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Functional analysis of the homeobox gene HOXB4 in primitive hematopoietic cells Antonchuk, Jennifer 2002

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FUNCTIONAL ANALYSIS OF T H E HOMEOBOX GENE HOXB4 IN PRIMITIVE HEMATOPOIETIC CELLS By JENNIFER A N T O N C H U K B.Sc., University of British Columbia, 1996  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY  In  THE F A C U L T Y OF G R A D U A T E STUDIES Genetics Graduate Program  We accept this thesis as_conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A April, 2002 © Jennifer Antonchuk, 2002  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  scholarly purposes may be her  representatives.  permission.  Bepailinfeiil of The University of British Columbia Vancouver, Canada  for  an advanced  Library shall make it  agree that permission for extensive  It  publication of this thesis for financial gain shall not  DE-6 (2/88)  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  is  granted  by the  understood  that  be allowed without  head of copying  my or  my written  ABSTRACT H e m a t o p o i e s i s i n v o l v e s the ordered p r o d u c t i o n o f mature b l o o d c e l l s from a rare p o p u l a t i o n o f undifferentiated, totipotent, stem c e l l s i n the b o n e m a r r o w . A p o o l o f stem cells is preserved t h r o u g h s e l f - r e n e w i n g d i v i s i o n s , to m a i n t a i n l i f e l o n g hematopoiesis. D e r e g u l a t i o n o f stem c e l l self-renewal a n d differentiation c a n h a v e  severe  clinical  consequences, i n c l u d i n g l e u k e m i a . H e m a t o p o i e t i c stem c e l l s are also h i g h l y v a l u a b l e for their therapeutic uses, as they c a n h e l p reconstitute the h e m a t o p o i e t i c system w h e n transplanted.  Y e t despite the central i m p o r t a n c e o f stem cells, w e k n o w v e r y little about  the regulatory m e c h a n i s m s c o n t r o l l i n g their b e h a v i o u r . HOXB4,  a m e m b e r o f the Hox f a m i l y o f t r a n s c r i p t i o n a l regulators, w a s p r e v i o u s l y  s h o w n to s e l e c t i v e l y enhance h e m a t o p o i e t i c stem c e l l g r o w t h in vivo. members  have  gene-specific roles i n hematopoiesis,  c o n t r o l l i n g differentiation a n d  p r o l i f e r a t i o n a l o n g s p e c i f i c lineages o r even c a u s i n g l e u k e m i a . o v e r e x p r e s s i o n o f HOXB4  Hox f a m i l y  Retrovirus-mediated  i n m u r i n e bone m a r r o w cells resulted i n enhanced stem c e l l  g r o w t h in vivo, w i t h o u t altering l y m p h o - m y e l o i d differentiation o r p r e d i s p o s i n g to l e u k e m i a . T h e major objective o f this thesis w a s to further e x p l o r e the effects  oiHOXB4  o v e r e x p r e s s i o n o n p r i m i t i v e h e m a t o p o i e t i c cells, and to test the u t i l i t y and l i m i t a t i o n s o f this strategy for stem c e l l e x p a n s i o n . C e l l s o v e r e x p r e s s i n g HOXB4  h a d a c o m p e t i t i v e g r o w t h advantage in vitro, and a  c o m p e t i t i v e transplant advantage in vivo.  HOXB4  o v e r e x p r e s s i n g bone m a r r o w c e l l  cultures s h o w e d increased p r o l i f e r a t i o n o v e r c o n t r o l c e l l cultures. M i c e transplanted w i t h //(9A34-transduced a n d control-transduced cells were s e l e c t i v e l y repopulated b y the HOXB4  o v e r e x p r e s s i n g cells.  Importantly, the g r o w t h enhancement i n d u c e d b y  HOXB4  ii  overexpression did not come at the expense of normal differentiation. Further analysis of the //C7A7J4-mediated enhancement to stem cell expansion in vivo revealed that the rate of expansion was elevated. Accelerated stem cell regeneration mediated by HOXB4 allowed stem cell levels to reach 100% of pre-transplant levels within the first three months post-transplant.  Stem cells did not expand beyond the  normal level in HOXB4 mice, suggesting retained responsiveness to negative feedback mechanisms. Thus, //CTAB^-transduced stem cells remained responsive to positive and negative feedback on expansion and to differentiation-promoting signals. This work also addressed the potential for HOXB4 to serve as a stem cell expanding factor ex vivo. Although significant biological and clinical advances await effective stem cell expansion regimes, previous efforts have been largely unsuccessful.  HOXB4  overexpression mediated rapid, extensive, and highly polyclonal stem cell expansions, resulting in over 1000-fold higher stem cell levels relative to controls and a 40-fold net stem cell expansion. These results show that HOXB4 can be used to expand stem cells ex vivo.  The results presented in this thesis add to the recognition of Hox genes as important hematopoietic regulators.  HOXB4 in particular can play a key role in the  regulation of the rate and/or probability of hematopoietic stem cell self-renewal. These studies further suggest the complex biomolecular pathways controlling stem cell fate, and point to new avenues to manipulate HSC expansion.  iii  T A B L E OF CONTENTS  ABSTRACT TABLE OF CONTENTS LIST OF TABLES AND FIGURES ABBREVIATIONS ACKNOWLEDGEMENTS PREFACE  ii iv vi vii xi xii  CHAPTER 1 INTRODUCTION 1.1 Hematopoiesis 1.1.1 Hematopoietic Hierarchy 1.1.2 Hematopoietic Assays and Purification/Enrichment Strategies 1.2 Hematopoietic Stem Cells 1.2.1 Ontological Sources 1.2.2 Totipotency 1.2.3 Stem cell plasticity 1.2.4 Self-renewal 1.2.5 Cell Cycle Status 1.2.6 Exhaustability of HSCs 1.2.7 Clinical importance of HSCs 1.2.8 HSC expansion ex vivo 1.3 Regulation of Hematopoiesis 1.3.1 Extrinsi c Factors 1.3.2 Intrinsic Regulation: transcription factors 1.4 Hox transcription factors 1.4.1 Hox discovery 1.4.2 Hox Gene Organization and Expression 1.4.3 Protein Structure and Function 1.4.4 Hox target genes 1.4.5 Hox function in mammalian development 1.5 Hox and hematopoiesis 1.5.1 Hox expression in hematopoietic cells 1.5.2 Leukemic effects 1.5.3 Hematopoietic effects in Hox loss-of-function models: 1.5.4 Hematopietic effects in Hox gain-of-function models 1.5.5 HOXB4 overexpression 1.5.6 HOXB4 overexpression: unresolved issues and thesis objectives  1 1 1 2 10 10 12 13 15 19 19 22 24 27 27 34 39 39 40 43 46 49 51 51 53 56 58 63 65  CHAPTER 2 HOXB4 Overexpression Mediates Very Rapid Stem Cell Regeneration and Competitive Hematopoietic Repopulation 67 2.1 Abstract 68 2.2 Introduction 69 iv  2.3.1 Retroviral Vectors 2.3.2 Mice 2.3.3 Transduction of Primary Murine Bone Marrow Cells 2.3.4 In Vitro Culture of Hematopoietic Cells 2.3.5 In Vivo Repopulation... 2.3.6 CFC Assay 2.3.7 CRU Assay 2.3.8 Proviral Integration Analysis 2.4 Results 2.4.1 Growth enhancing effects of HOXB4 in vitro 2.4.2 Competitive reconstitution of hematopoietic cells in vivo 2.4.3 Rapid regeneration of competitive repopulating units (CRU) 2.4.4 CRU regeneration at high transplant cell doses 2.5 Discussion 2.6 Appendix  73 74 74 75 76 76 77 78 78 79 81 85 89 91 95  CHAPTER 3 HOXB4-\MucQd. Expansion of Adult Hematopoietic Stem Cells ex vivo 97 3.1 Abstract 98 3.2 Introduction 98 3.3 Methods 100 3.3.1 Retroviral Vectors 100 3.3.2 Mice 101 3.3.3 Transduction of Primary Murine Bone Marrow Cells 101 3.3.4 Flow cytometry 102 3.3.5 CFC Assay 103 3.3.6 CRU Assay 103 3.3.7 Proviral Integration Analysis 104 3.4 Results : 104 ' 3.4.1 Ex vivo expansion of iftXYB^-overexpressing HSC 104 3.4.2 HOXB4-transduced HSCs are Competitive and Pluripotent 107 3.4.3 Polyclonal Expansion of HOXB4-Transduced HSCs 111 3.5 Discussion 113 CHAPTER 4 General Discussion 4.1 Mechanism of HOXB4-mod.idA.t6. stem cell expansion 4.2 Hox Gene Specificity 4.3 Target Genes 4.4 Stem Cell Plasticity 4.5 Therapeutic Applications  116 116 122 127 129 131  CHAPTER 5 BIBLIOGRAPHY  133  v  LIST OF T A B L E S A N D FIGURES  Figure 1.1: The hematopoietic hierarchy Figure 1.2: Models of stem cell plasticity '. Figure 1.3: Levels of HSC Regulation Figure 1.4: Hematopoietic cytokines and their location of action Figure 1.5: Transcription factors involved in hematopoietic lineage commitment Figure 1.6: HOXChromosomal Organization Figure 1.7: Hox Protein Structure Table 1.1: Hox target genes Figure 1.8: Retroviral lifecycle Table 1.2: Hox effects on primary hematopoietic cells  2 15 18 29 36 41 43 47 -.60 61  Figure 2.1: Construction and testing of the GFP and HOXB4-GFP retroviral vectors... 79 Figure 2.2: Expansion of transduced B M cells in liquid culture 81 Figure 2.3: Competitive reconstitution by HOXB4-GFP-tvansd\xced (Ly5.1 GFP ) and GFP-transduced (Ly5.T GFP ) cells 83 Figure 2.4: Competitive repopulation and normal differentiation 84 Figure 2.5: Repopulation by GFP- or GFP-transduced cells in sub-lethally irradiated W / W recipients 86 Figure 2.6: Kinetics of C R U expansion in vivo 88 Table 2.1. C R U regeneration at two weeks post-transplant 89 Figure 2.7: C R U regeneration following transplantation of high HOXB4-GFP transplant cell doses 90 Figure 2.8: Propidium iodide labelling of GFP and HOXB4-GFP cultures 95 Figure 2.9: Concordance between Ly5.1 and GFP expression 96 Figure 2.10: Polyclonal repopulation in HOXB4-GFP recipient mice 96 +  +  +  4 1  Figure Figure Figure Figure Figure Figure  4 1  3.1: Ex vivo expansion of mature cells and progenitors 3.2: Ex vivo expansion of HSCs 3.3: Lympho-myeloid repopulation by ex vivo expanded stem cells 3.4: Regeneration of HSCs following ex vivo expansion 3.5: Clonality of expanded HSCs 3.6: Model of HOXB4-mediated ex vivo HSC expansion  Figure 4.1: Potential mechanisms for regulation of HSC population size Figure 4.2: Model of Hox control over hematopoiesis  105 106 109 110 Ill 112  117 126  vi  ABBREVIATIONS  5-FU  5-flourouracil  AGM  aorta-gonad-mesonephros  ALL  acute lymphoid leukemia  AML  acute myelogenous leukemia  Antp  Antennapedia  ANT-C  antennapedia complex  ATRA  all-trans retinoic acid  bFGF  basic fibroblast growth factor  bl-ARE  Hoxb-1 autoregulatory element  [B6C3 ]  F1  F l hybrid of (C57B1/6J x C3H/HeJ)  BFU-E  blast forming unit-erythroid  bHLH  basic helix-loop-helix  BM  bone marrow  BMP  bone morphogenic protein  BX-C  bithorax complex  CB  cord blood  CBP  C R E B binding protein  CFC  colony forming cell  CFSE  carboxyfluorescein diacetate succinimidyl ester  CFU  colony forming unit  CFU-E  colony forming unit - erythroid  CFU-G  colony forming unit - granulocyte  CFU-GM  colony forming unit - granulocyte macrophage  C F U - G E M M colony forming unit - granulocyte, erythroid, m CFU-M  colony forming unit - macrophage  CFU-S  colony forming unit - spleen  CI  confidence interval  CKII  casein kinase II  CLL  chronic lymphoid leukemia  CLP  common lymphoid progenitor  CML  chronic myelogenous leukemia  CMP  -  common myeloid progenitor  CRU  competitive repopulating unit  Dhh  Desert hedgehog  DMEM  Dulbecco's modified Eagle's medium  dpc  days post-coitum  E  erythroid  Epo  erythropoietin  EB  embryoid body  ES  embryonic stem (cell)  FACS  fluorescence-activated cell sorting  FBS  fetal bovine serum  FL  fetal liver  Flt3L  Flt3 ligand  FOG-1  Friend of G A T A-1  G  granulocyte  G-CSF  granulocyte - colony stimulating factor  GFP  green fluorescent protein  GM-CSF  granulocyte macrophage - colony stimulating factor  GMP  granulocyte-macrophage progenitor  GVHD  graft versus host disease  HF  Hank's balanced salt solution with 2% FBS  HOM-C  homeotic complex  HPP-CFC  high proliferative potential colony forming cell  HSC  hematopoietic stem cell  LAP  intracisternal A particle  Ihh  Indian hedgehog  I-CAM  intercellular adhesion molecule  IC-Notch  intracellular domain of Notch  viii  IL  interleukin  IRES  internal ribosomal entry site  L-CAJVI  liver cell adhesion molecule  LIF  leukemia inhibitory factor  Lin  lineage markers  LTC-IC  long term culture-initiating cell  LTR  long terminal repeat  M  macrophage  M-CSF  macrophage-colony stimulating factor  MEP  megakaryocytic-erythroid progenitors  MfPlot  macrophage inflammatory protein 1 alpha  Mk  megakaryocyte  MMP  matrix metalloproteinase  N-CAM  nuclear-cell adhesion molecule  NcoR  nuclear co-repressor  NHL  non-Hodgkin's lymphoma  NK  natural killer  NOD/SCID  non-obese diabetes / severe combined immunodeficiency  NSC  neural stem cell  NUP98  nucleoporin-98  PB  peripheral blood  PE  phycoerythrin  Pep3b  C57B16/Ly-Pep3b  [PepC3]  F1  F l hybrid of (C57Bl/6Ly-Pep3b x C3H/HeJ)  PI  propidium iodide  PKA  protein kinase A  RA  retinoic acid  RAR  retinoic acid receptor  RBC  red blood cell  Rho  Rhodamine  RT-PCR  reverse transcription - polymerase chain reaction  RU  repopulation unit  SAGE  serial analysis of gene expression  Shh  Sonic hedgehog  SEM  standard error of the mean  SF  steel factor  SMRT  silencing mediator for retinoic acid receptor and thyroid hormone receptor  SRC  SCID repopulating cell  TALE  three amino acid loop extension  TCR  T-cell receptor  TGF(3  transforming growth factor beta  Tpo  thrombopoietin  TTF-1  thyroid transcription factor-1  Ubx  Ultrabithorax  uPA  urokinase-type plasminogen activator  VLA  very late antigen  w /w 41  41  C57Bl/6-W /W 4I  41  WGA  wheat germ agglutinin  YS  yolk sac  x  ACKNOWLEDGEMENTS  I am deeply indebted to my supervisor, Dr. R. Keith Humphries, for the opportunity to do graduate training in his laboratory.  I came to him with an interest in  Hox genes, and he gave me the freedom to pursue several aspects of Hox genetics in the hematopoietic system until I found my overwhelming interest in stem cell biology. Dr. Humphries also supported my attendance at several international conferences in hematology and stem cell biology, at which I experienced the shared enthusiasm for newly emerging concepts such as stem cell plasticity. I appreciate his fervent support and tireless guidance throughout my time in the lab. Cheryl Helgason and Cindy Miller were very helpful in teaching me the assays for hematopoietic cells.  I also thank Patty Rosten for technical assistance in molecular  biology, as well as Gayle Thornbury and Giovanna Cameron for cell sorting. Drs. Hugh Brock, Carolyn Brown and Fumio Takei served on my graduate committee, and I thank them for their assistance in project discussions. I would like to recognize the financial assistance I received, in the form of studentships from the National Sciences and Engineering Research Council of Canada and the University of British Columbia.  As well, this work was supported by the  National Cancer Institute of Canada, with funds from the Canadian Cancer Society and the Terry Fox Run; and from.the National Institute of Health (Grant No. R01DK48642). Finally, I send my thanks to all members of the Humphries lab (Nick, Sharlene, Patty, Christian, Andrew, Caroline, Suzan, Rewa, Cynthia, Sylvia and Ben) for making it such an enjoyable place to work.  xi  PREFACE  The work presented in Chapter 2 is essentially as reported in Antonchuk, J. Sauvageau, G. and Humphries, R. K, 2001, HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation, Exp Hematol 29(9): 1125-34. The work presented in Chapter 3 is essentially as reported in Antonchuk, J., Sauvageau, G. and Humphries, R. K, 2002, HOXB4-mducoA Expansion of Adult Hematopoietic Stem Cells ex vivo, Cell 109 (1): 39. For both of these chapters, Jennifer Antonchuk contributed a major portion of the work, including all of the data collection and shared responsibility on manuscript preparation.  CHAPTER 1  1.1  1.1.1  INTRODUCTION  Hematopoiesis  Hematopoietic Hierarchy Hematopoiesis is the process of blood cell development.  It begins in early  embryogenesis and continues throughout life. Approximately 200 billion red blood cells (RBCs) and 60 billion neutrophils are produced daily in adults, along with several million platelets, macrophages, B-cells and T-cells. Since the mature cells have limited lifespans and are largely unable to proliferate, they are replenished by differentiation and proliferation of more primitive progenitor cells, which are themselves replenished by differentiation and proliferation of a self-maintaining pool of totipotent hematopoietic stem cells (HSCs). A pool of HSCs is retained in the bone marrow (BM), and can amplify or differentiate as needed. These cells are critical for sustained blood cell production, as they have potency for all the different myeloid and lymphoid blood cell lineages. Hematopoiesis proceeds via a series of lineage commitment steps, from the most primitive HSC, to the myeloid- or lymphoid-restricted stem cells, to the multi-, bi- and uni-lineage progenitors, which finally mature into functional circulating end cells (Fig. 1.1).  1  Figure 1.1: The hematopoietic hierarchy. The totipotent HSC is shown at the top of the hierarchy, and the circular arrow represents self-renewal. Commitment to either myeloid or lymphoid lineages produces the common lymphoid progenitor (CLP) and common myeloid progenitor (CMP). Lineage restrictions produce progenitors with multipotent megakaryocytic-erythroid progenitors (MEP) and granulocyte-macrophage progenitors (GMP). Further commitment events produce unilineage progenitors, which mature into functional end cells in T-cell (T), B-cell (B), megakaryocyte (Mk), erythroid (E), macrophage (M) and granulocytic (G) lineages. Some hematopoietic assays are listed, aligned with the developmental level of the cell type they detect. Potentials for proliferation and self-renewal both decrease with differentiation.  1.1.2  Hematopoietic Assays and Purification/Enrichment Strategies Our current understanding of the hematopoietic hierarchy as outlined in Figure 1.1  has been established through the identification and characterization of cells with differing  2  proliferative and differentiative potentials. The development of functional assays and purification strategies have been central to uncovering key hematopoietic paradigms such as self-renewal, clonality and multipotentiality.  Here I present the key assays and  purification strategies, in bottom-to-top hierarchical order of the cell type being detected.  •1.1.2.1 Detection of Mature Cells Mature cells and their immediate precursors are the only hematopoietic cell types that can be unambiguously identified morphologically. These cells were thus identified very early, and the final stages of maturation for each blood cell type were mapped out. The relative size of cells, their degree of granulation, the precise size and shape of their nuclei, and their staining characteristics can be used to identify the lineage and maturation status of a terminally differentiating cell. Morphology and staining properties continue to be used today for routine diagnostics, such as for obtaining differential white blood cell counts. More recently, fluorescence-activated cell sorting (FACS) has been used for detection and/or purification of mature hematopoietic cells, by taking advantage of unique surface antigens expressed by cells in each hematopoietic lineage.  Commonly used  examples include T e r l l 9 for erythroid cells, Gr-1 for granulocytes, B220 for B-cells and CD4 and CD8 for T-cells.  Flourochrome-conjugated antibodies against these so-called  lineage markers (lin) are used with F A C S to detect cells in each lineage. The advantages of this technique are that it efficiently characterizes large numbers of cells, and it can be used for purification of live cells in a specific lineage.  3  1.1.2.2 Detection of Progenitor Cells The discovery of lineage-restricted progenitor cells in the late 1960s led to a model whereby cells become irreversibly committed to lineages, with progressive restrictions in potentiality, as they progress through hematopoietic differentiation. The colony forming cell (CFC) assay is a functional assay to test for the presence of clonogenic progenitor cells (Bradley et al, 1967; Ichikawa et al, 1966). In its basic form, cells are seeded onto semisolid media (eg. methylcellulose or soft agar) to prevent migration, and those cells with sufficient proliferative potential to generate colonies of >50 cells are termed colony forming units (CFU).  From the types of mature cells  generated by each C F U , its developmental potential can be inferred, as denoted by now standard designations.  For example, cells that generate colonies containing only  granulocytes are termed CFU-granulocyte (CFU-G), whereas those that generate colonies containing only macrophages are termed CFU-macrophage (CFU-M).  Primitive and  more mature erythroid progenitors can also be identified in this manner, by the generation of burst forming unit-erythroid (BFU-E) and CFU-E colonies, respectively. Intrinsic commitment of progenitor cells to a given lineage is indicated by the limited types of cells generated by each C F C in growth factor conditions which are supportive for all hematopoietic lineages. Colony assays have also allowed for identification of clonogenic progenitors with bi- or multi-lineage potentials.  Colonies containing granulocytes and macrophages  defined a C F U - G M progenitor, and delineated a separation of these lineages from the  4  other (erythroid and megakaryocyte) myeloid lineages. Even more primitive progenitors can be distinguished by their greater multilineage differentiation potential and high proliferative capacity. C F U - G E M M (granulocyte erythroid macrophage megakaryocyte) are capable of differentiation into at least 4 myeloid lineages, generate macroscopic colonies, and have the ability to form secondary colonies.  Clonogenic assays can  therefore distinguish cells within the B M that have extensive or more restricted lineage, proliferative and self-renewal potentials, thereby establishing a hierarchical model of hematopoietic differentiation. Recently, FACS-based strategies have been developed to purify bipotent granulocyte-macrophage progenitors (GMP) and megakaryoctye-erythroid progenitors (MEP) (Akashi et al, 2000), as well as multipotent common lymphoid progenitors (CLP) (Kondo et al, 1997) and common myeloid progenitors (CMP) (Akashi et al, 2000). For example, GMPs were shown to purify in the fraction of cells with no expression of lineage markers (lin), Sca-1 antigen or interleukin (IL)-7 receptor a, low expression of Fey receptor, and high expression of c-kit and CD34 antigens. The cells are therefore phenotypically defined as lin" IL7Ra" c-kit Sca-1" FcyR CD34 . Phenotypes such as +  hl  +  this can be used to determine the number of bipotent and multipotent progenitors in various model systems,  to study the regulatory mechanisms  of hematopoietic  differentiation.  5  1.1.2.3 Detection of HSCs In 1961, pioneering studies by Till and McCulloch identified a hematopoietic cell in mouse capable of producing mature erythroid, granulocytic, and megakaryocyte progeny cells in vivo. This discovery, which predated even the C F C assay, was the first indication that the disparate blood cell types derive from a common multipotential ancestor. The colony forming unit-spleen (CFU-S) assay was developed, whereby cells are injected into myeloablated recipient mice, and colonies that develop on the spleen after 9-14 days are enumerated (Till and McCulloch, 1961). The spleen colonies were found to contain over 10 cells and were proven to be of single cell (clonal) origin, 6  demonstrating the high proliferative capacity of the originating cell termed CFU-S. Also, they were capable of self-renewal, as recovered colony cells could generate more spleen colonies when injected into secondary mice (Siminovitch et al, 1963). The number of secondary colonies was highly variable, and from this a stochastic model of HSC regulation was proposed (Till et al, 1964). According to this model, regulation of HSC self-renewal occurs on the population as a whole, by controlling the overall probability of self-renewal versus differentiation outcomes. Although the CFU-S assay was seminal in establishing many of the fundamental HSC properties, it is no longer considered the defining assay for HSCs. Mature lymphoid cells were not detected in CFU-S colonies, indicating that the assay defines a myeloidrestricted early progenitor.  Moreover, with the development of transplantation-based  HSC assays it was possible to physically separate CFU-S from HSCs which gave longterm repopulation of transplant recipients (Jones et al, 1990; Ploemacher and Brons, 1989). HSCs can be identified through their unique ability to give long-term lympho6  myeloid reconstitution to myeloablated transplant recipients. Host mice are first exposed to lethal doses of ionizing radiation, and the donor graft is then delivered intravenously. Stem cells from the donor tissue home to the hematopoietic organs, where they reestablish multilineage hematopoiesis.  However, there is still heterogeneity within the  cells capable of multilineage reconstitution, and the most primitive subset can be further defined by the capacity to sustain donor-derived hematopoiesis long-term (several months).  Through these transplantation-based  assays, the abundance and relative  functional ability of HSCs can be determined. These assays have been used to define the genetic and biochemical factors regulating HSC number and activity. The repopulation unit (RU) assay developed by Harrison (1980) tests the relative ability of a cell sample to repopulate irradiated hosts relative to 10 normal B M cells. 5  The test sample is injected, along with a standard dose of freshly isolated and phenotypically distinguishable competitor cells, into a congenic irradiated murine recipient. After 3-4 months, the relative repopulation of myeloid and lymphoid cells by the two donor sources is calculated, and an R U value calculated by the formula: RU=(P*C)/(100-P),  where  P  is the  measured  percentage of test cell-derived  hematopoiesis, and C is the number of competitor cells used. The magnitude of the R U value reflects both stem cell number (quantity) and their proliferative contribution (quality).  This assay reveals subtle differences in repopulating ability between HSC  samples. A second widely used assay for HSCs is one based on limit dilution principles to detect and quantitate cells with lympho-myeloid repopulating ability. In the competitive repopulating unit (CRU) assay developed by Szilvassy et al. (1990), graded doses of a 7  test sample are injected together with a small number of distinguishable competitor cells that ensure survival of the recipients and provide a selective pressure to identify a very primitive class of HSC. Test cell-derived repopulation of lymphoid and myeloid cells is then monitored. Quantitation of HSCs is achieved by application of Poisson statistical analysis on the proportion of animals that test positive for test cell-derived repopulation at each cell dose transplanted, where the dose at which 37% of animals are negative is estimated to contain 1 H S C or 1 C R U . hi practice a threshold of >1% test cell-derived myeloid and lymphoid peripheral blood (PB) cells detected >12 weeks post-transplant has been shown to rigorously detect a long term lympho-myeloid repopulating cell. Quantitation of C R U has also recently been shown based on the percent test-derived repopulation per animal, as the relative repopulation and C R U number correlated at doses of less than 8 C R U (Audet et al, 2001). Immunocompromised mice bearing non-obese diabetes (NOD) and severe combined immunodeficiency (SCID) genotypes can tolerate hematopoietic grafts from human sources (Cashman et al, 1997).  This has allowed for the development of a  quantitative in vivo functional assay for human HSCs, known as the SCID repopulating cell (SRC) assay (Larochelle et al, 1996). Limiting doses of the test sample are injected into semi-lethally irradiated NOD-SCID hosts, and transplanted mice are scored as positive (engrafted) i f they contain a threshold level of human lymphoid and myeloid cells. As in the C R U assay, Poisson statistics are then employed to quantitate the SRC frequency.  8  1.1.2.4 Enrichment of HSCs Quantitative functional assays such as those described above have established that HSCs exist in the murine B M at a low frequency, approximately 1 in 16,000 cells or just 0.05% of the total B M . To study individual HSCs more effectively, purification strategies have been developed based on cell surface phenotype. It should be noted that all purification strategies yield heterogeneous populations, and some HSCs will inevitably be excluded from the selection. Moreover, separation of phenotype and function was demonstrated in murine B M transplantation experiments, where significant amounts of phenotypically defined HSCs but not long-term repopulating activity were reconstituted (Spangrude et al, 1995). Genetic modifications (eg. knockout models) could also affect the surface phenotype of HSCs. Nonetheless, by this method direct measurements on activation into and rate of cell cycle, and gene expression of HSCs have been possible. HSC purification strategies use FACS to select for expression or lack thereof of various cell surface antigens. HSCs generally lack expression of lineage markers (ie. they are lin"). In combination with lin" selection, early purification methods were based on binding to lectins such as wheat germ agglutinin ( W G A ) (Ploemacher et al, 1993), and +  exclusion of the mitochondrial dye Rhodaminel23 (Rhol23 ) (Uchida et al, 1996; l0  Udomsakdi et al, 1991; Zijlmans et al, 1995). However, a more highly enriched HSC population is generated by Sca-1 c-kit Thy-l'° selection (Spangrude et al, 1988). +  +  Human HSC purification usually involves selection for CD34 CD38" cells. +  Recently, another method based on the propensity of HSCs to exclude Hoescht 33342 dye has emerged. A unique side population of cells is observed when this staining is simultaneously observed at 2 wavelengths, and this population was found to be highly 9  enriched for CFU-S and multilineage repopulating cells (Goodell et al., 1996). Hoescht is actively excluded by the A B C transporter protein A B C G 2 in HSCs, and overexpression of ABCG2 led to expansion of SP type HSCs and a differentiation block at the HSC stage, indicating a functional role for this transporter in HSCs as well (Zhou et al, 2001).  1.2  Hematopoietic Stem Cells Using the above assays, key properties of the HSC were established: 1) they are  totipotent for all hematopoietic lineages; 2) they are capable of self-renewal; 3) they are quiescent; 4) they have limited regenerative capacity. In the following sections I will present each of these properties in detail, including the experimental observations which led to these conclusions, as well as some evidence that these properties are not always absolute.  1.2.1  Ontological Sources In the mouse, hematopoietic cells first emerge in extra-embryonic cells of the yolk  sac (YS) at 7.5 days post-coitum (dpc).  This is the exclusive site of "primitive"  (embryonic) hematopoiesis, which is largely restricted to the erythroid lineage. Primitive erythroid cells produced in the Y S have marked differences from "definitive" (fetal/adult) erythroid cells. They contain embryonic forms of hemoglobin (Wong et al, 1986), they are much larger, and they remain nucleated (reviewed in Zon, 1995). Progenitor cells from multiple myeloid lineages are found in the Y S , although these do not appear to undergo terminal differentiation in vivo (Johnson and Barker, 1985; Palis et al, 1999). 10  The presence of HSCs in the Y S is somewhat controversial. While Y S cells are not capable of repopulating adult transplant  recipients, they can give multilineage  hematopoietic repopulation to embryonic (Fleischman et al, 1982; Toles et al, 1989) or newborn (Yoder and Hiatt, 1997) transplant recipients, suggesting the existence of a distinct "embryonic H S C " (reviewed in Palis and Yoder, 2001). Definitive hematopoiesis originates in an intra-embryonic site known as the aorta-gonad-mesonephros (AGM) region (Medvinsky and Dzierzak, 1996), from which emerge HSCs (Muller et al, 1994), primitive progenitors such as the CFU-S, and lymphoid cells (Cumano et al, 1996). While early models of hematopoietic development proposed that HSCs migrated from primitive to definitive sites of hematopoiesis, this was disproved when it was shown that only A G M cells and not Y S or fetal liver (FL) cells gave rise to HSCs when the organs were separated at 9, 10, and 11 dpc (Medvinsky and Dzierzak, 1996). At 11 dpc, definitive hematopoiesis migrates to the FL, and this continues to be the main site of hematopoiesis until birth. A large hematopoietic expansion occurs within the FL, with 30-fold increases in cellularity and stem cell numbers (Ema and Nakauchi, 2000), and up to 250-fold increases in progenitors (Nicolini et al, 1999). Consistent with this large expansion, HSCs from F L are more actively cycling than are those from adult sources. This enhanced cell division may contribute to the higher repopulation activity of F L compared to adult B M cells. (Micklem et al, 1972; Pawliuk et al, 1996; Rebel et al, 1996a). Shortly after birth, hematopoiesis moves to the B M , where it continues for the remainder of the animal's life. Hematopoiesis in the adult B M is more steady-state than 11  in fetal or embryonic tissues. The HSCs are mostly quiescent, and there is very little change in HSC number throughout life. Analyses of differential gene expression in HSCs from fetal and adult sources have identified cell surface receptors, adhesion molecules, signaling intermediates and transcription factors (Ito et al, 2000; Oh et al, 2000; Phillips et al, 2000; Rebel et al, 1996b; Szilvassy et al, 2001). Further biomolecular analysis of these differentially expressed molecules will determine which are involved in defining the differences in stem cell behaviour between different ontological sources.  1.2.2  Totipotency  HSCs are the most primitive hematopoietic cell type, with complete potency for all hematopoietic lineages.  Totipotency of the HSC has been demonstrated in vivo  following transplantation, where both lymphoid and myeloid progeny cells were proven to have descended from a common parental stem cell.  The clonal nature of this  relationship was demonstrated first by radiation-induced chromosomal abnormalities (Abramson et al, 1977; Wu et al, 1968), and later by retroviral marking (Capel et al, 1989; Dick et al, 1985; Keller etal,  1985; Lemischka et al, 1986). Since the marking  occurs randomly, the presence of commonly marked cells in myeloid, B - and T-lymphoid lineages indicates that these all derived from a common totipotent stem cell clone. Thus, HSCs are capable of producing differentiated progeny in all hematopoietic lineages. However, not all HSCs are actively contributing to mature cell production at any given time. The clonal succession model first proposed by Kay (1965) stated that there was a serial fluctuation of actively differentiating H S C clones. Thus, one HSC  12  would exclusively provide differentiated progeny cells for a short time, after which its proliferative potential would be exhausted and the job would be taken over by another HSC clone.  Initial retroviral-marking studies appeared to support this theory, as  dominant marked clones appeared and declined following transplantation (Lemischka et al, 1986). However, more detailed experiments have since demonstrated the long-term persistence of some HSC clones in providing differentiated progeny (Harrison et al, 1988; Jordan and Lemischka, 1990; Keller and Snodgrass, 1990). The most recent data indicates that there is a high degree of heterogeneity within the H S C compartment, with variability in both the duration of clonal persistence and the size of their clonal outputs (Guenechea et al, 2001).  1.2.3  Stem cell plasticity  In recent years, evidence has accumulated which suggests"that the potency of stem cells might extend beyond the specific tissue in which they are found. Most examples involve regeneration of damaged tissue, suggesting that the injury response may involve recruitment of cells into the required lineage and that the microenvironment then directs the specific differentiation of cells according to the target organ. Thus BM-derived cells can reconstitute damaged heart (Orlic et al, 2001), lung (Krause et al, 2001), liver (Lagasse et al, 2000; Petersen et al, 1999), muscle (Ferrari et al, 1998) or brain (Kopen et al, 1999). Likewise, neural-derived cells can generate muscle, and muscle-derived cells can generate blood (Jackson et al, 1999). When multiple neural stem cells (NSCs) were implanted into blastocysts, NSC-derived progeny cells were found in mesodermal  13  lineages including heart, ectodermal lineages including CNS, and endodermal lineages including liver and intestine (Clarke et al, 2000). In all cases the newly formed cells appear to be fully mature and functional.  These studies led to the intriguing model  whereby stem cells could, under certain circumstances, undergo trans-determination and develop into disparate tissue types, even reaching outside the original germ layer of the donor tissue. However, trans-determination has yet to be proven, and alternate models of plasticity are equally possible (Fig. 1.2). The second model argues that multiple types of tissue-specific stem cells are present in a tissue. Thus for example liver-specific stem cells might exist in the B M but only be expressed when transplanted into the liver. A recent report showed that muscle-derived hematopoietic activity in mice previously transplanted with B M cells was found exclusively within the donor-derived portion of the muscle, suggesting that there is migration of tissue-specific stem cells (Kawada and Ogawa, 2001). Moreover, muscle cells with myogenic or hematopoietic repopulating activity can be physically separated based on expression of CD45 (Issarachai et al, 2001). A third model postulates that unrestricted stem cells exist but are directed by their local environments along tissue-specific lineages. Krause et al recently demonstrated that singly-transplanted BM-derived cells could contribute to multiple organs, including blood, lung, intestine and skin (Krause et al, 2001). Clonal repopulation of multiple tissue types strongly argues against the tissue-restrictive stem cell model. A n extension of this model is the possibility that tissue-restricted stem cells might be genetically reprogrammed back to a totipotent state. De-differentiation has been demonstrated in nuclear transfer experiments (Rideout et al,  2001; Wilmut et al,  1997), by in  vitro  14  culture of neural precursor cells (Kondo and Raff, 2000), and by the addition of msxl to myotubes (Odelberg et al, 2000).  Model 1: Trans-determination  totipotent stem cell  tissue-restricted stem cell  mature cells  o  o  o—  A A  oooo  oooo  Model 2: Distinct, tissue-restricted stem cells  o  o  o  o  A A  oooo  oooo  Model 3: De-differentiation to totipotent stem cells  A )o o o <  Figure 1.2: Models of stem cell plasticity. Regeneration of alternatetissues may occur due to trans-determination of tissue-restricted stem cells (Model 1), the presence of multiple different tissue-restricted stem cells in a tissue (Model 2), differentiation of a totipotent stem cell into multiple different tissue types (Model 3), or de-differentiation of tissue-restricted stem cells back to a totipotent state, followed by differentation to mutiple tissue types (Model 3).  Stem cell plasticity could potentially be exploited, to utilize the advantages of certain stem cell sources while avoiding the disadvantages of others (such as accessibility or ex vivo expandability). As a preliminary example of this, human fetal-derived neural stem cells, which are easily expanded ex vivo, can repopulate the hematopoietic system of SCID mice, and might therefore be used one day as a source of stem cells for treatment of hematological diseases (Shih et al, 2001).  1.2.4  Self-renewal Self-renewal is the generation of daughter cells that retain HSC function. During  ontogeny, there is a great expansion of all hematopoietic cells, including HSCs, to meet  15  the growing needs of the body. The murine F L at 12 dpc contains approximately 40 HSCs, as detected by the C R U assay. By 16 dpc this number has expanded 30-fold to 1500 HSCs (Ema and Nakauchi, 2000), and by adulthood a further 13-fold expansion brings the total HSC content up to 20,000 (Szilvassy et al, 1990). From this expansion we can infer that a large amount of self-renewal has occurred, however we require clonal analysis to prove that the adult HSCs descended directly from the fetal HSCs. HSC self-renewal also occurs in the B M following transplantation. There is an initial phase of hematopoietic recovery, when HSCs are stimulated to divide and replenish both mature and primitive hematopoietic compartments. "Regeneration" refers to the in vivo expansion of the HSC pool following transplantation.  It has been  demonstrated by recovery of cells capable of giving donor-derived long-term repopulation to multiple recipients from the B M of mice previously transplanted with only a single HSC (Brecher et al, 1993; Osawa et al, 1996). Quantitative analyses have shown that HSCs recovered from transplant recipients can be up to 100-fold higher than the number transplanted (Pawliuk et al,  1996).  Combining HSC regeneration with retroviral  marking studies, the clonal nature of self-renewal was demonstrated (Keller and Snodgrass, 1990). Improved HSC purification strategies and ex vivo culture techniques have allowed for the demonstration of self-renewal ex vivo. In an early study, an ex vivo culture which yielded a net HSC loss nonetheless demonstrated self-renewal, as multiple mice were repopulated by HSCs with common retroviral marking (Fraser et al, 1990).  More  recently, carboxyfluorescein diacetate succinimidyl ester (CFSE) has been used to track HSC divisions. This drug binds irreversibly to internal cell membranes and is diluted 16  with each division, thereby allowing for separation of cells based on their divisional history. After a 5-day culture of human F L or cord blood (CB) cells, the vast majority of HSC activity was present in the fraction that had divided at least once (Glimm and Eaves, 1999). Finally, direct evidence of self-renewal comes from Ema et al. (Ema et al, 2000), who seeded single cells enriched for HSC activity into tissue culture wells, and then demonstrated multilineage repopulation by the first division progeny.  When the two  daughter cells were separated, however, retention of long-term repopulating ability could sometimes be found in only one of the two wells, indicating that asymmetric divisions can occur. The stochastic model of HSC self-renewal, first postulated in 1964, states that HSCs have only an intrinsic probability  of self-renewal versus differentiation outcomes  (Fig. 1.3). This probability can be regulated on a population-wide basis, but the ultimate behaviour of an individual HSC cannot be determined with certainty. The model was first put forth to explain why not all CFU-S could seed secondary CFU-S (Till et al, 1964), and it has since be supported by studies on early progenitors, which showed that the self-renewal frequency fit an exponential, rather than a Poisson distribution (Humphries et al, 1981; Nakahata et al, 1982).  A probability of 0.5 (with either  symmetric or asymmetric divisions) would result in maintenance of HSC numbers, whereas even a slight increase in the probability of self-renewal would lead to net HSC expansion. A n alternative model for how HSC population size is controlled is the hematopoietic inductive microenvironment (HIM) model. By this model, there are a limited number of microenvironmental niches within the B M where HSCs are maintained 17  in an undifferentiated state (Ogawa, 1993).  Localized extrinsic factors including  interactions with stromal cells and cytokines in these HSC niches inhibit differentiation of HSCs.  Proponents of this model often cite the requirement to first myeloablate B M  transplantation recipients, presumably to free up HSC niches for the donor HSCs. However, when given in sufficient numbers, donor HSCs can engraft non-ablated recipient mice (Stewart et al, 1993). These studies suggest that there is competition for marrow space between host and donor HSCs (Quesenberry et al, 1997), but do not prove competition for sites of extrinsic HSC regulation.  Dead cell  Quiescent HSC  Figure 1.3: Levels of HSC Regulation. Control of H S C number can be exerted through activation into cell cycle, rate of cell division, apoptosis, and the probability of self-renewal versus differentiation outcomes of division. Large arrows indicate regulatory conditions favoring HSC expansion.  Differentiated Cell  18  1.2.5  Cell Cycle Status HSCs were initially considered to be quiescent, a property which could in theory  help maintain genomic integrity of this critically important population. This property was attributed to HSCs largely due to their ability to survive the cytotoxic effects of drugs such as 5-flourouracil (5-FU), a nucleotide analog which kills rapidly proliferating cells including progenitors and end cells by incorporating into S-phase D N A (Lerner and Harrison, 1990). However, more recent data indicates that the majority of HSCs in steady-state B M are cycling, albeit at a low rate. B y combining phenotypic identification of HSCs with BrdU incorporation, it was shown that 5% of HSCs were in S/G2/M phases at any one time, and over 90% of HSCs entered the cell cycle within 30 days (Cheshier et al, 1999). HSCs can be forced into more active cell division by in vivo stresses such as 5-FU administration (Harrison and Lerner, 1991) or B M transplantation (Nilsson et al, 1997). Certain hematopoietic cytokines can also activate HSCs (ie. recruit them into cycle from a quiescent state) (Leary et al, 1992; Suda et al, 1983). There are functional differences between cycling and quiescent HSCs, for they transiently lose engraftment potential during S/G2/M phases of the cell cycle (Glimm et al, 2000; Habibian et al, 1998). This is an important caveat to bear in mind for ex vivo HSC expansion and gene therapy strategies, which require both HSC division and subsequent engraftment.  1.2.6  Exhaustability of HSCs One of the important questions in stem cell biology has been whether there is a  19  limitation to the self-renewal ability of a stem cell. Does self-renewal provide an infinite source of HSCs, or does excessive self-renewal cause them to be "exhausted" and lose HSC activity? The self-renewal capacity of HSCs is clearly sufficient to support hematopoiesis for a full lifespan. However, by pushing the system beyond a single lifespan through serial transplantation, it initially appeared that HSCs became exhausted. After 4-5 rounds of transplantation and regeneration, B M cells were unable to give further donor-derived repopulation, suggesting that the HSCs may have reached their divisional limit (Ogden and Mickliem, 1976). Even within a single lifespan, studies suggested that HSC selfrenewal capacity decreased with aging.  F L HSCs have higher repopulation and  regeneration abilities than do those from adult sources (Micklem et al, 1972; Pawliuk et al, 1996; Rebel et al, 1996a), and HSCs from young mice regenerate to a greater extent then those from older mice (Chen et al, 2000). The Intrinsic Timetable model was put forth, which proposed that there were no truly self-renewing divisions, as incremental losses in HSC activity occurred with each cell division (Lansdorp, 1997). According to the model, H S C telomere lengths could serve as a barometer of divisional history, and as telomeres shorten so too does HSC activity decrease. Eventually the HSCs would reach their proliferative limit and senesce, similar to the observations of Hayflick (1965) on somatic cells ex vivo.  In support of this  model, shorter telomere lengths were demonstrated in CD34 CD38" cells from adult B M +  compared to F L (Vaziri et al, 1994). However, a direct connection between telomere length and HSC function has not been proven. Evidence against the exhaustability of HSCs has also been presented.  Most 20  importantly, it was shown that limitations in HSC regeneration following single or serial transplantations could be due to extrinsic inhibitory factors, rather than intrinsic selfrenewal limitations.  Quantitative analyses of HSC number revealed incomplete  regeneration of the HSC pool following transplantation, to approximately 10% of the normal size (Harrison et al, 1990).  Yet this failure to regenerate the entire HSC  compartment was not due to exhaustion of HSC self-renewal, as they could still regenerate when transplanted into secondary mice. Thus, serial transplantations failed due to successive dilutions of HSC numbers, and this failure could be overcome by increasing the transplant  cell doses to maintain input H S C numbers,  demonstrating retained self-renewal ability (Iscove and Nawa, 1997).  thereby  Pawliuk et al.  (1996) further demonstrated the incomplete realisation of self-renewal potential in regenerating HSC. Only a modest C R U regeneration occurred in mice transplanted with 1,000 FL-derived C R U , whereas over 100-fold C R U amplification occurred when just 10 C R U were transplanted.  The lower C R U (and total cell) dose may have extended the  recovery phase, thereby allowing greater self-renewal prior to the onset of negative feedback regulation. Decreases in HSC activity with increased divisional history have also been called into question. Loss of regenerative activity with age might only reflect a slower rate of proliferation, as has been documented for adult B M compared to F L (Lansdorp et al, 1993; Micklem, 1972). Moreover, equal or even improved repopulation ability has been reported with aging from 12dpc F L to 16dpc F L to young adult B M to old adult B M (Ema and Nakauchi, 2000; Harrison, 1983; Harrison et al, 1984; Harrison et al, 1989; Sudo et al, 2000). Therefore, HSC self-renewal does not appear to result in losses of either HSC 21  function or self-renewal ability, and forced amplification of HSC ex vivo need not inhibit HSC performance.  1.2.7  Clinical importance of HSCs HSC imbalances can have severe clinical implications. For example, loss of stem  cells in aplastic anemia patients causes drastic reductions in all hematopoietic cell types, and is often fatal (reviewed in Young et al, 2000). The disease pathophysiology involves T 1 cell-mediated autoimmune destruction of HSCs (Kook et al, 2001; Mathe et al, H  1970; Young and Maciejewski, 1997). Primitive cells, including CD34 cells, CFC, and +  long term culture-initiating cells (LTC-IC) are all drastically reduced to < 1% of normal at the time of presentation (Maciejewski et al, 1996). Mature blood cell counts are very low, and the B M becomes replaced with fat. The reduction in granulocytic neutrophils leads to recurrent infections, which can be fatal. Supportive treatment involves transfusion of RBCs and platelets, but more severe cases require immunosuppressive therapy or stem cell transplantation (Verbeek and Ganser, 2001). The severity of this disease underscores the central importance of HSCs. Hematological malignancies can also result from deregulated HSC self-renewal and differentiation. Overproduction of immature cells suppresses the production of end cells, causing susceptibility to infection (due to neutropenia), anemia (due to erythrocytopenia), and bleeding (due to thrombocytopenia). Because HSCs have self-renewal ability and persist for long periods, they are ideal targets for accumulation of mutations leading to deregulated  self-renewal, and several lines of evidence suggest that the initial  22  transformation event occurs at the stem cell level (reviewed in Reya et al, 2001). Cancers contain a heterogeneous population of cells, with different lineage marker expression phenotypes and different proliferation potentials, suggestive of a hierarchy. For example, only 1/100 to 1/10,000 murine myeloma cells have sufficient proliferative potential to form colonies in vitro (Park et al, 1971). In chronic myelogenous leukemia (CML), a hierarchy of transformed cells has been demonstrated, including transformed LTC-IC which retain full myeloid differentiation capacity (Jiang et al.,.2000; Udomsakdi et al, 1992). Rare leukemic stem cells, with high proliferative and self-renewal potential might be responsible for clonal expansion and metastasis.  Consistent with this, in acute  myelogenous leukemia (AML) patient samples only the CD34 CD38" cells could transfer +  the disease to NOD/SCID mice (Bonnet and Dick, 1997). Thus, H S C are the targets for transforming mutations, and leukemic stem cells drive cancer cell proliferation. Stem cell based therapies are used to treat a growing number of diseases, including but not limited to those involving HSCs in their pathophysiology. HSC transplantation following a myeloablative conditioning regimen is the only potentially curative treatment for aplastic anemia, hemoglobinopathies, and leukemias, as it results in a full or partial replacement of the diseased hematopoietic tissue with normal cells. However, there are significant risks associated with,HSC transplantation, including toxicity of conditioning regimens, graft versus host disease (GVHD) and mortality (reviewed in Anasetti et al, 2001). A promising new source of HSC donor material is CB, which gives a reduced incidence of G V H D due to the naive immunological state of the cells (Rocha et al, 2001). However, there is also a higher rate of mortality because of failed or delayed engraftment. This latter issue relates to the small size of CB grafts, and 23  their consequently low stem cell content. Higher H S C doses have consistently been found to correlate with improved disease-free survival and reduced transplant-related mortality (Mavroudis et al, 1996; Sierra et al, 2000). Until HSC expansion methods are dramatically improved, the use of CB material will remain restricted to children. HSC-based gene therapy is a growing treatment option for patients with hematological defects (reviewed in (Cavazzana-Calvo and Hacein-Bey-Abina, 2001). Introduction of corrective or replacement  genes into HSCs, followed by HSC  transplantation, garners life-long production of the therapeutic gene in vivo.  This  approach has been used on patients with A D A deficiency (Hoogerbrugge et al, 1996), X linked SCID (Cavazzana-Calvo et al, 2000), Fanconi anemia (Gregory et al, 2001; Liu et al, 1999b) and hemophilia (Kay et al, 2000). As well, animal studies are ongoing to establish gene therapy as a treatment for several other diseases. These are mostly single gene deficiencies, which can in theory be corrected by resumed production of the normal gene product.  Several obstacles remain, even in treating these "simple" diseases,  including low gene transfer to HSCs. Genomic integration of retrovirus-based vectors requires target cells to be proliferating (Sadelain et al, 2000), however activation of HSCs into cycle often results in loss of engraftment potential (Glimm et al, 2000). Separation of proliferation and differentiation will greatly improve the clinical outcomes of HSC-based gene therapies  1.2.8  HSC expansion ex vivo Ex vivo  expansion of HSCs has long been a goal of experimental hematology.  24  The ability to activate stem cells into division without causing their differentiation would be an immensely useful tool both for experimental and clinical uses. Experimentally, it will help elucidate the molecular mechanisms governing the decision to self-renew or differentiate. Gene expression profiles on purified HSC populations are helping to uncover HSC-specific genes (Phillips et al, 2000), and comparing HSC populations which are actively self-renewing will further define the molecular processes of selfrenewal. Clinically important gene therapy and transplantation applications would also benefit greatly from ex vivo HSC expansion, as outlined above. Yet despite the strong impetus for ex vivo HSC expansion, and the demonstrable proliferative capacity of these cells, relatively little success in H S C expansion has been achieved. Early studies on murine cells documented expansions of progenitors and total cells, but loss of repopulating ability.  Improved purification techniques, serum-free  medium, identification of early-acting cytokines (discussed below), and functional HSC assays have all contributed to systematic analyses of conditions that would support HSC maintenance or expansion. Maintenance of repopulating activity was reported by several groups (Matsunaga et al, 1998; Ramsfjell et al, 1999; Rebel et al, 1994; Yonemura et al, 1996; Yonemura et al, 1997). Interestingly, the same culture conditions which supported maintenance of B M HSCs could not support F L HSCs, indicating intrinsic differences between HSCs from different sources (Rebel and Lansdorp, 1996).  The  largest ex vivo expansion of murine HSCs to date using exogenous growth factors was a 4-fold C R U expansion and 7-fold LTC-IC expansion in 10-day cultures (Miller and Eaves, 1997). Several groups have also attempted to expand human HSCs ex vivo (Mayani et al, 25  1993; Ramsfjell et al, 1999; Robetamanith et al, 2001; Zandstra et al, 1997). As in the murine studies, a limited amount of expansion has been achieved.  The greatest  expansions reported for human cord blood HSCs using quantitative functional assays gave 2-4 fold increases over 4-8 days (Bhatia et al, 1997; Conneally et al, 1997). Growth on stromal cell lines also enhanced proliferation of HSCs with retention of SCLDrepopulating or LTC-IC activity (Bennaceur-Griscelli et al, 2001; Bennaceur-Griscelli et al, 1999; Yamaguchi et al, 2001). Further intrinsic differences in cytokine requirements from different sources of HSCs were demonstrated, since human HSCs have different optimal cytokine combinations from murine HSCs. Even under conditions where murine or human HSC proliferation was induced, there was a large accumulation of mature and progenitor cells, suggesting that differentiation was not prevented. Recent attention has focused on cell intrinsic factors, whose activation has in some cases resulted in retention or expansion of HSC ex vivo.  Overexpression of the P-  glycoprotein pump genes MDR1 or ABCG2 led to the expansion of side population cells with retained repopulation ability (Bunting et al, 1998; Bunting et al, 2000). Ex vivo maintenance of long-term repopulating cells and serial repopulation ability were both enhanced by the addition of dXX-trans retinoic acid (ATRA) to the cultures (Purton et al, 2000; Purton et al, 2001). Constitutive Notch activation in Sca lin" c-kit B M cells led +  +  to immortalization of blast-like cells which retained lympho-myeloid differentiation and long term repopulating ability (Varnum-Finney et al, 2000). Addition of soluble Sonic Hedgehog protein to liquid cultures on human B M cells led to at least 3-fold expansion of SCDD-repopulating cells via modulation of bone morphogenic protein (BMP)-4 levels (Bhardwaj etal, 2001). 26  1.3  Regulation of Hematopoiesis While much progress has been made in our understanding of the molecular  regulation of later stages of hematopoiesis, the decision of HSCs to self-renew or differentiate is still largely enigmatic. In the next section I will summarise our current understanding of the molecular regulation of hematopoiesis, with particular emphasis on regulation at the stem cell level.  1.3.1  Extrinsic Factors  1.3.1.1 Cytokines Extrinsic control of hematopoiesis comes in part from hematopoietic cytokines. These factors are secreted by B M stromal cells and bind to specific receptors on hematopoietic cells.  Cytokines promote survival and proliferation of hematopoietic  progenitors and cell lines in vitro.  Their role in lineage commitment, however, is still  unclear, and convincing arguments can be made for both permissive and instructive roles. Hematopoietic cytokines can be subdivided into 3 categories based on their relative location of action within the hierarchy: late-acting, intermediate-acting, and earlyacting.  A schematic representation of hematopoietic cytokines and their locations of  action is presented in Figure 1.4. Late acting-cytokines such as erythropoietin (Epo) and macrophage-colony stimulating factor (M-CSF) are required for proliferation and maturation along specific  27  pathways.  Mice lacking Epo or its receptor, Epo-R, produce near-normal numbers of  erythroid progenitors, but have impaired production of mature functional RBCs (Lin et al, 1996; Wu et al, 1995). Thus these late-acting'cytokines appear not to induce lineage commitment, but rather to promote amplification and maturation of committed progenitors. Further evidence that late-acting cytokine signals are permissive rather than instructive comes from receptor domain swapping experiments. When Epo-R  was  inserted into macrophage precursor cells, the addition of Epo stimulated the formation of macrophage colonies.  Activation of thrombopoietin (Tpo) (Kieran et al, 1996) or  granulocyte-colony stimulating factor (G-CSF) (Millot et al, 2001) signaling can rescue erythropoiesis in £po-i?-deficient mice, pointing to redundancy among cytokines. Evidence for an instructive role of cytokines comes from the differential development of bipotent granulocyte/macrophage progenitors along the granulocytic pathway in response to granulocyte macrophage-colony stimulating factor (GM-CSF) or G-CSF versus along the macrophage pathway in response to M - C S F (Heyworth et al, 1993; Metcalf and Burgess, 1982). Further to this, fusions of FK506 binding protein (FKBP) with either G-CSF or Flt3 receptor signaling domains and activation by F K B P resulted in macrophage cell emergence, whereas fusion of the same binding protein with the Tpo receptor M p l resulted in emergence of megakaryocyte cells and sustained growth of multipotent myeloid cells (Zeng et al, 2001). However, it is difficult to prove in these heterogeneous settings whether the cytokine directs lineage choices or differentially facilitates the survival of already committed progenitors.  28  3 fi«5^f-l 0  IL-6,  3  A> '  IL  -  3  JGM-CSF IL-4  MYELOID  LYMPHOID  /  1 Epo  J  I  GM-CSFI G-CSF, I GM-CSFI I CSF-1*  Red Blood Cells  GM-CSrl  Platelets  Neutrophil  (IL-1, 6) I IL-2, 4, 7*  1«i  1 1 Hi  Tpo I IL-4J  4  I  IL-4J  Monocyte/ Basophil/ Macrophage Mast Cell  IL-2, 3,4 5, 6, 7 (IL-11)  ft  1  1  1 1  I |  1  IL-1, 2, 4, 9 (IL-7)  Eosinophil  T-Cell  l  I  L  1.2. 3. 4,, 5, 6, 11  B-Cell  Figure 1.4: Hematopoietic cytokines and their location of action. Shown is a schematic representation of the hematopoietic hierarchy, and the various locations of action of some important cytokines.  Intermediate-acting cytokines,  such as IL-3 and GM-CSF increase the  proliferation of multipotent progenitors. IL-3 is a potent stimulator of progenitors, and was originally also thought to activate HSC self-renewal. It was subsequently shown that the presence of IL-3 resulted in loss of repopulation ability in murine cells after ex vivo culture (Yonemura et al, 1996). Yet more recent studies indicated that IL-3 enhanced multilineage reconstitution of cultured murine stem cells (Bryder and Jacobsen, 2000), and several groups have shown that it is required for maximal expansion of human stem 29  cells (Conneally et al,  1997; Robetamanith etal,  2001). Perhaps the answer to this  seeming contradiction lies in differential effects to different cytokine concentrations, as IL-3 was shown to be inhibitory to human LTC-IC growth only when present at high concentrations and when steel factor (SF) and Flt3 ligand (Flt3L) concentrations were low (Zandstrae*a/., 1997). Early-acting cytokines include IL-6, IL-11, G-CSF, leukemia inhibitory factor (LIF), Flt3L, and SF. These support survival of primitive hematopoietic progenitors and HSCs, cause them to divide, and maintain them in the undifferentiated state. None of these factors can induce HSC self-renewal on their own, but they act synergistically to promote survival and proliferative activation of HSCs. IL-6, IL-11 and LIF share a common gpl30 signal transducer, which binds to the activated receptor and propagates a signal via the J A K / S T A T pathway.  Inhibition of  gpl30 by neutralizing antibodies inhibited the expansion of progenitors in hyperIL-6 (ie. IL-6 fused to soluble IL-6 receptor) plus SF-stimulated cultures of human CD34 C B +  cells (Ebihara et al, 1997; Sui et al, 1995). Flt3 (the receptor for Flt3L) and c-kit (the receptor for SF) belong to the tyrosine kinase receptor family. Mice lacking either SF (the W locus) or c-kit (the SI locus) have reduced CFU-S and long-term repopulating cells as well as non-hematopoietic defects. Mice deficient for expression of Flt3L  (McKenna et al,  2000) or its receptor  (Mackarehtschian et al, 1995) have reduced repopulation ability and lymphoid defects. Both SF and Flt3L have potent effects in vitro on human and murine repopulating cells, and are included in most HSC expansion cultures. SF synergizes with a large number of other early-acting factors, including IL-6, IL-11, Flt3L, and G-CSF (Bhatia et al, 1997; 30  Conneally et al, 1997; Ema et al, 2000; Miller and Eaves, 1997; Ramsfjell et al, 1999; Rebel et al, 1994; Sui et al, 1995; Yonemura et al, 1997; Zandstra et al, 1997). Similarly, Flt3L synergizes with IL-3, IL-6, G-CSF or SF to increase cloning efficiency and colony size (Hudak et al, 1995). Tpo is unique among hematopoietic cytokines, in that it has properties of both early- and late-acting cytokines (Drachman, 2000).  It promotes  megakaryocyte  proliferation and platelet production, and mice lacking expression of Tpo (de Sauvage et al, 1996) or Mpl (Gurney et al, 1994) have 80-90% fewer platelets and megakaryocytes. However, Tpo signaling also promotes  survival and proliferation of primitive  hematopoietic cells, as demonstrated both by repopulation deficiencies in knockout mice (Kimura et al, 1998) and by ex vivo expansion of primitive cells in cultures containing Tpo (Hunnestad et al, 1999; Liu et al, 1999a; Luens et al, 1998; Murray et al, 1999; Piacibello et al, 1998; Piacibello et al, 1999; Solar et al, 1998; Yagi et al, 1999). HSC self-renewal is dependent not only on the types of cytokines present but also on their relative concentrations. Cultures of Flt3L, SF, IL-6, IL-3, and G-CSF maximally expanded progenitor, cells at 30 times lower concentrations than those required for maximal expansion of LTC-IC (Zandstra et al, 1997). The ligand-receptor threshold model postulates that HSC self-renewal requires maintenance of critical signals above a threshold level. Thus the ligand, its receptor, and signaling intermediates must all be available to the cell at threshold concentrations to allow self-renewal, and i f any of these is reduced the outcome will instead be differentiation (Zandstra et al, 2000). Negative hematopoietic regulation also involves cytokines. Inhibitory cytokines such as macrophage inflammatory protein 1 alpha (MEPla) and transforming growth 31  factor beta (TGFP) act antagonistically with early-acting cytokines to inhibit proliferation of primitive progenitors (Batard et al, 2000; Eaves et al, 1991; Keller et al, 1994). These could act to facilitate maintenance of HSC pool sizes, and confer negative feedback on HSC regeneration following B M transplantation (Pawliuk et al, 1996).  1.3.1.2 Developmental Signaling Molecules A number of developmental^ important extrinsic signaling pathways also play a role in hematopoiesis.  This is perhaps not surprising, since processes that govern  embryonic development, such as progenitor cell proliferation, lineage commitment, differentiation and maturation are ongoing in the hematopoietic system as well. The Wnt family of genes encode secreted glycoproteins which bind receptors of the Frizzled family, activating a pathway which leads to stabilization of (3-catenin, which can then translocate to the nucleus with TCF/Lef and activate transcription of target genes (for reviews see Hunter, 1997; Miller and Moon, 1996; Willert and Nusse, 1998). Wnt and Frizzled genes are expressed on primary murine (Austin et al, 1997) and human (Van Den Berg et al,  1998) hematopoietic and stromal cells.  Addition of Wnt-  conditioned media to primitive F L or B M cells led to increased proliferation of multipotent progenitors (Austin et al, 1997). Inhibition of Wnt signaling by addition of antisense Wnt 11 reduced or abolished differentiation of a quail mesodermal cell line along monocytic or erythroid lineages, and these same lineages were reciprocally elevated when Wnt 11 or Wnt5a was administered to HSC-enriched B M cultures (Brandon et al, 2000). Thus Wnt signaling appears to function in hematopoietic regulation, to modulate  32  the diversity of hematopoietic cell types. Notch signaling in development is important for specification of cell fates. Notch binding to Jagged or Delta ligands on adjacent cells releases the intracellular portion of Notch (IC-Notch), and ultimately inhibits transcription of lineage-specific genes. Lateral inhibition via Notch signaling allows adjacent cells to respond differently to common environmental stimuli. Notch activation inhibits differentiation along a primary fate, leaving it available to adopt an alternative fate (for reviews see Kojika and Griffin, 2001; Milner and Bigas, 1999). Similarly, in hematopoiesis Notch activation specifies lineage choices by inhibiting differentiation along alternate lineages. It does so at several stages of development, including the HSC, the C L P , and various stages of T-lymphopoiesis. Overexpression of a constitutively active form of Notchl  (IC-Notchl)  promotes  development of T-lymphopoiesis (Pui et al, 1999) and of a(3 T-cell receptor (TCR) (Washburn et al, 1997) and C D 8 (Robey et al, 1996) T cells. Conversely, loss-of+  function Notch mutants have accumulations of B-cells (Radtke et al, 1999), and most Tcells are y5 T C R (Washburn et al, 1997) and C D 4 (Robey et al, 1996). The role of +  Notch in myelopoiesis is more controversial, but most evidence supports a role in inhibition of cytokine-induced differentiation, leading to expansion of undifferentiated blast cells. IC-Notchl inhibits G-CSF-induced granulocytic differentiation of 32D cells (Bigas et al, 1998; Milner et al, 1996; Tan-Pertel et al, 2000), hemin-induced erythroid differentiation of K562 cells (Lam et al, 2000), and GM-CSF-induced macrophage differentiation of bipotent macrophage/dendritic cell precursors (Ohishi et al, 2001). In murine HSC-enriched populations, addition of IC-Notch4 (Ye and Moore, 2000) or the Notch ligands Deltal (Ohishi et al, 2001) or Jaggedl (Bartelmez et al, 2000) delayed 33  the acquisition of lineage markers. Most dramatically, addition of IC-Notchl led to the generation of immortalized cytokine-dependent cell lines with retained lympho-myeloid differentiation ability in vitro and in vivo (Varnum-Finney et al, 2000). The TGFB superfamily includes TGFfi, BMP and activin members. TGFP is a potent inhibitor of cycling in murine (Sitnicka et al, 1996) and human (Batard et al, 2000; Garbe et al, 1997) stem cells, as addition of this factor reduced output of mature and progenitor cells while maintaining primitive cells.  Conversely, addition of  neutralizing antibodies to TGF(3 promoted proliferation of primitive cells (Hatzfeld et al, 1991; Imbert et al,  1998).  This effect of TGFp is accomplished in part through  inhibition of expression of both SF and c-kit (Heinrich et al, 1995). The mammalian hedgehog family, whose target genes include BMP4, consists of Sonic hedgehog (Shh), Desert hedgehog (Dhh) and Indian hedgehog (Ihh). Addition of soluble Dhh or Shh to cultures of human CD34 CD381in" cells resulted in expansion of cells with a primitive +  phenotype and at least 3-fold expansion of multilineage SRCs (Bhardwaj et al, 2001). This effect was mediated by modulating the local effective concentration of BMP4 (Bhatia et al, 1999), through induced expression of both BMP4 and its specific inhibitor Noggin.  1.3.2  Intrinsic Regulation: transcription factors While extrinsic factors are largely permissive for hematopoietic survival and  maturation, intrinsic factors act deterministically, to program lineage commitment and hematopoietic cell fates. A hematopoietic cell's developmental state is reflected by its  34  complement of expressed genes. Experiments have shown that multipotent progenitor cells express a wide variety of lineage-associated genes at low levels (Hu et al, 1997). Development along a given lineage thus occurs by both activation of lineage-specific maturation factors, and repression of genes associated with alternate lineages. Transcription factors act in both these processes, thereby promoting lineage choices. Through the study of c/s-binding factors at lineage-specific genes and genes activated in leukemias, hematopoietic transcription factors have been identified.  These act at all  hematopoietic branch points, including specification to the hematopoietic fate, branching of the major lymphoid and myeloid lineages, and commitment of bipotent progenitors to single lineages (for reviews see Orkin, 2000; Shivdasani and Orkin, 1996; Sieweke and Graf, 1998). Some of the major hematopoietic transcription factors, and their locations of action, are outlined in Figure 1.5. Genes required for specification of hematopoietic cell fate include SCL (also called tal-]) and LM02 (also called rbtn2), which encode basic helix-loop-helix (bHLH) and LEVI-domain type transcription factors, respectively. Mice lacking either of these genes have complete absences of primitive hematopoiesis and die at approximately 10 dpc (Porcher et al, 1996; Warren et al, 1994). Moreover, chimeric mice generated from injection of SCX-null ES cells had an absence of SCL-null blood cells in any lineage, indicating a requirement for this transcription factor in definitive hematopoiesis as well (Porcher et al, 1996). SCL and L M 0 2 proteins interact physically (Larson et al, 1996; Wadman et al, 1994), and their similar loss-of-function phenotypes suggest cooperative transcriptional control of hematopoiesis-specific genes. Both proteins are further required for erythroid differentiation, where they form a complex together with the transcription 35  factors GATA1, E2A and Ldbl (Valge-Archer et al, 1994; Visvader et al, 1997; Wadman et al, 1997).  Ectopic expression of SCL or LM02 in T-cells, either via  chromosomal translocations in human cells (Boehm et al, 1991; Brown et al, 1990; Finger et al, 1989; Royer-Pokora et al, 1991) or transgenic mouse models (Larson et al, 1996), lead to the generation of T-cell acute lymphoid leukemia (ALL).  platelets Figure 1.5: Transcription factors involved in hematopoietic lineage commitment. A schematic representation of the hematopoietic hierarchy and the primary locations of action for some hematopoietic-specific transcription factors (underlined).  36  Ikaros is an intrinsic factor which acts at the lympho-myeloid branching point. This zinc finger protein promotes specification to the lymphoid lineage. Mice lacking Ikaros lack all B-lymphocytes and precursors, as well as fetal T-lymphocytes although a few C D 4 T-cells are aberrantly produced (Wang et al, 1996). A more severe phenotype, +  involving complete absence of all T, B , and N K cells and their progenitors, was seen in mice with a dominant negative Ikaros mutation (Georgopoulos et al, 1994), possibly due to the inability of the dominant negative protein to interact with its homologous partner Aiolos (Morgan et al, 1997). Ikaros and Aiolos repress transcription of non-lymphoid genes via recruitment of histone deacetylase (HDAC) complexes to specific promoters (Kim et al, 1999; Koipally et al, 1999), thereby altering chromatin accessibility. Ikaros also appears to have HDAC-independent repressive function, through interaction with the co-repressor C-terminal binding protein (CtBP) (Koipally and Georgopoulos, 2000). Further specification within the lymphoid lineage comes from factors such as Pax5. Pax5 is a paired-box transcription factor which directs cells along the B-lymphoid lineage by repressing differentiation along alternate lineages. Pax5 knockout mice lack mature B-cells and precursors (Urbanek et al, 1994), and pro-B cells from these mice will reconstitute T but not B lymphoid cells in transplanted mice (Rolink et al, 1999). PaxJ-deficient pro-B cells can differentiate to a wide variety of mature cell types in vitro, including macrophages,  osteoclasts, dendritic cells, granulocytes, and N K cells.  However, when Pax5 is added back, the pro-B cells are once again restricted to the B lineage, with associated restrictions to gene expression (Nutt et al, 1999). Pax5 represses target gene expression through interaction with the co-repressors of the Groucho family (EberhardeM/., 2000). 37  Lineage specification within the myeloid system comes in part from crossantagonism  between  GATA-1  and  PU.l.  GATA-1  promotes  erythroid and  megakaryocyte differentiation. Loss of either GATA-1 or its cofactor Friend of GATA-1 (FOG-1) (Tsang et al, 1997) resulted in blocked erythropoiesis at the proerythroblast stage (Fujiwara et al, 1996; Pevny et al, 1995; Pevny et al, 1991; Simon et al, 1992), and defects in megakaryocyte development (Shivdasani et al, 1997; Tsang et al, 1998; Vyas et al, 1999). The Ets family transcription factor P U . l (also called Spi-1) acts in disparate lymphoid and myeloid lineages. Mice lacking PU.l have reduced or absent monocytes, granulocytes, T-lymphocytes and B-lymphocytes (Anderson et al, 1998; McKercher et al, 1996; Scott et al, 1997; Scott et al, 1994). Forced expression of GATA-1 in a chicken multipotential progenitor line led to erythroid, megakaryocyte or eosinophil differentiation (Kulessa et al, 1995), whereas forced expression of PU.l in the same line led to myeloid differentiation (Nerlov and Graf, 1998). GATA-1 and P U . l directly interact with and inhibit one another (Nerlov et al, 2000; Rekhtman et al, 1999). GATA-1 represses P U . l by inhibiting its association with the coactivator c-Jun (Zhang et al, 1999). P U . l in turn inhibits the binding of GATA-1 to D N A (Zhang et al, 2000). Thus the ultimate lineage choice will be decided and then reinforced by the relative levels of these two transcription factors. Hox family transcription factors are emerging as important regulators of hematopoiesis, acting at various levels of the hematopoietic hierarchy. The following sections will discuss the roles and mechanism of action of these proteins.  38  1.4  1.4.1  Hox transcription factors  Hox discovery  Over 100 years ago, Bateson (1894) described a homeotic phenotype, where one region is transformed "into the likeness of another".  More recently, Lewis (1978)  discovered a group of 3 genes in Drosophila melanogaster that gave homeotic phenotypes when mutated, and named these the homeotic genes. For example, in flies lacking the Ultrabithorax (Ubx) gene, the thoracic balancing organs called halteres are transformed into a second set of wings. The 3 genes identified by Lewis make up the bithorax complex (BX-C). Later a second complex of 5 homeotic genes was identified: the antennapedia complex (ANT-C) (Schneuwly et al, 1986; Scott et al, 1983). These genes also gave homeotic phenotypes, such as the transformation of antennae to legs in Antennapedia (Antp) mutants. Drosophila  Together the 2 groups of clustered genes make up the  homeotic complex {HOM-C).  HOM-C  products give antero-posterior  positional identity, thereby specifying which structures a segment should produce. Misexpression of an HOM-C gene alters segmental identity, leading to the ectopic production of morphological structures. Beyond their phenotypic similarities, the HOM-C genes were found to share a 183 base pair sequence dubbed the homeobox, which encodes a 61 amino acid homeodomain. The genes are thus often referred to as homeobox genes, which led to their naming as Hox genes in other organisms including vertebrates. The clustered Hox genes are called the Class I Hox family, to distinguish them from the many non-clustered and divergent homeobox-containing genes. These latter are called Class II homeobox genes, and are  39  organized into over 12 gene families based on shared sequence motifs, including caudal, bicoid, engrailed, even skipped, Pit-Oct-Unc (POU), empty spiracles, and orthodenticle (Cillo et al, 2001). This thesis deals solely with the Class I Hox genes.  1.4.2  Hox Gene Organization and Expression Mammalian genomes contain 39 Hox genes, organized into clusters A through D,  which arose through duplications of a single ancestral cluster (Pendleton et al, 1993). Genes can be aligned by sequence similarities into 13 paralog groups, indicating conservation of gene order within each cluster. Standard nomenclature now includes both cluster and paralog group identities, as in human HOXA1 or murine Hoxa-1 (Scott, 1992). Remarkably, gene order has been conserved throughout Hox cluster evolution, as vertebrate genes follow the same order as their Drosophila counterparts (Graham et al., 1989) (Fig. 1.6).  Because all mammalian Hox genes (and most Drosophila HOM-C  genes) are transcribed in the same orientation, the clusters can be said to have 5' to 3' orientation, with lower numbered paralog groups at the 3' ends of the clusters. Hox genes have a remarkable expression pattern, which may in large part explain the extreme conservation of gene order.  The genes are expressed colineaf with  chromosomal order. This was first identified in Drosophila, where genes at the 3' ends of the clusters were found to be expressed earliest in development, with more 5' genes expressed later.  Colinearity also extends to the spatial domains of H O M - C gene  expression, with 3' genes .expressed in more anterior structures and more 5' genes having sequentially more posterior expression domains. This temporal and spatial colinearity  40  also holds true for mammalian Hox gene expression during development (Dolle et al, 1989; Duboule and Dolle, 1989; Graham et al, 1989; Izpisua-Belmonte and Duboule, 1992; Izpisua-Belmonte et al, 1991), suggesting that gene order might have been conserved in order to maintain this tightly linked expression pattern.  Potential  mechanisms for colinearity of Hox expression include sequential activation of more 5' genes by 3' gene products, 3' to 5' morphogen affinity gradients, and progressive opening of the chromatin from 3' to 5'.  HOX A  J A1  HOX B J  H  A2  B1  W  B2  A3  A4  H  H B3 H B4 H B5 H  HOXC  A7  B6  C6  HOXD 1 DD1 1 I  .  D4  D3 I L  J I  ,  A9  A11  H B7 H B8 HB9B9  B13  C8 C8 H C9 HC10HC11HC12HC13  D8 H D9 HD10HD11HD12HD13 I L  J L  i Drosophila-  lab  3'  pb  early anterior  A13  DfdHScr H Antp k-//-HUbxHAbdA^ AbdA Direction of gene transcription  r AbdB  5'  late posterior  Figure 1.6: HOX Chromosomal Organization. The four mammalian HOX clusters (AD) are shown on top, with alignments (blue lines) to the Drosophila ANT-C (left) and BXC (right). Genes at the 3' ends of the clusters are expressed earliest and most anterior, with sequentially later and more posterior expression of more 5' genes.  41  T h e clustered organization o f Hox genes m a y also be a consequence o f shared regulatory sequences (for a r e v i e w see D u b o u l e , 1998). between  E n h a n c e r sequences located  t w o Hox genes can i n some cases activate expression o f b o t h genes. F o r  example, shared enhancers have been found for  Hoxb-3 and Hoxb-4 ( G o u l d et al., 1997),  and for Hoxb-4 and Hoxb-5 (Sharpe et al, 1998), w i t h refinements i n expression c o m i n g from additional gene-specific regulatory elements.  T h e r e are also g l o b a l enhancers,  w h i c h act over several genes i n a manner analogous to the g l o b i n l o c u s c o n t r o l r e g i o n . A region just outside the  5' e n d o f the HoxD cluster appears to delay a c c e s s i b i l i t y to 5 '  located genes, i n c l u d i n g an ectopically located  Hoxd9-lacZ transgene ( K o n d o a n d  D u b o u l e , 1999; van der H o e v e n et al, 1996). L o s s o f these shared regulatory sequences by cluster disruption c o u l d cause gross transformations, w h i c h m i g h t not be c o m p a t i b l e with survival. There is also a hierarchical functional pattern w h i c h correlates w i t h gene order (for a r e v i e w see D u b o u l e and M o r a t a , 1994). T h e more 5 ' (posterior) genes appear to be functionally d o m i n a n t over m o r e 3 ' (anterior) genes. regulation o f n e i g h b o u r i n g 3* gene expression.  T h i s occurs i n part f r o m d o w n -  H o w e v e r , there is also functional  i n h i b i t i o n , because ubiquitous expression o f Hox genes i n transgenic flies o r m i c e g i v e homeotic effects  o n l y anterior to their normal expression d o m a i n s .  T h i s results i n  posterior transformations o f the b o d y p l a n , as regions take o n the identity o f the most 5 ' gene expressed. C o n v e r s e l y , loss o f Hox expression affects o n l y structures at the anterior limits o f a Hox expression d o m a i n , i n d i c a t i n g that i t has l i m i t e d function i n more posterior regions where other (more 5 ' ) Hox genes are co-expressed.  H o w e v e r , whether  42  this functional hierarchy is a cause or a consequence of the conserved gene order is still unclear.  1.4.3  Protein Structure and Function  Hox proteins are DNA-binding transcription factors.  They have domains for  D N A binding, and for protein-protein interactions. A general schematic of Hox protein structure is shown in Fig. 1.7.  Meis interaction NH  2  Casein Kinase II phosphorylation  I  Pbx interaction: ANV HOMEOYPWM DOMAIN  N-term a helix 1 arm  COOH  a helix 2  a helix 3  Figure 1.7: Hox Protein Structure. Shown is a representative Hox protein structure, demonstrating the important domains for protein-protein interation (Meis and Pbx interaction domains, grey), D N A binding (homeodomain, striped), and post-translational modification (CKII phosphorylation site, black). The homeodomain region is represented below, showing the N-terminal arm and 3 a-helices which contact the D N A .  The most prominent structure of all Hox proteins is their homeodomain (reviewed in Gehring et al, 1994). The structure of the homeodomain has been deduced by N M R (Billeter et al, 1990; Qian et al, 1989) and X-ray crystallography (Kissinger et al, 1990; L i et al, 1995). At the N-terminal end is a flexible arm, followed by alpha helix I, which is connected by a loop to alpha helix II. The helix-turn-helix sequence connecting helices 43  II and HI forms a highly conserved structure common to many D N A binding proteins. Footprinting, E M S A , and trans-activation assays have shown that Hox proteins bind D N A as monomers to a 5'-TAAT-3' core motif (Kalionis and O'Farrell, 1993). Helix IH acts to recognize this sequence and binds D N A in the major groove. The flexible N terminal arm binds to bases in. the minor groove, and the loop between helices II and III binds to the D N A backbone (Otting et al., 1990). Hox proteins can also bind D N A in association with cofactors of the three amino acid loop extension (TALE) family of divergent homeodomain-containing proteins. Motifs required for interaction with these proteins are located N-terminal to the Hox homeodomain.  Hox proteins in paralogs 1 through 10 bind Pbx cofactors via either  Y P W M (paralogs 1-8) (Piper et al., 1999) or A N V (paralogs 9-10) amino acid sequences, and those in paralogs 9 through 13 bind Meis cofactors via their N-terminal ends (Shen et al, 1997).  These interactions only occur in the presence of D N A , where Pbx-Hox  (Chang et al, 1995; Penkov et al, 2000) or Meis-Hox (Shen et al, 1997) cooperatively bind bipartite recognition sequences.  Cofactor binding thereby increases Hox target  specificity, by increasing the length of the recognition sequence. Cofactor binding also increases the strength of D N A binding by Hox proteins (Green et al, 1998). Moreover, binding  to  Pbx modulates  interactions  between  the  N-terminal arm of Hox  homeodomains with D N A , thereby establishing different D N A binding specificities for Hox proteins from different paralog groups (Chang et al, 1996). Meis and Pbx can also interact with each other in the presence or absence of D N A (Chang et al, 1997b), and this interaction facilitates nuclear translocation of Pbx by blocking a nuclear export signal (Abu-Shaar et al, 1999; Affolter et al, 1999; Berthelsen et al, 1999). Finally, trimeric 44  Meis-Pbx-Hox complexes have been demonstrated, and have enhanced D N A binding stability (Jacobs et al, 1999 Shanmugam, 1997 #224; Shen et al, 1999). Thus, cofactor binding can significantly contribute to specificity and activity of Hox D N A binding. Although Hox proteins have inherent fr-a/w-activation activity (Vigano et al, 1998), Hox-Pbx heterodimers can also regulate transcription through recruitment of transcriptional co-activators or co-repressors to Hox target genes. Trans-activation can be enhanced by recruitment of C R E B binding protein (CBP) via interaction with Pbx (Asahara et al, 1999; Chariot et al, 1999; Saleh et al, 2000). This co-activator has acetyltransferase activity, and confers an open chromatin state by acetylation of histone N-terminal tails.  Conversely, Hox-Pbx can also recruit a co-repressor complex  containing SMRT (silencing mediator for retinoic acid receptor and thyroid hormone receptor) and nuclear co-repressor (NcoR) (Asahara et al, 1999; Saleh et al, 2000), which Nhas deacetylase activity.  Saleh et al. (2000) further demonstrated that cell  signalling via protein kinase A (PKA) could switch Hox-Pbx interactions from corepressors to co-activators. Hox  proteins might also be regulated by post-translational modifications.  Phosphorylation of Drosophila Antp by casein kinase II (CKII) inhibits its activity (Jaffe et al, 1997), and the same type of regulation might also occur with mammalian Hox proteins, as many contain putative CKII phosphorylation sites.  Phosphorylation of  HOXA10 by an unknown kinase decreased its ability to bind D N A and to repress transcription in vitro (Eklund et al, 2000). Hoxb7 inhibits differentiation of the myeloid cell line 32D, and both the homeodomain and the Pbx interaction motif are strictly required for this function. Conversely, mutation of the Hoxb7 CKII sites causes the gain45  of-function phenotype of enhanced differentiation, indicating that phosphorylation at these sites normally inhibits Hoxb7 function (Yaron et al, 2001).  1.4.4  Hox target genes Surprisingly little is known about the target loci of Hox proteins. One class of  targets is clearly the Hox genes themselves, through auto- and cross-regulation (Zappavigna V . A . , 1991). For example, the Hoxb-1 autoregulatory element (bl-ARE) regulates Hoxb-1 expression in rhombomere 4 of the hindbrain. Hoxb-1 binds to this region with Pbx, and positively controls its own expression. Hoxa-1 can also bind to this region and activate Hoxb-1 expression in response to retinoic acid, and this crossregulation also requires Sox/Oct heterodimer binding to the b l - A R E (Di Rocco et al, 2001). Outside of the Hox genes themselves, Hox proteins activate or repress transcription of a variety of regulatory and effector molecules.  Target genes of the  Drosophila H O M - C proteins include transcription factors (eg. empty spiracles, teashirt), signalling molecules (wingless, decapentaplegic), structural molecules (eg. centrosomin) and adhesion molecules (eg. connectin) (reviewed in Graba et al, 1997).  46  Table 1.1: Hox target genes Target Gene  Hox Gene  Target Gene Function  Cell Type  Reference  N-CAM  HOXB8 HOXB9 HOXC6 Hoxa-1 HOXD3 HOXD9  adhesion  fibroblast line; lung cancer line  (Chalepakis et al., 1994; Hamada et al, 2001; Jones et al.,  L-CAM E-CAD integrin a V p 3  Hoxa-9 HOXD3 HOXD3  1993; Jones etal, 1992) adhesion adhesion adhesion  integrin P3 integrin a 3  fibroblast line thymocytes; lung cancer line vascular endothelium; lung cancer line  integrin a 4 mgl-1  HOXC8  uPA  HOXD3  MMP2  HOXD3  osteopontin  HOXA9 HOXC8 HOXB7 HOXA5  bFGF progesterone receptor TTF-1 GATA-2 p53 AP-1 complex (Jun-B, Fra-1) p21  HOXB3 Hoxb-1 HOXA5 HOXB4 HOXA10  putative adhesion (Ig-hke) matrix degrading enzyme matrix degrading enzyme bone growth  vascular endothelium lung cancer line  (Jones etal, 1992)  (Izonetal, 1998); (Hamada et al, 2001) (Boudreaue/a/., 1997); (Taniguchi et al., 1995); (Hamada et al, 2001) (Tomotsune et al., 1993) (Boudreauc^a/., 1997); (Hamada et al, 2001) (Hamada et al, 2001)  lung epithelium line breast cancer line breast cancer line  (Sbiet a/., 2001);  fibroblast line hindbrain  (Guazzi etal, 1994) (Pata etal, 1999)  apoptosis proliferation  breast cancer line fibroblast line  cell cycle arrest  myelomonocytic line  ( R a m a n s a l , 2000a) ( K r o s l and Sauvageau, 2000) (Bromleigh and Freedman, 2000)  growth factor growth factor receptor transcription factor transcription factor  (Shi etal, 1999) (Care et al, 1998) (Raman et al, 2000b)  , Mammalian Hox proteins also regulate the expression of a number of adhesion and extracellular matrix molecules (Table 1.1). These include integrin family members a5f33 (Boudreau et al, 1997), a2J31, a5/31, and a6(jl (Cillo et al, 1996), as well as Immunoglobulin (Ig) superfamily genes nuclear-cell adhesion molecule (N-CAM) (Jones et al, 1992), intercellular adhesion molecule (I-CAM), very late antigens (VLA)-2, -5,  47  and -6 (Cillo et al, 1996), and a2V collagen (Penkov et al, 2000). Other extracellular matrix molecules regulated by Hox include cytotactin (Jones et al, 1992) and the matrix degrading urokinase-typeplasminogen activator (uPA) (Boudreau et al, 1997). Hoxa-9 represses transcription of the bone matrix protein osteopontin, and that repression is relieved when Hoxa-9 is displaced by Smadl in response to TGFp stimulation (Shi et al, 2001). Regulatory molecules are also targets of Hox proteins in mammals. For example, H O X B 7 can activate basic fibroblast growth factor (bFGF) expression, which is an important angiogenic factor (Care et al, 1998).  Hoxb-1 activates expression of the  GATA-2 gene in the developing mouse hindbrain (Pata et al, 1999). A n elegant study by Raman et al (2000b) recently demonstrated that H O X A 5 can trans-activate the p53 gene, and that inactivation of HOXA5 often substitutes for p53 inactivation in breast cancer cells.  The first direct link between a Hox protein and cell cycle regulation came from  evidence that HOXA10 can activate p21 expression, resulting in cell cycle arrest and differentiation of U937 myelomonocytic cells (Bromleigh and Freedman, 2000). similar link between HOXB4 and cell cycle regulators has been reported.  A  In RAT-1  fibroblasts engineered to express high levels of HOXB4, there is a correlative rise in cyclin Dl expression and a shortened G i phase. H O X B 4 regulates cyclin Dl expression indirectly, however, by modulating expression of AP-1 transcription factor complex members Jun-B and Fra-1 (Krosl and Sauvageau, 2000).  48  Hox function in mammalian development  1.4.5  As in Drosophila, Hox genes play key roles in patterning the developing mammalian embryo. They are expressed colinearly 3' to 5' and anterior to posterior (or proximal to distal) along the developing hindbrain, trunk and limbs. In general, genes in paralog groups 1 through 4 control formation of hindbrain and cervical regions, those in paralog groups 5 through 8 control thoracic regions, and those in paralog groups 9 through 13 control limbs and sacral regions. With so many Hox genes expressed in development, how important is any one gene? Within a cluster, Hox genes have sequential anteroposterior expression domains, and therefore function in different regions of the developing embryo.  Moreover,  functional hierarchy is conserved, as the structures affected in loss-of-function mutants correspond to the anterior-most limits of expression. For example, mice deficient for Hoxb-4 have defects restricted to upper cervical vertebrae, even though the gene is normally expressed posteriorly to the end of the spinal cord (Ramirez-Solis et al, 1993). Similarly, gain-of-function mutants have homeotic transformations only in regions anterior to their normal expression domains. For example, mice with ectopic expression of Hoxa-7 under the control of the (3-actin promoter had multiple craniofacial abnormalities, but no posterior defects even though the gene was almost ubiquitously expressed (Balling et al, 1989). Thus, within a given Hox cluster, each gene controls the development of a particular body region, according to its relative location within the cluster. Between clusters, however, there does appear to be some overlap in function. Within a paralog group, genes have similar anterior expression limits, due to conserved 49  c/s-regulatory elements, and therefore function in common regions.  Comparing the  phenotype of mice lacking two different genes in the same paralog group, we often see some common aspects and some unique aspects. For example, while both the Hoxb-4 and Hoxd-4 knockout mice displayed partial transformation of the second (C2, axis) to first ( C l , atlas) cervical vertebrae, the structural changes were gene-specific. The Hoxb-4 null mouse had a broadened axis neural arch (Ramirez-Solis et al, 1993), while the Hoxd-4 null mouse also had a reduced axis dens (Horan et al, 1995a), both of which are characteristic of the atlas. Mice deficient for multiple genes in the same paralog group exemplify the partial redundancy, as they have both additive and novel phenotypes. For example mice doubly deficient for Hoxb-4 and Hoxd-4 exhibit defects not seen in either single mutant (eg. C3 to C l and C3 to C2 transformations), as well as increased penetrance and severity of defects seen in single mutants (eg. C2 to C l transformation) (Horan et al, 1995b). Further dose-dependency is demonstrated in mice triply deficient for Hoxa-4, Hoxb-4 and Hoxc-4, which exhibit transformations of C2, C3, C4, and C5 toward C l identity (Horan et al, 1995b). The limits of functional redundancy were tested recently, by replacing the Hoxa-3 coding region with that of Hoxd-3, and vice versa. Although the individual gene knockout phenotypes are completely non-overlapping, either gene could completely rescue the null phenotype of the other when moved to its respective endogenous location (Greer et al, 2000). There are no completely redundant genes; all have individual phenotypes. However the uniqueness appears to have more to do with subtle evolved differences in c/s-regulation than with specific protein functions.  50  1.5  Hox and hematopoiesis  1.5.1  Hox expression in hematopoietic cells Based on the involvement of Hox genes in development, and the many  correlations between developmental regulatory pathways and hematopoietic regulatory pathways, several groups have examined whether Hox proteins might also have a role in hematopoiesis. Several early studies examined expression of particular Hox genes in murine and human hematopoietic cell lines. A general pattern emerged from these studies, whereby Hox A cluster genes are expressed predominantly in myelomonocytic cell lines (Magli et al, 1991; Vieille-Grosjean et al, 1992b), whereas Hox B cluster genes are expressed predominantly in erythroid cell lines (Magli et al, 1991; Shen et al, 1989; VieilleGrosjean et al, 1992b). Hox C cluster genes are expressed in both myeloid and erythroid lines (Magli et al, 1991), and with a single exception (Taniguchi et al, 1995) Hox D cluster gene expression has never been detected in hematopoietic cell lines (Magli et al, 1991).  However, there are multiple exceptions to the above expression patterns,  including expression of several Hox A (Kongsuwan et al, 1988; Vieille-Grosjean et al, 1992b) and B (Petrini et al, 1992; Shen et al, 1989) cluster genes in B - and T-lymphoid cell lines. Hox expression was then analyzed in primary hematopoietic tissues. Expression of Hox A, B, and C cluster genes were detected by RT-PCR in progenitor-enriched (CD34 ) populations of human B M cells (Moretti et al, 1994; Vieille-Grosjean et al, +  1992a).  Moreover, Sauvageau et al. demonstrated developmental stage-specific Hox  51  expression patterns in human B M cells (Sauvageau et al, 1994). Genes located at the 3' ends of the HOX A and B clusters (eg. HOXB3) were expressed only in the most primitive, LTC-IC-enriched (CD34  CD45RA  +  10  CD71 ) population, whereas those 10  located at the 5' ends of the clusters (eg. HOXA10) had persistent expression into a progenitor-enriched (CD34 CD45RA CD71 ) population. Expression of all genes (ie. +  10  hl  HOXB3 or HOXA10) was extinguished in the mature (CD34") cell population.  Thus,  there appears to be a pattern of Hox gene down-regulation with early hematopoietic development, which is colinear with 3' to 5' gene order. This down-regulation might be necessary for cells to progress through the early stages of hematopoiesis. Other groups have also demonstrated temporal colinearity of Hox gene activation when hematopoietic cells are induced to terminally differentiate.  When human PB  progenitor cells were induced to differentiate along granulocytic or erythrocytic pathways, a sequential activation of HOXB3, HOXB4, HOXB5, and (granulocytic lineage only) HOXB6 was observed (Giampaolo et al, 1994). Similarly, a 3' to 5' wave of activation of HOXB1 through HOXB9 was observed when PB T-lymphocytes were activated by P H A (Care et al, 1994), or when PB natural killer (NK) cells were activated by IL-2 and IL-1 (3 (Quaranta et al, 1996). Inhibition of Hox gene activation might therefore prevent terminal differentiation. Finally, the expression of HOX genes in malignant hematopoietic cells was analyzed. HOXA10 expression was found in all acute and chronic myeloid leukemia (ie. A M L or C M L ) samples, but not in acute or chronic lymphoid leukemia (ie. A L L or CLL) samples (Lawrence et al, 1995), consistent with its myeloid-restricted expression in cell lines. Several HOX B cluster genes, but not HOX A, C, or D cluster genes were detected 52  in C M L patient samples (Celetti et al, 1993). In contrast to the pattern seen in normal B M , HOXA9, HOXA10, HOXB3 and HOXB4 all failed to be down-regulated with hematopoietic maturation in patients with AJVIL (Kawagoe et al, 1999).  Lymphoid  leukemias also expressed Hox genes. Multiple HOX genes from throughout the HOX A, B, and C clusters were detected in B - or T-cell A L L and C L L samples (Celetti et al, 1993). HOXC4 and HOXC6 were expressed in the majority of non-Hodgkin's lymphoma (NHL) patient samples, but HOXC5 expression was restricted to anaplastic large T-cell lymphomas (Bijl et al, 1997a; Bijl et al, 1997b). Thus both myeloid and lymphoid leukemias often have aberrant HOX expression patterns, which might be a cause of their failure to differentiate, as outlined in the next section.  1.5.2  L e u k e m i c effects  Misexpression of a Hox gene, or altered Hox protein function can cause deregulated growth and differentiation of hematopoietic cells. This was first demonstrated in the leukemic cell line WEHI-3B, in which both the Hoxb-8 and IL-3 genes are activated by insertion of intracisternal A particle (LAP) endogenous retroviruses upstream of their coding regions (Blatt et al, 1988).  It was subsequently demonstrated that  activation of both the Hox and the cytokine genes were required for transformation. A fraction (22%) of the mice transplanted with B M cells overexpressing Hoxb-8 (see below) developed A M L after approximately one year (Perkins and Cory, 1993). Likewise, overexpression of IL-3 alone resulted in myeloproliferation, but it was not transplantable and the mice survived 45 days. However, when both Hoxb-8 and IL-3  53  were overexpressed, a highly aggressive myeloid leukemia developed, killing the mice within 20 days (Perkins et al, 1990). A recent report revealed that Meis2 expression is also elevated in the WEHI-3B cell line, although there is no detectable IAP in the region (Fujino ^ a / . , 2001). ' The ability of a Hox gene to transform hematopoietic cells in vivo was first demonstrated in the B X H - 2 mouse strain. This strain is highly susceptible to leukemias, due to mutagenesis by an actively transposing endogenous retrovirus. B y cloning and sequencing the region surrounding the proviral genome in leukemic samples, protooncogenes can be identified. By this method it was first demonstrated that co-activation of a Hox gene and a Hox cofactor gene could be leukemogenic. 15% of the mice with A M L had Meisl activations, and 95% of those also had either Hoxa-7 or Hoxa-9 activations (Moskow et al, 1995; Nakamura et al, 1996). The ability of HOXA9 and Meisl  to transform murine cells was further demonstrated by retrovirus-mediated  overexpression of both genes (Kroon et al, 1998). The importance of this model to human leukemias was underscored by recent studies demonstrating the frequent coexpression of HOXA9 and Meisl in human A M L cells (Lawrence et al, 1999) and cell lines (Afonja et al, 2000). HOXA9 was also the only gene of over 6,000 tested to correlate with failure to respond to chemotherapy (Golub et al, 1999). Proto-oncogenes can be activated either by amplification of gene expression, or by the generation of a novel fusion gene through chromosomal rearrangement.  Two Hox  genes are directly involved in leukemia-associated translocations with the nuclear pore gene nucleoporin-98 (NUP98).  Translocations t(7;ll)(pl5;pl5) and t(2;ll)(q31;pl5)  fuse the amino-terminal portion of NUP98 with the carboxy-terminal portions of HOXA9 54  (Borrow et al, 1996b) and HOXD13 (Raza-Egilmez et al, 1998), respectively. The F G repeat region of NUP98 is retained in the fusion, and acts as a potent ^raws-activator through interaction with CBP and p300 (Kasper et al, 1999). The Hox sequences retain their homeodomains and, in the case of HOXA9, Pbx interaction sequences. The fusion protein therefore likely causes deregulated expression of Hox target genes, by Hoxmediated D N A binding and NUP98-mediated transcriptional activation. Several leukemic fusion genes also indirectly involve activation of Hox pathways. The E2A-Pbx fusion generated by translocation t(l;19)(q23;pl3) is present in one quarter of all cases of childhood pre-B A L L (Kamps et al, 1990).  E 2 A is an important  transcription factor in B-cell development, and its fra/M-activation domain is retained in the fusion protein. The Pbx homeodomain is also retained in the fusion, and is required for frans-activation of a Hox-responsive element and to block differentiation of myeloid progenitors (Kamps et al, 1996). E2A-Pbx can bind D N A co-operatively with Hox proteins (Chang et al, 1995), and the Hox cooperativity motif is critical for transactivation and transformation (Chang et al, 1997a). Thus transformation is modulated by Hox interaction, suggesting that it occurs through ectopic activation of Hox target genes. Interestingly, although in humans this fusion gene transforms cells in the B lineage, mice transgenic for E2A-Pbx developed T-cell leukemia (Dedera et al,  1993), and  overexpression of E2A-Pbx in a murine B M transplantation model led to A M L (Kamps, 1997). Upstream regulators of Hox expression can also be oncogenic, by causing ectopic Hox expression. Milis a homo log of the Drosophila trithorax gene, which acts as part of the trithorax complex to maintain active Hox expression through higher order chromatin 55  remodelling (for a review see Mahmoudi and Verrijzer, 2001).  Mil is involved in  translocations associated with many different forms of leukemia, with at least 30 different fusion partners identified to date (Buske and Humphries, 2000). Similarly, retinoic acid can activate Hox expression in the correct 3' to 5' gene order (Flagiello et al, 1997), and ectopic retinoic acid can cause homeotic defects (Kessel and Gruss, 1991). The retinoic acid receptor alpha (RARoc) protein binds retinoic acid outside the cell, then translocates to the nucleus where it acts as a transcriptional activator. The PML-RARa  fusion gene  present in acute promyelocyte leukemia has RARa-mediated D N A binding, and P M L mediated fraws-activation, without the proper regulatory controls on either protein (Look, 1997).  1.5.3  Hematopoietic effects in Hox loss-of-function models Loss-of-function models have 'documented a requirement for Hox gene expression  in normal hematopoiesis. The earliest studies used antisense oligonucleotides to inhibit expression of Hox genes in hematopoietic cells and cell lines.  It should be noted,  however, that the antisense oligonucleotides used may have cross-reacted with other Hox genes, and therefore the effects may not be gene-specific.  Transfection of antisense  Hoxb-6 decreased myeloid features in K562 cells and increased erythroid features in H E L cells (Shen et al, 1992). Similarly, introduction of antisense HOXA5 reduced myeloid colony formation and enhanced erythroid colony formation in human B M cells (Fuller et al, 1999). Exposure of human B M progenitors to antisense Hoxc-6 specifically inhibited erythroid colony formation (Takeshita et al, 1993), whereas exposure of murine B M cells  56  to antisense Hoxb-7 selectively inhibited myeloid colony formation (Wu et al, 1992). These studies  indicate a requirement  for Hox gene expression in blood cell  differentiation, and suggest that specific Hox genes direct lineage decisions. While a great number of Hox knockout models have been generated, only a few have reported hematopoietic defects, suggesting that there may be functional redundancy within the Hox family.  Mice lacking Hoxa-9 have hematopoietic defects in multiple  myeloid and lymphoid lineages. Mature granulocyte and T- and B-lymphocyte counts are reduced, pursuant to reduced production of committed myeloid and B-cell progenitors. A reduction in erythroid progenitors was also noted, although circulating R B C counts and hematocrits were normal (Lawrence et al, 1997). There is no functional assay for T-cell progenitors, but the expression of surface antigens revealed defective T-lymphopoiesis at an early stage, with delayed progression from the triple negative (CD4~ CD8" TCR") to double positive (CD4 CD8 ) stage, and failure to rearrange TCRP or form a pre-TCR +  complex (Izon et al,  +  1998).  Like Hoxa-9, deficiency for Hoxc-8 led to reduced  progenitor production, as homozygous null mice had decreased C F U - G M and BFU-E contents in their F L and B M (Shimamoto et al, 1999). Conversely, mice lacking Hoxb-6 had a specific 2- to 5-fold enlargement of the erythroid progenitor (BFU-E) compartment (Kappen, 2000). Very recently, a new Hoxb-4 deficient mouse model was generated and examined for hematopoietic effects (Bjornsson et al, 2001a).  These mice had slightly reduced  circulating R B C counts, and decreased B M and splenic cellularity with normal lineage distributions. They had a reduced number of high proliferative clonogenic progenitors, suggesting a reduction of primitive cell number or proliferation. B y mating these mice 57  with mice deficient for Hoxb-3, the phenotype was greatly exaggerated (Bjornsson et al, 2001b). Moreover, phenotypically selected HSCs from the doubly mutant mice were 2fold reduced in proliferative activation.  Perhaps because of this reduced HSC  proliferation, repopulating cells from the double mutant were better able to survive 5-FU administration. Thus, Hoxb-4 and Hoxb-3 are involved in proliferation of primitive cells, and mice lacking these genes have cellular reductions across all hematopoietic lineages. Finally, a human B M failure and skeletal malformation syndrome has been attributed to loss of HOXA11.  Patients in two separate families presented with  amegakaryocytic thrombocytopenia and radio-ulnar synostosis. A nonsense mutation in the homeodomain of HOXA11 was identified in affected individuals (Thompson and Nguyen, 2000).  1.5.4  Hematopietic effects in Hox gain-of-function models A number of studies have utilized retroviral gene transfer to permanently  introduce a Hox gene under the control of a ubiquitous promoter into hematopoietic cells. Retroviruses are able to enter mammalian cells through interactions between viral envelope proteins and specific cell surface receptors.  The viral R N A genome is then  reverse transcribed into a double stranded D N A (dsDNA) copy, and upon nuclear envelope breakdown at cell division the dsDNA integrates randomly into the host cell genome (Fig. 1.8). These aspects of the retroviral lifecycle are exploited for gene transfer purposes, by cloning experimental (ie. Hox) and reporter genes into viral vectors and packaging the sequences in viral proteins.  The majority of viral genes have been  58  removed, however, rendering the particles incompetent to replicate and carry out the lytic phase of the retroviral lifecycle. These //ax-encoding retroviruses are cultured together with murine or human hematopoietic cells, thereby generating a population of cells with very high expression of the Hox gene specified.  The consequences  of Hox  overexpression can then be examined in both in vitro culture systems and in vivo transplantation models (Table 1.2). Retrovirus-mediated overexpression of Hoxb-8 in a myeloid cell line inhibited granulocytic differentiation and enhanced macrophage differentiation (Krishnaraju et al, 1997). Hoxb-8 was also overexpressed in murine B M cells, and a myeloproliferative syndrome developed which was greatly accelerated by IL-3, as detailed above (Perkins et al, 1990). Mice transplanted with murine B M cells engineered to overexpress HOXB3 had a very different phenotype. They had increased myelopoiesis, as shown by elevated levels of mature granulocytes and progenitors. As well, the elevated ratio of myeloid cells was further contributed to by defects in T- and B-lymphopoiesis. T-cell development was blocked at the double positive (CD4 CD8 ) to double negative (CD4" CD8") transition, +  +  with a build-up of cells expressing the y8TCR. B-lymphopoiesis was similarly blocked at an early stage, as transduced cells did not contribute pro-B progenitors, and reductions were observed in all PB B-cell compartments (Sauvageau et al, 1997). HOXB3 mice eventually came down with myeloproliferative syndrome, however the latency period was very long (250 days). Surprisingly, overexpression of Meisl but not its natural cofactor Pbxl  greatly  accelerated  the  myeloproliferative syndrome  caused  by HOXB3  overexpression (Thorsteinsdottir et al, 2001). 59  viral DNA  Figure 1.8: Retroviral Iifecycle. Shown are the replicative (top) and infectious (bottom) cycles of the retroviral Iifecycle. Retroviral packaging cell lines express viral proteins, which allow vector-coded RNAs to be packaged into virions. These are then used to transduce target cells, where the vector sequence is reverse transcribed and integrated into the genome. Neither the target cell nor the vector sequence encodes viral proteins, preventing the replicative cycle from occurring in these cells.  60  Table 1.2: Hox effects on primary hematopoietic cells. Hox Gene  Genetic Alteration  Target Cells  Phenotypic Effects  HOXA5  R V gene transfer  human  i BFU-E i myeloid differentiation  Hoxa-7 Hoxa-9  R V insertion  mouse  A M L (when activated with M e i s l )  Hoxa-9  Knockout  mouse  I T-cells and progenitors I B-cells and progenitors i CFU-E  HOXA9 HOXA10  t ( 7 ; l l ) fuses HOXA9-NUP98 R V gene transfer  human  AML  mouse  T megakaryocyte/blast progenitors ICFU-M I B - c e l l progenitors late A M L  HOXA11  nonsense mutation  human  I megakaryopoiesis  HOXB3  R V gene transfer  mouse  T granulocytes and progenitors I T-lymphopoiesis I B-lymphopoiesis late A M L  Hoxb-4  Knockout  mouse  •I B M , spleen cellularity  HOXB4  R V gene transfer  mouse ES  T primitive progenitors ( C F U - G E M M ) able to repopulate transplanted mice  HOXB4  R V gene transfer  mouse  T all progenitors  HOXB4  R V gene transfer  human  T H S C regeneration in vivo repopulation advantage  HOXB7  R V gene transfer  human  T proliferation o f myeloid cells  Hoxb-8  R V insertion or R V gene transfer  mouse  A M L (when activated with IL-3)  Hoxb-8  Knockout  mouse  t BFU-E  I H S C proliferation  T primitive progenitors ( S R C ) T primitive progenitors ( L T C - I C )  HOXC4 Hoxc-8  i R V gene transfer Knockout  ; human mouse  T primitive progenitors ( L T C - I C ) i CFU-GM i BFU-E  HOXD13  t(2; 11) fuses HOXD13-NUP98  human  AML  61  Overexpression of HOXA10 once again gave a distinct phenotype from that of Hoxb-8 or HOXB3.  Myelopoiesis was skewed, with increased production of  megakaryocyte and blast cell colonies, and absence of macrophage colonies.  B-  lymphopoiesis was also affected, as transduced cells did not contribute any pro-B progenitors. However, T-lymphopoiesis was normal in this model. Most of the mice developed A M L within 20 to 50 weeks post-transplant.  The long latency period and  mono- or bi-clonality of the leukemias indicated that secondary genetic events were required for transformation (Thorsteinsdottir et al., 1997). The nature of the secondary event was not dissected at the time, but it has since been revealed that combined overexpression of HOXA9 with its cofactor Meisl can greatly accelerate leukemic transformation (Kroon et al, 1998; Thorsteinsdottir et al, 2001) and reduce cytokine dependence (Calvo et al, 2001). Both the Pbx and Meis interaction domains of H O X A 9 are required for transformation (Schnabel et al, 2000). HOXA10 overexpression was subsequently examined in human hematopoietic cells. In vitro culture of HOXA10-transduced cells generated as much as 100-fold more blast cells than control cultures.  Replating of myeloid colonies also generated  significantly more secondary colonies and a large proportion of these contained blast cells, again indicating reduced ability to differentiate. Lymphopoiesis was also perturbed, as mice transplanted with HOXA 10-transduced cells had reduced lymphoid to myeloid ratios in the transduced cell compartment (Buske et al, 2001). Enforced expression of high HOXB7 in human PB CD34  +  cells enhanced  proliferation of cells induced to differentiate along the granulocyte or macrophage lineages, but had no effect on cells induced to erythrocyte or megakaryocye lineages. 62  H0XB7 overexpression also enhanced growth of primitive progenitors by 2-4 fold, as assessed by high proliferative potential (HPP)-CFC or LTC-IC assays (Care et al, 1999). HOXA5 differentiation.  overexpression in human  CD34  +  B M cells inhibited erythroid  A reduced number of BFU-E was noted, and those colonies that did  develop contained largely undifferentiated cells.  Myelopoiesis in liquid cultures was  shifted toward granulocyte and macrophage lineages (Crooks et al, 1999).  This is  consistent with the reduced erythroid differentiation observed in K562 cell line when HOXA5 was added, and the opposite effect of antisense HOXA5 on human B M (Fuller et al, 1999).  1.5.5  HOXB4 overexpression Retrovirus-mediated gene transfer was used to study the effects of HOXB4  overexpression on embryonic stem (ES) cells. Overexpression of HOXB4 did not alter the ability of ES cells to form embryoid bodies (EBs), but it did significantly enhance the hematopoietic output per EB. Multilineage myeloid progenitors (CFU-GEMM) were the most effected, with 6-fold elevations over control EBs. There was also a slight increase in the number of CRU-E per E B , but no change in C F U - G M or B F U - E production (Helgason et al, 1996). Thus HOXB4 overexpression specifically enhanced growth of progenitors with high myeloid lineage potential in this system. Unlike other Hox overexpression models in murine B M , HOXB4 did not skew or perturb differentiation along any hematopoietic lineage.  Myeloid progenitors were  present in all lineages, with no change in number or distribution. Mice transplanted with  63  //QZB4-transduced cells had normal red and white blood cell counts, and normal numbers and distributions of cells expressing lineage markers in the B M or spleen (Macl, T e r l l 9 , B220) and thymus (CD4, CD8). Recipient mice did have elevated myeloid progenitor cells, with 2-5 fold CFC increases in the B M and 10-30 fold C F C increases in the spleen after 12-20 weeks respectively (Sauvageau et al, 1995). However, the most dramatic effect observed in HOXB4 recipients was the greatly enhanced stem cell regeneration post-transplant.  Control-transplanted mice regenerated  the C R U pool to approximately 5% of the normal level, consistent with previously published data using unmanipulated B M .  i/ClYB^-transplanted mice, however,  regenerated the C R U compartment to 100% of normal, up to 50-fold higher than controls (Sauvageau et al, 1995). Furthermore, when the C R U content of iftQAB^-transplanted mice was assessed any time between 16 and 52 weeks post-transplant, it was always found to lie within the normal range (Thorsteinsdottir et al, 1999). Although hematopoietic cell transformation was never observed in these studies, HOXB4  overexpression has been  shown to transform  RAT-1 fibroblast  cells.  Transformation was enhanced by co-transduction of the RAT-1 cells with Pbxl (Krosl et al, 1998). Human C B cells overexpressing HOXB4 had a competitive growth advantage in vivo. Mice transplanted with a 1 to 2 ratio of //QZB4-transduced to control-transduced cells had nearly 5-fold higher B M repopulation by the HOXB4 cells (Buske et al, submitted; Schiedlmeier et al, 2001). HOXC4,  which is in the same paralog group as HOXB4,  was recently  overexpressed in human CD34 B M cells. HOXC4 overexpression mediated a large (13+  64  fold) increase in primitive cells, as measured by the LTC-IC assay, and moderate increases in more mature progenitors (Daga et al, 2000). These results are consistent with an effect on HSC growth, similar to the HOXB4 effect on murine cells.  This  demonstrates the functional redundancy between genes of the same paralog group, and suggests that the HOXB4 overexpression effect might reflect gain-of-function for multiple 4 paralog group genes. th  HOXB4 was recently overexpressed in murine Y S and ES cells, with surprising results. After overexpressing HOXB4, investigators were able to detect definitive HSCs, capable of long-term multilineage repopulation to adult recipients from both of these cell types (Kyba et al, 2001).  As discussed earlier, the Y S is a site of primitive  hematopoiesis, as was not believed to contain any definitive HSCs. Similarly, ES cells recapitulate early hematopoietic development, and can be induced to undergo primitive hematopoiesis but cannot normally engraft adult recipients. These studies shed new light on the development of the hematopoietic system, as they suggest that definitive potential is a latent property of embryonic HSCs, regulated by proteins such as HOXB4.  1.5.6  HOXB4  overexpression: unresolved issues and thesis objectives  Around the same time as these HOXB4 overexpression studies were being carried out, it was revealed that regeneration of normal HSCs is limited not by reaching proliferative exhaustion, but rather by feedback inhibition after a short expansion phase. The question remained, therefore, whether the enhanced C R U regeneration by HOXB4transduced cells was due to an extended expansion phase (eg. by over-riding the  65  inhibition) or an elevated C R U growth rate during the expansion phase, or both. Resolving this issue was the first objective of my thesis, as it would determine whether HOXB4 overexpression rendered the HSCs more sensitive to positive stimuli during the regeneration phase, or i f it rendered them less sensitive to negative stimuli during negative feedback phase. A second unresolved issue was the relative ability of i/QZB^-transduced HSCs to . generate mature progeny.  Could individual 7/&YZ?4-transduced HSCs repopulate  transplanted mice to a higher level than control HSCs? And would they be better able to reconstitute the hematopoietic system in a competitive setting? These questions surround the issue of whether HOXB4 overexpression alters the quality of HSCs or just their quantity. Finally, could HOXB4 be used as a stem cell expansion factor ex vivo? The vast experimental and therapeutic applications of HSC expansion make it an important, yet largely unrealized goal. The ability of HOXB4 overexpression to enhance HSC growth in vivo makes it an attractive candidate for ex vivo HSC expansion. In this study I examine the effect of HOXB4 on ex vivo C R U survival and self-renewal.  66  CHAPTER 2  HOXB4  OVEREXPRESSION  MEDIATES  VERY  RAPID STEM C E L L REGENERATION AND COMPETITIVE HEMATOPOIETIC REPOPULATION  67  2.1  Abstract  Objective:  H o x t r a n s c r i p t i o n factors  hematopoiesis.  have  emerged  as  important  regulators  of  I n particular, w e have s h o w n that o v e r e x p r e s s i o n o f HOXB4 i n m o u s e  bone m a r r o w c e l l s c a n greatly enhance the l e v e l o f h e m a t o p o i e t i c stem c e l l regeneration a c h i e v e d at late times (>4 m o n t h s ) post-transplantation. T h e objective o f this study w a s to resolve i f HOXB4 increases the rate and/or d u r a t i o n o f stem c e l l regeneration; a n d also to see i f this enhancement w a s associated w i t h i m p a i r e d p r o d u c t i o n o f e n d c e l l s o f w o u l d lead to c o m p e t i t i v e r e c o n s t i t u t i o n o f a l l compartments.  Methods:  R e t r o v i r a l vectors  w e r e generated w i t h the GFP reporter gene ± HOXB4 to enable the i s o l a t i o n a n d direct t r a c k i n g o f transduced c e l l s i n culture o r f o l l o w i n g transplantation. S t e m c e l l r e c o v e r y w a s m e a s u r e d b y l i m i t d i l u t i o n assay for l o n g t e r m c o m p e t i t i v e r e p o p u l a t i n g c e l l s ( C R U ) . Results:  HOXB4 o v e r e x p r e s s i n g c e l l s have enhanced g r o w t h in vitro, as demonstrated b y  their r a p i d d o m i n a n c e i n m i x e d cultures a n d their shortened p o p u l a t i o n d o u b l i n g t i m e . F u r t h e r m o r e , M 7 A 7 J 4 - t r a n s d u c e d c e l l s have a m a r k e d c o m p e t i t i v e r e p o p u l a t i n g advantage  in vivo i n b o t h p r i m i t i v e a n d mature compartments. C R U r e c o v e r y i n HOXB4 recipients w a s e x t r e m e l y r a p i d , r e a c h i n g 2 5 % o f n o r m a l b y 14 days post transplant o r s o m e 8 0 - f o l d greater than c o n t r o l transplant recipients, a n d attaining n o r m a l n u m b e r s b y 12 w e e k s . M i c e transplanted w i t h e v e n h i g h n u m b e r s o f #QA2>W-transduced C R U regenerated u p to but not b e y o n d the n o r m a l C R U levels.  Conclusion:  HOXB4 i s a potent enhancer o f  p r i m i t i v e h e m a t o p o i e t i c c e l l g r o w t h l i k e l y b y i n c r e a s i n g s e l f - r e n e w a l p r o b a b i l i t y but w i t h o u t i m p a i r i n g homeostatic c o n t r o l o f h e m a t o p o i e t i c stem c e l l p o p u l a t i o n size or the rate o f p r o d u c t i o n a n d maintenance o f mature e n d cells.  68  2.2  Introduction  Hematopoietic stem cells (HSCs) play a pivotal role in the establishment and subsequent lifelong maintenance of hematopoiesis. A critical property of HSCs is their ability to undergo self-renewal - a feature that is the cornerstone of stem cell transplantation-based therapies, in which HSC are required not only to differentiate to repopulate mature hematopoietic tissues but also to regenerate a functional totipotent stem cell compartment.  Studies in the murine model using rigorous and quantitative  assays for HSCs have interestingly revealed that the level of HSC recovery following bone marrow (BM) transplantation is incomplete. Even at relatively high transplant doses with adult B M cells, recovery reaches only approximately 10% of the normal level (Harrison et al, 1990).  Somewhat higher levels of recovery are apparent following  transplantation of stem cells from fetal liver (Chen et al, 1999; Pawliuk et al, 1996; Rebel et al, 1996a), but again regeneration of the stem cell compartment is incomplete, in contrast to apparently normal reconstitution of later progenitor and end cell compartments. A number of studies suggest that both intrinsic and extrinsic regulatory mechanisms underlie HSC behaviour.  Thus, for example, the increased proliferative  capacity of fetal compared to adult-derived HSCs is suggestive of some intrinsic genetic determinants of self-renewal potential. The observed incomplete regeneration of the HSC compartment in primary transplant recipients, and a limited ability to serially transplant B M cells (Harrison, 1979) have also suggested an inherent exhaustability of HSC selfrenewal, possibly indicative of an intrinsic mitotic clock (Vaziri et al, 1994). Limitations  69  in stem cell expansion, however, may also reflect the presence of negative extrinsic regulatory mechanisms, since the regenerated HSCs can be shown to retain significant self-renewal potential (Iscove and Nawa, 1997). A greater understanding of the genes and processes controlling HSC self-renewal could provide important new strategies for achieving enhanced or even selective HSC expansion. Recent attempts to identify stem cell control genes have included linkage analysis on mouse strains with differing HSC functional abilities (Chen et al, 2000), and expression profiling on hematopoietic populations with differing HSC contents (Phillips et al, 2000). While these studies have identified an expanding list of potential HSC regulators, the involvement of these genes in control of H S C self-renewal remains unclear. Candidate HSC regulators have also been identified based on phenotypic effects in loss- or gain-of-function mouse models. For example, overexpression of the antiapoptotic gene Bcl-2 led to increased numbers of Thy-1.1 Sca-l 10  hl  c-kit l i n hl  neg  cells,  which have long-term multilineage repopulation potential (Domen et al, 2000), while absence of the cell cycle regulator p21 led to reduced radioprotection upon serial transplantation (Cheng et al, 2000b). A number of recent studies have brought attention to the Hox family of transcription factors as potential hematopoietic regulators. Multiple gene members of the Hox A, B, and C clusters are expressed in hematopoietic cells, and this expression appears to be restricted to the most primitive cell types, such as human CD34 CD38" B M cells, +  and down-regulated in more mature compartments (Sauvageau et al, 1994).  These  patterns of expression are suggestive of Hox functional roles in early hematopoietic cells.  70  Consistent with this, lack of a functional Hoxa-9 gene led to impaired myeloid and lymphoid differentiation (Izon et al, 1998; Lawrence et al, 1997). In contrast, numerous studies have revealed myeloproliferative effects of overexpression of several different Hox genes (Sauvageau et al, 1995; Sauvageau et al, 1997; Thorsteinsdottir et al, 1997). Interestingly, these studies have demonstrated Hox gene-specific effects at multiple levels of hematopoiesis. For example HOXB3 overexpression impaired lymphoid but enhanced myeloid development (Sauvageau et al, 1997), while HOXA10 overexpression altered megakaryocyte, macrophage, and B-cell differentiation, and was associated with a myeloproliferative syndrome (Thorsteinsdottir et al, 1997). Of particular interest in the context of HSC regulation are our previous findings from overexpression of HOXB4 (Sauvageau et al, 1995). Observed in vitro effects of HOXB4 overexpression include increased clonogenic progenitor replating and increased recovery of colony forming units in the spleen (CFU-S) after culture, indicating enhanced growth of primitive cells (Sauvageau et al, 1995). In vivo assays also reflected enhanced growth at the primitive cell level. While there was no change in the proportion or number of end cells in mice transplanted with //QA2?4-overexpressing cells, and only a modest increase in clonogenic progenitors, there was a marked increase in the level of HSC regeneration (Sauvageau et al, 1995). Polyclonal competitive repopulating unit (CRU) levels in HOXB4 recipients reached the normal (ie. pre-transplant) level by 4 months post-transplant, and were sustained at this level for at least one year, in sharp contrast to the limited regeneration  in control-transplanted  mice (Sauvageau  et al,  1995;  Thorsteinsdottir etal, 1999).  71  In earlier studies, our goal was to determine the maximal level of HSC regeneration, and whether the enhancement would be associated with stem cell exhaustion; we therefore analyzed mice at late times post-transplant (up to one year). In the current study we have focused on very early times post-transplant, so as to assess the effect of HOXB4 on the rate of HSC regeneration. We found significant regeneration in the first 2 weeks post-transplant, arguing that the enhanced recovery of HSCs results mainly from an increased rate and/or probability of self-renewal, rather than a prolonged period of recovery. Further to this, we also assessed regulation of H S C growth with HOXB4 overexpression, by examination of C R U regeneration following transplantation of very large cell doses. The HSC population size remains strictly controlled, as C R U contents do not rise above the normal level even over a 30-fold range of transplant cell doses.  Finally, we questioned whether the //QZB4-mediated HSC self-renewal  enhancement  was accompanied by a compensatory inhibition of differentiation.  Although recipients of non-selected i7QA2?4-transduced B M cells developed mature blood cells in all lineages, we were unable with previous constructs to directly assess the contribution by transduced cell progeny to the various hematopoietic lineages. In the current study we have exploited the green fluorescent protein (GFP) marker gene to facilitate the isolation and tracking of transduced cells, allowing us to directly test the competitive growth potential of HOXB4 transduced cells, and to directly assess whether HOXB4 HSCs retain full differentiative potential. These studies reveal that HOXB4 overexpression confers a marked competitive growth advantage on hematopoietic cells in vitro, and a competitive repopulation advantage in vivo. v  Differentiation was not  72  compromised, as all lineages of mature blood cells contained normal distributions of HOXB4-GFP  +  cells. These studies highlight HOXB4 as a candidate HSC regulator that  can be exploited to enhance the growth of primitive hematopoietic cells without deleterious effects on HSC regulation or differentiation, and to further our understanding of the processes controlling HSC growth.  2.3  2.3.1  Materials and Methods  Retroviral Vectors The M S C V 2.1 (Hawley et al, 1994) vector (kindly provided by Dr. R. Hawley,  American Red Cross, Rockville, M D ) , was first modified by replacing the pgk-neo cassette with a sequence containing the internal ribosomal entry site (IRES) sequence derived from the encephalomyocarditis virus and the gene for enhanced green fluorescent protein (GFP) (this cassette was kindly provided by Dr. P. LeBoulch, Massachusetts Institute of Technology, Cambridge, M A ) . This M S C V IRES GFP vector (GFP vector) served as a control and backbone for cloning of a HOXB4 c D N A upstream of the IRES, to create M S C V HOXB4 IRES GFP (HOXB4-GFP  vector).  Production of high-titre  helper-free retrovirus was carried out by standard procedures (Pawliuk et al, 1994), using virus-containing supernatants from transfected amphotropic Phoenix packaging cells (Kinsella and Nolan, 1996) to transduce the ecotropic packaging cell line GP E86 +  (Markowitz et al, 1988). The retroviral titres of the GFP and HOXB4-GFP  producer  cells were 3 x 10 /ml and 2 x 107ml respectively, as assessed by transfer of GFP 5  73  expression to NTH-3T3 cells. HOXB4-GFP  Absence of helper virus generation in the GFP and  producer cells was verified by failure to serially transfer virus conferring  GFP expression to NTH-3T3 cells.  2.3.2  Mice Parental strain mouse breeders were originally purchased from The Jackson  Laboratory (Bar Harbour, ME) and subsequently bred and maintained at the British Columbia Cancer Research Centre animal facility. They were housed in microisolator units and provided with sterilized food, water, and bedding. Irradiated animals were additionally provided with acidified water (pH 3.0).  Strains used as B M transplant  donors were either C57B16/Ly-Pep3b (Pep3b) or the F l hybrid of (C57Bl/6Ly-Pep3b x C3H/HeJ) ([PepC3] ), and those used as recipients were either C57Bl/6-W /W 41  41  F]  (W /W ) 41  41  o r  the F l hybrid of (C57B1/6J x C3H/HeJ) ([B6C3 ] ). Donor and recipient F1  strains are phenotypically distinguishable on the basis of allelic differences at the Ly5 locus: donor Pep3b are Ly5.1 homozygous and donor [PepC3] F I are Ly5.1/5.2. 41  n-tjA\  heterozygous, whereas recipient W '  2.3.3  w  and [B6C3 ]FI are Ly5.2 homozygous.  Transduction of Primary Murine Bone Marrow Cells Primary mouse BM cells were transduced as previously described (Kalberer et al,  2000; Sauvageau et al, 1995). Briefly, BM cells were extractedfrommice treated 4 days previously with 150 mg/kg 5-fluorouracil (Faulding) and cultured for 48 hrs in DMEM 74  supplemented with 15% fetal bovine serum (FBS), 10 ng/ml human interleukin (hIL)-6, 6 ng/ml murine (m)IL-3, and 100 ng/ml mSF. Media and serum were purchased from StemCell Technologies (Vancouver, B C , Canada), and cytokines were expressed in COS cells and purified in the Terry Fox Laboratory.  The cells were then harvested and co-  cultured with irradiated (1500 cGy X-ray) GP E86 viral producer cells for 48 hrs in the +  same medium with the addition of 5 pg/ml protamine sulfate (Sigma, Oakville, ON, Canada). Loosely adherent and nonadherent cells were recovered from the co-cultures and incubated a further 48 hrs in the same medium without protamine sulfate. Retrovirally-transduced  B M cells were selected based on GFP expression using a  FACStar (Becton Dickinson, Mississauga, O N , Canada).  2.3.4  In Vitro Culture of Hematopoietic Cells Ly5.1-positive B M cells transduced with HOXB4-GFP  and Ly5.2-positive cells  transduced with GFP vectors were cultured after GFP selection in D M E M supplemented with 15% FBS, 10 ng/ml hTL-6, 6 ng/ml mIL-3, and 100 ng/ml mSF. We tracked the proportion of Ly5.1- and GFP-positive cells over time in cultures initiated with mixtures of GFP-transduced Dickson).  and HOXB4-GFP-transduced  cells using a FACScan (Becton  Total cell numbers were evaluated from cultures initiated with 5 x l 0  4  cells/well, by harvesting cells from 3 wells every 4 days and counting on a hematocytometer.  75  2.3.5  In Vivo Repopulation Recipient [PepC3] i or W / W 4 1  F  4 1  mice were irradiated with 900 cGy or 450 cGy of  137  Cs y-radiation, respectively. FACS-selected GFP-positive B M cells were then injected into the tail vein of irradiated recipient mice. Peripheral blood (PB) cell progeny of transduced cells were tracked at various intervals post-transplant by expression of GFP and Ly5.1. 100 u.1 of blood was extracted from the tail vein, and the erythrocytes were lysed with ammonium chloride (StemCell Technologies). Leukocyte samples suspended in Hank's balanced salt solution (StemCell Technologies) with 2% FBS (HF) were incubated sequentially on ice with 6u.g/ml of 2.4.G2 (anti-Fc receptor antibody produced in Terry Fox Lab), then biotinylated anti-Ly5.1 (Pharmingen, Mississauga, ON, Canada), and finally phycoerythrin (PE)-labeled streptavidin (SA) (Pharmingen). A l l samples were washed with HF and lpg/ml propidium iodide (PI; Sigma) prior to analysis on FACScan, FACSort, or FACScalibur (Becton Dickinson) flow cytometry machines. Expression of Ly5.1 identified donor-derived cells, and expression of GFP identified retrovirallytransduced cells. At the time of animal sacrifice, B M samples were analyzed by F A C S in the same manner.  2.3.6  CFC Assay Hematopoietic clonogenic progenitor frequencies were determined by plating  suitable aliquots of B M or spleen cells in methylcellulose medium (HCC-3334, StemCell Technologies) containing 3U/ml erythropoietin, and supplemented with 50 ng/ml mSF,  76  10 ng/ml hIL-6, and 10 ng/ml mIL-3, and then scoring the resultant colonies after 10 days of incubation.  2.3.7  C R U Assay  H S C s were detected and evaluated using a limit-dilution transplantation-based assay for cells with competitive, long-term, lympho-myeloid repopulation function. The basic procedure (Szilvassy et al, irradiated W / W 4 1  4 1  1990), and a modification employing sublethally  recipients (450 c G y  1 3 7  C s y-radiation) as a source o f endogenous  competitor cells (Miller and Eaves, 1997), have been described i n detail previously. Briefly, irradiated W / W 4 1  4 1  recipients were injected with 10 to 2 x 10 cells, and the 2  5  blood obtained by tail vein bleeding of these mice was analyzed by F A C S >12 wks posttransplant for evidence o f lympho-myeloid repopulation. M i c e which had >1% donorderived ( G F P ) cells in both lymphoid (SSC* +  0  FSc'°) and myeloid (SSC* ' FSC* ') 1  1  subpopulations were considered to be repopulated with transduced cells. Discrimination by flow cytometry o f myeloid and lymphoid cells was confirmed using cell surface staining to detect lineage-specific markers (Gr-1, Mac-1 versus B220). C R U frequencies in the test B M sample were calculated by applying Poisson statistics to the proportion of negative  recipients at different dilutions using L i m i t Dilution Analysis (StemCell  Technologies) software.  77  2.3.8  Proviral Integration Analysis Genomic D N A was extracted from producer cell lines or primary hematopoietic  tissues using DNAzol (GibcoBRL, Burlington, ON, Canada). Proviral size was examined by digestion of D N A with Xbal, which cleaves within the M S C V LTRs.  Unique  integrations were identified by digestion of D N A with HindUI, which cleaves once within the provirus. Digested D N A was then separated in 1% agarose gel by electrophoresis and transferred to zeta-probe (Bio-Rad, Mississauga, O N , Canada) membranes by standard Southern blotting techniques (Sambrook et al, 1989). Membranes were probed with 32  a P-dCTP-labeled GFP sequence.  2.4  Results To gain further insight into the nature and magnitude of HOXB4-mduced alterations  on hematopoietic cell growth, we examined the effects of HOXB4 overexpression in murine adult B M cells following retroviral transduction.  To facilitate isolation and  tracking of transduced cells and their progeny, we incorporated the HOXB4 gene into a vector also carrying the GFP reporter gene.  As shown in Figure 2.1.A, the M S C V  retroviral vector backbone was modified to support viral LTR-driven expression of GFP alone (GFP vector) or of a bicistronic cassette encoding HOXB4 and GFP (HOXB4-GFP vector). Retrovirus-mediated gene transfer was then used to generate hematopoietic cells that overexpressed HOXB4 (Fig. 2.1.B), and the growth of these cells was assessed both in vitro and in vivo. We confirmed the biological activity of this new vector through 78 its  ability to confer enhancement of CFU-S growth in vitro (not shown), as previously reported by our group (Sauvageau et al, 1995).  x GFP (2.8 kb)  LTR  H  IRES  GFP  Figure 2.1: Construction and testing of the GFP  LTR  and HOXB4-GFP (4.0 kb)  H LTR  HOXB4  IRES  GFP  B  LTR  HOXB4-GFP  retroviral vectors. (A) Structure and expected sizes of integrated provirus for the two vectors used in this study, including Xbal (X) and Hindlll (H) restriction sites. (B) Representative histogram showing 80% gene transfer to murine BM cells after transduction with the HOXB4-GFP vector. Transduced, GFP cells (indicated by G l gating) can be selected by FACS. +  GFP  2.4.1  Growth enhancing effects of HOXB4 in vitro Incorporation of the GFP reporter gene allowed us to perform a simple yet  quantitative assessment of the relative growth of transduced cells in a series of mixed cultures.  Liquid cultures were initiated with equivalent total cell numbers but varying  proportions of HOXB4-GFP-transduced (Ly5.1 ) and GFP-transduced (Ly5.1") cells, and +  79  the proportion of ifOZB^-overexpressing cells was monitored by Ly5.1 expression (Fig. 2.2.A). Cultures in which the HOXB4-GFP-transduced cells initially contributed only 10 or 50% rapidly became dominated by HOXB4 overexpressing cells. For example, the proportion of HOXB4-GFP-dehved  cells in a culture initiated with just 10% of these cells  increased to a majority within 20 days, and reached 75% by the end of the culture period. The predominance of i/(9X54-GFP-transduced cells was confirmed by Southern blot analysis of D N A taken from 3 week cultures (Fig. 2.2.B). These findings additionally indicate that the growth advantage conferred by HOXB4  overexpression is cell  autonomous, as only the 7-fQZB^-transduced cells were enhanced in these mixed cultures. Analysis of population doubling times were determined to be 26.4 hours for GFPtransduced cells versus 22.4 hours for HOXB4-GFP-txansd\xcQd  cells (Fig. 2.2.C).  Neither culture had a significant number of apoptotic cells, as measured by propidium iodide staining (data not shown; Appendix Fig 2.8). These data suggest that HOXB4 overexpression did not lead to a block in apoptosis, and that the growth rate of the culture was enhanced, possibly reflecting an increased division rate or an increased proportion of dividing cells. The data presented here suggest a cell autonomous growth enhancing effect of HOXB4 on late hematopoietic progenitor cells as found in short-term liquid cultures. Furthermore, the data provide a straightforward assay for some aspects of HOXB4 proliferative effects.  80  Figure 2.2: Expansion of transduced B M cells in liquid culture. (A) Cultures were initiated with 0 (A), 10 (•), 50 (•), or 100 (0) percent HOXB4GFP-transduced Ly5.1 cells, and the reciprocal percentage control-transduced Ly5.1" cells. The graph depicts the rise in the proportion of HOXB4transduced (Ly5.1 ) cells over time. (B) Southern blot analysis of D N A taken from mixed cultures at 30 days, digested with Xbal and probed with GFP, showing disappearance of the 2.8kb control GFP proviral band. (C) Growth of sorted, transduced cells in culture. The HOXB4-GFP culture has a shorter doubling time than the control GFP- transduced culture, as calculated from the slopes (dotted lines) of the GFP (0) and HOXB4-GFP (•) growth curves. +  10 20 Time in culture (days)  30  +  B 4.0 kb  2.8 kb  HOXB4-GFP j.J T =22.4hrs d  T =26.4 hrs d  •  0  2.4.2  1  5 Time in Culture (days)  r  10  Competitive reconstitution of hematopoietic cells in vivo To directly assess the growth enhancing effects of HOXB4 on earlier stages of  hematopoiesis, we next examined its ability to enhance reconstitution.  In initial 81  experiments to address this, mice were transplanted with various mixtures of GFPtransduced (Ly5.1") and //QZB^-GFP-transduced (Ly5.1 ) cells, and the relative +  hematopoietic contributions of the two populations were monitored over time. Keeping the total transplant dose constant, competitions of either equal numbers of GFPtransduced and HOXB4-GFP-transduced  cells, or a 20-fold greater number of GFP-  transduced cells were inoculated. Representative PB F A C S profiles from recipients of both input transplant ratios are shown for early and late times post-transplant (Fig. 2.3), and data compiled from all mice in the 5:95 transplant group are summarized in Figure 2.4. As early as 6 weeks post-transplant, the vast majority of the PB cells (>75%) were HOXB4-GFP-derived  (Ly5.1 GFP ), even for recipients in which HOXB4 cells were in  competition with a 20-fold greater number of control cells.  Furthermore, this  repopulation advantage demonstrated by PB analysis was sustained to at least 35 weeks post-transplant, the last time point analyzed. The data in Figure 2.4 are derived from the ratio of M)Z84-transduced (Ly5.1 GFP ) to GFP-transduced (Ly5.1" GFP ) cells in the +  +  +  PB, to exclude residual host cells from the calculations. At 6 weeks there was a large host (Ly5.1" GFP") cell ^contribution, which diminished by the week 35 analysis. However, there were also a number of HOXB4-GFP-transduced  cells in which GFP  expression from the viral LTR was reduced or absent (Ly5.1 GFP"). These cells were +  also not included in the calculations for Figure 2.4, which may therefore slightly underestimate the magnitude of the HOXB4 competitive advantage. HOXB4 HSCs retained normal differentiation to end cells. experiment, detailed analysis of mice transplanted exclusively with  In a separate HOXB4-GFP-  82  transduced o r control-transduced c e l l s c o n f i r m e d n o r m a l p r o p o r t i o n s o f c e l l s i n l y m p h o i d and m y e l o i d lineages.  A s s h o w n i n F i g u r e 2 . 4 . B , there w a s n o significant difference i n  the p r o p o r t i o n s o f c e l l s e x p r e s s i n g B 2 2 0 , G r - 1 , M a c - 1 , or T e r l l 9 b e t w e e n  HOXB4-GFP  transduced a n d G F P - t r a n s d u c e d P B compartments.  50:50 input I  6.40  Figure 2.3: reconstitution  5:95 input  Competitive by HOXB4-  GFP-transduced  75.56  (Ly5.1  +  G F P ) and GFP-transduced (Ly5.r GFP ) cells. Representative PB FACS profiles from n=4 mice transplanted with 50:50 (left) or 5:95 (right) HOXB4-GFP to control GFP transplant cell ratios. A t 6 weeks posttransplant, the vast majority o f the G F P P B cells were derived from the HOXB4GFP-transduced cells ( L y 5 . 1 , upper right quadrant). B y 35 weeks post-transplant, the host contribution (Ly5.1~ G F P " , lower left quadrant) was diminished, and there was a clear dominance o f HOXB4G F P - d e r i v e d cells i n the white blood cell compartment, i n mice from both the 50:50 and 5:95 transplant ratios. B M at 35 weeks was made up almost exclusively of HOXB4 overexpressing cells. Secondary transplant recipients (n=4 per group) also showed a predominance o f HOXB4-GFP derived cells i n the P B . 4  A  6 week PB  2 • •'  : Bra?f>'  1,  ''fife  12.70  73.41  24.07  55.75  Hi*  8 month PB  4  +  +  ; ;  lUffi^ 10° 10  '  10  1  2  1.08  '.'3.02  ;  10  10  3  4  . 96.93  10°  10'  ' '".-\/' y:7() ;  :  10  2  10  3  10  1  87.37  5.00  8 month BM  .  0.67- • 10° 10'  10*  10.52  1.32  10  3  76.03  1.33 •• 10° 10'  24.61  10  2  .6.30 10 10 3  4  60.29  2° mice PB  IT)  .-.:i>SS.>. 0.78 io  1  io GFP  GFP  1  io  J  io  2.11  83  A  100  -,  CQ  £ 80 -a a> a 3 60 -a £ o  4  I  0  20  3  u  OH  10 20 Time post-transplant (weeks)  ca +  B  o  Figure 2.4: Competitive repopulation and normal differentiation. (A) Relative proportions of HOXB4-GFP (•) or GFP (•) transduced cells in circulating white blood cells from recipients of 5xl0 HOXB4-GFPtransduced cells and 9.5xl0 GFP-transduced cells (5:95 cell ratio), calculated as the proportion #QA7i4-GFP-transduced (Ly5.1 ) or GFPtransduced (Ly5.T) cells within the G F P fraction. n=4 mice were analyzed every 6-12 weeks, and sacrificed after 35 weeks for transplantation into secondary recipients. (B)  30  40  100'  4  +  o o  80  >  60  QJ  a  nnn  40  20  MAC-1  GR-  B220  TER119  +  Mean proportion of G F P cells that expressed each marker, ±S.E.M in n=12 GFPtransduced (light bars) or n=15 HOXB4-GFP-transduced (dark bars) B M recipient mice. There was no significant difference (p<0.1) in expression of Gr-1, Mac-1, B220, or Terl 19 within the G F P compartments of these two groups. +  +  Analysis of the B M cells at 8 months post-transplant demonstrated that the dominant contribution by HOXB4-GFP-transduced  cells also extended to more primitive  compartments. As shown in Fig. 2.3, virtually all B M cells from both transplant groups were Ly5.1 G F P , and thus derived from //"QZS^-GFP-transduced repopulating cells. +  +  These data together provide the first direct evidence that HOXB4 overexpression confers complete dominance in reconstitution of both mature end cells in the PB and primitive 84  precursors in the B M . Importantly, this competitive advantage appears to extend to the most primitive HSC compartment, since secondary mice transplanted with B M cells from this primary transplant also showed nearly exclusive HOXB4-GFP-derived PB repopulation (Fig. 2.3).  2.4.3  Rapid regeneration of competitive repopulating units (CRU) The above results are consistent with HOXB4 conferring a marked growth  advantage at the repopulating cell level, with no impairment of later differentiation steps. To examine more closely the effects of HOXB4 on C R U regeneration, we assessed the magnitude and kinetics of regeneration in the B M using the limit dilution assay for competitive, long-term, lympho-myeloid repopulating cells. In previous studies, the earliest time C R U had been examined was 16 weeks posttransplant.  To examine early stages of reconstitution we began to assess C R U  regeneration beginning 2 weeks after transplantation, and at 2-4 week intervals up to 16 weeks post-transplant. Primary recipients received 225,000 GFP-transduced or HOXB4GFP-transduced and FACS-selected cells, a dose estimated to contain 50 C R U per recipient. Sublethally irradiated W / W 4 1  4 1  mice were used as recipients in this study, as  they provide compromised, endogenous competitor cells for the C R U assay (Miller and Eaves, 1997). Both primary and secondary transplants were done in this competitive setting, to minimize "contaminating" C R U from primary hosts while allowing transplant engraftment. At intervals spanning 2 to 16 weeks post-transplant, four cohorts from each  85  group were sacrificed and analyzed for transplant engraftment, progenitor content, and C R U content.  o m  100 -i  +  ~.  80 60  +  a,  40  O  20  CL,  nririnn 2  4  8  12  Time Post-transplant (weeks)  16  Figure 2.5: Repopulation by GFPor HOXB4-GFPtransduced cells in sub-iethally irradiated W AV recipients. Shown is the mean (± SEM) donor-derived repopulation of PB (top graph) or B M (middle graph) by HOXB4-GFPtransduced (dark bars) or GFP-transduced (light bars) cells in n=4 recipient cohorts at each time point. The HOXB4 overexpressing cells repopulate more rapidly and to a greater extent in this competitive setting. Mean (± SEM) B M CFC recovery is elevated at all time points in the HOXB4GFP transplant group (bottom graph). 41  41  Time Post-transplant (weeks)  Engraftment was measured by analysis of Ly5.1 and GFP expression in PB and B M by F A C S .  As shown in Fig. 2.5, transduced cell progeny were detected in the  circulation of both HOXB4-GFP  and GFP transplant recipients as early as 2 weeks, and  rose to plateau values at approximately 8 weeks.  HOXB4-GFP  recipients showed a  modest acceleration of PB reconstitution, most notable at 4-8 weeks post-transplant. 86  Accelerated repopulation of the B M by HOXB4-GFP-transduced  cells was more  pronounced. At 2 weeks post-transplant, 74% of the B M cells in HOXB4-GFP recipients were GFP-positive, compared to only 42% of the cells in control recipients. Furthermore, HOXB4-GFP  transduced cells had a complete competitive advantage over the  endogenous W / W 4 1  4 1  B M cells, as these cells reached nearly 100% GFP-positivity,  compared to only 60% in the control mice.  The HOXB4-mediated repopulation  advantage was further reflected in the magnitude and rate of B M clonogenic progenitor recovery.  Recipients of HOXB4-GFP  cells had between 2- and 10-fold more B M  progenitors than control recipients, and progenitor content approached a plateau earlier in the HOXB4-GFP recipients compared to the GFP recipients. The rapid hematopoietic recovery in HOXB4-GFP  recipients was most dramatic  at the C R U level. This assay quantitates stem cell numbers by transplantation of a test sample at limiting dilutions, and assaying for long-term, lympho-myeloid repopulation. However, our a priori estimation of C R U recovery in the control mice overestimated the recovery rate, and as a result the B M dilutions transplanted at 4 and 12 weeks were too low. None of the C R U test mice were repopulated by the test samples, and we were unable to get an accurate quantitation of the C R U frequencies at these two times. At two weeks post-transplant, the B M C R U content in control mice was only. 0.2% of normal, and gradually increased to a maximum of 24% at 16 weeks (Fig. 2.6). In contrast, the C R U levels in HOXB4-GFP recipients had already reached 25% of normal by two weeks post-transplant, and were within the normal range by 12 weeks. expansion in HOXB4-GFP  The rapid C R U  transplant recipients was confirmed in a second trial, where  87  expansion in HOXB4-GFP  transplant recipients was confirmed in a second trial, where  CRU expansion was 10-fold greater in HOXB4-GFP (Table 2.1). HOXB4  recipients at 2 weeks post-transplant  overexpression is thus associated with increases in both the rate of  Normal Range  10,000 _  1,000 J  100 J  10 J  0  I 8  10  12  14  16  18  Time Post-transplant (weeks) CRU recovery and the magnitude of maximum recovery.  Figure 2.6: Kinetics of CRU expansion in vivo. The number of CRU in femurs of 4 cohorts of HOXB4-GFP (•) or GFP (•) were evaluated at 2, 4, 8,12 and 16 weeks posttransplantation using the CRU assay. At 4 and 12 weeks, the CRU values in control GFP mice were below the level of detection, as no GFP 3° recipients were repopulated by transduced cells. The CRU values at these times are below the points indicated (-1) on the graph. For all other time points, the results are expressed as the average +SEM of the CRU numbers in one femur of HOXB4-GFP or GFP control mice. The dashed lines represent the normal number of CRU in 1 femur of an unmanipulated mouse, based on previous data (Pawliuk et al., 1996). CRU regeneration by HOXB4-GFP-transduced cells was significantly greater than by GFP-transduced cells at 2 weeks (pO.OOl), 8 weeks (p<0.05), 12 weeks (pO.OOl) and 16 weeks (p<0.05). 88  Table 2.1. CRU regeneration at two weeks post-transplant. Expt No. 1  Transplant type HOXB4-GFP  C R U frequency (1/x) (95% C.I) 2.8 x 10 (1.2 x 10 - 6.7x 10 ) 4.9 x l O (2.0 x 1 0 - 1.1 x 10 )  No. CRU/femur % of (95% C.I.) normal 620 (260-1,500) 25 6 (1-46)  0.2  5.7 x l O (2.3 x 1 0 - 1.4x 10 ) <4.7xl0  400 (160-1,000)  16  <30  <1.2  4  4  GFP  4  5  5  2  HOXB4-GFP  6  4  4  GFP  2.4.4  5  5  CRU regeneration at high transplant cell doses In the above experiments, HOXB4-GFP  recipient mice were transplanted with  approximately 50 C R U , resulting in regeneration of C R U to within the normal range of unmanipulated mice.  To assess whether similar C R U levels would be achieved with  higher transplant doses, thus suggesting a ceiling on C R U population size, or whether "super" plateau levels might be achieved, we tested the effect of transplant dose on C R U recovery. Mice were transplanted over a 30-fold cell range, from 3 x l 0 cells to 10 cells. 5  7  At all three doses, C R U recovery at 8 months post-transplant was within the normal range (Fig. 2.7). Thus, transplant innoculums spanning an estimated range of 60 to 2,400 C R U all resulted in C R U levels within, but not beyond, the level found in unmanipulated mice. We confirmed that the regenerated C R U were derived from the HOXB4-GFP-Xransa\ucQa' cells, as there was complete concordance between mice reconstituted by donor (Ly5.1 ) +  cells and those expressing GFP (data not shown; Appendix Fig. 2.9). A high degree of polyclonality was found in the B M from mice at all cell doses (data not shown; Appendix  89  Fig. 2.10), confirming that multiple CRU clones had expanded in vivo.  1,000,000  ,  100,000  CD  CD  O  §1 ID CD  *  J  10,000  Normal range  I:  1,000 100  E 10 1  1—I I I I Mil  10  100  1—I  I I Mill  1—1 I I I Mil  1,000  10,000  Number CRU Transplanted  Figure 2.7: CRU regeneration following transplantation of high HOXB4-GFP transplant cell doses. CRU frequency in the transplant innoculum was determined to be approximately 1/4,000, giving the input CRU numbers shown on the X-axis. CRU content in the BM of n=4 recipient mice was determined at 8 months post-transplant, and is represented as the mean (± 95% confidence interval; CI) CRU content per mouse. At each dose tested, CRU recovery is within the 95% CI of the CRU content determined from an unmanipulated control mouse.  90  2.5  Discussion In this study we undertook a detailed analysis of the ability of HOXB4 to effect the  growth of hematopoietic cells, in vitro and in vivo. Previous studies demonstrated that HOXB4 overexpression enhanced plateau levels of C R U recovery, as assessed at relatively late times post-transplant (Thorsteinsdottir et al, 1999). In this study we have focused instead on very early times post-transplant, and found a striking enhancement on the rate of C R U regeneration. Furthermore, this enhancement of H S C self-renewal does not appear to come at the expense of either responsiveness to HSC regulation or terminal differentiation. By incorporating GFP as a selectable marker in retroviral vectors we were able to isolate control- or /fQZB^-transduced cells and rigorously track their growth.  We  provide clear evidence of a competitive growth advantage of i/QZB^-transduced cells in vivo, manifest in both mature and primitive hematopoietic cell populations. Transplant innoculums  containing just  5% - //QZB4-transduced cells resulted  in complete  repopulation of both primitive and mature hematopoietic compartments by HOXB4overexpressing cells. This competitive repopulation argues strongly that the effects of HOXB4  are cell autonomous, as the growth advantage  was not conferred to  accompanying cells present in the same transplant innoculum. This was further supported by in vitro data, where only the HOXB 4-transduced cells showed increased growth in mixed cultures. Furthermore, we provide direct evidence that HOXB4 overexpression does not inhibit hematopoietic differentiation, as evident by normal recovery and maintenance of circulating blood cells of all lineages, cells proven to be derived from  91  H0XB4-GFP-transduced  cells by the presence of GFP.  HSC recovery was accelerated in recipients of HOXB4-GFP-transduced  cells.  HSC regeneration in recipients of GFP control-transduced marrow was barely detectable at 2 weeks post-transplant, reaching only 0.2% of the normal level, and even by 16 weeks had reached plateau levels of only 24%.  In contrast, C R U number in recipients of  //OXijV-GFP-transduced B M had already recovered to 80-fold higher levels (or 25% of normal) by 2 weeks, and reached plateau levels within the normal range as early as 12 weeks post-transplant. The HOXB4-inducQd acceleration in HSC regeneration could reflect a number of possible mechanisms. One possibility is that HOXB4 overexpression may shorten the stem cell cycle time, and/or increase the proportion of stem cells actively dividing. The latter explanation seems unlikely, given observations that virtually all H S C are actively proliferating shortly after B M transplantation (Iscove and Nawa, 1997). Studies on RAT1 fibroblasts engineered to overexpress HOXB4  revealed increased proliferation  associated with up-regulation of cell cycle regulators of the A P I complex as well as cyclin D (Krosl and Sauvageau, 2000). A shortening of cell cycle is further supported by the significantly shorter population doubling time in bulk B M cultures. While the degree of C R U growth in the first few weeks in vivo is beyond that accountable solely by the enhanced growth kinetics demonstrated in vitro, these cultures contained a vast majority of more mature cells, which might not be effected by HOXB4 overexpression. A second possibility is that HOXB4 levels directly alter the probability of HSC self-renewal. The acceleration of C R U expansion is consistent with this, particularly i f  92  we suppose that both control and HOXB4 HSCs are maximally cycling. Additionally, HOXB4 may alter the responsiveness of HSCs to extrinsic regulators.  Increased  responsiveness to self-renewal stimulation is suggested by the accelerated growth demonstrated in the early period post-transplant, and a decreased responsiveness to negative regulation is suggested by the ability to reach much higher plateau levels. Intriguingly however, even with large transplant cell doses, HSC regeneration did not exceed normal levels, indicating that HOXB4 does not override extrinsic mechanisms that appear to control HSC population size. This is in contrast to the findings seen with B M cells engineered to overexpress MDR1, which had continued HSC growth in vivo, leading to a myeloproliferative syndrome (Bunting et al, 1998; Bunting et al, 2000). Another intriguing, although purely speculative, possibility is that HOXB4 might allow for recruitment of other cell types into a stem cell fate, by forcing somewhat more mature hematopoietic cells to "de-differentiate" to hematopoietic totipotency. A number of recent studies have demonstrated that lineage commitments can be reversible given sufficient alternate stimulus.  Examples include "trans-differentiation" of muscle  (Jackson et al, 1999), neural (Bjornson et al, 1999), or hematopoietic (Ferrari et al, 1998; Gussoni et al, 1999; Kopen et al, 1999; Lagasse et al, 2000; Pereira et al, 1998; Periera et al, 1995) stem cells when transplanted to alternate body environments, as well as a recent report of oligodendrocyte precursor cell "de-differentiation" into neural stem cells (Kondo and Raff, 2000). If we speculate that the effects demonstrated here represent an exaggeration of the normal role of HOXB4, then this transcription factor could play a critical role in HSC  93  regulation. While we cannot rule out the possibility that the very high levels of HOXB4 in this setting are mimicking the effects of another, closely related gene, it appears as though HOXB4 levels could regulate stem cell self-renewal under steady state conditions, and be a limiting factor in HSC regeneration. This is in agreement with Hox expression data, which showed Hox genes - particularly those at the 3' end of the clusters, such as HOXB4 - to be preferentially expressed in primitive cells and down-regulated with hematopoietic differentiation (Sauvageau et al, 1994). The growth-enhancing effects demonstrated here in vitro and in vivo raise the possibility of exploiting HOXB4 as a stem cell amplifying factor, for example to provide a selective growth advantage to transduced cells in gene therapy models. These data raise interesting speculation on possible growth enhancing effects of HOXB4 on HSCs in vitro, a property that has yet to be tested. Additionally, downstream genes whose expression is affected by HOXB4 are also potentially important HSC effectors, and identification of such genes will be of interest to further our understanding of HSC regulation.  94  2.6  Appendix  I present here data not included in the original manuscript.  80 70 ^  H  60 • apoptosis  O 50  c  • G0/G1  40  • S  o 30 i—  • G2/M  20 10  GFP  HOXB4-GFP  Figure 2.8: Propidium iodide labelling of GFP and HOXB4-GFP  cultures. B M cells  transduced with GFP or HOXB4-GFP were sorted for GFP expression and plated at 10 cells/well on 24 well plates. After 8 days of liquid culture, 6 wells were harvested and stained with propidium iodide. There was no significant difference in the proportion of apoptotic or cycling cells between the two cultures.  4  95  Figure 2.9: Concordance between Ly5.1 and GFP expression. Representative FACS profiles from C R U test mice are shown. A l l mice which scored positive for donorderived (Ly5.1+) lympho-myeloid repopulation also scored positive for transduced cellderived (GFP+) lympho-myeloid repopulation, even at limit dilution (mice 1-2). Primary C R U doses and secondary cell doses are: 1 = 60 C R U , 20,000 cells; 2 = 600 C R U , 20,000 cells; 3 - 2,400 C R U , 200,000 cells.  3 x 10 cells 60 C R U 5  3 x 10 cells 600 C R U  '  ^0  u..  10 cells 2,400 C R U  6  mm.  7  ^ j  Figure 2.10: Polyclonal repopulation in HOXB4-GFP recipient mice. Southern blot of genomic D N A harvested from B M cells of individual mice transplanted 30 weeks previously with high doses of HOXB4-GFP transduced cells, digested with Hindlll and probed with radiolabeled GFP. Each band represents a unique retroviral integration site, and therefore a unique repopulating cell clone. All mice display highly polyclonal repopulation.  Wm  if gMk  wmW  96  CHAPTER 3  H0XB4-INVVCET)  HEMATOPOIETIC STEM CELLS  EXPANSION  OF  ADULT  EX VIVO  97  3.1  Abstract Hox transcription factors have emerged as important regulators of primitive  hematopoietic cell proliferation and differentiation. In particular, HOXB4 appears to be a strong positive regulator of hematopoietic stem cell self-renewal. Here we demonstrate the potency of HOXB4 to enable high level ex vivo hematopoietic stem cell expansion. Ten to 14 day cultures of non-transduced or GFP-transduced murine bone marrow cells experienced large stem cell losses. In sharp contrast, cultures of #QZB4-transduced cells achieved rapid, extensive and highly polyclonal stem cell expansions, resulting in 40-fold net stem cell growth. Importantly, the expanded HOXB4 stem cells retained full lymphomyeloid  repopulating  potential  and  enhanced  in  vivo regenerative  potential,  demonstrating the feasibility of achieving significant ex vivo expansion of hematopoietic stem cells without impairment of functional properties.  3.2  Introduction The relative inability to expand hemopoietic stem cells (HSCs) ex vivo imposes  major limitations on the current use of HSC transplantation. This is especially true in cases where the number of available stem cells is limiting (eg. cord blood derived stem cells for transplantation into adults).  While studies have shown that self-renewal is  clearly possible in vitro (Ema et al, 2000; Fraser et al, 1990; Glimm and Eaves, 1999), most culture conditions nonetheless differentiation  result in net HSC losses, indicating that  is favoured over expansion.  Several studies  have  documented  98  maintenance and moderate (3-fold) expansions of HSCs in serum-free cultures initiated with highly purified HSC populations and high concentrations of early-acting cytokines (Conneally et al, 1997; Miller and Eaves, 1997). However, optimization of cytokine combinations does not seem to be sufficient to induce clinically significant HSC expansions, as it appears they serve a more permissive role in HSC survival and proliferation rather than directing self-renewal probability. Recent attention has focused on cell intrinsic pathways, whose activation has caused some HSC expansion ex vivo. Overexpression of the P-glycoprotein pump genes MDR1 or ABCG2 led to the expansion of side population cells with retained repopulation ability (Bunting et al, 1998; Bunting et al, 2000). Activation of retinoic acid receptor signalling by addition of exogenous all-trans retinoic acid resulted in retention of longterm repopulating activity after 14 days in culture (Purton et al, 2000). Constitutive Notch activation in Sca lin"c-kit bone marrow cells led to immortalization of blast-like +  +  cells which retained lympho-myeloid differentiation and long term repopulating ability (Varnum-Finney et al, 2000). Addition of soluble Sonic Hedgehog protein to liquid cultures of human bone marrow cells led to at least 3-fold expansion of SCIDrepopulating cells via modulation of BMP4 levels (Bhardwaj et al, 2001). However, none of these ex vivo systems have achieved HSC expansions comparable to the up to 100-fold HSC regeneration demonstrated in vivo following bone marrow transplantation (Pawliukera/., 1996). We have been interested in the Hox family of transcription factors, whose deregulated expression have effects on hematopoietic proliferation and differentiation ranging from blocked lymphoid development (Sauvageau et al, 1997) to acute myeloid 99  leukemia (Borrow et al, 1996a; Buske and Humphries, 2000; Lawrence et al, 1999; Thorsteinsdottir et al,  1997).  In particular, retroviral overexpression of HOXB4  significantly enhanced in vivo HSC regeneration, with -1000-fold net increases of transduced H S C in both primary and secondary recipients (Antonchuk et al, 2001; Sauvageau et al,  1995; Thorsteinsdottir et al,  1999).  HOXB4  overexpression  specifically enhanced the rate of HSC expansion without impairing normal differentiation or causing the cells to become transformed. Using long-term in vivo repopulation of mice and principles of limiting dilution to quantitatively determine H S C expansion, we now demonstrate that HOXB4 has the unique capacity to induce HSC expansion ex vivo to values measured at more than 3 logs over controls in short term liquid cultures. The expanded stem cell population remained competitive and multi-potent (B, T and myeloid) in primary and in secondary recipients.  3.3  3.3.1  Methods  Retroviral Vectors Generation of the M S C V - I R E S - G F P and MSCV-HOXB4-TKES-GFP  vectors and  the GP E86-based producer cells were described previously (Antonchuk et al, 2001). +  Retroviral titres of the GFP and HOXB4 producer cells were 3 x 10 /ml and 2 x 10 /ml 5  5  respectively, as assessed by transfer of GFP expression to NIH-3T3 cells.  100  3.3.2  Mice Parental strain mouse breeders were originally purchased from The Jackson  Laboratory (Bar Harbour, M E ) and subsequently bred and maintained at the British Columbia Cancer Research Centre animal facility according to the guidelines of the Canadian Council on Animal Care.  They were housed in microisolator units and  provided with sterilized food, water, and bedding. Irradiated animals were additionally provided with acidified water (pH 3.0). Bone marrow transplant donors were C57B16/LyPep3b (Pep3b) and express Ly5.1, and recipients were C57B1/6-W41/W41 (W ) and 41  express Ly5.2.  3.3.3  Transduction of Primary Murine Bone Marrow Cells Primary mouse bone marrow cells were transduced as previously described  (Antonchuk et al, 2001). Briefly, bone marrow cells were extracted from mice treated 4 days previously with 150 mg/kg 5-fluorouracil (Faulding, Vaudreuil, PQ, Canada) arid cultured for 48 hrs in D M E M supplemented with 15% fetal bovine serum (FBS), 10 ng/ml hIL-6, 6 ng/ml mIL-3, and 100 ng/ml mSF. Media and serum were purchased from StemCell Technologies (Vancouver, B C , Canada), and growth factors were expressed from cloned cDNAs in COS cells and purified in the Terry Fox Laboratory.  After  stimulation, the cells were harvested and infected by either co-cultivation with irradiated (1500 cGy X-ray) GP+E86 viral producer cells or by the addition of virus-containing supernatant from the GP+E86 producer cells. Both infection protocols involved 48 hours growth on tissue culture plates with the above cytokine combinations and with the  101  addition of 5 pg/ml protamine sulfate (Sigma, Oakville, ON, Canada). Loosely adherent and nonadherent cells were recovered from the co-cultures, or all cells were recovered from the supernatant infections. Following the infection procedure, bone marrow cells were cultured a further 6 to 10 days in the same medium, without protamine sulfate. On days 0, 2, and 4 during the infection, and days 6, 10 and 14 following the infection, cells were counted on a hematocytometer, and an aliquot was removed for F A C S , CFC, and C R U assays. A l l of the remaining cells from days 0 or 2, or 10 cells from each culture 7  on days 4, 6 and 10 were replated at 2 x l 0 cells/ml. 5  3.3.4  Flow cytometry Approximately 10 cells from each liquid culture sample were washed once with 5  Hank's balanced salt solution (StemCell Technologies) with 2% FBS (HF), then once with HF and lpg/ml propidium iodide (PI; Sigma) prior to analysis of GFP expression on a FACScalibur (Becton Dickinson, Mississauga, ON, Canada). For analysis of transplant recipients, 100 pi of blood was extracted from the tail vein, and the erythrocytes were lysed with ammonium chloride (StemCell Technologies). Leukocyte samples suspended HF were incubated sequentially on ice with biotinylated anti-Ly5.1, then APC-labeled streptavidin and either phycoerythrin (PE)-labeled B220 or a combination of PE-labeled Gr-1 and PE-labeled Mac-1 (all antibodies from Pharmingen, Mississauga, ON, Canada). A l l samples were washed with HF and lpg/ml PI prior to analysis on FACScalibur. '  102  3.3.5  CFC Assay Hematopoietic clonogenic progenitor frequencies were determined by plating  suitable aliquots of bone marrow or spleen cells in methylcellulose medium (HCC-3334, StemCell Technologies) containing 3U/ml erythropoietin, and supplemented with 50 ng/ml mSF, 10 ng/ml hIL-6, and 10 ng/ml mIL-3, and then scoring the resultant colonies after 10 days of incubation.  3.3.6  CRU Assay HSCs were detected and evaluated using a limiting dilution transplantation based  assay for cells with competitive, long-term, lympho-myeloid repopulation function. The basic procedure (Szilvassy et al, irradiated W  4 1  1990), and a modification employing sublethally  recipients (450 cGy  137  C s y-radiation) as a source of endogenous  competitor cells (Antonchuk et al, 2001; Miller and Eaves, 1997), have been described in 137  detail previously.  Briefly, sublethally (450 cGy of  4  ,  Cs y-radiation) irradiated W  recipients were injected with 10 to 9.5 x 10 cells, and their blood was analyzed by F A C S >16 wks post-transplant for evidence of lympho-myeloid repopulation.  Mice  which had >1% donor-derived (GFP ) cells in both lymphoid (B220 ) and myeloid (Gr-1 +  +  +  or Mac-1 ) subpopulations were considered to be repopulated with transduced cells. C R U +  frequencies in the test bone marrow sample were calculated by applying Poisson statistics to the proportion of negative recipients at different dilutions using Limit Dilution Analysis (StemCell Technologies) software.  103  3.3.7  Proviral Integration Analysis. Genomic D N A was extracted from primary hematopoietic tissues using DNAzol  (GibcoBRL, Burlington, O N , Canada). Unique proviral integrations were identified by digestion of D N A with H i n d m , which cleaves once within the provirus, and at various distances within the genome. Digested D N A was then separated in 1% agarose gel by electrophoresis and transferred to zeta-probe membranes (Bio-Rad, Mississauga, O N , Canada) by standard Southern blotting techniques (Sambrook et al, 1989). Membranes 32  were probed with a P-dCTP-labeled GFP sequence.  3.4  Results  3.4.1  Ex vivo expansion of //QXZ?4-overexpressing HSC Ex vivo expansion of HSCs was determined in cultures of non-transduced,  control-transduced, and MXYB^-transduced cells using a modification of the C R U assay as previously described (Antonchuk et al, 2001). Mouse bone marrow cells harvested 4 days after intravenous injection of 5-fluorouracil (5-FU) were grown for 2 days in the presence of serum and hematopoietic cytokines.  The non-transduced group then  continued to be cultured in the same media, while the other two groups (hereafter referred to as the GFP and HOXB4 cultures) were transduced with retroviruses carrying either GFP alone or HOXB4 and GFP for an additional 2 days, resulting in infection efficiencies of 44% and 33% respectively. The 3 groups were then cultured for an additional 10 days, without selection for GFP-expressing cells. Growth of total cells, G F P cells, clonogenic progenitors and HSCs were monitored at intervals of 2-4 days. 104 +  HOXB4 overexpression gave a moderate 2-3-fold growth advantage to total cells and progenitors during the 14-day culture (Fig. 3.1a,c).  This was due to a selective  expansion of transduced cells, demonstrated by the rapid predominance of G F P cells and +  progenitors which occurred only in the HOXB4 culture (Fig. 3.1b,d).  Figure 3.1: Ex vivo expansion of mature cells and progenitors, (a) Growth of total  nucleated cells over 14 days in non-transduced (•), GFP (•) and HOXB4 (A) cultures, (b) Proportions of transduced (GFP ) cells in GFP and HOXB4 cultures. The dramatic increase in G F P cells in the HOXB4 culture reflects a cell autonomous growth advantage conferred by HOXB4. (c) Growth of clonogenic progenitors (CFC) in non-transduced ( • ) , GFP (•) and HOXB4 (A) cultures over 14 days, (d) G F P and GFP" C F C after 14 days of culture. While the proportion of G F P CFCs reflects the input transduction efficiency in the GFP culture, the predominance of G F P CFCs in the HOXB4 culture reflects a HOXB4-med.iaXeA growth advantage. +  +  +  +  +  105  a  stim  .2  1O  inf  liquid culture  1  ! 1  -|  0  I  2  r—  4  — i —  6  —I—  —r—  8  10  i  12  — i —  14  1  16  Time in Culture (days)  2  1003  liquid culture  100/  O.Of  8 Days in culture  10  O.OOf  Exptl  Expt2  Expt3  Expt4  Figure 3.2: Ex vivo expansion of HSCs. (a) Change in H S C number over 14 days in non-transduced (•), GFP-transduced (•) or //CTAB^-transduced (A) B M cultures. Shown are the absolute number of C R U (mean ± 95% confidence intervals) in the culture. While the control cultures experienced significant HSC decreases, the HOXB4 cultures achieved a 40-fold net HSC increase, (b) A second, 10-day experiment confirmed the net changes in HSC number. Values are shown relative to starting numbers (set to 1). Asterix in a and b indicate a significant difference (pO.OOOl) between HOXB4 and GFP cultures, (c) The rapid HSC expansion was confirmed in 4 independent experiments, with HOXB4 cultures undergoing approximately a 6-fold HSC expansion by day 6, in contrast to an average 17-fold reduction seen for GFP cultures. 106  HSC numbers in these cultures were measured every 2-4 days by in vivo limiting dilution analysis. Although the HSC contents of the 3 cultures were initially equivalent at approximately 7,000 C R U , they rapidly increased in the culture containing HOXB4transduced cells, with 8-fold expansion by day 6 and a further 5-fold expansion by day 14 when the experiment was terminated. As a result, there was a net 41-fold HSC expansion over 14 days in the HOXB4 culture (Fig 3.2a). This strikingly contrasted with control non-transduced and GFP cultures, in which a constant decrease in HSC content resulted in 29- to 58-fold HSC reductions, respectively, by day 14. Combining the net HSC loss in the GFP culture and the net HSC gain in the HOXB4 culture resulted in an overall difference of over 1000-fold HSC content between these two cultures. This expansion was restricted to //QA34-transduced cells, as there was complete concordance between the detection of donor-derived (Ly5.1 ) and //QZB^-transduced (GFP ) cells in +  +  repopulated mice, even at limit dilution. These results were reproduced in a second experiment, and again indicated a similar difference in HSC values between the GFP and the HOXB4 cultures (Fig 3.2b). Two additional experiments (i.e. Expts 3 and 4, Fig. 3.2c), evaluating HSC content at day 6 of culture (i.e. 4 days after initial exposure to retrovirus) showed the reproducibility of these findings (Fig 3.2c).  3.4.2  //OXZW-transduced HSCs are Competitive and Pluripotent. To confirm that the expanded HSCs retained full multilineage repopulation  ability, F A C S analysis was performed on bone marrow, spleen, and thymus samples of  107  mice transplanted 16 weeks previously with cells harvested at day 14 from the cultures described above.  The presence of transduced (GFP ) cells in marrow myeloid and +  erythroid cells, splenic B-cells, and thymic T-cells from representative recipients indicated that the expanded cells from either the GFP or HOXB4 cultures were not compromised in their capacity to differentiate along myeloid and lymphoid lineages (Fig. 3.3a). Clonal analyses based on proviral integration sites were performed using purified subpopulations of bone marrow myeloid cells (Gr-1 or Mac-1 ), splenic B cells (B220 ) +  +  +  and thymic T cells (CD4 or CD8 ) (Fig. 3.3b). The identical proviral integration sites in +  +  myeloid, B and T lineages of a representative mouse transplanted with cells from the HOXB4 culture demonstrated the pluripotent nature of an expanded HSC clone. The proliferative potential of the expanded //ClYB^-transduced HSCs was further evaluated by measuring their ability to regenerate the HSC compartment in vivo. To that end, we performed C R U assays on bone marrow cells from mice transplanted 4 months previously with cells from the 14 day GFP or HOXB4 cultures. While the overall level of reconstitution was roughly equivalent for the two groups (Fig. 3.3a), the level of HSC regeneration was much higher in the H O X B 4 mice. We determined that each GFPtransduced HSC expanded approximately 70-fold in vivo (95% confidence intervals (CI): 10 - 500 CRU), while each 7/OY34-transduced HSC expanded approximately 5,000-fold (95% CI: 1,400 - 22,400 CRU) (Fig. 3.4b).  Thus, #OZB4-transduced HSCs, which  previously expanded 40-fold ex vivo (as a population), were still capable of massive in vivo expansion, regenerating the HSC compartment to near-normal size. Furthermore, the in vivo expanded HOXB4 HSCs still provided significant reconstitution of the lymphoid and myeloid lineages. 108  a  GFP  H0XB4  B S T  B S T  «p  _ s  H0XB4 B  g m  s  B220  T  48  n# mm  HP iH  mm  •  Figure 3.3: Lympho-myeloid repopulation by ex vivo expanded stem cells,  (a)  Representative F A C S profiles demonstrating multilineage repopulation by transduced cells. GFP-expressing cells were present in (from left) myeloid (Gr-1 or Mac-1 ) and erythroid (Terl 1 9 ) bone marrow cells, B-lymphoid ( B 2 2 0 ) splenocytes, and T-lymphoid (CD5 ) thymocytes of mice transplanted 9 months previously at limit dilution_from 6 - 1 0 day GFP or HOXB4 cultures. Representative GFP (top panels) and HOXB4 (bottom panels) mice show equivalent multilineage repopulation, consistent with unimpaired differentiative ability, (b) Representative Southern blots (8 mice per group analyzed, with similar results) demonstrate common proviral insertion sites in reconstituted myeloid and lymphoid tissues from mice transplanted at limit dilution. Common proviral bands are present in enriched or purified myeloid, T-lymphoid, and B-lymphoid populations from each mouse, indicating that a common repopulating cell clone repopulated all lineages. B = bone marrow cells, S = splenocytes, T = thymocytes, B , M = G r - 1 or M a c - 1 bone marrow cells, SB22O = B 2 2 0 splenocytes, T^g = C D 4 or C D 8 thymocytes. +  +  +  +  +  G  +  +  +  +  +  109  a Primary Transplant type  gpp  No. HSC transplanted  HOXB4  1 2.09  HOXB4  1 59.03  37  2.78  64.79  5.76  16.01  3.94  76.10  t in  W^.  •  16.95  21.93  • IP 16.42  ' V ? •.  14.20  GFP  ^  100,000  4-i  ro ^  | ^ 10,000 fl) o 0) CD i-  l_  CD  co 3  o  E E i_ 3 C  O  CD Q.  1,000 J 100 10  Primary Transplant type  gpp  No. HSC transplanted  HOXB4  1  37  Figure 3.4: Regeneration of HSCs following ex vivo expansion, (a) Representative FACS profiles of bone marrow from mice transplanted with (from left) 1 GFP HSC, 1 HOXB4 HSC or 37 HOXB4 HSCs from the 14-day cultures reveals them all to be highly repopulated by transduced (GFP ), donor-derived (Ly5.1 ) cells. Ly5.1"GFP cells represent transduced cell progeny in the erythroid lineage, which do not express Ly5.1. (b) Calculated HSC (CRU) content in pooled bone marrow from the above groups of mice (n=2 GFP or 3 HOXB4 primary mice) demonstrated that only the HOXB4 mice had regenerated HSCs to near the normal range (dotted line). +  +  +  110  3.4.3  Polyclonal Expansion of J/0XB4-Transduced HSCs To resolve whether the documented HOXB4 growth advantage occurred on a large  number of HSCs versus on a restricted subset of such cells, we sought to estimate the number of different HSC clones that were expanded during the ex vivo cultures described in the first section. For this, we assessed the number of different transduced clones that contributed to long-term repopulation when multiple HSCs harvested at day 10 or day 14 from GFP or HOXB4 cultures were transplanted.  Qpp 1 1  1 2  1 3  1 4  2.1 2.2 2.3 2.4  fli^  %0  Figure 3.5: Clonality of expanded HSCs. The degree of clonality in repopulating cells recovered from the cultures was determined by Southern blot analysis of bone marrow from mice transplanted with HOXB4 or  fr° y The first number denotes the experiment number (ie. 1 or 2), and HP Ml the second number denotes the d individual mice. GFP mice 1.1-1.4 «tf fcf w received 0.5 transduced HSC (2.5 x 10 cells) on day 10, and GFP mice 2.1-2.4 received 10 transduced HSCs (2 x 10 cells) on day 10. HOXB4 mice 1.1-1.5 received 15 1.1 1.2 1.3 1.4 1.5 1 6 1.7 i s 2.1 2.2 23 2.4 transduced HSCs (2.5 x 10 cells) on day 10, HOXB4 mice 1.6-1.8 received 37 transduced HSCs (1.3 x 10 cells) on day 14, and HOXB4 mice 2.1-2.4 received 47 transduced HSCs (6 x 10 cells) on day 10. | ^ Iff GFP mice from each of two experiments have common banding patterns, indicating that few HSC clones survived. However, HOXB4 mice have differing banding patterns, revealing the persistence of multiple transduced repopulating cell clones after the ex vivo expansion. G  F  P  c c , l s  m  1 0 - 1 4  d a  c u l t u r e s  6  7  H  Q  X  B  4  5  6  3  111  Cultures containing GFP-transduced cells contained approximately 300 and 400 transduced HSCs at day 10 in experiments 1 and 2 respectively. Yet recipients of high doses of GFP-transduced cells had clonal to oligo-clonal reconstitution (Fig. 3.5, upper panels) at 4 months post-transplant, suggesting that while a few selected clones expanded, the vast majority of the HSC population was lost in these cultures. In contrast, recipients of similar numbers of //QZB^-transduced HSCs (but much lower cell doses, see figure legend) showed a highly polyclonal reconstitution (Fig. 3.5, lower panels), indicating that 7/QA34-tranduced HSCs survived and expanded during the 14 day culture (see Fig. 3.6). Thus, the enhanced HSC expansion in HOXB4 bone marrow cultures can be explained at least in part by an increased retention of stem cell clones.  Death of most HSC, expansion of few Net decline  • •• • • ••• • • #  #  More HSC clones able to expand,  •••••/.-.•  More expansion per HSC  Initial HSC Population Net Expansion Post culture  Figure 3.6: Model of /70XB¥-mediated ex vivo HSC expansion. The initial culture contained multiple transduced HSC clones. Without HOXB4 overexpression, the majority of the HSC were lost from the culture, due to differentiation or death. A few surviving clones did undergo self-renewal, but the end result was still net HSC decline. With HOXB4 overexpression, more HSC clones were able to survive and expand in the culture and this, along with more expansion per HSC, caused a net HSC expansion. 112  3.5  Discussion  Based on previous studies showing the potential of intrinsic factors to control HSC self-renewal decisions, and our previous results showing marked enhancement of HSC regeneration mediated by HOXB4  in vivo, we hypothesized that HOXB4  overexpression might enable enhanced HSC recovery after ex vivo culture. A simple bulk bone marrow cell culture containing serum, IL-3, IL-6 and SF was used, and in this system we documented a //QZZ?4-mediated net HSC expansion of greater than 40-fold in 14 days. Moreover, the expansion occurred quite rapidly, with significant HSC growth recorded in just a few days. These cultures were initiated with non-selected populations containing approximately 30% //QATi^-transduced cells and 70% non-transduced cells. Yet the HSC expansion was confined to the HOXB4-GFP  +  cell population, suggesting  that the 40-fold net expansion reported here might even be an underestimate of the true level of expansion achieved by /7QZB4-transduced HSCs. The quality of the expanded HSCs was not impaired, as demonstrated by their ability to fully repopulate all lineages. Significantly, all mice transplanted with HOXB4expanded HSCs remained healthy, and without any manifestations of hematopoietic disorder (eg. myeloproliferative disease) for extended observation periods (9 months). Moreover, we confirmed that expanded HSCs retained significant proliferative potential. We previously demonstrated enhanced HSC regenerative ability in i/67A34-transduced bone marrow cells (Antonchuk et al, 2001; Sauvageau et al, 1995; Thorsteinsdottir et al, 1999).  We now examined the regenerative ability in //QATi^-transduced cells  following 40-fold ex vivo HSC expansion. We show here that the hyper-regenerative potential of HOXB4 is retained after ex vivo expansion, thus the extensive ex vivo HSC 113  growth did not impair the ability of HOXB4 cells to expand extensively in vivo. In fact, by this measurement the //QZS^-expanded HSCs  are qualitatively better than  unmanipulated HSCs. HOXB4 overexpression may also increase cell proliferation. It has been argued that the morphological changes seen in Hox mutants may be due to modification of local growth rates (Duboule, 1995).  Studies in RAT-1 cells showed that HOXB4  overexpression activated the expression of AP-1 complex genes Fra-1 and Jun-B, with subsequent up-regulation of cyclin Dl (Krosl and Sauvageau, 2000). We previously demonstrated that HOXB4 overexpression enhanced ex vivo growth of total bone marrow cultures, and that this effect was due to increased proliferation rather than reduced apoptosis (Antonchuk et al., 2001). We now show that the //OXiM-mediated growth advantage is largely restricted to the most primitive hematopoietic cell populations: in 14 days there was a 2-fold enhancement to total (mostly mature) cell growth, a 3-fold enhancement to progenitor cell growth, and a 1000-fold enhancement to HSC growth. The primitive cell-specific growth advantage suggests enhanced HSC proliferation and/or an enhanced probability of self-renewal mediated by HOXB4 overexpression. The potential use of HOXB4 or a downstream effector in human therapeutic applications including HSC transplantation is buoyed by recent findings in the human system. Overexpression of HOXC4 in human bone marrow cells led to ex vivo expansion of clonogenic progenitors and long-term culture initiating cells (LTC-IC) (Daga et ah, 2000).  HOXC4 is a paralog of HOXB4, and the similar overexpression phenotype  suggests an overlapping function between members of the 4 paralog group in promoting th  a primitive state. Ongoing studies indicate that overexpression of HOXB4 also allows the 114  expansion of human HSCs as detected by their capacity to repopulate immunocompromised mice (R.K.H. submitted), giving further promise to potential therapeutic applications of HOXB4 overexpression. Recent findings indicate that HOXB4 may also reprogram mouse embryonic stem cells into long-term repopulating cells, opening new avenues to therapeutic cloning of hemopoietic stem.cells (Kyba et al, 2001). These results reinforce our current study which demonstrates that ex vivo HSC expansion is achievable under conditions which include high expression levels of HOXB4.  In addition, the rapid ex vivo HSC expansion  induced by HOXB4 shown here, combined with new avenues of engineering direct protein delivery to cells (e.g. HIV TAT tags; Nagahara et al, 1998) suggest that it should be possible to develop a HOXB4 protein that can act as a mitogen specific for HSCs. Such findings open new exciting avenues for cellular and. genetic manipulation of HSCs.  115  CHAPTER 4  GENERAL DISCUSSION  The results presented in this thesis clearly show that overexpression of HOXB4 promotes the growth of HSCs in vitro and in vivo. HSCs maintain production of all blood cell lineages throughout life, and can reconstitute a complete hematopoietic system in transplanted recipients.  Moreover, not only are they required for steady-state  hematopoiesis and B M transplant strategies, but their absolute numbers are critical in both these settings. Strategies to control HSC population sizes will benefit treatments for many hematological diseases including leukemias, and improve gene therapy applications for a growing number of diseases. Yet despite more than three decades of research, the biomolecular mechanisms that regulate HSC survival, proliferation and differentiation remain poorly understood. I have demonstrated here that intrinsic factors can regulate HSC growth, and point to HOXB4 as a key modulator promoting H S C self-renewal.  4.1  Mechanism of //OXB^-mediated stem cell expansion HSC population size is controlled in vivo by concerted regulation over  proliferation, differentiation and apoptosis (Fig. 4.1). HSCs are largely quiescent, but can be induced to proliferate by extrinsic signals. However, it is the intrinsic factors that determine H S C fate, and therefore enable self-renewal. Several in vitro studies have demonstrated the limited ability of extrinsic cytokines to cause HSC expansion, as they are strongly mitogenic but also promote differentiation (Fraser et al., 1990; Rebel and Lansdorp, 1996). For HSC expansion to occur therefore, extrinsic conditions must favor proliferation while intrinsic determinants of cell fate maintain the undifferentiated state 116  and prevent apoptosis (Domen and Weissman, 2000).  Dead cell  A  i  Quiescent HSC  Cycling HSC  HSCs  Figure 4.1: Potential mechanisms for regulation of HSC population size. HOXB4 could increase HSC activation, cycling rate or probability of self-renewal, or decrease apoptosis. These options are discussed in the text.  Si ft)  i.  o 3  Differentiated Cell  Activation of HSCs into cycle is a clear requirement for expansion. These cells are largely quiescent, but can be stimulated to divide following in vivo stresses such as 5FU administration or BM transplantation, or the in vitro addition of early-acting cytokines.  Mice deficient for the anti-proliferative factor p21 have a more actively  cycling HSC population and higher numbers of primitive cells (Cheng et al., 2000a). HOXB4 overexpression may also act to enhance HSC activation, thereby increasing the proliferating fraction of HSCs. However, previous studies have shown that the HSCs are maximally activated in both the in vivo and ex vivo settings used in these studies. The 117  early recovery phase post-transplantation is a time of HSC expansion for normal adult BM cells, with up to 100-fold increases in HSC numbers (Pawliuk et al, 1996). During this time up to 80% of long-term repopulating cells are susceptible to 5-FU killing, indicating that the population is actively proliferating (Iscove and Nawa, 1997). Also, an ex vivo study using a cytokine combination similar to that used here found that essentially all long-term repopulating cells were induced into cycle (Trevisan et al, 1996). Thus, it is likely that the HSCs were already maximally activated by extrinsic factors, and HOXB4 overexpression did not induce their proliferation. Moreover, if HOXB4 overexpression had caused an intrinsic activation of HSCs it would be very difficult for the cells to respond to negative extrinsic stimuli (ie. forcing them back into Go), and the mice would likely have succumbed to leukemia. Yet, in the experiments presented here HOXB4transduced HSCs were responsive to negative feedback regulation ceasing expansion at the normal HSC level, and the mice did not develop leukemias. Consistent with this, FACS-selected HSCs from Hoxb-4 knockout mice showed no reduction in their ability to be activated into proliferation by a number of different cytokine combinations (Bjornsson et al, 2001a). However, to answer this question directly, individual FACS-selected HSCs with and without HOXB4 overexpression could be assessed for their proliferative response to early-acting cytokines ex vivo (ie. proportion of cells dividing and time to first division).  Also, in vivo 5-FU toxicity and BrdU incorporation experiments could  determine the proportion of HSCs actively cycling in mice receiving transplants of control or //QZB4-transduced cells. Once activated into cycle, the rate of HSC division could also affect the level of expansion. This is particularly true for the regenerating HSC population following BM 118  transplantation, where there is a limited period of expansion. According to the model put forth by Pawliuk et al. (1996) and Iscove and Nawa (1997), there is strong proliferative stimulation following transplantation, in order to repopulate the post-irradiation hematopoietic system. Progenitor and mature cell populations reach normal levels earlier than the HSC population, due to loss of HSC progeny through differentiation. Yet once progenitor and mature cell populations have recovered, negative feedback returns the system to steady state, where HSC numbers are maintained rather than expanded. HOXB4 overexpression could be increasing the rate of self-renewing HSC divisions, thereby increasing the maximal HSC level achieved in vivo prior to the onset of negative feedback. The in vitro growth advantage mediated by HOXB4 on total B M cells shown here further suggests an accelerated proliferative response, possibly mediated by hyperresponsiveness to mitogenic stimuli. Consistent with this is the finding that Jun-B and Fra-1 - nuclear factors involved in proliferation - are direct transcriptional targets of HOXB4 in a rat fibroblast cell line (Krosl and Sauvageau, 2000). There is thus strong evidence that HOXB4 overexpression increases the rate of HSC division. To test this hypothesis, CFSE could be utilized to track the rate of cell division in individual FACSselected HSCs in vitro. It will also be interesting to determine the proliferative curve of 7/QZB4-transduced cells in response to various cytokine concentrations. Finally, analysis of HOXB4 target genes might reveal direct control over expression of cell cycle regulators (discussed below). HSC division can produce either self or differentiated progeny (Till et al, 1964), and the probability of self-renewal affects the degree and rate of HSC expansion. For 119  example, a self-renewal probability of 0.3 would result in net HSC loss, whereas probabilities of 0.6 or 0.8 would result in net HSC gains, with the latter population expanding faster. The accumulation of differentiated progeny would be proportionately less as the probability of self-renewal increased. The probability of self-renewal in the ex vivo cultures of i/QA2?4-transduced cells must have been greater than 0.5, as net HSC population increases were observed. Ignoring apoptosis for the moment, control cultures would have had self-renewal probabilities of less than 0.5, resulting in net HSC losses. Thus HOXB4 appears to directly enhance the probability of self-renewing divisions. To test this ex vivo, HSCs could be marked with CFSE, and this could then be used to purify populations of cells that had undergone one or more divisions. Increased retention of long-term repopulating ability in the divided fraction would indicate an increased probability of self-renewal. According to the model of HSC regeneration discussed above, we might expect that acceleration of HSC division without modification of self-renewal probability would merely cause all hematopoietic populations to recover faster, thereby initiating negative feedback earlier but still premature of the normal HSC level. In fact, the rate of PB repopulation was higher in recipients of //CTZS^-transduced cells, suggesting that the entire hematopoietic system may have recovered faster due to increased proliferation. However, the enhanced level of HSC regeneration in HOXB4 mice also indicates that the probability of self-renewal was increased. Since the alternative outcome to self-renewal is differentiated progeny, does the enhancement  to  self-renewal  mediated  by  HOXB4  translate  into  decreased  differentiation? Clearly HOXB4 overexpression does not block HSC differentiation, as 120  mature /TQAB^-overexpressing cells were found in all lineages at normal frequencies. Also, as mentioned above, the recovery rate of progenitors and mature cells in recipients of //QATi^-transduced cells was equal to or better than their control counterparts. Similarly, the rate of progenitor and mature cell production in ex vivo HOXB4 cultures was slightly increased over control cultures.  Taken together these data suggest that  HOXB4 overexpression moderately increases the self-renewal probability, yet mature cell production is not diminished, as the reduced differentiative probability is offset by an accelerated proliferation rate. HSC apoptosis is the final way in which HSC population size can be controlled. A transgenic murine line with elevated Bcl-2 expression had reduced apoptosis leading to approximately 2-fold higher levels of short- and long-term repopulating cells (Domen et al, 2000). Hematopoietic cytokines prevent apoptosis in vitro (Willert and Nusse, 1998), and Bcl-2 overexpression appears to mimic cytokine stimulation thereby allowing cultures of phenotypically-defined HSCs to survive and expand in SF alone (Domen and Weissman, 2000). HOXB4 might also enhance HSC expansion through inhibition of apoptosis.  While we failed to detect a significant difference in the proportion of  apoptotic cells in total BM cell cultures, we cannot rule out an anti-apoptotic effect of HOXB4 at the HSC level. To test this possibility, ex vivo cultures of FACS-selected HSCs with and without HOXB4 overexpression could be tested for their level of apoptosis (eg. by TUNEL assay). In addition, future studies should examine expression of known apoptotic regulators in HSCs following HOXB4 transduction. Overall, it is difficult to separate effects on proliferation, differentiation, and apoptosis based on the studies presented here. HSC expansion involves all of these 121  processes, and HOXB4 may oversee HSC expansion by controlling all three processes. It will be important to separate these effects and determine the role of HOXB4 in each. Many of the effects seen in this HOXB4  overexpression model could be  accentuated by an enhanced engraftment effect. Stem cell transplantation models rely on the ability of HSCs injected intravenously to home to and engraft in the BM. Typically 17 % of injected cells find their way to the BM (Oostendorp et al, 2000) in a process mediated by adhesion molecules such as I-CAM and VLA-4 (Quesenberry and Becker, 1998).  As adhesion molecules are common targets of Hox-mediated transcriptional  activation (Cillo et al, 1996), it is possible that HOXB4 overexpressing HSCs express higher levels of these adhesion molecules and therefore more efficiently home to the BM. This could explain in part the competitive engraftment advantage of //QZB^-transduced cells.  The competition assay effectively determines the relative ability of HSCs to  compete for marrow space, and homing to the B M is an important determining factor in that competition. Alternatively however, the enhanced proliferation mediated by HOXB4 overexpression could inhibit engraftment, as there is a documented loss of engraftment ability during cell cycle transit (Glimm et al, 2000). The relative homing ability of //QA7J4-transduced cells could be determined by analyzing the number of GFP-positive cells in the BM at various times (1-12 hours) after injection of control GFP- or HOXB4GFP-transduced cells.  4.2  Hox Gene Specificity To what extent can we conclude that the results presented here represent an  122  exaggeration of the normal role of HOXB4? HOXB4 expression is normally quite low, and restricted to primitive cells (Sauvageau et al, 1994). The retroviral MSCV 2.1 LTR promoter used to drive HOXB4 expression in these studies directs high and long-term expression in primitive murine hematopoietic cells as well as their mature progeny. Expression of the transgene is therefore both higher and more persistent than the endogenous gene. Augmentation of normal HOXB4 function is suggested by the fact that the effects are restricted to the same cell type where HOXB4 is normally expressed (Sauvageau et al, 1994), and by the reduced HSC proliferation documented in mice lacking Hoxb-4 (Bjornsson et al, 2001a). There are significant similarities in sequence among Hox genes, particularly between those in the same paralog group. Moreover, similar phenotypes in loss-offunction mouse models for two paralogous Hox genes also suggest a functional redundancy between Hox genes.  We must therefore question whether the results  presented here are due to enhanced/persistent HOXB4 function (ie. increased expression of normal HOXB4 target genes) or whether the high levels of HOXB4 interfere with or mimic the function of other HOX proteins. Several other HOX genes have been overexpressed in murine BM cells, with effects clearly different from those reported here. HOXA10 overexpression enhanced the formation of megakaryocytic progenitors, blocked macrophage and B-lymphoid differentiation, and predisposed to leukemic transformation (Thorsteinsdottir et al, 1997). Overexpression of HOXB3 blocked production of CD4 CD8 T-lymphocytes, +  +  suppressed early B-lymphoid development and led to a myeloproliferative syndrome (Sauvageau et al, 1997). These results are markedly different from the enhanced HSC 123  expansion phenotype resulting from HOXB4  overexpression, suggesting Hox  specific (or at least paralog-specific) effects on hematopoiesis.  This likely reflects their  regulation o f different target genes during hematopoietic, development. comprehensive lists o f Hox  overexpression phenotypes  gene-  A s more  and H o x target genes are  compiled, the extent o f Hox specificity w i l l become more apparent. However, there are also significant similarities among the  overexpression  phenotypes o f paralogous Hox genes. Human cells engineered to overexpress had increased numbers o f primitive cells, similar to the HOXB4 murine cells. W e require direct comparisons o f HOXB4 the same  system, with quantitative assay  HOXC4  overexpression effect on  and HOXC4  overexpression i n  for stem cell expansion (such as the  experimental procedure used here to assess murine H S C expansion ex vivo) to determine how similar these two phenotypes are. A thorough examination o f progenitor and mature cells in various hematopoietic lineages in a murine HOXC4 necessary to determine i f HOXC4 HOXB4-\ike  overexpression model is also  might have a role i n differentiation separate from the  effect. If no differences are seen in a direct comparison (and also possibly  comparing HOXA4  and HOXD4),  then functional redundancy between these paralogous  Hox genes would be indicated. It would then be interesting to determine i f the deficient phenotype would be enhanced by combined Hoxb-4 elucidate whether Hoxc-4  and Hoxc-4  normally functions in H S C regulation.  direct comparison of HOXB4  and HOXC4  Hoxb-4  deficiency, to  O n the other hand, i f a  reveals phenotypic differences, then domain  swapping experiments could reveal critical sites involved i n the H S C expansion mechanism (eg. sites o f protein-protein interaction or post-translational modification). A related unresolved issue is how Hox  genes might co-ordinately regulate 124  p r o l i f e r a t i o n a n d differentiation o f h e m a t o p o i e t i c c e l l s i n m u l t i p l e lineages a n d v a r i o u s j stages o f d e v e l o p m e n t . development,  where  M o s t o f o u r k n o w l e d g e o f Hox f u n c t i o n c o m e s from e m b r y o n i c they  provide temporal  a n d spatial  identity v i a c o l i n e a r  gene  expression. H e m a t o p o i e s i s , although a n o n g o i n g process, also i n v o l v e s a n ordered series o f events as c e l l s d e v e l o p from the H S C through the h i e r a r c h y to mature e n d c e l l s . T h e differential 3 ' to 5 ' e x p r e s s i o n o f Hox genes w i t h h e m a t o p o i e t i c d e v e l o p m e n t suggests that they m i g h t p r o v i d e p o s i t i o n a l identity w i t h i n the h i e r a r c h y ( F i g u r e 4.2). T h u s 3 ' located genes s u c h as HOXB4 that are o n l y expressed i n the m o s t p r i m i t i v e h e m a t o p o i e t i c cells c o u l d g i v e p r i m i t i v e c e l l identity, w h i l e 5 ' genes s u c h as  HOXA10 that have  persistent e x p r e s s i o n into the progenitor compartment c o u l d g i v e a m o r e mature identity. Persistence o f a p r i m i t i v e identity w i t h i n HOXB4 o v e r e x p r e s s i n g c e l l s w o u l d e x p l a i n the increased p r o b a b i l i t y o f self-renewal i n d i c a t e d b y the H S C e x p a n s i o n reported Expansion  o f megakaryocyte  progenitors  and uncontrolled  here.  myeloproliferation i n  HOXA10 o v e r e x p r e s s i o n studies are l i k e w i s e consistent w i t h a r o l e for this gene i n i d e n t i f i c a t i o n o f a m y e l o i d progenitor state. dominance  by 3'  genes,  as a l l Hox  genes  H o w e v e r , this m o d e l requires are expressed  i n the m o s t  functional primitive  compartment, thus o p p o s i n g the data f r o m d e v e l o p m e n t a l studies s h o w i n g d o m i n a n c e b y 5 ' genes.  125  t  5' Figure 4.2: Model of Hox control over hematopoiesis. The hematopoietic hierarchy is shown above, proceeding left to right. Shown below is a schematic representation of Hox gene expression and relative location of action within the hierarchy. The expression (shaded boxes) of three hypothetical Hox genes are represented, from 3 ' , middle, and 5 ' (top to bottom) regions of a Hox cluster. Their locations of action (striped arrows) could correspond to their mature boundaries of expression, by giving hierarchical positional identity to developing hematopoietic cells.  HOXB3 overexpression results at first appear not to fit this model, as overexpression of this 3 ' gene blocked T- and B-cell development, and did not enhance 126  HSC regeneration. However, recent loss-of-function studies did reveal a role of Hoxb-3 in HSC proliferation. HSCs deficient in Hoxb-3 and Hoxb-4 had a reduced proliferative capacity (Bjornsson et al, 2001b), a phenotype which represented an exaggeration of that in the single Hoxb-4 knockout (Bjornsson et al, 2001a). Thus HOXB3 has a redundant role in HSC proliferation, and must be down-regulated for normal differentiation to occur. This model of Hox control over the hierarchical position of hematopoietic cells could be achieved via spatially colinear Hox expression within the 3-dimensional marrow environment.  HSCs reside in specific (as yet undefined) niches, and cells progress  toward marrow sinuses and subsequently into the circulation as they mature. The identification of adhesion molecules as transcriptional targets of Hox proteins supports a role of these factors in maintaining 3-dimensioal structure. Further studies are required to address the spatial profile of Hox gene expression in BM (eg. by in situ hybridization).  4.3  Target Genes  The fundamental mechanism of HOXB4 function is modulation of gene expression. Identification of HOXB4 target genes will therefore help determine the ultimate mechanism of //(9A34-mediated HSC expansion. regulated by HOXB4  Some of the genes up- or down-  overexpression are the effectors of HSC self-renewal, so  identification of HOXB4 target genes will help define the biomolecular pathways involved in this poorly understood process. Once identified, these effector molecules  127  could also potentially be used in place of HOXB4 for gene therapy and HSC expansion applications. The /YQAW-mediated ex vivo HSC expansion procedure presented in Chapter 3 generates an actively self-renewing population of HSCs. These cells are thus the ideal cell population for comparisons of gene expression (with non-expanding, controltransduced HSCs). Isolation of these cells from the liquid culture first requires that they be phenotyped however, as we cannot assume them to have the same surface phenotype as normal HSCs. Once phenotyped, populations of //OAB^-transduced HSCs (or even single cells) can be purified by FACS, and RNA can be extracted for analysis of gene expression. Differential expression of candidate genes in 7/OATi^-transduced and control HSCs could be assessed by methods such as Northern blot, RNase protection, or real-time (quantitative) RT-PCR.  Candidate genes that should be examined include cell cycle  regulators (eg. p21, cyclins), apoptotic regulators (eg. Bcl-2) and adhesion molecules (eg. I-CAM, VLAs), as discussed above. Also, expression of other Hox genes should be examined, as Hox proteins commonly cross-regulate each other.  Developmental  regulatory molecules such as Notch (Varnum-Finney et al, 2000), Wnt (Austin et al, 1997) and Shh (Bhardwaj et al, 2001) are emerging as HSC regulators, and it will be important to determine whether their expression is controlled by HOXB4. Shh has been shown to activate expression of certain 5' located Hox genes (Roberts et al, 1995), which can then activate Shh expression in a positive feedback loop (Knezevic et al, 1997). Further connections are mediated by Bmp-4, whose expression is also activated by Shh (Roberts et al, 1995) and whose downstream effector Smadl can interact with and alter 128  the activity of Hoxc-8 (Shi et al, 1999). The expression levels of these genes in HOXB4transduced HSCs will help elucidate these pathways and their importance for HSC selfrenewal. Global analysis of gene expression in control- and //QAS^-transduced HSCs can be used to identify novel genes that may be involved in self-renewal. Experimental approaches to identify all genes differentially expressed in these two cell types include differential display, serial analysis of gene expression (SAGE) and microarray. DNA microchips are becoming increasingly available and cost-effective, making microarray analysis the ideal method for high-throughput gene expression analysis. There is even a DNA microchip being produced currently which contains an array of HSC-specific murine genes (Phillips et al, 2000). By whatever method, a list of target genes regulated by HOXB4 in HSCs should be assembled, and then compiled with existing databases of HSC-specific genes (eg. the stem cell database at http://stemcell.princeton.edu/). In this way we are establishing a comprehensive record of HSC-specific genes, which can be used to decipher the complex regulatory networks that control HSC fate decisions.  4.4  Stem Cell Plasticity  The ability of a tissue-specific stem cell such as an HSC to change into a nonspecific or alternate tissue-specific stem cell (eg. NSC) is a property known as plasticity. Future experiments should address the role of HOXB4 in both de-differentiation and trans-differentiation plasticity events of HSCs.  129  The HSC expansion mediated by HOXB4 may have been caused in part by recruitment of non-HSCs into an HSC fate.  That is, committed cells engineered to  overexpress HOXB4 may have de-differentiated back into an HSC fate.  This is  particularly evident in the ex vivo setting, where 18-fold HSC expansion occurred in the first 48 hours after exposure to HOXB4-Qncoa\mg  retrovirus, which calculates to 4.2  population doublings or an HSC division rate of 11.4 hours. Furthermore, all CRU detected at the end of the transduction were GFP , so if we suppose 6 hours for retroviral +  entry, integration and expression (Coffin et al, 1997), and a gene transfer efficiency of 30%, we now have 5.9 doublings in 42 hours or an HSC division rate of just 7 hours. This is in contrast to previous data showing that HSCs normally divide approximately once every 12 hours under maximally activating conditions ex vivo (Reddy et al, 1997). Therefore, future experiments should discern whether the ex vivo expansion documented here was due to accelerated cycling of HSCs or to recruitment of cells into an HSC fate. By starting the cultures with approximately one HSC and determining the output HSC clonality (by Southern blotting of BM from transplanted mice) one could assess whether the number of clones increased over time in the cultures. Alternatively, the ability to generate HSCs from an HSC-depleted (eg. lin ) population would also suggest that +  HOXB4 could facilitate de-differentiation of cells to an HSC fate. Another interesting question is whether HOXB4 could increase the potential of HSCs (or other tissue-specific stem cells) to trans-differentiate.  Thefrequencyof  plasticity events is currently quite low (Ferrari et al, 1998; Orlic et al, 2001), which limits the potential utility of alternate-source stem cells for therapeutic purposes. The acquisition of definitive HSC function from primitive cells was recently demonstrated 130  when H0XB4 was overexpressed in YS or ES cells (Kyba et al, 2001). While this may represent realization of latent properties within embryonic HSCs, it could also represent trans-determination from one developmental stage-specific stem cell to another. HOXB4 overexpression might therefore increase the frequency of plasticity events, as measured by the number of donor-type cells regenerated in the alternate organ.  4.5  Therapeutic Applications The results presented in this thesis show great promise for therapeutic applications  of HOXB4. Ex vivo HSC expansion mediated by HOXB4 can potentially be used for gene therapy and BM transplantation applications. As outlined in Chapter 1, HSC numbers currently limit the ability to use CB grafts in adult patients. Also, procedures which require ex vivo culture of hematopoietic cells, including gene therapy and leukemic cell purging of autologous grafts, often result in significant stem cell losses. As HSC number directly controls the efficacy of BM transplantation procedures (Mavroudis et al, 1996; Sierra et al, 2000), the ability to expand these cells ex vivo will be enormously useful. Secondly, the competitive advantage of HOXB4 cells could be exploited for selective engraftment and/or in vivo amplification of gene-modified cells. Gene therapy models are often hampered by poor gene transfer and engraftment efficiencies, thus HOXB4 might help to generate complete reconstitution of an hematopoietic system by gene-corrected cells. One approach toward achieving selective engraftment has been to fuse the extracellular domain of a growth factor receptor (eg. tamoxifen-responsive estrogen binding domain or Epo receptor) to the intracellular domain of a potent  131  mitogenic receptor (eg. G-CSF receptor). By administration of the ligand (ie. tamoxifen or Epo), selective expansion of gene-modified cells can be achieved (Hanazono et al, 2000; Kume et al, 2001; Ueda et al, 2001). However, success with this approach has been limited, as the effect is transient and requires repeated drug administrations. Also, there appears to be some bias in amplification of certain lineages (eg. granulocytic). HOXB4 has the advantage of giving a long-term selective advantage while not requiring drug administration. Most importantly, HOXB4 overexpression does not bias lineage representation. However, further studies into the safety and efficacy of using this factor in the human setting are warranted. Early results indicate that HOXB4 overexpression does give a competitive transplant advantage to human cells (Schiedlmeier et al., 2001), although its effect on human HSC expansion ex vivo has not been quantitatively assessed. Studies into the safety of using HOXB4 on human cells are also essential. Although HOXB4 overexpression has never led to leukemias in mice, it did transform RAT-1 fibroblasts (Krosl et al,  1998), and other Hox gene alterations have been associated with  leukemogenesis (eg. HOXA9; Kroon et al, 1998). Transient transfer of HOXB4 could be achieved either through an inducible system (eg. tetracyclin-inducible; Kyba et al, 2001), or by transfer of the protein (eg. as an HIV TAT fusion; Nagahara et al, 1998). Modifications such as these might allow more effective and/or safe utilization of the HOXB4 advantages.  132  CHAPTER 5  BIBLIOGRAPHY  Abramson, S., Miller, R. G. and Phillips, R. A. 1977. 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