SF (10)  3 (n=6) 4 (n=5) 5 (n=4)  12.6 20.6 7.1  38 ± 13 49 ± 15 27 ± 8  0.4 0.3 0.5  M-KIT post-culture  SF (300)  4 (n=6) 5 (n=6) 6 (n=6)  4.1 2.7 3.8  50 ± 13 37 ± 11 36 ± 9  1.4 1.6 1.5  A total of 6 independent experiments were performed (no. 1 to 6)  Values are related to the uncounted derivative cells from the number of initial 5-FU B M cells shown. Donor-derived cells were detected as Ly5.1 cells and were measured 4 months post-transplant. +  H S C frequencies were calculated per 10 starting 5-FU B M cell equivalents assuming 1 HSC on average reconstitutes 8.3% of the WBCs present 4 months post-transplant.  d  1.0 0.8 0.9  SF(10)  b  0  d  4  MIG post-culture  M-KIT pre-culture  a  b  Donorderived WBCs (Ave. ±SD) 41 ± 2 1 28 ± 7 22 ± 6  4  97  Table 4.5. Data used to calculate the expansion in vitro of M-FLT-IG-transduced HSCs cultured with different concentrations of FL Experimental group  Cytokine (ng/mL)  Donorderived WBCs (Ave. ±SD)  Estimated HSC frequency (per 10 cells)  1 (n=4) 2 (n=4) 5 (n=4)  No. of starting equivalent cells injected per mouse (X 10 ) 4.5 4.6 4.2  45 ± 1 2 39 ± 15 32 ± 10  1.2 1.0 0.9  Experiment no. (no. of recipient mice)  a  c  d  4  4  MIG pre-culture  MIG post-culture  FL(1)  5 (n=3) 6 (n=3) 7 (n=3)  7.1 7.5 7.5  5±2 6±3 7±2  0.1 0.1 0.1  MIG post-culture  FL (10)  1 (n=4) 2 (n=6) 5 (n=4)  1.7 1.8 1.1  18 ± 7 10 ± 5 17 ± 10  1.2 0.6 1.9  1 2 5 6  (n=4) (n=3) (n=4) (n=3)  4.5 4.6 4.2 3.0  53 ± 15 42 ± 15 25 ± 8 14±6  1.4 1.1 0.7 0.6  M-FLT-IG pre-culture  M-FLT-IG post-culture  FL(1)  5 (n=3) 6 (n=3) 7 (n=3)  7.1 7.5 7.5  27 ± 13 21 ± 5 17 ± 7  0.5 0.3 0.3  M-FLT-IG post-culture  FL (10)  1 (n=4) 2 (n=6) 5 (n=6)  1.7 1.8 1.1  35 ± 6 27 ± 8 13 ± 3  2.5 1.8 1.4  a  A total of 6 independent experiments were performed (no. 1 to 6)  b  Values are related to the uncounted derivative cellsfromthe number of initial 5-FU BM cells shown.  0  Donor-derived cells were detected as Ly5.1 cells and were measured 4 months post-transplant. +  H S Cfrequencieswere calculated per 10 starting 5-FU BM cell equivalents assuming 1 HSC on average reconstitutes 8.3% of the WBCs present 4 months post-transplant. d  4  98  a) CRU, after 7 days in culture 2.0 H  KIT MIG  1.0 H  KIT MIG  0)  D) C o.o (0  o  *  I  1  1  T  .X.  1  10  SF (ng/ml)  I  300  o 2.0  1.0  0.0 10  FL (ng/ml) Figure 4.10. Effect of overexpression of c-kit and flt3 on the maintenance of HSCs in vitro Day 4 5-FU B M cells were transduced with M I G (white bars), M - K I T (black bars) or M F L T - I G (gray bars) and 24 hours after the second exposure to virus were resuspended in medium (initial cell density ~ 4 x l O cells/mL) containing 20 ng/mL IL-11 and different concentrations of murine SF (a) or human F L (b) for 7 days. Aliquots of the cultured cells were then transplanted into sublethally irradiated W41 recipients. The H S C content was estimated from the level of donor-derived blood cells present 4 to 6 months post-transplant (assuming 1 H S C generates 8.3 % of the reconstituted WBCs). The fold-expansions were calculated by dividing the H S C number detected at the end of the culture period by the H S C number present in the cells used to initiate the cultures. Results are reported as mean ± S E M from 3 independent experiments. * indicates a significant difference (p<0.05) from the M I G value. 3  99  4.3. Discussion Previous in vitro HSC expansion studies have indicated that high concentrations of either SF or F L in the presence of an intermediate concentration of IL-11 are associated with enhanced HSC self-renewal (Audet et al. 2002). In Chapter 3, I demonstrated that an increased level of expression of c-kit or flt-3 in BaF3 cells could increase the ability of these cells to mount a proliferative response to the cognate ligand.  I therefore hypothesized that  overexpression of c-kit or flt-3 in HSCs might similarly enhance the in vitro responsiveness of these cells to the same ligands.  Since optimal expansion in vitro of normal H S C requires  exposure to very high levels of SF or FL, it seemed likely that this response might be limited by the concentrations of SF or F L that are practical to test and engineering such cells to produce higher levels of c-kit or flt-3 might allow even higher HSC expansions to be achieved at lower SF or F L concentrations. In this chapter, a highly efficient retroviral transduction method (giving 30 to 90% gene transfer to HSCs) was used to overexpress c-kit or flt-3 in primitive hematopoietic cells. This method then made it possible to examine the effects of increased c-kit and flt-3 expression on the SF or FL-dependent regulation of HSC responses. were assessed.  Two strategies to optimize viral titres  One was to co-transfect the packaging cells with 2 other retroviral protein  expression plasmids (one encoding the retroviral env and one the gal-pol proteins).  This,  together with the test M S C V plasmid, resulted in a 5-fold increase in retroviral titers. The second was to evaluate the use of different promoter or enhancer elements in the viral LTR. As shown in Figure 4.1, 3 different retroviral vectors with hybrid M S C V / M M L V 5' and M M L V / E F - l a / M S C V 3' LTRs were constructed. However, from the viral titers measured and transgene expression levels obtained in transduced BaF3 target cells (Table 4.1), the original M S C V vector proved to be superior with no indication of a benefit from any of the hybrid LTRs tested. The functionality of the transduced c-kit and flt-3 receptors in primitive primary hematopoietic cells was first demonstrated by comparing their responses to SF and F L (together with 20 ng/mL IL-11) in short term (4 to 7 day) cultures using either total cell or C F C amplification as the endpoint. Previous studies have shown that the effective stimulation of primitive cells requires synergism between the downstream signaling effects of 2 or more activated receptors. In the present experiments, it was desirable to select a cytokine that would 100  interact strongly with either SF or F L to show effects on HSCs as well as CFCs and total cells. The use of 20 ng/mL IL-11 had been suggested from previous studies (Audet et al. 2002) to be suitable for this purpose. The specific cytokine dose-response curves generated for c-kit and flt-3-overexpressing cells provided clear evidence of an acquired increased receptor-specific sensitivity to SF (by the M-KIT-transduced cells) and F L (by the M-FLT-transduced cells). Moreover, this conferred on these cells a selective growth advantage in vitro so that they rapidly outgrew co-existing control-transduced cells when mixtures of these cells were cocultured under conditions designed to specifically favour the receptor-expressing cells. Interestingly, this increased cytokine sensitivity was accompanied, at least in the case of the ckit-transduced cells, by a depressed responsiveness to very high levels of the corresponding ligand (100 to 300 ng/mL SF), a phenomenon also seen in the BaF3 cell model (Chapter 3). It should also be noted that the effects seen in these experiments would be underestimated since the transduced cells were not isolated at the beginning of the culture when they would have constituted from only 30% to 90% of the cells present (Table 4.2). To determine whether similar effects might also be demonstrable in HSCs, an experimental design that could maximally display differences between control and test conditions under a limited (1 or 2) conditions was required. I chose a 7 day culture period to ensure that the cells had sufficient time to complete several divisions (based on a 24 hour cycle) of HSCs (C. Eaves, personal communication) but not longer because this inevitably leads to an increasing loss of HSCs (Antonchuk et al. 2001). The doses of SF and F L selected were chosen because they had been previously found to cover the range of suboptimal to optimal stimulation of HSC amplification by normal cells in vitro. Consistent with the total cell and C F C expansion data, HSCs expressing artificially increased levels of c-kit (or flt-3) appeared able to execute more self-renewal  divisions at lower levels of SF or F L  concentrations (in synergy with 20 ng/mL of IL-11). This effect persisted at the high SF concentration, but was of reduced magnitude so that overall a significant net amplification of HSCs ex vivo was not achieved using this strategy. Nevertheless, it may be of interest to future bioprocess applications in terms of reducing the amounts of cytokines required and alleviating HSC losses due to culture-related decreases in cytokine concentrations (Zandstra et al. 1997a). Previous studies demonstrated that F L supports the proliferation of early murine B-cell progenitors whereas  SF also  supports the growth of myeloid, erythroid, mast and  101  megakaryocyte progenitors (reviewed in (Galli et al. 1994; Lyman and Jacobsen 1998)). However, numerous other studies of murine B M cells forced to express various types of hematopoietic growth factor receptors have shown acquired alterations in their proliferation control but not in their pattern of differentiation (Lu et al. 1996; Lu et al. 1998; Stoffel et al. 1999; Yan et al. 1999) (Krosl 1997). The findings reported here showed no effect of overexpression of c-kit or flt3 on the differentiation of the transduced cells, nor was there any evidence of perturbation in their numbers generated in vivo (Table 4.3, Figure 4.8). In summary, using a protocol that enabled a large proportion of HSCs to be transduced and resultant effects on their cytokine responses in vitro could then be assessed without prior selection of the transduced cells. Receptor overexpression resulted in a significant increase in ligand sensitivity for all endpoints measured (total cell, C F C and H S C expansion).  The  expansion of HSCs was assessed by quantitative in vivo transplant assays and this expansion of the H S C compartment was not accompanied by any perturbation of the mechanisms that regulate their output of different types of WBCs. However, induced expression of high levels of the c-kit or flt-3 receptors depressed responsiveness to very high SF or F L concentrations such that no significant further amplification of HSCs in vitro was achieved.  102  CHAPTER 5: THE ROLE OF C-KIT AND FLT-3 IN HSC EXPANSION IN VTVO 5.1. Introduction Previous studies have shown that large (up to ~100-fold) expansions of HSCs can be obtained in transplanted irradiated mice (Osawa et al. 1996b; Pawliuk et al. 1996). Endogenously produced SF and F L likely contribute to the support of this process since inactivating mutations  of either  of these  factors  or their receptors  inhibit H S C engraftment  (Mackarehtschian et al. 1995; Miller et al. 1997). In addition it has been reported that radiation treatment causes blood levels of SF to increase (Hunt et al. 1992). However, whether H S C expansion in vivo is limited by the innate ability of these cells to respond to SF and F L is not known. In Chapter 4,1 showed that HSCs could be efficiently engineered to overexpress c-kit or flt-3 and that this sensitized them to respond in vitro to lower concentrations of SF and F L respectively. It was therefore of interest to determine whether such engineered HSCs would behave differently from normal HSCs in vivo due to their enhanced sensitivity to the levels of SF and F L present in the mice at different times post-transplant. The results presented in Chapter 4 also demonstrated that the numbers and types of mature WBCs produced by c-kit and flt3-transduced HSCs in vivo were not significantly altered. However, it is possible that there was an effect on HSC expansion but this was not manifested as an increase in mature cell output.  Second, it is possible that, in vivo, transcription of the receptor transgene was  spontaneously shut down (or mutated). Finally, it is possible that the concentrations of SF and F L in the B M micro-environment are already sufficient to maximize the self-renewal response of normal HSCs in which case an increased sensitivity to these cytokines would not offer any advantage.  The experiments described in this chapter were designed to investigate these  possibilities. An in vivo competitive reconstitution protocol was used to determine the relative engraftment potential of test versus control HSCs and evaluate if and how this might change from the input ratios of test and control HSCs over an 8-month period. As an adjunct to this experiment the magnitude of in vivo expansion of the test and control HSCs in the primary mice was also compared (in secondary transplant experiments). To demonstrate if functional receptors continued to be produced by the transduced HSCs, B M cells were harvested from the primary recipients and then tested in an in vitro proliferation assay to determine whether they had retained (or lost) their heightened sensitivity to SF (or FL). Finally, in vivo SF injections 103  were performed in an attempt to provide exogenous stimulation of the receptor-overexpressing HSCs in vivo. To overcome the possible problem that injected cytokines are cleared rapidly in vivo, a chimeric receptor FF3 consisting of the extracellular domain of c-fms (the M-CSF receptor) and the intracellular domain of flt-3 was constructed and then used to transduce murine B M cells. This FF3 chimeric receptor was expected to provide an opportunity to assess engineered H S C responses to the high levels of endogenous M-CSF production that can be induced by injection of bacterial lipopolysaccharide (LPS).  104  5.2. Results 5.2.1. In vivo competitive reconstitution experiments Figure 5.1 shows a schematic outline of the design of the long-term competitive in vivo experiment used in this study. B M cells from 5-FU-treated mice and then transduced with either the M-KIT or M-FLT-IG virus were mixed in a ratio of 1:4 with simultaneously MIGtransduced cells to create MIG/MIG, KIT/MIG and FLT/MIG populations. In each case the "test" cells were Ly5.1 and the "competitor" cells and recipients were Ly5.2 . +  +  The cell  mixtures were made immediately post-transduction and then transplanted into sub-lethally irradiated W41 recipients. The ratios of Ly5.1 :Ly5.1"GFP cells in the peripheral blood as +  +  determined by FACS analysis were then monitored over the subsequent 8 months. A n example for each of the cell combinations tested at the 4-month post-transplant time point is illustrated in Figure 5.2  105  Day 4 5-FU mouse BM cells (Ly5.1 cells)  Day 4 5-FU mouse BM cells (Ly5.2 cells)  +  4 days  +  MIG, M-KIT or M-FLT-IG transduction  ~ 9 HSCs (Unsorted)  MIG 4 days transduction ~ 36 HSCs (Unsorted)  mixed populations inject into sublethally irradiated W41 recipients (Ly5.2+)  Peripheral blood samples collected 1, 2, 4 and 8 months post-transplant for FACS analyses Secondary BM transplantation Figure 5.1. In vivo competitive reconstitution experimental design 106  Figure 5.2. FACS analysis of WBCs from recipients of mixed populations of control and receptor-transduced BM cells MIG, M-KIT or M-FLT-IG-transduced Ly5.1 B M cells were mixed with MIG-transduced Ly5.1" B M cells in a 1 : 4 ratio to create the MIG vs M I G (top), M-KIT vs M I G (middle) and M-FLT-IG vs M I G (bottom) mixed populations (9 X 10 test cells + 36 X 10 control cells, initial ratio = 0.2). These populations contained approximately 45 HSCs (9 test: 36 control HSCs) and were injected into sublethally irradiated W41 recipients. WBCs were stained with anti-Ly5.1 Ab conjugated with Cy5 for F A C S analysis. Representative FACS profiles at 4 months post-transplant are shown. The two dash-squared areas in each profile represent the regions of Ly5.1 and Ly5.1" G F P cells used to determine the relative contribution of the test and control-transduced HSCs to the hematopoietic system of the recipients. 4  4  +  107  +  As shown in Figure 5.3, the ratio of test cell-derived WBCs to control cell-derived WBCs in the primary recipients of all combinations of transduced cells was similar to the initial value over the 4-8 months of follow-up (4 months for the MIG vs MIG controls, 8 months for the other combinations).  To determine whether the observations made on the  mature WBCs produced mirrored the ratios of regenerated HSCs, B M cells were harvested from the primary mice at 4 to 6 months post-transplant and injected into secondary W41Ly5.2 recipients. As shown in Figure 5.4, the ratio of test cell-derived WBCs produced in the +  secondary recipients 4 months later was similar to the ratio of the same genotypes of WBCs in the primary recipients. From the level of test cell-derived WBCs obtained in the secondary recipients, and a knowledge of both the fraction of primary B M cells injected per secondary recipient (assuming a total mouse B M cellularity of 10 cells) and the number of transduced HSCs originally 8  transplanted, it was possible to calculate the net expansion of transduced HSCs in the primary recipients. These calculations showed that the self-renewal behaviour of the transduced-HSCs was intact with no differences between the test and control groups (Table 5.1).  108  0.50  MIG vs MIG - transduced HSCs  T i m e p o s t - B M T (months) 109  Figure 5.3:  Time course study of the  competitive reconstitution of  primary recipients of receptor-transduced cells  MIG, M-KIT or M-FLT-IG-transduced Ly5.1 B M cells were mixed with MIG-transduced Ly5.1" in a 1 : 4 ratio to create mixed populations of MIG vs MIG (hollow bar), M-KIT vs MIG (black bar) and M-FLT-IG vs MIG (gray bar) (9 X 10 test cells + 36 X 10 control cells, initial ratio = 0.25). These populations contained approximately 45 HSCs (9 test: 36 control HSCs) and were transplanted into sublethally irradiated W41 recipients. WBCs collected 1, 2, 4 and 8 months post-transplant were stained with anti-Ly5.1 Abs conjugated with Cy5 for FACS analysis. The measured ratios of Ly5.1 : Ly5.1" G F P W B C s in the primary recipients are shown. Results for the 1, 2 and 4-month time point observations are reported as mean ± SEM of values from individual mice (n=7) from 2 independent experiments. Results for the 8-month observations are reported as mean ± S E M from a single experiment (n=4). +  4  +  110  4  +  + Q. LL  o  LO  CD  >> Q. c IO  CD O  o o (0  vs MIG  MIG  KIT  vs  MIG  FLT  vs  MIG  Figure 5.4: Competitive HSC-reconstituting activity of receptortransduced HSCs in primary recipients assessed by transplantation into secondary recipients Secondary B M transplantations were performed with cells harvested (6 months posttransplant) from recipients of the primary transplants from mice described in Figure 5.3. WBCs from the secondary recipients were collected 4 months post-transplant for determination of the ratio of L y 5 . 1 : Ly5.1" G F P cells. Results are reported as mean ± S E M of values for 9 mice from a single experiment. MIG vs MIG (hollow bars), M-KIT vs MIG (black bars) and M-FLT-IG vs MIG (gray bar). +  +  Ill  Table 5.1: Calculated expansion of transduced HSCs in primary mice Transduction  No.  group  of initial HSCs No.  generated  injected  a  MIG  of final HSCs  HSC expansion  0  1.0  125 ±51  -26  5.9 ±0.4  180 ±40  -31  5.1 ±1.3  157 ±92  -31  4.9  ±  . (n=4) M-KIT  (n=4) M-FLT-IG  (n=3)  The number of initial HSCs transplanted was calculated by dividing the level of Ly5.1 engraftment at 4 months post-transplant by 8.3, based on the assumption that output of a single H S C is 8.3% of the total WBCs. Results are shown as mean ± S E M .  a  The final number of HSCs regenerated in the primary recipients by 4 months posttransplant of the HSCs originally injected was determined by dividing the Ly5.1 engraftment in the secondary recipients at 4 month post- transplant by 8.3%. This is based on the assumption that an 8.3% contribution of each regenerated H S C to the W B C count and the fact that approximately 1% of the B M cells from primary recipients was transplanted (i.e. 10 cells out of a total of ~ 10 ). Results are shown as mean ± S E M . b  6  c  8  H S C expansion = (number of initial donor HSCs) / (number of final donor HSCs)  112  To investigate whether the transduced receptor genes continued to be expressed and remain functional in the cells regenerated in primary recipients, B M cells were harvested from the primary recipients and tested in vitro for persistent increased sensitivity to SF or FL. As shown in Figure 5.5, B M cells from recipients of M-FLT-IG-transduced cells were more sensitive to the mitogenic action of F L than those from recipients of MIG-transduced cells. Similarly, B M cells harvested from recipients of M-KIT-transduced cells were more sensitive to the mitogenic action of SF than those from recipients of MIG-transduced cells.  113  10  d) c  0 100  10  0.1  1000  SF (ng/ml) •  2  o  2  b)  1J  - - - X v« -  0 0.1  1  100  10  1000  FL (ng/ml) Figure 5.5: Cytokine sensitivities of BM cells from mice engrafted with receptor-transduced HSCs B M cells from mice that showed high levels (>50%) of donor-derived WBCs 4 months post-transplant were suspended at a density of ~10 cells/mL in medium containing 20 ng/mL IL-11 and different concentrations of murine SF (a) or human F L (b). Total viable cells were counted after 4 days. The fold-expansion was calculated by dividing the final number of viable cells in each culture by the number used to initiate the cultures. Results shown are the averages of 2 independent experiments, with recipients of MIG-transduced cells (crosses), M-KIT-transduced cells (triangles) or M-FLT-IG-transduced cells (circles). 5  114  5.2.2. Effect of in vivo injection of stimulatory factors In a final series of experiments, recipients of M-KIT-transduced cells were injected intraperitoneally every other day for 2 weeks (6 times in total) with 10 mg of mSF and the ratio of Ly5.1:Ly5.1GFP WBCs before and after were compared. As shown in Table 5.2, the +  injected SF gave no competitive advantage to the M-KIT transduced cells. Large quantities of F L were not available for injection, necessitating a different strategy be used to try to selectively promote the growth in vivo of M-FLT-transduced cells. This was achieved by creation of a vector encoding a chimeric receptor composed of the extracellular domain of the M-CSF receptor (c-fms) and the intracellular domain of flt3 (called fms-flt-3, FF3) (Figure 5.6a). The substituted ligand binding domain was used to allow the transduced cells to respond to the elevated levels of endogenously produced M-CSF that are maintained over 24 hours after LPS injection in vivo. The structure of the FF3 vector was confirmed by restriction digestion analysis and also by D N A sequencing. This revealed an altered nucleotide in the FF3 receptor as compared to the published sequence in the GenBank Database (see Section 2.2.2). Nevertheless, expression of the chimeric receptor encoded in the FF3 vector could still be detected by staining transduced cells with an anti-c-fms Abs (Figure 5.6b) and a mitogenic response of FF3-transduced BaF3 cells to M-CSF could also be demonstrated (Figure 5.6c). Therefore it was assumed that the altered amino acid in the transmembrane domain region of the chimeric receptor did not have a significant impact on its functional properties. Ly5.1 B M cells were then transduced with M-FF3-IG and again mixed with MIG +  transduced Ly5.1" cells in a ratio of 1:4. The mixed population of FF3/MIG cells thus created was then injected directly into mice to compare their relative contributions to the WBCs produced in the same type of competitive transplantation experiment used for the other vectors. Table 5.2 shows the ratios of Ly5.1 /Ly5.1"GFP WBCs present in these recipients 4 months +  post-transplant  +  Oust prior to starting the LPS  injections - every other day for 2 weeks) and 2  and 30 days after the last LPS injection. These showed no significant deviation at either time point from the original 1:4 ratio in the cells transplanted.  115  Table 5.2:  Level of engraftment in recipients of mixture of receptor and MIG- transduced cells before and after injection of SF or LPS  Group  Injected"  Mouse#  M-KIT vs MIG  SF  1 2 3  0.39 0.11 0.24  0.36 (0.32) 0.09 (0.9) 0.26 (0.22)  PBS  1 2 3  0.24 0.45 0.08  0.23 0.41 0.06  LPS  1 2 3 4  0.37 0.11 0.13 0.23  0.36 (0.35) 0.08 (0.12) 0.12(0.11) 0.20 (0.21)  PBS  1 2 3 4  0.13 0.17 0.11 0.17  0.11 0.17 0.13 0.16  M-FF3-IG vs MIG  Ratio of L y 5 . 1 / L y 5 . 1 ( G F P ) After injection Before injection +  +  b  SF (10 ug per mouse) and LPS (15 ug per mouse) was injected every other day for 2 weeks (total of 6 times).  a  "The ratios of Ly5.1 / Ly5.1" GFP in the WBCs of each mouse were measured by FACS on the same day as the first injection (before) and 2 days after the last injection. Mice from the SF and LPS injected groups were reassessed another month later and the results shown in parentheses.  116  a) N  S E A  E E  MSCV LTR  C)  S  mm  1 1  M-FF3-IG  N  X  FF3 cDNA  IRES-GFP MSCV LTR  6000 M-FF3-IG 4000 H  Q_ O  2000 H MIG 0.001  0.01  0.1  1  10  M-CSF (ng/ml)  117  100 1000  Figure 5.6.  Structure, expression and  function of a retroviral vector  encoding a c-fms-flt3 chimeric receptor  a) Structure and size of the fms-flt3 (FF3) vector (M-FF3-IG). Only the region between the M S C V retroviral LTR elements is shown. Restriction sites important for the identification and construction of this vector were as follows: Apal (A), EcoRI(E), Nhel (N), Smal(S) and Xhol(X). The structure of the vector was confirmed by restriction digestion analysis and D N A sequencing, b) Representative FACS profiles of M-FF3-IG-transduced BaF3 cells. The cells were stained either with a control IgG isotype Ab (left panel) or anti-c-fms Ab (right panel), and a secondary Ab conjugated with PE. The squared areas in the profiles represent the regions in which transduced receptors were detected, c) 1(T M-FF3-IGtransduced polyclonal BaF3 populations were cultured in 0.1 mL of medium containing different concentrations of M-CSF for 48 hours. H -thymidine was added to the culture in the last 8 hours of incubation. Each point was averaged from triplicates and fit with a sigmoidal model. Data are representative of 2 independent experiments. 3  118  5.3. Discussion In this chapter, the ability of forced overexpression of c-kit or flt-3 in HSCs to affect their reconstituting and self-renewal in irradiated recipients was examined.  Continued  expression of functional receptors on the progeny of the transduced HSCs could be clearly documented and their regenerative and self-renewal was shown to be intact.  Nevertheless,  there was no evidence that the receptor-transduced HSCs had acquired a selective growth advantage in vivo, even when the appropriate ligand was administered exogenously or induced endogenously. These findings do not support the concept that either SF or F L levels in vivo limit HSC expansion either immediately post-transplant into sublethally irradiated animals or later after hematologic recovery is complete. Certainly the fact that the progeny of the transduced cells could be shown to be expressing functional c-kit and flt3 receptors at levels to give them an increased specific sensitivity to SF and FL, respectively, would counter the argument that the lack of growth advantage displayed by these cells in vivo was simply due to a post-transplant silencing of the transduced genes. Rather these findings suggest that the normal level of c-kit and flt3 on HSCs is sufficient to optimize their response to the levels of SF and F L that these cells encounter in vivo in the adult.  This could be anticipated if the effective local  concentrations of SF and F L in vivo were relatively high, in which case, an increased expression of c-kit or flt3 would not necessarily be anticipated to translate into a competitive advantage.  Indeed, the in vitro dose response data from Chapters 3 and 4, suggested that  stimulation of cells overexpressing c-kit or flt-3 with very high levels of cognate ligands would inhibit rather than promote a proliferative response. These studies underscore the poor understanding we still have of how H S C survival, proliferation and self-renewal responses are molecularly controlled by the environment in vivo in the adult. It is interesting, however, to note that during early fetal life, SF does not appear to play any essential role in the generation and expansion of the H S C compartment (Ikuta and Weissman 1992) although elimination of SF or c-kit-mediated signaling is lethal due to the suppressive effects this has on the later development of erythropoietic cells (Paulson and Bernstein 1995). Very recently, evidence of the importance of Wnt-activated signaling in the regeneration of transplanted HSCs in adult mice has been reported (Reya et al. 2003). Thus it  119  may well be that novel factors as yet not well defined may synergize with SF and/or F L ways that have still to be elucidated.  120  CHAPTER 6: GENERAL DISCUSSION In the last decade, various approaches have been undertaken to demonstrate the feasibility of manipulating HSC self-renewal.  The most successful to date has been the  retroviral-mediated overexpression of HoxB4 (Sauvageau et al. 1995; Antonchuk et al. 2002), a transcription factor that is elevated in primitive hematopoietic cells and down-regulated when they differentiate (Sauvageau et al. 1995). When HoxB4 levels are forced to remain high, H S C self-renewal is favored and HSC numbers increase more rapidly to reach higher levels both in  vitro and in vivo than are seen with normal HSCs. Even in the absence of HoxB4 transduction, it can be inferred that HSC self-renewal (as well as HSC proliferation status) is altered when the hematopoietic system is stimulated to regenerate after myeloablative treatments.  This is  associated with a net expansion of HSCs to return their numbers to -10% of normal values, whereas the slow turnover of HSCs required to sustain normal W B C production does not lead to any change in their numbers throughout adult life. Therefore, it seems likely that changes in the local concentration of cytokines active on HSCs might be regulators not only of H S C proliferation but also of HSC self-renewal.  Indeed, evidence that SF and F L can have such  effects have obtained from in vitro experiments with highly purified HSC populations. In this thesis, I investigated the possibility that such HSC self-renewal responses might be enhanced by increasing the sensitivity of the ability of HSCs to repond to extracellular SF or F L and that these responses might be limited by achievable interactions with their cognate receptors.  I first showed that retroviral vectors encoding functional c-kit and flt3 receptors  could be generated and using a cell line model demonstrated that responsiveness to both SF and F L could be selectively increased by forced overexpression of the c-kit and flt-3 receptors in a receptor level-dependent fashion (Chapter 3). Interestingly, a decreased response to very high concentrations of ligand was also seen. This suggests that lack of availability of other downstream mediators might limit further enhancement of ligand-stimulated responses or, alternatively, that excessive formation of ligand-receptor complexes can lead to different internal signaling events. I then showed that the results obtained in BaF3 cells could be extrapolated to the immediate progeny of transduced 5-FU-treated primary adult mouse B M cells (Chapter 4). 5F U treatment selectively kills mature hematopoietic cells and progenitors (Lerner and Harrison 1990) and thus provides a crude but simple and effective method for obtaining an enriched 121  population of primitive cells. After 4 days in vitro in the presence of FL, SF and IL-11 at the concentrations used in this study, these cells undergo some differentiation, but remain enriched in their progenitor content. Using such target cells, it was possible to demonstrate that all cell types produced from these primitive transduced cells, including HSCs, had acquired an increased responsiveness to SF or F L according to the vector used for their transduction. Thus, they displayed a greater response than control cells at lower cytokine concentrations but also showed a depressed response to very high cytokine concentrations, a finding also seen with the receptor transduced BaF3 cells. The high efficiencies of gene transfer to HSCs (estimated to be ~30 - 50%) which made it possible to detect effects on the transduced cells both in vitro and in vivo without their prior selection.  This was an important practical consideration, since full expression of the  transgene requires another 24 - 48 hours, during and after which time, HSCs numbers would become increasingly difficult to sustain based on historical experience (Miller and Eaves 1997; Audet et al. 2001; Antonchuk et al. 2002). A final series of experiments were then undertaken to determine whether increased ckit or flt-3 receptor expression on HSCs (and their progeny) would alter their growth or differentiation in vivo. parameters was detected.  In contrast to the results anticipated, no change in any of these The competitive transplantation procedure used here is the most  sensitive type of experiment known to detect cell autonomous changes in the in vivo behaviour of transplantable hematopoietic cells (Harrison et al. 1993). In the present study no differences were discerned at either the level of the output of mature WBCs or at the level of H S C regeneration.  It thus appears that neither c-kit nor flt-3 receptor levels on HSCs limit the  capacity of these cells to respond to the signals that regulate their self-renewal in vivo. Moreover, since c-kit and flt3-overexpressing HSCs were more sensitive to SF and F L , respectivly, it is logical to imply that neither the concentrations of SF nor F L are the limiting factors for in vivo HSC regeneration in irradiated receipients. A number of similar studies of the effects on murine B M cell behaviour of the forced overexpression of other cytokine receptors have been carried out usually with the goal of determining whether unique responses could thus be elicited. These include experiments with the receptor signaling domains of c-mpl, the G-CSF receptor, and Flt-3. It was found that only mpl supported the sustained growth of transduced B M cells, with a dramatic expansion of 122  multipotential progenitors in vitro (Jin et al. 1998; Jin et al. 2000; Zeng et al. 2001). However, B M cells transduced with a cytokine receptor such as c-mpl or EPO were found to be pathogenic in vivo (Cocault et al. 1996) (Krosl 1997).  In fact, most, if not all, such recipients rapidly  developed a myeloproliferative-like disease (e.g., with evidence of hepatosplenomegaly and/or a high W B C count) and died 9 to 12 weeks after the transplant. However, histological analysis showed that the spleen, liver, and peripheral blood were invaded by erythroblasts at every stage of differentiation and hence death may not have involved a dysregulation of HSCs. There was also no significant change in the peripheral leukocyte count, and the number of CFU-Es and GM-CFCs and multi-CFCs were even found to be reduced 2- to 3-fold in the B M of the c-mpl overexpressing mice (Yan et al. 1999). It is also interesting to note that abnormal expression or mutation of c-kit and especially flt-3 has been commonly found in "naturally arising" human leukemia (see section 1.7.4); however, in the present study only one leukemogenic event was detected in all of the transduced populations studied.  The leukemia obtained arose in a mouse that had been  transplanted with flt-3-transduced B M cells and was propagated by a cell that had been transduced. Although the mechanism of leukemogenesis is not known, it seems likely that it was attributable to an insertional mutagenesis event affecting a neighbouring gene. The concept that certain intracellular signaling thresholds stimulated by exogenous cytokines are important in determining different biological responses has been observed in other systems. For example, recent data from studies of EGFR stimulation suggest that the biological outcome of T K signaling strongly depends on the timing, duration, and amplitude of activation of signaling components (Marshall 1995; Shymko et al. 1997; Downward 1998). It has likewise been shown that cytokines such as E G F can elicit different outcomes in the context of different levels of expression of the corresponding receptor (Traverse et al. 1994; Millot et al. 2002). In other cell systems, signaling through the same pathway in the same cell type can result in completely different outcomes, such as proliferation versus differentiation, depending on the amplitude and persistence of activation of signaling intermediates. It is thus likely that the levels and accessibility of particular signaling intermediates are also key determinants in these decisions (Schlessinger and Ullrich 1992; Yoshida et al. 1994; Heinrich et al. 1998; York et al. 1998). Parameters that affect the down-regulation of receptor-induced signaling events would also be expected to affect biological outcomes; for example, through  123  modulation of phosphatases that dephosphorylate receptor kinases, factors that regulate the rate of internalization of receptor-ligand complexes, and/or degradation of the receptor. 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