"Medicine, Faculty of"@en . "Medical Genetics, Department of"@en . "DSpace"@en . "UBCV"@en . "Bowie, Michelle Beatrice"@en . "2010-01-16T20:43:35Z"@en . "2006"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "To characterize the extent and timing of changes in hematopoietic stem cell (HSC) properties during ontogeny, experimental strategies were developed to allow quantitative assessment of their proliferative activity, self-renewal potential and differentiation behaviour in vivo. All HSCs in the fetal liver [i.e. foetal liver] were found to be cycling and following their transplantation into irradiated adult hosts, they rapidly generated daughter HSCs and produced large numbers of granulopoietic progeny. In contrast, adult HSCs, which are predominantly quiescent, regenerated new HSCs more slowly and produced fewer granulopoietic progeny. They also showed a coordinated change in expression of several transcription factors that regulate HSC functions. Interestingly, HSCs retained a fetal phenotype with respect to all these features until 3 weeks after birth and then, within one week, acquired an adult HSC phenotype. Additional studies of serially transplanted HSCs indicated that this switch also took place within the same time frame in adult mice reconstituted with fetal or 3-week post-natal HSCs, suggesting the switch is intrinsically programmed. To further investigate the mechanism of this switch, an in vitro model suitable for monitoring the survival, proliferation and self-renewal activity of highly purified fetal liver HSCs was developed. Using this model, I found that the cell cycle transit time of optimally stimulated fetal HSCs and adult HSCs is the same, but with lower Steel factor requirements for fetal HSCs. This suggested that the fetal-to-adult switch involves a decreased response to c-Kit activation. Interestingly, the self-renewal behaviour of fetal HSCs expressing a defective form of c-Kit mimicked adult +/+ HSCs, both in vitro and in vivo, but showed no difference in cycling activity, suggesting that Steel factor responsiveness specifically regulates HSC self-renewal responsiveness in vivo. Future studies of changes in gene expression during the switch, including analyses of c-Kit-defective HSCs as well as normal HSCs, may help to link the observed changes in Steel factor responsiveness to the molecular mechanisms that control changes in HSC self-renewal and cycling control during ontogeny."@en . "https://circle.library.ubc.ca/rest/handle/2429/18465?expand=metadata"@en . "A N A L Y S I S OF D E V E L O P M E N T A L L Y P R O G R A M M E D C H A N G E S IN H E M A T O P O I E T I C S T E M C E L L S by M I C H E L L E B E A T R I C E B O W I E B . S c , Queen's University, 2001 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF D O C T O R OF P H I L O S O P H Y in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Genetics) T H E U N I V E R S I T Y OF B R I S T I S H C O L U M B I A July 2006 \u00C2\u00A9 Michelle Beatrice Bowie, 2006 ABSTRACT To characterize the extent and timing of changes in hematopoietic stem cell (HSC) properties during ontogeny, experimental strategies were developed to allow quantitative assessment of their proliferative activity, self-renewal potential and differentiation behaviour in vivo. A l l H S C s in the fetal liver were found to be cycling and following their transplantation into irradiated adult hosts, they rapidly generated daughter HSCs and produced large numbers of granulopoietic progeny. In contrast, adult HSCs , which are predominantly quiescent, regenerated new H S C s more slowly and produced fewer granulopoietic progeny. They also showed a coordinated change in expression of several transcription factors that regulate H S C functions. Interestingly, H S C s retained a fetal phenotype with respect to all these features until 3 weeks after birth and then, within one week, acquired an adult H S C phenotype. Additional studies of serially transplanted H S C s indicated that this switch also took place within the same time frame in adult mice reconstituted with fetal or 3-week post-natal HSCs , suggesting the switch is intrinsically programmed. To further investigate the mechanism of this switch, an in vitro model suitable for monitoring the survival, proliferation and self-renewal activity of highly purified fetal liver H S C s was developed. Using this model, I found that the cell cycle transit time of optimally stimulated fetal H S C s and adult H S C s is the same, but with lower Steel factor requirements for fetal HSCs . This suggested that the fetal-to-adult switch involves a decreased response to c-Kit activation. Interestingly, the self-renewal behaviour of fetal H S C s expressing a defective form of c-Kit mimicked adult +/+ HSCs , both in vitro and in vivo, but showed no difference in cycling activity, suggesting that Steel factor responsiveness specifically regulates H S C self-renewal responsiveness in vivo. Future studies of changes in gene expression during i i the switch, including analyses of c-Kit-defective HSCs as well as normal HSCs , may help to link the observed changes in Steel factor responsiveness to the molecular mechanisms that control changes in H S C self-renewal and cycling control during ontogeny. i i i TABLE OF CONTENTS Abstract i i Table of Contents iv List o f Tables v i i List o f Figures v i i i List o f Abbreviations x Acknowledgements x i i CHAPTER 1 I N T R O D U C T I O N 1 1.1 General overview of hematopoiesis 1 1.1.1 Definition and identification of HSCs 1 1.1.2 The hierarchical organization of hematopoiesis 3 1.2 Development of hematopoiesis 6 1.2.1 Generation of hematopoietic cells in the mouse embryo 6 1.2.2 Generation of hematopoietic cells from murine ES cells in vitro 9 1.3 Differences between fetal and adult hematopoiesis 10 1.3.1 Mature cells 10 1.3.2 H S C s 11 1.3.2.1 Kinetic properties 11 1.3.2.2 Phenotypic properties 12 1.3.2.3 Homing and mobilization 13 1.3.2.4 Self-renewal 16 1.3.2.5 Growth factor responsiveness 18 iv 1.4 Genes that regulate HSCs 19 1.4.1 Genes critical to both fetal and adult HSCs 19 1.4.2 Genes more critical to fetal than adult HSCs 20 1.4.2.1 Rural 20 1.4.2.2 Notch 22 1.4.2.3 Sci 23 1.4.3 Genes more critical to adult than fetal HSCs 25 1.4.3.1 Bmi-1 25 1.4.3.2 c-Kit 27 1.4.3.3 Gfi-1 28 1.4.3.4 Tie2 29 1.5 Experimental objectives 30 CHAPTER 2 HEMATOPOIETIC STEM CELLS PROLIFERATE UNTIL AFTER BIRTH AND SHOW A REVERSIBLE PHASE-SPECIFIC ENGRAFTMENT DEFECT 80 CHAPTER 3 FETAL-TO-ADULT CHANGE IN HEMATOPOIETIC STEM CELL SELF-RENEWAL PROPERTIES OCCURS AS AN ABRUPT AND PROGRAMMED POSTNATAL EVENT 120 CHAPTER 4 STEEL FACTOR P L A Y S A DISTINCT ROLE IN THE SELF-RENEWAL PROPERTIES OF HEMATOPOIETIC STEM CELLS F R O M DIFFERENT STAGES OF D E V E L O P M E N T 153 CHAPTER 5 CONCLUSION A N D RECOMMENDATION FOR FURTHER W O R K 172 vi LIST OF TABLES Tables in Chapter 1 Table 1.1: Phenotypic comparison between fetal liver and adult bone marrow H S C s 36 Tables in Chapter 2 Table 2.1: Limit ing dilution data for C R U frequency determinations for E l 8.5 fetal liver and bone marrow cells 106 Table 2.2: Limit ing dilution data for C R U frequency determinations for lin\" bone marrow cells from 3 and 4 week-old mice 107 Tables in Chapter 3 Table 3.1: C R U frequency determinations for purified (1/10) single fetal liver HSC-derived clones under two conditions (pooled data from 2 experiments) 143 v i i L I S T O F F I G U R E S Figures i n Chapter 1 Figure 1.1: Hematopoiesis 33 Figure 1.2: Competitive repopulating unit (CRU) assay 34 Figure 1.3: Limit ing dilution analysis ( L D A ) 35 Figures in Chapter 2 Figure 2.1: A l l fetal H S C s are sensitive to cell cycle-specific drugs 99 Figure 2.2: F A C S profiles o f the distribution of different lin\" populations in Go, G i and S / G 2 / M . . .100 Figure 2.3: The cycling activity of C R U s is down-regulated between 3 and 4 weeks of age 101 Figure 2.4: Hoechst/Pyronin-sorted HSCs display an absolute but transient S/G2/M engraftment defect 102 Figure 2.5: The engraftment defect of H S C s in S / G 2 / M is corrected by treatment of the host, but not the cells, with SDF-1G2 103 Figure 2.6: Donor-derived repopulation of SDF-lG2-treated mice 104 Figure 2.7: Gene expression analysis of the G i and S / G 2 / M subsets of highly purified lin\" S c a l + C D 4 3 + M a c l + HSCs from fetal liver and 3-week bone marrow 105 v i i i Figures in Chapter 3 Figure 3.1: Fetal liver H S C s self-renew to a greater extent than bone marrow H S C s in a transplant 137 Figure 3.2: H S C s switch from fetal liver-like self-renewal to adult bone marrow-like self-renewal abruptly and intrinsically 138 Figure 3.3: Fetal liver-like and adult bone marrow-like HSCs generate distinct P B lineage contribution patterns when transplanted into irradiated recipients 139 Figure 3.4: Purification and culture of fetal liver HSCs reveals a similar cell cycle length as adult bone marrow H S C s 140 Figure 3.5: New H S C developmental switch discovered 141 Figure 3.6: Gene expression analysis of purified fetal liver, 3-wk, 4-wk and 10-wk-old bone marrow H S C s 142 Figures in Chapter 4 Figure 4.1: Comparison of the effects of different growth factor cocktails on fetal liver C R U self-maintenance in vitro 163 Figure 4.2: Steel factor dose response curves for the in vitro self-renewal of+/+ and W41 C R U s from fetal liver and adult bone marrow 164 Figure 4.3: Comparison o f c-Kit expression on fetal liver and adult bone marrow H S C s 165 Figure 4.4: Cel l cycle analysis of W41 fetal liver HSCs 166 Figure 4.5: W41 fetal liver HSCs self-renew at the adult bone marrow H S C self-renewal rate 167 ix LIST O F ABBREVIATIONS 3 H-Tdr - tritiated-thymidine 5 - F U - 5-fluorouracil a4int - a4-integrin A G M - aorta-gonado-mesonephros A M L - acute myeloid leukemia A n g - Angiopoietin A P C - allophycocyanin A T F - activating transcription factor A T M - ataxia-telangiectasia mutated BIT - bovine serum albumin, insulin and transferrin B F U - E - burst forming unit -erythroid B M - bone marrow B m i - B cell-specific Moloney murine leukemia virus integration site C A F C - cobblestone area forming cell C b f - core-binding factor C D K - cyclin-dependent kinase C L P - common lymphoid progenitor C F C - colony-forming cell C F U - S - colony-forming unit-spleen Cgy - centigray C M P - common myeloid progenitor C R U - competitive repopulating unit C S F - colony stimulating factor D N A - deoxyribonucleic acid E - embryonic day E B - embryoid bodies ES - embryonic stem F A C S - fluorescence activated cell sorter F ITC - fluorescein isothiocyanate F L - fetal liver G - g a p Gapdh - glyceraldehyde-3-phosphate dehydrogenase G E M M - granulocytes, erythroblasts, megakaryocytes and macrophages Gf i - growth factor independent G M P - granulocytes and monocytes progenitors H A - hyaluronic acid H S C - hematopoietic stem cell HF /2 - Hanks balanced salt solution containing 2% F C S H S C - hematopoietic stem cell Hst - Hoechst 33342 IGF-2 - insulin-like growth factor 2 IL - interleukin LIF - leukemia inhibitory factor L T C - I C - long-term culture-initiating cell M - mitosis M E M - minimum essential medium M P D - myeloproliferative disorders m R N A - messenger ribonucleic acid N O D / S C I D - nonobese diabetic/severe combined immunodeficient N K - natural killer O P N - osteopontin P B S - phosphate buffered saline P E - phycoerythrin p.c. - post coitus P c G - polycomb group PI - propidium iodide PI3K - phosphatidylinositol 3'-kinase Pias - protein inhibitor of activated Stat3 P L C - phospholipase C Pre - polycomb repressive complex P-Sp - paraaortic splanchnopleure Py - pyronin Y Rho - Rhodamine 123 R N A - ribonucleic acid R T K - receptor tyrosine kinase Runx - runt-related transcription factor S - synthesis SCF - stem cell factor SDF-1 - stromal cell-derived factor-1 SF - Steel factor S F M - serum-free medium SP - side population S T A T - signal transducer and activator of transcription T F - transcription factor T P O - thrombopoietin W B C - white blood cell W G A - wheat germ agglutinin x i A C K N O W L E D G M E N T S I would like to acknowledge expert technical assistance from Gayle Thornbury and Lindsey Laycock o f the Flow Cytometry Facility of the Terry Fox Laboratory and the staff of the Animal Resource Centre of the B C Cancer Research Centre. Thank you to Debra Wytrykush for her secretarial assistance in the preparation of this thesis and the derived manuscripts. I was the recipient of a Stem Cel l Network Studentship and a Studentship funded by the Canadian Institute of Health Research and the Michael Smith Foundation for Health Research. This work was also supported by grants from the National Cancer Institute of Canada (with funds from the Terry Fox Foundation), the Stem Cel l Network and P01 HL-55435 from the N H L B I / N I H . Thank you to my committee members, Robert Kay, Pamela Hoodless and Hugh Brock for their assistance with this Thesis. Particularly, thank you to my supervisor, Connie Eaves. x i i CHAPTER 1 INTRODUCTION 1.1 General overview of hematopoiesis Hematopoiesis refers to the process of blood cell production, which occurs throughout life and in the mouse involves the regulated output every day of millions of cells of many different types. These include neutrophils, eosinophils, basophils/mast cells, monocytes/macrophages, erythrocytes, megakaryocytes, platelets, B-lineage cells, T-lineage cells and natural killer (NK) cells for long periods of time (see Figure 1.1). The production of these cells involves the execution of a complex series of molecular changes within more primitive hematopoietic precursors. In addition, these changes have to be co-ordinated with mechanisms that regulate cell proliferation and survival to ensure that the final numbers of new blood cells produced meet physiological demands both during and after normal growth and in response to injury. Understanding the process of hematopoiesis thus requires a description of the complex cellular and molecular mechanisms that allow hematopoietic cells to differentiate into different blood cell types, the regulation of the changes that occur, as well as the elucidation of how these mechanisms interface with signals from the environment. 1.1.1 Definition and identification of HSCs Blood cell production is sustained throughout life by the proliferative activity of a small population of pluripotent cells with long-term self-renewal potential, referred to as hematopoietic stem cells (HSCs). Initial evidence for the existence in adult hematopoietic tissue of cells with these two properties; namely, pluripotency and self-renewal ability (i.e. the ability to divide without restriction or activation of this pluripotentiality) was provided by 1 morphological studies of the cells present in the bone marrow of patients with myeloproliferative disorders (MPD). These revealed that, although only a single type of mature blood cell was elevated in the circulation, precursors of the other lineages were also often found at high levels in the bone marrow which led to the suggestion that these disorders all originate in a common pluripotent precursor (1). The first direct support for this concept came from experiments that showed individual transplant-derived spleen colonies containing multiple lineages of myeloid cells carried the same unique cytogenetic marker, inferring the derivation of each from a single pluripotent cell, the so-called colony-forming unit-spleen (CFU-S) (2). Shortly thereafter, clonal marking of longer term transplants in mice provided definitive evidence of a single adult bone marrow cell with lympho-myeloid differentiation potential that could be serially transplanted (3-6). With the discovery in the 1980s that most CFU-S have, at best, limited self-renewal activity (7-9) and that most CFU-S can be separated prospectively from cells with long-term repopulating activity (7), quantitative transplant-based assays were devised with greater specificity for murine HSCs defined by their long-term lympho-myeloid repopulating activity (10). These assays now typically make use of congenic donor-recipient pairs of mice, that express distinct CD45 epitopes referred to as Ly5.1 and Ly5.2 to enable definitive identification of donor-derived cells among the lymphoid and myeloid cells regenerated in individual irradiated recipients. To allow the frequency of HSCs to be determined in a given \"test\" cell suspension, the test cells are injected in varying numbers until a limiting dose is achieved - i.e., a transplant dose that causes only a fraction of the mice injected to show continued test-cell derived contributions to their lymphoid and myeloid blood cells long term, i.e., for at least 4 months (10). To enable the \"negative\" mice to survive, all recipients must be independently protected by a minimal dose of HSCs. These can either be co-transplanted 2 from a donor o f the same genotype as the recipient or can be provided endogenously; for example, using sublethally irradiated recipients with genetically compromised HSCs (11). HSCs detected using this methodology are called competitive repopulating units (CRUs) (Figure 1.2) and their frequencies are derived by limiting dilution analysis of the proportion o f negative recipients obtained in a given set of experiments (Figure 1.3) (10). C R U s thus defined constitute a very small percentage (~.01%) of the cells in the bone marrow of normal adult mice (12; 13). The validity of this approach is based on the assumption that the donor-derived lymphoid and myeloid progeny originated from a single common pluripotent donor cell that was likely to have undergone extensive self-renewal, as indicated by early retroviral marking experiments demonstrating the generation o f clonal populations o f multilineage cells containing the same common inserts (3-5). Additional evidence that C R U constitute a biologically discrete population have come from more recent experiments showing that C R U s can be purified to near homogeneity using several phenotype-based cell strategies. These have allowed both their pluripotent and self-renewal properties to be demonstrated by tracking the progeny produced in recipients of single cell transplants (14-19). It has also been possible to use cell separation approaches to demonstrate that C R U can be prospectively separated from transplantable pluripotent cells with less durable repopulating ability (17;20-22). 1.1.2. The hierarchical organization of hematopoiesis The process by which H S C eventually give rise to mature blood cells proceeds as an extensive series of progressive changes spanning multiple cell generations. This is the basis of the creation of a developmental hierarchy in which different intermediate cell types can now be distinguished by a variety o f robust, quantitative assays that detect differences in the 3 proliferation and differentiation capabilities o f these cells in vitro or i n vivo. Two important features o f these assays is their ability to measure the number (rather than just the activity) o f the input cell type being evaluated and the fact that there is a range of cell doses over which the output is linearly related to input. Nevertheless they all have the disadvantage that they detect cells retrospectively by virtue o f the number and type of progeny produced under defined (optimized) conditions and after a specified length of time. A s a result, they are slow, labour-intensive and are generally less precise than direct detection methods. The long-term culture-initiating cell (LTC-IC) assay and the related cobblestone area forming cell ( C A F C ) assay are in vitro methods that can detect a cell population that overlaps with C R U s (15;23). These exploit the ability of H S C s and their immediate progeny to be maintained and to proliferate to varying degrees exclusively when cocultured in contact with stromal feeder layers. The principle o f these assays is to extend the time o f culture to the point that it can be safely assumed that the cells being produced must have derived from H S C s in the original input. This assumption has not been rigorously examined, but correlative studies indicate that the detection of myeloid colony-forming cell (CFC) progenitors (or C A ' s ) beyond 4-5 weeks depends on initiating the cultures with cells that share features with H S C s and that can support their expansion (15;24;25). Many types o f more differentiated cells can be characterized by their ability to form colonies o f mature progeny in semi-solid medium containing specific growth factors, hence the term C F C . This type of methodology can detect pluripotent cells that generate colonies containing granulocytes, erythroblasts, megakaryocytes and macrophages ( C F C - G E M M ) (26;27) as wel l as different types of lineage-restricted C F C s . Cel ls that generate only granulocytes and macrophages are termed C F C - G M (28;29) and similar assays and nomenclature exist for cells that generate small or large colonies containing erythroid 4 progeny ( C F C - E and B F U - E , respectively) (30) or megakaryocytes ( C F C - M k and B F U -M k , respectively) (31). More recently, the expanded availability o f monoclonal antibodies and improved machines for multi-parameter sorting of viable cells in a sterile fashion has led to the identification o f surface profile phenotypes that can be used to directly identify cells at different stages of hematopoietic differentiation in defined tissues. However, it should be noted that most of the markers used to establish these profiles do not show fidelity in their expression when the cells are stimulated. The use of phenotypic markers is desirable because of its speed and immediacy, but also has greater limitations of sensitivity. The first differentiation steps that H S C s undergo usually lead to a loss of either lymphoid or myeloid potential resulting in the generation of either a common lymphoid progenitor ( C L P ) (32) or a common myeloid progenitor ( C M P ) (33). C L P s are characterized as L i n ~ I L - 7 R + T h y - l ~ S c a - l l o c - K i t l 0 cells in adult mouse bone marrow and these markers identify cells that can produce T, B and N K cells, but not myeloid cells. Conversely, C M P s are FcRy'\u00C2\u00B0CD34 + cells and can generate megakaryocytes, eryrthrocytes, granulocytes and macrophages, but not lymphoid cells. C M P s probably overlap with C F U - S and C F C - G E M M . Later cell types that are restricted to the production of megakaryocytes and erythrocytes only (MEPs; functionally identified as B F U - E ) or to the production of granulocytes and monocytes progenitors (GMPs ; functionally as C F U - G M s ) can also be prospectively isolated by cell surface phenotype from both the bone marrow and the fetal liver (34). However, while this described hematopoietic hierarchy is applicable to the majority, it is not necessarily always the case (35). Examples of such rare cell types were first seen when single cells were plated in culture; the colonies generated contained diverse combinations of cell types not predicted from a rigid hierarchy (36). There are now a number of cells that do not follow the standard 5 hematopoietic hierarchy briefly outlined in Figure 1.1, such as lympho-myeloid cells that lack erytho-megakaryocytic potential (37) or a mature cell type with the potential to generate both B cells and macrophages (38). 1.2 Development of hematopoiesis 1.2.1. Generation of hematopoietic cells in the mouse embryo The first cells with transplantable H S C activity appear in the mouse embryo in the yolk-sac (39;40) and aorta-gonado-mesonephrous ( A G M ) regions (41) on embryonic days (E) 9 and 10 of gestation, respectively. Since these cells have direct access to the circulation, it is thought that they are the origin of the HSCs that appear in the fetal liver on E l 1. The frequency of H S C s (CRUs) reaches a peak in the fetal liver on E l 4.5 with maintenance of their numbers until day 16 (42) after which these numbers steadily decline, possibly due to an exodus o f H S C to the spleen and bone marrow (43). This redistribution of HSCs into the spleen and bone marrow occurs over a period of several days. HSCs are first detected in the fetal spleen on E13 (44), which then increases over the next 2-3 days (E15.5-16.5), and on E17.5, HSCs are first detected in the fetal bone marrow (45). After birth, H S C numbers continue to decline i n the liver and spleen but increase in the bone marrow where H S C s then persist for the lifetime of the mouse. This shift in the tissue location of hematopoiesis appears to be an evolutionarily conserved process and, while the tissues involved differ, it can be seen in organisms as primitive as drosophila and as complex as humans (46). The murine yolk-sac is a splanchnopleure, which means that it is composed of 2 layers o f tissue, extraembryonic mesodermal cells and visceral endoderm cells. This structure develops between E7 and E7.5 (47;48) and is the first site of blood cell production. The inner layer appears to be the origin of primitive macrophages and erythroblasts and endothelial cells 6 can be seen to arise from the outer layer (49). The simultaneous appearance of erythroblasts and endothelial cells that share expression of F lk -1 , CD34, Tie-2, G A T A - 2 , L M O - 2 and S C L has suggested that these 2 lineages arise from a common hemangioblast precursor within the developing yolk-sac (50). The E8 para-aortic splanchnopleure (P-Sp)/aorta-gonad-mesonephros ( A G M ) region is another important and independent site of hematopoietic cell genesis in mammalian embryos. In mice, this is the first intra-embryonic site in which C F U - S can be detected between from E l 0 until E l l . After this time, the fetal liver takes over as the major hematopoietic tissue (41). It appears that the A G M is not conducive to the generation of H S C s that can be detected by transplantation assays into adult recipients and only after cells from the A G M have further developed in the fetal liver can transplantable H S C s be detected (44). The fetal liver begins to form on E9 and, at that time, the only hematopoietic cells it contains appear to be committed myeloid precursors (51-53). However, by E l 1, the fetal liver contains both C F U - S and C R U s (13;54;55). These cells are thought to be derived from colonizing cells that originated in the yolk-sac and/or A G M regions rather than representing the progeny o f mesenchymal precursors that are also present in the fetal liver (56). The fetal liver remains the major site o f hematopoiesis from E12 until birth (57). The development of bone marrow begins in the fetus with the formation of a cellular matrix in the bone cavities, followed by their population by an influx of HSCs , most likely emanating from the fetal liver and released into the circulation, starting around E l 7 . Bones that are hematopoietically active are characterized by a particular type of cellular matrix, produced by mesenchymal precursors that migrate into the marrow cavity to generate the stroma of the bone marrow and induce the migration of H S C into these sites (58). This model is supported by the finding that primitive hematopoietic cells (CFU-S) that are genetically 7 unable to produce stromal derived factor-1 (SDF-1) or its receptor ( C X C R 4 ) fail to colonize the bone marrow of fetal mice in spite of apparently normal hematopoiesis in the fetal liver. However, it is important to note that most of these data are limited to studies of C F U - S (45) and comprehensive quantitative data on the frequency and rate o f expansion o f H S C s in the fetal bone marrow have not been reported. Recently, the murine placenta has also been found to contain H S C activity, with a peak of activity between E12 and E l 4 , followed by a rapid drop in stem cell frequency up to E l 5.5, as the liver expands (59). The large number of H S C s found in the placenta raises the question as to whether this is a site of independent stem cell generation or if, similar to the fetal liver, it is a transient niche supporting H S C expansion. A significant feature o f the development of the hematopoietic system in the embryo is the apparent delayed appearance o f cells with the properties of HSCs . In fact, the first recognizable hematopoietic cells that arise directly from mesenchymal derivatives are terminally differentiated cells which do not appear to be derived from cells with extensive pluripotent or proliferative ability. This stands in marked contrast to the conventionally recognized hierarchy of hematopoietic cell types present in the adult (described above). These findings have led to the concept that different molecular programs may exist to regulate the generation o f hematopoietic cells at different stages of ontogeny (60). It could therefore be informative to characterize more fully the differences that exist between fetal and adult HSCs in order to elucidate the mechanisms by which one evolves from the other. Interestingly, a recent study provided evidence suggesting that all HSCs in the adult are derived from cells detectable as H S C s in the embryo by using the stem cell leukemia (SCL) locus to direct the expression o f a tamoxifen inducible Cre recombinase in hematopoietic and endothelial cells (61). They showed that the progeny of S C L expressing 8 cells in the E l 0-11 embryo contribute to the bulk o f HSCs in both the fetal liver and adult bone marrow. However, as not all o f the cells in the fetus and adult were marked, some de novo generation of H S C s in these tissues can not be ruled out (62). 1.2.2. Generation of hematopoietic cells from murine ES cells in vitro Embryonic stem (ES) cells are derived from the inner cell mass of the embryonic blastocysts and are capable of differentiating into all three primary germ layers of the embryo (endoderm, mesoderm and ectoderm) and therefore to all cell types found in an adult (63). These cells propagate extensively and can be maintained indefinitely ex vivo in an undifferentiated state in the presence of L I F (64). These cells can be genetically modified and reintroduced into blastocysts to allow the identification o f critical roles played by genes in all tissue types. To assess the role of a gene at a later stage of development or in a particular tissue specifically, conditional knock-outs can be generated, where the gene is essentially shut-down under the control of a tissue-specific promoter or upon the addition of an exogenous signal at a chosen time during development (65;66). The generation of the hematopoietic lineage from ES cells shows similar kinetics in vitro to that seen during normal embryonic development and proceeds through similar intermediates, from hemangioblasts to the long-term and short-term repopulating hematopoietic cells and to committed progenitors of the myeloid and lymphoid lineages, which has allowed ES cells to be used to derive various hematopoietic lineages (67-73). 9 1.3 Differences between fetal and adult hematopoiesis 1.3.1 Mature cells Many features of terminally differentiating myeloid cells remain the same throughout ontogeny and, as noted above, appear to be produced by cells undergoing a similar sequence of changes at the molecular level. Nevertheless, some differences are known, the most notable examples being the types of haemoglobins produced by erythroblasts in fetal and adult mice (74) and the speed with which these cells transit the cell cycle (75;76). Human fetal progenitors also demonstrate a faster growth rate than their adult bone marrow counterparts, as demonstrated by the speed and size o f colonies they generate in vitro (76). In addition, cell tracking studies have shown directly that human fetal liver-derived C D 3 4 + cells divide in vitro with a shorter cell cycle time than do those derived from cord blood (77). In the case of lymphoid cells, differences in the programs of maturing T-cells, B-cells and N K cells have also been described. These result in differences in the T-cell receptor gene rearrangements exhibited by fetal and adult T-cells (78), differences in the surface marker profiles of fetal and adult B-lineage cells (79) and differences in the receptors expressed by fetal and adult N K cells. In addition, there appears to be marked changes in the growth factor requirements of fetal and adult lymphoid cells (80). For example, interleukin-7 is critical to T-and B-cell development, as demonstrated by mice with a homozygous null mutation of the IL-7 gene (81). However, these same mice have detectable B lymphopoiesis in the fetus that is not seen in the adult, indicating the operation of compensatory mechanisms that can support fetal B-cell differentiation that are either absent or not effective in the adult (82). 10 1.3.2 HSCs A number o f differences in the way more primitive hematopoietic cells may differentiate are also well documented. Summarized below are the differences in kinetic, phenotypic, homing, self-renewal and growth factor responsiveness properties of fetal and adult HSCs . 1.3.2.1 Kinetic properties Transplants o f murine fetal liver cells ( E l 4-18) repopulate the bone marrow and spleen o f irradiated recipients faster than do transplants of adult bone marrow cells (54) with a 16-hour vs. a 25-hour doubling time of the cell populations produced in the bone marrow in the first 3 weeks post-transplant (75). These classical experiments clearly point to differences in parameters that regulate the output of cells from hematopoietic cells with repopulating activity but do not discriminate between differences in H S C self-renewal potential, cell cycle transit times and or possible differences in rates o f cell loss due to cell death. Early studies also showed that primitive cells in the fetal liver and adult bone marrow differ in their proliferative activities, as indicated by the proportion that these cells are in cycle. For example, comparison of the cycling status of C F U - S from fetal liver and adult bone marrow revealed that those from fetal liver are mostly in cycle, whereas those from adult bone marrow are mostly quiescent (54). Subsequent studies confirmed that these differences extend to the H S C compartment with >80% of adult bone marrow HSCs being resistant to in vivo treatment with 5-fluorouracil (5-FU), a cytotoxic agent, and 40% of the cells in a subset of fetal liver cells that contains the HSCs being found to be in the S/G2/M phase of the cell cycle (83;84).' A number o f genes alter the cycling characteristics of H S C , including p21, p27, p i 8, cycl inD (85-90) and Gfi-1 (91) . Various genes appear to regulate the proliferation of fetal 11 liver and adult bone marrow H S C differently. A s discussed below in detail, these include genes for Steel factor (SF), c-Kit (92-94) and Tie-2 (95) that control H S C interactions with their microenvironment, as well as a number of intrinsic signaling molecules and transcription factors that appear to be involved in regulating H S C function (see Section 1.4). 1.3.2.2 Phenotypic properties Fetal and adult H S C s have also been found to display a different spectrum o f markers on their cell surface and to exhibit differences in their dye efflux and adhesion properties (Table 1.1). Notable examples include a poor ability of fetal liver HSCs to adhere to fibronectin and a positive expression o f C l q R p (96), a C-type lectin-like type I transmembrane protein recognized by the monoclonal antibody A A 4 . 1 (97). Fetal liver HSCs also express readily detectable levels of CD1 l b (Mac l ) , an integrin component that is commonly found on mature macrophages from all developmental stages, but not on adult bone marrow H S C s (43;98). The demonstration that Mac-1 is expressed on fetal liver H S C s was one o f the first indications o f the lack of differentiation stage-specificity of many phenotypic markers. This highlights their inherent unreliability as surrogate direct indicators of HSCs and, in particular, the problems caused by the indiscriminate use of standard lineage-negative antibody cocktails to obtain enriched populations of H S C s from manipulated cell suspensions (96;99;100). Rebel et al. (1996) avoided this, and carefully characterized the phenotypic similarities between fetal and adult stem cells as being the expression of Sca-1, c-Kit , M H C class I and CD43 antigens, as well as high binding to wheat germ agglutinin (WGA+) and the lack of expression of B220, Gr-1, L y - 1 , Thy-1, CD71 and Fall-3. A s differences, they found that fetal liver H S C s stain positively with monoclonal antibodies against A A 4 . 1 , Mac-1 and C D 4 5 R B and fetal liver also retain Rhodamine 123 (are Rho l23 + ) whereas adult stem cells do 12 not (98). In mice, CD34 and CD38 are also developmental^ regulated H S C cell surface markers. Murine fetal liver H S C s are CD34 + CD38\" whereas adult bone marrow HSCs are CD34\"CD38 + (101-104). The receptor tyrosine kinase Tie-2 may also be differentially expressed by H S C s during ontogeny, with reports suggesting that murine fetal liver H S C s express Tie-2 but adult H S C s do not (105-107). However, the Tie-2 antibody is not yet a reliable material and may prove to label both adult and fetal HSCs . H S C s from the fetal liver also do not efflux Hoechst 33342 and are thus not detectable within the gate used to identify adult H S C s as side population (SP) cells using fluorescent activated cell sorting ( F A C S ) (108). Wi th a few exceptions (e.g. c -Ki t (92)), most developmentally regulated cell surface markers expressed on H S C s change in adult H S C s as a function of an alteration in their proliferative activity and/or cytokine activation. Therefore, it seems likely that a similar explanation might apply to the phenotypic differences exhibited by fetal liver HSCs . For example, 5 -FU treatment of mice leading to activation of their HSCs in vivo (109) and/or direct activation of adult H S C s in vitro with cytokines causes the CD34\", C D 3 8 + , Mac-1\", SP, Rho'\u00C2\u00B0 phenotype of quiescent HSCs to acquire a C D 3 4 + , CD38\", M a c - 1 + , non-SP, Rho + phenotype, identical to that characteristic o f fetal liver H S C (102;104;108;110-112). A l so noteworthy is the lack of consistency in the phenotypic profiles of murine and human HSCs . For example, human fetal liver HSCs are C D 3 4 + (113), sharing the murine CD34 expression pattern of fetal liver HSCs , but bearing the SP phenotype of adult bone marrow HSCs. 1.3.2.3 Homing and mobilization Another important property of HSCs is their motility and ability to migrate in response to specific chemoattractants. H S C s migrate from one site to another throughout life; i.e., from the fetal liver to the fetal spleen and bone marrow in the embryo and later a small percentage 13 of HSCs are constantly found in the peripheral circulation (114). This property underlies the ability of H S C s from both mice and humans to be transplanted intravenously. The mechanisms that attract and retain HSCs in the bone marrow, as well as those that allow their release, are still not fully understood although a number of molecular ligand-receptor pairs controlling H S C adhesion and migration have been identified. V L A - 4 ((X4P1 integrin) expressed on H S C s and its receptor V C A M - 1 on bone marrow stromal cells are critical mediators o f adhesion and mobilization, as shown by the fact that treatment o f mice with antibodies to V L A - 4 and V C A M - 1 results in H S C mobilization. This appears to involve signaling through the c-Kit pathway, with both c-Kit and V L A - 4 being downregulated on H S C s when they are mobilized (115-125). Most recently, the 014 integrin component of V L A - 4 has been shown to be required for the competitive function and self-renewal of H S C s in vivo due to their reduced retention within the niche in its absence (126). C - K i t is also thought to play a direct role in H S C adhesion to the endosteal bone, but not necessarily in their migration (127). CD44 is another adhesion molecule expressed by HSCs (128). CD44 has many ligands one of which is hyaluronic acid ( H A ) , the major component of the E C M in the bone marrow (129) and E-selectin, which is expressed by endothelial cells (130). Some reports have suggested that antibodies to CD44 could affect the homing of H S C s (131 ;132), yet CD44\" / _ mice are healthy and fertile with normal numbers o f in vivo-homing H S C s (133). Nevertheless, in vitro, the SDF-1 -mediated induction of human progenitor cells to migrate on H A was associated with their assumption of a polarized morphology and the formation of pseudopodia at the leading edges o f which C D 4 4 was concentrated (132). Interestingly, high levels of CD44 expression on tumour cells mediates their metastatic spread (134). 14 Matrix-bound cytokines, such as Steel Factor (SF) and Flt3-ligand (135-137) may also serve as adhesion molecules for their respective receptors (c-Kit, the receptor for SF, and Flt-3, the receptor for Flt3-ligand), both of which are expressed on the surface o f HSCs . Certainly membrane-bound SF expressed on stromal cells can cause the adhesion of c-Kit-expressing HSCs to them (138) and thus play a role in H S C retention in the bone marrow, that when reversed leads to their mobilization (139). SDF-1 may be a major regulator o f H S C homing and is produced in large amounts by bone marrow stromal cells (140). Conversely, administration of antagonists of SDF-1 into normal adult mice (or humans) causes the H S C s in the bone marrow to detach from their niche and enter the circulation - a process referred to as mobilization (141). SDF-1 is a member o f the chemokine family o f molecules but is unique in its ability to bind to a single receptor, C X C R 4 , a member of the G-coupled receptor family. C X C R 4 is also the only chemokine receptor found to be expressed on HSCs thus conferring its response specifity to SDF-1 (142). However, C X C R 4 is not restricted in its expression to primitive hematopoietic cells and is highly expressed on B-lineage cells and many other cell types including numerous kinds of metastatic tumor cells (143-145). In vitro, the chemoattractant properties of SDF-1 on H S C s (and derivative progenitor types) have been well documented using transwell assays (140). Activation o f C X C R 4 leads to the activation of phosphoinositol-3 kinase which in turn activates PKC-zeta , whose downstream targets include Pyk-2 and E R K 1 / 2 (146). Interestingly, the levels o f C X C R 4 expression on the cells it chemoattracts can vary widely and this parameter is not predictive of either their in vitro chemoattractant responsiveness (147-149) or their in vivo homing activity (150). Mice in which either the SDF-1 or C X C R 4 genes have been inactivated die at a late stage of embryogenesis with gross bone marrow failure in spite of apparently normal hematopoiesis in the fetal liver, likely due 15 to an impaired ability of the fetal liver H S C to migrate into or be retained within in the bone marrow (147; 151-154). SDF-1 elicits multiple responses in CXCR4-expressing cells including the induction o f a chemoattractant migratory response by H S C s (142;155), inhibition of H S C cycling (156), and H S C mobilization (155;157). Additional mediators of H S C chemotaxis are l ikely to be found, such as the recent discovery o f the P2Y-l ike receptor, GPR105,(158). Migration of H S C s across the endothelium occurs preferentially in the Go/Gi phase o f the cell cycle and fetal H S C s are more efficient than adult mobilized P B HSCs at this process (159). Similarly, the mobilized HSCs found in the circulation are predominantly Go/Gi cells (160; 161) even when the method of mobilization is shown to initiate their prior entry into division within the bone marrow (162). Interestingly, adult H S C s stimulated to proliferate in vitro or in vivo display an engraftment defect during their period of transit through S/G2/M (84; 163; 164), although this defect could not be demonstrated for human fetal liver H S C s (165). Some differences in the expression of adhesion and homing molecules were found when G1/G0 vs S/G2/M cells enriched in their content of HSCs were compared, such as a higher expression of V L A - 4 and a lower expression of CD44 in the G\/GQ cells (163). Adhesive interactions between HSCs and their microenvironment also play a role in the regulation o f H S C survival and proliferation as well as in their homing and mobilization. 1.3.2.4 Self-renewal A s noted above, after the first appearance of H S C on the 9 t h and 10 t h days of embryogenesis, it is thought that the later expansion of this compartment occurs by the execution of symmetric self-renewal divisions; i.e., cell cycles in which competency for multi-lineage differentiation is maintained but not activated in both daughter cells. Self-renewal divisions are the hallmark 16 attribute of all stem cells and much effort has been devoted to elucidating this process with a view of devising ways to manipulate it. Controlled enhancement of H S C self-renewal ex vivo offers the potential both to develop strategies for obtaining amplified H S C populations and their mature progeny for a variety of therapeutic applications and to create models for investigating mechanisms of leukemogenesis (166; 167). Fetal liver HSCs have garnered interest as a population with seemingly greater intrinsic self-renewal potential than bone marrow HSCs , based on the sustained larger P B cell outputs by fetal liver H S C s as compared to bone marrow H S C s (99; 168; 169). In addition, fetal liver-derived cells reconstitute more daughter C R U s after a given period (168; 169) and can typically be serially transplanted more times than adult bone marrow cells. However, it appears that H S C numbers are not fully regenerated in irradiated transplanted mice, even though mature blood cell output is fully restored (170;171) and when the transplant dose is corrected for the actual content of C R U , huge expansions o f adult bone marrow C R U can be documented (172). A s yet, similar experiments have not been performed with H S C s regenerated in primary recipients of fetal liver-derived HSCs . Thus, it has been difficult to discern the separate contributions of increased self-renewal activity and greater survival or shorter cell cycle times of fetal liver H S C s on the greater regenerative activity they have been found to display in experiments that do not measure these responses independently. Notably, only a single paper has been published describing the kinetics of normal H S C expansion in mice transplanted with adult bone marrow cells (172) and similar data does not exist for fetal liver HSCs . A s a result, it is not known when recipients of primary transplants should be assessed for secondary C R U content in order to provide a quantitative index of the self-renewal activity of the cells transplanted. 17 1.3.2.5 Growth factor responsiveness There is now definitive evidence that the self-renewal of H S C s can be modulated by exposure to different extrinsic factors, both in vivo and in vitro. In vivo evidence is seen following virtually every H S C transplant, where the number of HSCs initially injected can be shown to increase at least 10-20 fold (169; 172), compared to their maintenance at a constant level in normal adult mice. The same shifts seen in H S C self-renewal also occur when normal (+/+) HSCs are transplanted into ff-deficient recipients (defective in normal c-Ki t signaling) and are stimulated to expand their numbers (94; 173). Significant efforts have been made to identify factors that influence H S C self-renewal in vitro, although these have been focused primarily on H S C s from young adults (174; 175) or from samples of human cord blood (176) and relatively little is known about self-renewal control in fetal liver HSCs . The difficulty in finding one or more growth factors that w i l l promote an expansion of H S C is not due to an inability to identify factors that can optimize the entry of H S C s into division (77), but rather because of the relative inability of any growth factors identified to date to effectively sustain the stem cell potential o f the stimulated H S C . Interestingly, certain GFs have been shown to stimulate H S C expansion but only over a narrow range (i.e., IL-6), while others (e.g. Steel factor, SF) attain their maximal effects only at high doses (>300ng/mL) (175). In the case of fetal liver HSCs , even less is known about factors that may promote H S C self-renewal but some additional candidates have been identified (e.g., Fibroblast growth factor-1 (177), thrombopoietin (174), Wnt proteins (178), Sonic hedgehog (179) and T A T - H o x B 4 (180). 18 1.4 Genes that regulate HSCs 1.4.1 Genes critical to both fetal and adult HSCs Studies of the generation and activity after transplantation of HSCs from mice in which specific genes have been modified or completely inactivated, or are overexpressed, have provided a significant body of information about specific intrinsically expressed receptors, signaling intermediates and transcription factors that appear to be important in controlling H S C proliferation and self-renewal. Many transcription factors regulate the self-renewal, proliferation, and/or the early differentiation of HSCs . Those shown to play a critical role (as demonstrated by inactivation studies) include: Activating transcription factor (ATF) 4, required for high-level proliferation in the fetal liver (181), Core-binding factor p, required for fetal liver H S C emergence and normal maturation (182; 183), c-Myb and p300, required for self-renewal and differentiation, respectively (184-186), c-Myc, required for differentiation (187), Gata-2, for normal proliferation, especially in adult bone marrow H S C s after cytotoxic treatment (188;189), Ikaros, required for normal numbers of H S C (190-192), Lmo-2, required for emergence o f H S C s (193; 194), meis l , required for the proliferation/self-renewal of H S C s (195; 196), M E F , required for normal maintenance of adult H S C quiescence (197), mel-18, required to negatively regulate proliferation, by binding to cyclinD2 (198-200), mixed-lineage leukemia, required for emergence of fetal liver H S C s (201), and those shown to play an important role (as demonstrated by overexpression studies): Hox genes (202-204), and rae-28 (205), both resulting in enhanced self-renewal. There are also non-transcription factors that play critical roles in stem cells, such as signaling intermediates S T A T 3 , required for self-renewal (206;207), S T A T 5 , required for normal competitive repopulating ability (208-212), and the cyclin-dependent kinase inhibitors described above p21, p27, p i 8, required to maintain quiescence, progenitor proliferation and to inhibit self-renewal, respectively (85-19 87;90); receptors, such as the retinoic acid receptor y, that mediates cell cycle arrest (213) and the receptor for thrombopoietin, c-mpl, required for normal numbers of H S C s (214); as are secreted factors such as transforming growth factor-|32, a positive regulator of competitive repopulation in adult H S C s (215) and wnt, a positive regulator of proliferation and expansion (167;178;216;217). Through studies of chimeras and using conditional gene knock-out strategies, it has also become possible to determine whether identified candidates act in a cell autonomous fashion and whether they may differentially affect fetal and adult HSCs , particularly where marked deleterious effects on fetal cells are obtained. In the last decade, with the advent of methods to obtain highly purified populations of murine H S C s from both fetal and adult sources, it has also been possible to determine whether candidate regulators are differentially expressed in these populations. It is interesting to note that many of the genes found to have important effects on H S C were first identified because of their involvement in fusion proteins created by translocations associated with different types o f acute myeloid leukemia ( A M L ) in humans (218). This is not surprising, given that such diseases are thought to originate after sufficient accumulation of genetic deregulation in HSCs . 1.4.2 Genes more critical to fetal than adult HSCs 1.4.2.1 Runxl Runxl (runt-related transcription factor 1) is the murine homolog of a human gene originally called AML-1 to reflect its discovery on chromosome 21 from the cloning of a common breakpoint in human A M L cells that carry a 8;21 translocation (219). Runxl encodes the DNA-binding subunit of a member of the core-binding factor (Cbf) family of transcription 20 factor complexes and has primarily activating functions in myeloid cells (220-222). It has, therefore, also been called Cbfa2. Target genes identified include CyclinD3, IL-3, GM-CSF and RAG1 and TCRa (223-227). A critical role for Runxl in early normal hematopoietic development was first revealed by the generation of two Runxl-deficient mouse lines, both o f which died mid-gestation as a result of bleeding in the central nervous system and soft tissues, with complete loss of fetal liver hematopoiesis (228;229). A l l hematopoietic cell lineages were found to be affected by the loss of Runxl, with the exception of primitive erythropoiesis in the yolk-sac, which was minimally affected. Similarly, in vitro, Runxl'A ES cells could generate normal numbers of primitive erythroid precursors but were impaired in their ability to generate blast colony-forming cells (cells that have properties of hemangioblasts) and failed to produce definitive hematopoietic C F C s (230). In the fetal liver of Runxl heterozygotes, hematopoietic progenitor numbers were found to be reduced by comparison to wild-type controls, suggesting that Runxl has a dose effect on mechanisms that regulate the production of primitive hematopoietic cells in the fetus. Nevertheless, in adult haploinsufficient animals, no phenotype was detected, implying the operation of different control mechanisms in the adult or an ability o f other genes to substitute for, or compensate for, a lack of R u n x l (231). A n inducible gene-targeting strategy for Runxl was then pursued in order to assess directly the effect of an absence of R u n x l in adult cells that had been generated from cells that produced R u n x l at normal levels (232-234). A complete absence of Runxl in the adult did not impair continued production of hematopoietic cells suggesting that it is dispensible for the maintenance o f adult H S C activity although enhanced granulopoiesis and defective megakaryocytpoiesis and early lymphopoiesis was noted. Competitive repopulation assays confirmed that Runxl'A adult bone marrow cells contain elevated short-term myeloid 21 repopulating activity (<2 months) but are similar to +/+ bone marrow cells in their ability to repopulate irradiated mice with mature neutrophils, monocytes and megakaryocytes for more prolonged periods. Using a \"knock-in\" marker strategy, R u n x l was shown to be expressed in mesenchymal and endothelial cells at all sites in the yolk-sac and embryo proper where the first definitive hematopoietic cells first appear, as well as in all o f the H S C s that emerge during this process. However, when expression of R u n x l , itself, was deleted, a complete block was seen in the generation of hematopoietic cells at these locations (235;236). Taken together, these findings indicate an essential role of R u n x l in the generation o f fetal HSCs from hemangioblasts in the embryo but not for their later self-renewal. However, it is interesting to note that forced expression of RUNX1-ETO (a fusion protein shown to act as a potent dominant negative inhibitor of R u n x l during development (228;229) in C D 3 4 + human cord blood cells enhanced the long term retention of lympho-myeloid differentiation potential as well as an ability to repopulate irradiated N O D / S C I D mice (237). Thus, although Runxl may be dispensible for the maintenance and initial differentiation of adult HSCs , these cells appear to remain sensitive to pathways that can be modulated by changes in R u n x l activity. 1.4.2.2 Notch Notch genes were discovered almost 80 years ago in Drosophila and found to encode type 1 transmembrane glycoprotein receptors that are cleaved after activation by binding their specific ligands, either Jaggedl or 2, or Delta (238). Cleavage results in the translocation of intracellular Notch to the nucleus where it can act as a transcriptional activator, for example for the c-myc promoter (239) . O f the 4 mammalian notch homologs, Notch-1 has been most extensively studied and its ability to inhibit H S C differentiation (promote H S C expansion) 22 documented (240-242). Homozygous inactivation of Notch-] causes embryonic lethality by day 9.5 (243;244) and at this time HSCs could not be detected when they were assayed for their ability to repopulate conditioned newborn mice (245). Further studies showed that in vitro, Notch- Y1' E S cells proliferated normally and were not impaired in their ability to generate either E B s or EB-derived definitive erythroid and myeloid C F C s . Following injection into wild-type blastocysts, they contributed initially to the C F C and C F U - S populations produced up to E l 0.5 of gestation. However, after that time, these cells rapidly disappeared and were not replaced even though a high degree of chimerism persisted in other organs (246). In contrast, elimination of either Jaggedl or Notch 1 or both in adult H S C s using an inducible Cre-loxP-mediated inactivation strategy revealed no effects on the ability of the affected H S C to exhibit their self-renewal or multipotentiality either postransplant or endogenously after treatment of the mice with 5 -FU (247). These findings strongly suggest that Notch 1 plays a differential role in regulating the ability of fetal and adult HSCs to execute self-renewal divisions when stimulated to proliferate. 1.4.2.3 Sci The stem cell leukemia (Sci) gene encodes a basic helix-loop-helix family member transcription factor, that can either activate or repress transcription, depending on the composition o f the multicomponent complexes it forms and plays an important role at multiple stages of hematopoietic cell differentiation, as reviewed in (248). The Sci protein would thus be expected to be able to play different roles through interactions with different partners present in varying levels in specific cell types (248). Some of these are known to be critical for hematopoiesis, such as E 2 A , and the bridge protein L M O - 2 , which also binds G A T A - 1 23 (see below). This type of complex can activate the c-Kit promoter, through functional interaction with S p l (249). Although studies of cells from Sc l + / \" heterozgotes with a lacZ/w reporter gene knocked into one of the Sci genes have now shown that Sci is expressed in a number of tissues, it has also been possible to establish that, within the hematopoietic system, Sci is expressed at the highest levels in the most primitive cells, including H S C s (61;250;251). The Sci gene was first targeted for disruption in 1995 by 2 groups, who both found that this resulted in embryonic lethality by E9.5, with complete loss of blood formation in the yolk-sac (252;253). 5c f A E S cells are also unable to contribute to hematopoiesis in mouse chimeras, demonstrating the cell-autonomous requirement for Sci in the generation of both primitive and definitive hematopoiesis in the fetus (254;255). In vitro, Sc^' E S cells were able to generate f l k - l + blast-CFC but the colonies then produced contained only vascular smooth muscle cells and no detectable endothelial or hematopoietic cells, suggesting that Sci may be required for the first steps in the transition from a mesodermal precursor to a hemangioblast (250;256). Recent m R N A knockdown studies in zebrafish suggest that Sci may also play important roles in the generation of endothelial and hematopoietic cells in this organism but not until after the hemangioblast stage when each of these lineages has become distinct (257). To assess the role of Sci in adult hematopoiesis, conditional knockout mice have been generated and studied. In one such study, analysis of bone marrow cells from targeted adult mice revealed that Sci, like Runxl and Notch-1, is dispensable for continued H S C function. However, in the case o f the ScT A HSCs , the ability to generate differentiated myeloid and lymphoid progeny was preserved, but the generation of mature erythroid and megakaryocyte precursors was compromised (258). Similar results were reported by the second group who 24 found that Sci was required for the short-term repopulation of irradiated recipients but did not appear necessary for H S C self-renewal (259;260). Interestingly, transduction of H S C s with vectors encoding either a wild-type Sci c-D N A or a dominant-negative (dn) form also had downstream effects on hematopoiesis without altering the long-term repopulating capacity of the transduced H S C s . The specific effects were the promotion of short-term myeloid repopulation by the SW-transduced cells and promotion o f lymphoid contribution by the dnSW-transduced cells (261). 1.4.3 Genes more critical to adult than fetal HSCs Genes critical to adult bone marrow H S C s but not fetal liver H S C s may be considered much easier to study as knock-outs of these might be anticipated to allow the organism to initially develop normally with the consequences of the mutant phenotype occurring later after birth. However, it is not clear when this point would be expected, since very little is known about the timing or relevance o f extrinsic vesus intrinsic factors in regulating changes in the H S C compartment between fetal life and adulthood. Nevertheless, some gross differences have become apparent from single generation transplants suggesting that genes more critical to adult than fetal hematopoiesis can be identified. 1.4.3.1 Bmi-1 B cell-specific Moloney murine leukemia virus integration site-1 (Bmi-1) is a gene encoding a member of the Polycomb group (PcG) of transcription factors. Two general types of multimeric P c G complexes are recognized. Both are involved in silencing genes through directing the modification o f chromatin structure, one in the initiation o f this process (called polycomb repressive complex 2 or Prc2) and the other in the stable maintenance of gene 25 repression (called polycomb repressive complex 1 or P rc l ) , and as a result, both are considered strong candidates for playing important roles in orchestrating the epigenetic changes in gene regulation that must occur during the self-renewal and initiation of lineage restrictive events that occur in stem cells (262;263). Bmi-1 is a member of the P r c l complex and other members of the P r c l complex are Mel-18, Rae28, R i n g l and M33 . Bmi-1 is expressed at elevated levels in highly purified H S C populations isolated from both fetal liver and adult bone marrow and acts to repress the C D K inhibitor p l 6 I n k 4 a and p i9^ (263 ;264) . A s predicted for a gene that differentially targets the self-renewal o f adult H S C s , homozygous deletion of Bmi-1 allows the affected mice to survive until about 1-2 months after birth at which point they die with hypocellular marrows. Assessment of the cellular composition o f the fetal liver of Bmi'1' mice showed these have a normal cellularity with unaltered numbers of c - K i t + S c a l + T h y l . l'\u00C2\u00B0 lin\" cells, a population that is normally enriched in the fetal liver HSCs . However, when Bmi-1'1' fetal liver cells were transplanted into adult irradiated mice, no B-lineage cells were produced and overall multi-lineage repopulating activity was reduced initially and declined irreversibly to undetectable levels within 8 weeks (265) unless Bmi-1 expression was restored, in which case H S C at near normal frequencies could be demonstrated (264;265). This demonstrates that the initial generation and expansion of fetal liver H S C s is not impaired by a lack of Bmi-1, but later, this transcription factor becomes increasingly critical for maintenance of the H S C compartment both when the cells are allowed to develop endogenously or when transplanted directly into an adult irradiated environment. Consistent with this interpretation was the finding that cells with the c - K i t + S c a l + Thy 1.110 lin\" H S C phenotype were still detectable in the bone marrow of 4-5 week-old Bmi-1\" 7\" mice but at 10-fold reduced numbers and these were even more defective in their ability to reconstitute primary recipients (264;265). Interestingly, the greater biological effect 26 o f a Bmi-1 deficiency in adult bone marrow cells was mirrored by similar differences in the extent of activation obtained on Bmi-1 target genes including p l 6 t a k 4 a and p i 9 ^ (265), although later experiments showed that these did not translate into detectable effects on proliferative activity or H S C apoptosis (199). The latter investigation also showed that forced over-expression of Bmi-1 could enhance the frequency of symmetrical divisions by primitive pluripotent hematopoietic cell; stimulated by cytokines to proliferate in vitro, corroborating the hypothesis that Bmi-1 acts maintain the competency o f H S C s (199). 1.4.3.2 c-Kit C-Kit is a proto-oncogene that encodes a type 3 cell-surface tyrosine kinase receptor also referred to CD117 (266). The ligand that binds to and activates c-Kit has been variably designated as c-Kit-ligand, Stem cell factor (SCF) and Steel factor (SF) because it is expressed on HSCs and is the product of the Steel gene. Both c-Kit and its ligand are produced as transmembrane proteins that can be variably cleaved proteolytically to yield an additional soluble form with full binding activity (267;268). SF binding induces homodimerization and tyrosine phosphorylation of the receptor, forming docking sites for SH2 domain-containing signaling intermediates (269). These include phosphatidylinositol 3'-kinase (PI3 K ) and phospholipase Oy-1 (270), the Src family kinases Src, Lyn , Fyn (271), the GTPase activating protein G A P (272), proteins of the p 2 1 R a s - M A P K pathway (269) and S T A T 1 , 3 and 5 (273-275). Additional signaling molecules that couple to c-Kit , including V a v (276), Jak2 (277), Dok (278), Tec (279) and the tyrosine phosphatase SHP-1 (246). C - K i t is expressed on all H S C s and hematopoietic progenitors throughout embryonic and adult life and signaling downstream of the c-Kit receptor is required for many aspects of normal hematopoiesis. 27 The W (encoding c-Kit) and Steel (encoding Steel factor) loci were first identified from early genetic studies o f spontaneously arising mutant white spotted mice that shared the same profile of pleiotropic phenotypes affecting coat colour, sterility and red blood cell production (280) . These phenotypes are caused by abnormalities in the generation of melanocytes, germ cells and erythroid cells. Severe W mutations conferred a cell-intrinsic defect on primitive hematopoietic cells normally detected as C F U - S , whereas severe Steel mutations conferred a defect that was not transplantable and restricted to the environment of hematopoietic tissues (281) . T h e W 4 , / W 4 1 mouse has a single nucleotide substitution i n the coding region of the c-K i t gene that results in partial impairment of the tyrosine kinase activity of the SF receptor. The fetal liver of these mice contains a normal number of transplantable H S C s but their longterm competitive activity is slightly reduced (94). Moreover, i f they are simply allowed to expand without being transplanted, the numbers attained in the bone marrow by early adulthood is ~10-fold lower than that of wild-type mice and the repopulating activity per W 4 1 / W 4 1 H S C is also impaired (93). Similarly, the fetal liver o f Sl/Sl mice, which do not express a functional SF protein, have been found to contain - 4 0 % of the normal number o f cells with a S c a l + Thy 1.110 lin\" phenotype on E l 3 and these then increased at the same rate as their counterparts in wild-type embryos until E l 5 when these embryos all die from anemia (92). 1.4.3.3 Gfi-1 The growth factor independent-1 (Gfi-1) gene encodes a zinc-finger transcription factor with repressor activity and was identified as a common site of Moloney murine leukemia virus integration in lymphoma cell lines that acquire IL-2 independence (282). Gfi-1 is expressed in H S C s and C L P s and granulopoietic progenitors but not in C M P s or megakaryocyte-erythroid 28 progenitors (283). The Gfi-1 protein interacts with protein inhibitor of activated Stat3 (Pias) to mediate proliferative responses to cytokines and can enhance Stat3 signaling in primary T cells (284). There have not yet been any reports of experiments to measure H S C numbers or activity in the fetal liver of Gfi-1*'\" mice, but the fact that young Gfi-1\"7\" mice are not anemic and have sustained myelopoiesis suggests that Gfi-1 is not critical for the initial appearance and early expansion of H S C s (285). However, adult G f i - 1 7 ' bone marrow cells showed a greatly reduced ability to repopulate primary irradiated recipients and an almost complete failure to generate cells that w i l l repopulate secondary recipients (91;283). Interestingly this late appearance o f defective H S C self-renewal differed from that elicited by deletion of Bmi-1 where the H S C s conversion to a quiescent population was not altered (199). In contrast, in the case of Gf i - l \" ' \" mice, both Hoechst 33342/PyroninY staining of the l in\"Sca l + c-Ki t + population isolated from the bone marrow of adult G f i - l ' 7 \" mice and +/+ controls (164; 165) and B r d U labelling studies (286;287) of these cells tracked over time showed an increased proportion o f the H S C s from the adult Gfi-1' '\" mice to be proliferating (91;283). In these cells G A T A - 2 was slightly upregulated and the cell cycle inhibitor p 2 1 C i p l A V a f l was 10-fold down-regulated (91). This is a significant observation since adult p 2 1 c i P i / w a n _ n u l l m i c e h a y e a s i m i l a r H S C d e f \u00C2\u00A3 c t ( g 5 ) 1.4.3.4. Tie2 Tie2 is a tyrosine kinase receptor expressed on the surface of endothelial cells and both fetal liver and adult H S C s (105;106;288;289). Tie2 and its ligands, the angiopoietins, have been suggested to play a role in the recruitment and mobilization of adult H S C s from the bone marrow (290). Initial observations indicated that Tie-2\" / _ embryos display reduced hematopoietic differentiation as cultured explants from early stages of development (from the 29 P-Sp) in Tie2' /\" embryos did not generate expected numbers of differentiated cells (291). Nevertheless, fetal liver hematopoiesis was not significantly affected by inactivation o f the Tie2 gene whereas marked failure of Tie2\"/\"hematopoiesis was seen in the bone marrow o f adult mice (95). The reason for the adult dependence of H S C on Tie-2 signaling may be related to the nature of the niche occupied by H S C s in the adult. HSCs are known to be concentrated at the periphery o f the marrow cavity adjacent to osteoblasts that secrete Angiopoietin-1 (Ang-1), a ligand for Tie-2, that can maintain adult H S C s in vitro (292) and promotes adult H S C quiescence (293). 1.5. Experimental objectives A s reviewed above, strong evidence of differences in the proliferative status and regenerative potential of fetal liver and adult bone marrow H S C s has been found, although neither of these putative differences have been rigorously documented or quantified. Surprisingly, attempts to maintain fetal liver H S C s in vitro have shown that these HSCs are even more difficult to sustain than adult bone marrow H S C s (294;295) in spite of the fact that both the proliferative activity and the regenerative ability of fetal liver H S C s in vivo is thought to be greater. These latter findings suggested that effects of specific growth factors on the ability of fetal liver H S C s to maintain their undifferentiated status might be different from the results obtained for adult bone marrow HSCs (175) and predicted by studies of human cord blood and adult bone marrow L T C - I C expansion in vitro (296). Based on this information, I set out to define more precisely how the cycling and self-renewal activity o f the H S C s present at different stages o f development change over time, to determine how these might relate to changes in other potential indicators of H S C function (i.e., output o f different types of differentiated cells), and to examine whether any changes 30 observed were intrinsically or environmentally regulated and to try to determine how they might be regulated at a molecular level. M y overall working hypothesis was that the reported greater in vivo regenerative activity of transplanted fetal liver H S C s is due to intrinsic differences in the self-renewal potential of these cells as compared to their adult counterparts. This required designing experiments that would allow their self-renewal activity to be measured independently of their cycling activity. Accordingly, I first executed a series of experiments to quantify the proportion of functionally defined H S C s that are in Go (quiescent) vs G1 /S /G2 /M (actively proliferating) using a variety of existing methodologies that could be directly coupled to assessing C R U frequencies. These methods were then applied to H S C s from different sources and stages o f development o f the mouse starting with E l 4.5 fetal liver and finishing with bone marrow cells from 10 week-old young adult mice. The results of these experiments are presented in Chapter 2. I then designed experiments to determine whether the self renewal properties of fetal liver HSCs are truly different from adult bone marrow HSCs , and, i f so, to identify when they change. The first step was to delineate the precise kinetics of H S C regeneration in vivo from a fixed number of transplanted HSCs. These data were used to define a time when differences in H S C regenerative activity could best be compared and then applied to transplants of H S C s from the same sources assessed for changes in their proliferative activity in Chapter 2. To test whether H S C from fetal liver and adult bone marrow can transit the cell cycle at different rates (i.e. have different cell cycle transit times) a method for purifying fetal liver H S C s to near homogeneity was developed and H S C proliferation was then monitored by direct visualization 31 of these cells in single cell cultures. The results of these experiments are presented in Chapter 3. A final series of experiments was undertaken to explore a possible mechanism for the changes in H S C behaviour documented in Chapters 2 and 3. Given the importance of SF-stimulation of adult HSCs to maximize the self-renewal of adult HSCs in vitro (175) in contrast to an apparent weak effect of deficient SF signaling on fetal H S C s (92;94) a change i n SF signaling sensitivity might underlie the changes observed in H S C cycling control and self-renewal potential. To test this hypothesis, I first undertook experiments to compare the dependence of fetal liver and adult H S C s on SF stimulation in vitro and then asked how this dependence might be altered in HSCs from W ^ / W 4 1 mice that have defective c-Kit signaling and whether any differences observed might be predictive of their self-renewal behaviour as assessed using the secondary transplant endpoint developed in Chapter 2. The results of these experiments are presented in Chapter 4. 32 Lymphoid Stem Cell Stem Cell Myeloid Stem Cell T Lymphocyte B Lymphocyte NK Lymphocyte Neutrophils Eosinophils Basophils / mast cells Monocyte/ macrophages Erythrocyte Megakaryocyte/ platelets Figure 1.1: Hematopoiesis A simplified overview of the concept of hematopoiesis that progresses from stem cells to mature blood cells. Subsets of lineages are shown. 33 400 cgy ^W*i/W<1-Ly5.2 mice Ly5.1 cells (graded doses) Multi-lineage Ly5.1+ cells i Figure 1.2: Competitive repopulating unit ( C R U ) assay. Schematic of the principle components of a C R U assay: recipients are irradiated and transplanted with graded doses of congenic cells; 16 weeks must elapse before the presence o f multilineage donor-type cells detected in the peripheral blood of the recipient can indicate whether a C R U was present in the test cell population or not. A low dose of irradiation (400 cGy instead of 900 Cgy) eliminates the need to ensure recipient survival by co-injection of helper cells with the test population, but does not eliminate the competing endogenous stem cell pool. W41 recipients have compromised H S C s (attenuated c-Kit signaling), and can therefore be used in combination with a low dose of irradiation. 34 100 log-linear relationship characteristic of a single-37\u00C2\u00B0>^n^qatjyes^. hit process 1 ^ \u00E2\u0080\u00A2 10 [ I \u00E2\u0080\u00A2 1 frequency of cell of interest within test population test cell dose (linear scale) Figure 1.3: Limit ing dilution analysis ( L D A ) to determine the frequency of C R U within a test population. Poisson statistics describe the probability distribution of random counts and state that the frequency of negative outcomes (recipients that are not repopulated in all lineages long-term) is equal to e\"\"^ (where n equals the number of cells tested, and f equals the frequency of stem cells (1 in f)). The log linear relationship of the frequency of negatives = -n/f. so when n=f, the frequency of negatives =e_1 = 0.37 C R U assay results can thus be plotted based on the above to determine at which test cell dose the proportion of negative outcomes is equal to 37%. 35 Table 1.1: Phenotypic similarities and differences between fetal liver and adult bone marrow H S C s Fetal Liver Adult Bone Marrow H S C s B220' G r l \" L y l \" T h y - l \" CD71\" Fall-3\" T e r l l 9 \" N k l . 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J.Haematol. 101:770-778. 79 CHAPTER 2 HEMATOPOIETIC STEM CELLS PROLIFERATE UNTIL AFTER BIRTH AND SHOW A REVERSIBLE PHASE-SPECIFIC ENGRAFTMENT DEFECT The work presented in this Chapter was accepted for publication in the Journal of Clinical Investigation. M . B . Bowie, K . D . McKnight , D . G . Kent, L . McCaffrey, P . A . Hoodless, C. J. Eaves Kristen McKnight isolated E l 8.5 fetal bones, David Kent isolated R N A and generated c D N A from my samples, and Lindsay McCaffrey assisted with C R U analysis. 80 INTRODUCTION Hematopoietic stem cells (HSCs) are defined as cells with multi-lineage hematopoietic differentiation potential and sustained self-renewal activity. Operationally, HSCs are detected by their ability to regenerate longterm multi-lineage hematopoiesis in myeloablated recipients. H S C numbers can be quantified by endpoints that measure this regenerative activity in genetically distinguishable, radio-protected hosts transplanted with limiting numbers of H S C s (1). HSCs are also characterized by extensive heterogeneity. Variability in many H S C properties is dictated by changes in their state of activation and the consequent changes in these properties are thus reversible. For example, most of the H S C s present in normal adult mice are in a deeply quiescent (Go) state (2-4) and, in association with this status, they express CD38 but not CD34 or M a c l (5;6). These Go H S C s also actively exclude certain fluorescent dyes, such as rhodamine-123 (Rho) (7;8) and Hoechst 33342 (Hst) (9). The latter property underlies the detection of adult mouse HSCs as \"side population\" (SP) cells (10). However, when HSCs are activated, they rapidly down-regulate expression of CD38 (6;11), increase expression o f CD34 (12) and M a c l (13; 14) and acquire a Rho-bright, non-SP phenotype (15). In association with these changes, some of the HSCs begin to differentiate and hence permanently lose their longterm repopulating activity, but many do not, in spite of their transiently altered phenotype (16). Another property of H S C s that appears to vary reversibly is their ability to exit from the circulation into the bone marrow and re-initiate hematopoiesis. Quiescent adult mouse H S C s can execute this process at near unit efficiency in suitably myelosuppressed hosts, as shown by their ability to be detected at purities of >20% following intravenous injection (15;17;18). However, notable changes in H S C engrafting potential have also been found to accompany the progression of HSCs through the cell cycle both in vitro and in vivo (3; 19-21). Specifically, H S C activity was not detectable in suspensions of adult or 81 neonatal hematopoietic cells in S/G2/M, even when substantial H S C activity could be found in the corresponding G i cells. The transient nature of the silencing of H S C homing activity during the progression of these cells through S/G2/M is inferred from the fact that the populations studied did not contain Go HSCs , or were expanding their H S C content, although formal documentation of the re-acquisition of repopulating activity by incapacitated S/G2/M H S C s was not documented. The molecular mechanisms that control the bone marrow-homing activity of H S C s are not fully elucidated, although a number of cell surface ligand-receptor interactions with known involvement in cell adhesion and chemotaxis have been implicated. These include stromal cell-derived factor-1 (SDF-1 V C X C R 4 , Steel factor (SF)/c-kit, CD44/Hyaluronic acid ( H A ) or osteopontin (OPN), V L A - 4 / V - C A M T and P2Y-l ike receptor and an unknown ligand (22-25). The expression and activity of some of these appear to be variably affected by cytokine exposure (26;27); however, their specific involvement in the engraftment defect of H S C s in S / G 2 / M has remained unclear. In the mouse embryo, pluripotent hematopoietic cells with longterm repopulating ability first appear in the aorta-gonado-mesonephros ( A G M ) region on the 9th day of gestation (28). These cells then migrate to the fetal liver and later to the bone marrow with continuing expansion of their numbers until young adulthood is reached (29). Most of the HSCs in the embryonic day (E) 14.5 liver have phenotypic characteristics of activated adult HSCs (CD38\", M a c l + , C D 3 4 + , Rho + , non-SP) (13;14), as might be expected for an expanding H S C population. The proportion of HSCs in the E14.5 fetal liver that are proliferating has been previously estimated from phenotyping studies to be - 4 0 % (3), although a more direct measurement of this fraction as not been reported. In this study, we sought to investigate the possibility that the H S C S / G 2 / M engraftment defect is constant through ontogeny, while the cycling status of HSCs is not. 82 A s a first step towards elucidating the mechanisms that regulate changes in H S C turnover and engraftment properties during ontogeny, we designed experiments to assess their proliferative status in mice at different stages of development. Our results show that the entire H S C population remains in cycle until the 3rd week after birth regardless of the tissue in which the H S C s are located. Then within a week, the majority of the HSCs switch abruptly from an actively dividing to a quiescent state. Unt i l this switch occurs, those H S C s that are in S/G2/M show the same engrafting defect previously demonstrated for adult H S C s that have been stimulated to divide. Interestingly, prior to the establishment of a quiescent H S C population, the HSCs in S/G2/M were found to express higher levels of SDF-1 than those in G i and their defective engrafting activity could be completely reversed, either by holding them ex vivo for a few hours until they re-entered G i , or by pre-treating the host with a specific antagonist o f stromal cell-derived factor-1 (SDF-1). RESULTS AU HSCs in the E14.5 fetal liver are rapidly proliferating To measure the proportion of HSCs that are in cycle in the E l4 .5 fetal liver, we used 3 complementary strategies. In the first, we injected pregnant mice on E l 3 . 5 with 100 mg/kg o f 5-fluorouracil (5-FU) and then removed the fetuses 16 hours later, prepared cell suspensions from the fetal livers and measured the number of HSCs present using a limiting dilution transplantation assay for longterm (16-week) competitive repopulating units (CRUs) (1). In these experiments, we detected very few C R U s in the fetal livers of the 5-FU-treated embryos (Figure 2.1 A , left panel, 3 experiments). A comparison of the yields of C R U s from the 5-FU-treated fetal livers with the control fetal livers from pregnant mice injected on E l 3 . 5 with 83 phosphate buffered saline (PBS) indicated that the 5-FU treatment had reduced the expected C R U population in vivo by more than 1000-fold. We then assessed the cycling status of H S C s in the E l 4 . 5 fetal liver by measuring the proportion of C R U s that survived a 16-hour exposure to high-specific activity 3H-thymidine ( 3 H-Tdr) (30). Sixteen hours was anticipated to be sufficient to allow all cycling HSCs to enter S-phase, as confirmed later (see below), with minimal exit of any quiescent cells from Go (31), as demonstrated for adult bone marrow HSCs , most of which are in Go (Figure 2.1 A , right panel). For these experiments, the Ter l 19 + (erythroid) cells were first removed from the fetal liver cells to give a 10-fold enrichment in H S C content and the cells were then incubated in a serum-free medium supplemented with 50 ng/ml SF only. This growth factor condition was chosen based on other data demonstrating that freshly isolated E14.5 fetal liver C R U s are maintained at input numbers for 16 hours under these conditions (Chapter 4). The results of the 3 H - T d r suicide experiments showed that this treatment reduced the number of C R U s in the suspensions of E14.5 fetal liver cells more than 100-fold (P<.001, Figure 2.1A, middle panel), whereas the same treatment had no significant effect on the recovery of C R U s in similarly treated lineage-marker-negative (lin\") bone marrow cells from young adult (10 week-old) mice by comparison to either control cells (incubated without 3 H-Tdr , Figure 2.1 A , right panel, P=0.17) or the starting values (data not shown). We then assessed the distribution of C R U s between the Go and G1/S/G2/M fractions of E14.5 Ter l 19\" fetal liver cells. These subsets were isolated by fluorescence activated cell sorting ( F A C S ) on the basis of their staining with Hst and pyronin Y (Py) (32). A representative F A C S profile of the Hst and Py-stained cells is shown in Figure 2.2A. The combined results of in vivo assays of the sorted cells from 4 independent experiments are shown in the left part of Figure 2.3. These indicate that all of the transplantable C R U activity 84 was confined to the G1/S/G2/M fraction. Based on the total number of Go cells assayed, the proportion of quiescent H S C s could be estimated to be less than 0.02%. HSCs undergo a complete and abrupt change in cycling activity between 3 and 4 weeks after birth Since H S C s are known to be present in the bone marrow of mice at later times of gestation, it was of interest to investigate whether H S C s first become quiescent in the fetus at that site. To address this question, we used the 16-hour 3 H-Tdr suicide assay to determine the cycling status of the C R U s present in the bone marrow of mice at E l 8.5. For comparison, we also evaluated the cycling status of C R U s in the E l 8.5 fetal liver. The frequency of C R U s in these 2 tissues was 1 per 10 5 and 1 per 7 x 10 4 total nucleated cells (Supplementary Table 1); i.e., ~5 and 3.5-fold lower than in adult bone marrow (1 per 2 x 10 4 total nucleated cells (33)), and 6 and 4-fold lower than in the E14.5 fetal liver (1 per 1.7 x 10 4 total nucleated cells (33)). After overnight exposure to high-specific activity 3 H-Tdr , no C R U s could be detected in the suspensions of either the E l 8.5 fetal bone marrow cells or the E l 8.5 fetal liver cells, in contrast to the control cells incubated in the same medium without H-Tdr (Figure 2.1B). Thus all H S C s in the fetus, irrespective of their location, appear to be rapidly proliferating. To further investigate the pace and timing of the transition of H S C s into a largely quiescent population, we analyzed the cycling status of C R U s in lin\" bone marrow cell suspensions from 3 and 4 week-old (weanling) mice. In initial experiments, the frequencies of C R U s in the lin\" bone marrow cells obtained from the 3 and 4 week-old mice were found to be the same (1 per 6.5 x 10 3 and 1 per 6.3 x 10 3 lin\" cells; Supplementary Table 2) and ~2-fold lower than in the l in ' bone marrow cells from 10 week-old (young adult) mice (1 per 2.9 x 10 3 lin\" cells). Bone marrow cells from 3 and 4 week-old mice were then fractionated by F A C S 85 into their component Go and G]/S/G2/M subsets based on the gates shown in the left panels of Figure 2.2B and 2.2C, and the sorted Go and G1/S/G2/M cells were assayed separately for C R U activity. Re-analysis o f the sorted Go and G i / S / G 2 / M fractions after staining for Ki67 confirmed that the cells expressing this proliferation marker were confined to those we had designated as G1/S/G2/M (see representative profiles in the right panels of Figure 2.2B). Remarkably, the results of the in vivo assays showed that all of the C R U s detected in the bone marrow of 3 week-old mice were also confined to the G1/S/G2/M fraction, whereas >98% of the C R U s in the bone marrow of 4 week-old mice were found in the Go fraction (Figure 2.3). Thus, there is a rapid down-regulation of C R U proliferative activity in the bone marrow of mice between 3 and 4 weeks of age with little change in C R U numbers. HSCs in S/G2/M show a specific and reversible engraftment defect regardless of their developmental origin or route of injection into assay recipients Given the previously reported engraftment defect of adult H S C s stimulated to enter S/G2/M (19), it was o f interest to determine whether the number of proliferating H S C s present early in development might be routinely underestimated due an inability of those in S/G2/M to be detected. To investigate this possibility, the G1/S/G2/M population o f E14.5 Ter l 19\" fetal liver cells was subdivided into its component G i and S/G2/M fractions and then each of these 2 subsets was assayed separately for C R U s . In this case, the gate settings chosen to separate the G i (2n D N A ) and S-phase cells (>2n D N A ) were validated by the profiles obtained when the sorted cells were stained with propidium iodide (PI) and reanalyzed by F A C S (Figure 2.4A, left panel). A l l C R U activity detectable in the G1/S/G2/M fraction of Ter l 19\" E14.5 fetal liver cells was confined to the G i subset (left bars in Figure 2.4B and control values in the left side of 86 Figure 2.5A). Similar experiments performed with lin\" bone marrow cells from 3 week-old mice showed that the C R U s in the G1/S/G2/M population from this source were likewise confined to the G i fraction (control values in the right side o f Figure 2.5 A and data not shown). It is of interest to note that the S/G2/M defect was specific to repopulating cells able to produce progeny in all lineages for at least 16 weeks. In contrast, cells with short term repopulating activity (8 weeks) were readily detected in the S / G 2 / M fraction as well as in the G i fraction, thus confirming the restriction o f this cell-cycle-dependent engrafting defect to cells with sustained multi-lineage repopulating activity (34;35). To determine whether the apparent engraftment defect o f proliferating C R U s was reversible, we first assayed the C R U content of aliquots of the same isolated G i and S/G2/M cells after they had been incubated for 6 hours at 37\u00C2\u00B0C in serum-free medium containing 50 ng/ml of SF. During this time, many of the G i cells progressed into S/G2/M and many of the S/G2/M cells moved into G i , as seen by their altered PI (Figure 2.4A, right panel) or Hst (data not shown) staining profiles. In vivo assays showed that C R U activity reappeared when the S/G2/M cells were cultured for 6 hours, whereas the C R U activity originally present in the G i cells was partially lost (middle and left bars of Figure 2.4B). We next asked whether the inability o f intravenously transplanted C R U s in S/G2/M to engraft recipient mice might be overcome by injecting the cells directly into the femoral bone marrow space. However, intrafemoral injection did not enable any C R U s in this subset of Te r l 19\" E l 4 . 5 fetal liver cells to be detected (Figure 2.4B) even though the frequency of C R U s measured in the corresponding G i E l 4 . 5 fetal liver cells after intrafemoral injection was the same as when the latter were transplanted intravenously (1 per 3.6 x 10 3 cells versus 1 per 3.8 x 10 3 cells). 87 The S/G2/M engraftment defect of HSCs can be overcome by pretreatment of the host with a SDF-1 antagonist Previous reports have shown that SDF-1 can promote both the mobilization (36) and the homing (37-39) o f HSCs . However, the mobilization o f primitive hematopoietic cells can also be stimulated by blocking SDF-1 / C X C R 4 signaling, as achieved by in vivo administration o f A M D 3 1 0 0 , a SDF-1 antagonist (40). In addition, it has recently been shown that in vivo administration of A M D 3 1 0 0 can increase the competitive engrafting ability o f transplanted marrow cells in unirradiated hosts (41). These suggested that targeting the S D F - 1 / C X C R 4 pathway might also influence the variable engraftment properties of cycling HSCs , either by influencing the H S C s themselves or the transplanted host. To investigate these alternatives, we asked whether either pretreating the hosts or the cycling HSCs to be transplanted with a specific antagonist of SDF-1 might alter the level of repopulation obtained 16 weeks later. The SDF-1 antagonist used in these experiments was SDF-1 G2 (also called P 2 G because it is identical to SDF-1 except that the proline at position 2 has been converted to glycine (42)). SDF-1 G2 is thus structurally quite different from A M D 3 1 0 0 but similar in its ability to block SDF-1 from binding to C X C R 4 without activating C X C R 4 (42;43). SDF-1 G 2 also shares with A M D 3 1 0 0 an ability to elicit effects on primitive hematopoietic cells both in vitro and i n vivo (44). When mice were injected with 10 pg of SDF-1 G2 (or PBS) 2 hours prior to the transplantation o f FACS-sorted Gj or S/G2/M cells and then analyzed for the presence of donor-derived blood cells 16 weeks later, the results for E l 4 . 5 fetal liver and 3-week mouse bone marrow cells were similar (Figure 2.5A). Treatment of recipients with SDF-1 G2 had no effect on the repopulating activity o f C R U s in G i . In contrast, SDF-1 G2 pretreatment of recipients o f S/G2/M cells enabled long-term multi-lineage repopulation to be readily detected (7 and 4 o f 10 mice transplanted with fetal liver and 3-week bone marrow S/G2/M cells, 88 respectively, vs. 0 of 10 in both sets of controls injected with PBS , in a total of 3 experiments). Moreover, the SDF-1 G2 pretreated hosts showed levels of repopulation by both sources of S/G2/M cells that were indistinguishable from those seen in mice transplanted with Gj cells (Figure 2.6). On the other hand, i f the SDF-1 G2 treatment was applied directly to the cells to be transplanted for 30 minutes before they were injected, no difference in the engrafting activity of the transplanted G i or S/G2/M cells was seen by comparison to untreated controls over a wide range of SDF-1 G2 and SDF-1 concentrations tested, either with or without added SF (Figure 2.5B). HSCs in S/G2/M express higher levels of SDF-1 transcripts than HSCs in Gi To begin to understand the mechanism behind the observed H S C S/G2/M engraftment defect and how it might be overcome by SDF-1 G2 pre-treatment of the host, we isolated highly purified populations of H S C s from E l 4 . 5 fetal livers and from the bone marrow of 3 week-old mice (lin\" Sca-1 + C D 4 3 + M a c l + cells representing - 2 0 % pure H S C s , Bowie, M B and Eaves, C J , manuscript in preparation) and sorted these into their corresponding Go/Gi and S/G2/M fractions as revealed by Hst staining. Aliquots of from -200 to 800 cells were collected from each fraction in 3 independent sorting experiments and transcript levels for Gapdh, c-Kit, c-mpl, CD44, a4-integrin (a4int), VCAM-1, CXCR4 and SDF-1 were measured by quantitative real-time analysis of the c D N A s prepared from the isolated R N A extracts, as described in the Methods. Transcripts for all of these genes were consistently detected in both the Go/Gj and S/G2/M fractions of the highly purified suspensions of HSCs cell populations from fetal liver and 3 week bone marrow, including SDF-1, which had not previously been shown to be expressed by H S C s (Figure 2.7). Interestingly, SDF-1 was also the only one of the genes 89 assessed that was found to be expressed at significantly different levels in Go/Gi and S/G2/M H S C s (9-fold higher in the latter, P<.05). DISCUSSION This study presents 2 new and clinically relevant features of H S C regulation. The first is the unanticipated sudden and complete change in H S C proliferative activity that occurs in juvenile mice between 3 and 4 weeks of age. Both the abruptness and the reproducibility of this change suggest an underlying mechanism that is tightly controlled and broadly active. It is notable that this change was not linked to the migration of H S C s during late embryogenesis from the microenvironment of the fetal liver to that of the bone marrow, but rather, was strictly associated with the developmental status o f the donor. Thus, although differences between bone marrow and fetal liver niches and stromal cells have been sought and described (45-48), these differences do not appear to directly determine the cycling activity of the H S C s they are thought to regulate. The present data are more consistent with a model in which the mechanism of H S C cycling control in vivo is indirectly controlled by external cues, perhaps v i a changing stimulation of the stromal cells that then alter the signals they deliver, as suggested by studies o f the longterm marrow culture system (44;49) and of elements of the bone marrow microenvironment in vivo (25). However, internally programmed changes in H S C responsiveness to external factors could also contribute to a developmentally controlled alteration i n H S C cycling activity. In humans, an abrupt change in H S C proliferative activity at an analogous point in development (2-4 years) has been inferred from measurements of the rate of decline in telomere length of circulating leukocytes (50). This suggests that the mechanisms involved in regulating H S C proliferative activity during ontogeny may be preserved across these species 90 and the mouse w i l l be a relevant model for their future elucidation. It is interesting to note that, in the mouse, a number of other changes in hematopoietic cell properties or output parameters have already been found to change during ontogeny in concert with this transition of the H S C compartment from a predominantly cycling to a predominantly quiescent population. These changes include the initial acquisition of an SP and R h o d u \" phenotype by H S C s (15), and the completion of appearance and rapid cycling of adult-type (Ly49 + ) natural killer cells and peripheral T-cells (51;52). Many other differences in the properties of fetal and adult H S C s and the programs they dictate have also been described (13;53;54). It w i l l clearly be of interest to determine the extent to which these may be programmatically linked to the mechanisms that precipitate the change in H S C cycling that occurs in mice between 3 and 4 weeks of age. Several genes have been implicated in the differential control of H S C behavior at different stages of development. These include genes encoding various transcription factors, i.e., Runxl (55), Notch (56), Sci (57), bmil (58;59) and Gfi-1 (60;61), as well as the growth factor receptors, c-Kit (62;63) and Tie2 (64). Further delineation of the molecular basis of the unique programs operative in fetal H S C s and how these regulate fetal HSCs cycling are of major interest as this information could provide new strategies for expanding H S C s and offer potential insights into mechanisms of leukemogenesis. The second significant set of findings emanating from our studies are the universality and pronounced extent of the engraftment defect found to characterize cycling H S C s in the S/G2/M phases of the cell cycle, the specificity of this effect for hematopoietic cells with prolonged versus short term repopulating activity, and the reversibility of this defect either following their progression into G i , or by pre-treating the host with a specific antagonist of SDF-1 . Interestingly, the corrective effect of in vivo administered SDF-1 G2 could not be 91 replicated by treatment of the cells with this agent prior to injection. The in vivo effect o f SDF-1 G2 could also not be mimicked by intrafemoral injection of the test cells. The inability of intrafemoral injection to overcome the defective engraftment of H S C s in S/G2/M suggests that this defect is likely mediated by events that affect the transplanted H S C s after they have entered the bone marrow environment. Quantitative analysis of the level of expression of 7 candidate genes in the G i and S/G2/M subsets of purified cycling H S C s from both fetal liver and 3 week bone marrow sources confirmed the expected expression of c-Kit, c-mpl, CD44, a4int, VCAM-1 and CXCR4 and further revealed that these cells also all contain SDF-1 transcripts. Moreover, although the transcript levels were not different between the G i and S/G2/M fractions for c-Kit, c-mpl, CD44, a4int, VCAM-1 and CXCR4, a 9-fold increase in SDF-1 expression was noted in the S/G2/M H S C s . Previous work has suggested that the ability of transplanted HSCs to reach a niche within the bone marrow that can support their self-maintenance may depend on the strength o f the SDF-1 gradient they encounter within the bone marrow space causing them to migrate towards the osteoblasts that line the bone (22). According to such a model, the ability o f H S C s to express varying levels of SDF-1 in the absence of changes in their expression of C X C R 4 , might be anticipated to regulate their ability to respond to other more distal sources o f SDF-1 . Up-regulated expression of SDF-1 during the progression of H S C s through S/G2/M, as demonstrated here, might then be sufficient to interfere with an appropriate intra-bone marrow migratory response resulting in the rapid differentiation, death or irreversible sequestration o f these cells in a site where they could not be stimulated to divide. Timed blockade of C X C R 4 on cells within the bone marrow by injected SDF-1 G2 might then be envisaged to increase in the level of intra-marrow SDF-1 to a point that transiently restores an effective chemoattractant gradient for the otherwise insensitive HSCs in S/G2/M. Such a 92 possibility has, in fact, recently been modeled in the zebrafish, where overexpression of SDF-1 in the germ cells was found to prevent the normal migration of these cells towards endogenous SDF-1 signals (65). In the hematopoietic system, it is interesting to note that longterm repopulating SDF-1' ' ' H S C s could engraft irradiated hosts whereas only short term repopulation was obtained from CXCR4'1' cells (66;67). In addition, as would be predicted from the explanation we have advanced, forced overexpression of C X C R 4 in retrovirally-transduced (i.e. proliferating) human H S C s was able to enhance the in vivo engrafting activity o f these cells (68) and, conversely, treatment with antibodies to C X C R 4 had the opposite effect (69). However, S D F -1 levels in the bone marrow are also subject to regulation, for example, as occurs following the administration of granulocyte colony-stimulating factor (G-CSF) (70). Recently, Chen et al. (41) found that administration of A M D 3 1 0 0 pre-transplant can produce a modest improvement in the engraftment of quiescent adult bone marrow HSCs transplanted into non-irradiated hosts. The.mechanism proposed was that the injected A M D 3 1 0 0 initiated the mobilization of endogenous H S C s within the marrow thereby improving the ability of the incoming transplanted H S C s to compete for niche occupancy. The studies o f Chen et al thus differed in several respects from those described here where we have observed an enhanced engraftment by H S C s that was more marked and exclusive to H S C s that were in S/G2/M at the time o f injection. Therefore, it seems unlikely that the mechanisms responsible for the enhanced engraftment seen in both experimental models are similar, in spite of the fact that they are both mediated by treatment of the host with an SDF-1 antagonist. The fact that proliferating human H S C s show the same engraftment defect when they transit S/G2/M is noteworthy (20) and underscores the clinical implications of these findings. 93 For example, our results predict that intrafemoral injection of transplants is unlikely to be a useful strategy for improving the therapeutic effectiveness of H S C s induced to expand in vitro. To date, interference of SDF-1 action by specific C X C R 4 inhibitors has been used primarily for enhancing the yield of H S C s from donors for transplantation into myeloablated patients (71;72). Another application of such inhibitors suggested by the findings reported here could be to treat recipients of transplants of cycling cells. Thus significant benefit might also be derived by pretreatment of the host, particularly when transplants of genetically modified or cultured cells are to be administered since half of the HSCs in an asynchronously dividing population would be expected to be in S/G2/M. MATERIALS AND METHODS Mice. Ly5-congenic strains of C57B1/6 mice were used as donors and recipients. A l l recipients were also homozygous for the W41 allele. Mice were bred and maintained in microisolators with sterile food, water and bedding at the B C Cancer Research Centre according to protocols approved by the University of British Columbia Animal Care Committee. Cells. Single cell suspensions were prepared in Hank's balanced salt solution containing 2% fetal calf serum (FCS) (HF/2, StemCell Technologies). Enriched populations of HSCs were obtained by immunomagnetic removal of Ter l 19 + or l i n + cells from fetal liver and bone marrow cell suspensions, respectively (using EasySep\u00E2\u0084\u00A2, StemCell Technologies). Antibodies used for isolation of lin\" cells between 4 and to 10 weeks of age were anti-B220, Ter l 19, anti-G r l , an t i -Lyl and anti-Macl (StemCell Technologies). To isolate lin\" cells from 3 week-old 94 mice, the M a c l antibody was omitted because M a c l was known to be expressed on fetal and cycling H S C s (13;14;73). Tritiated3H-Tdr suicide assay. Cells were suspended at 10 6/ml in Iscove's medium containing 5 x 10\"5 mol/12-mercaptoethanol, a serum substitute (BIT\u00E2\u0084\u00A2, StemCell Technologies) and 50ng/ml murine SF (StemCell Technologies). Equal volumes were then incubated at 37\u00C2\u00B0C, in 5% CO2 in air for 16 hours in 35 mm petri dishes in the presence or absence o f 20 uCi /ml of 3 H-Tdr (25 uCi/mmol; Amersham). The cells were then harvested, washed twice with Iscove's medium containing 2% F C S and limiting dilution C R U assays performed. FACS isolation and analysis of cells in different cell cycle phases. Cells were suspended in H F / 2 containing 1 pg/ml Hst (Molecular Probes/Invitrogen) either 10 pmol/1 fumitremorgin C (a gift from Dr. Susan Bates, N I H , Bethesda, M D ) or 50 pmol/1 reserpine (Sigma Chemicals) and then incubated at 37\u00C2\u00B0C for 45 minutes. PyroninY (Py; Sigma) was added at 1 pg/ml and the cells incubated for another 45 minutes at 37\u00C2\u00B0C. Cells were washed twice in HF/2 with 1 pg/ml PI (Sigma) in the second wash and were finally resuspended in HF/2 with PI and kept on ice in the dark until being sorted (PT cells only) on a 3 laser F A C S Vantage (Becton Dickinson). For analysis of D N A content, cells were either re-stained with Hst only using the same protocol, or with PI at least 1 hour after storage at 4\u00C2\u00B0C of cells that had been washed twice in ice-cold P B S with 0.1% glucose and fixed in 1 ml of ice-cold 70% ethanol. To stain the cells with PI, cells were washed twice with 2% P B S and resuspended in P B S with 0.1% glucose, 5 pg/ml PI and 200 ug/mL R N A s e A . Cells were then incubated for at least 1 hour at 4\u00C2\u00B0C and then analyzed directly on a FACSCal ibur (Becton Dickinson). To stain sorted cells 95 for K i 6 7 , the cells were washed and resuspended in 50 ul of ice-cold 80% ethanol and then incubated at -20\u00C2\u00B0 C for at least 2 hours. The fixed cells were washed twice in 300 ul of P B S with 1% F C S and 0.09% NaN3 (pH=7.2). Fluorescein isothiocyanate (FITC)-conjugated anti-human K i 6 7 antibody (Becton Dickinson) was then added and the cells incubated for 30 minutes at room temperature in the dark. Cells were then analyzed by F A C S , using cell stained with a FITC-conjugated mouse IgGi (Becton Dickinson) as a control. In vitro treatment ofS/G2/MHSCs. Sorted cells were incubated at 37\u00C2\u00B0C in 5% CO2 in air in the wells o f a round-bottom 96-well plate in serum-free media (as for the H-Tdr suicide assays) with one of the following 6 additions: 100 ng/ml SDF-1 (a gift from I. Clark-Lewis, Biomedical Research Centre, University of British Columbia, Vancouver, B C , Canada) or 300 ng/ml SDF-1 G2 but no SF, or 50 ng/ml SF alone, or 50 ng/ml SF plus 100 ng/ml SDF-1 or 300 ng/ml SDF-1 or 300 ng/ml SDF-1G2. Cells were then harvested from the wells and equal aliquots injected into recipient mice such that each mouse received the equivalent of either 4 x 10 3 starting G i cells or 1.2 x 10 4 starting S / G 2 / M cells. CRU assay. Recipient (W41/^1) mice were sublethally irradiated with either 400 cGy of 1 3 7 C s y-rays or 360 c G y o f 250 k V p X-rays and then injected intravenously with the test cells except when injected intrafemorally as indicated. Intrafemoral injections were performed as described (74). C R U s were identified by their ability to have generated >1% donor Ly5-type blood cells including L y l + (T-cell), B 2 2 0 + (B-cell) and G r l / M a c l + (granulocyte/monocyte) subsets that could be detected >16 weeks after transplantation (75). C R U frequencies were calculated using the L-calc program (StemCell Technologies) from the proportions of mice that were negative for this endpoint. Recipients treated with SDF-1 G2 (a gift from I. Clark-96 Lewis) were injected intravenously with 10 |xg per mouse of SDF-1 G2 dissolved in PBS 2 hours after being irradiated and were then transplanted another 2 hours later. This schedule was used in an attempt to minimize direct interaction of the injected H S C s with SDF-1 G2 in the circulation (based on the likely rapid clearance of SDF-1 G2) and maximize any potential effect on the host by keeping the interval between injecting the SDF-1 G2 and the transplant as short as possible. Controls were injected with P B S instead of the SDF-1 G2. Real-time PCR. Cells were sorted into 1 ml HF/10 and R N A was isolated using the PicoPure\u00E2\u0084\u00A2 R N A Isolation K i t (Arcturus Biosciences Inc.) as recommended by the supplier including a 15 minute D N A s e l treatment (Qiagen) on the column at room temperature. R N A was eluted into an 11 ul volume and stored at -80\u00C2\u00B0C. A c D N A preparation was then generated using the Superscr ip t\u00E2\u0084\u00A2 III First-Strand Synthesis System for R T - P C R (18080093, Invitrogen) again as recommended by the manufacturer, with the reaction scaled up to use 25ul. Quantitative real-time P C R was performed using the following primer pairs (5' to 3'): a4int (NM_010576.2) forward primer A G G A C A C A C C A G G C A T T C A T , reverse primer C C T C A G T G T T T C G T T T G G T G ; CD44 (NM_009851.1) forward primer C T T T A T C C G G A G C A C C T T G G C C A C C , reverse primer G T C A C A G T G C G G G A A C T C C ; c-Kit (NM_021099.2) forward primer A C A A G A G G A G A T C C G C A A G A , reverse primer G A A G C T C A G C A A A T C A T C C A G ; c-mpl (NM_010823.1) forward pimer A G T G G C A G C A C C A G T C A T C T , reverse primer G A G A T G G C T C C A G C A C C T T ; GXCR4 (NM_009911.2) forward primer C G G A G T C A G A A T C C T C C A G T , reverse primer C T G G T C A G T C T C T T A T A T C T G G A A A A ; Gapdh (NM_008084) forward primer A A C T T T G G C A T T G T G G A A G G , reverse primer A T G C A G G G A T G A T G T T C T G G ; SDF-1 (NM_001012477) forward primer G A G C C A A C G T C A A G C A T C T G , reverse primer 97 C G G G T C A A T G C A C A C T T G T C ; VCAM-1 (NM_011693.2) forward primer T G A T T G G G A G A G A C A A A G C A , reverse primer A A C A A C C G A A T C C C C A A C T T . The relative expression changes were determined with the 2 \" A A C T method (76), and the housekeeping glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene transcript was used to normalize the results. Statistical analyses. Comparisons were made using the Wald test. 98 CM o 0) o. on o 25 20 15 10 5 E14.5 FL 5-FU E14.5 FL 3H-Tdr Wk 10 BM ^H-Tdr E18.5 FBM 3H-Tdr a> I 100 > a 10 o a> $ 1 c a> > 100-1 50-1 0 G^S/Gj/M 60% 10% 0 60 100 150 200 260 C 4 week-old Lin- BM 260-200 > .E 160 c o >> 100 0. 60 0 \u00E2\u0080\u00A2 l \u00E2\u0080\u00A2 w \u00E2\u0080\u00A2 ^^23% mm 0 60 100 160 200 260 Hoechst 33342 o f M , l w , r o , , \u00E2\u0080\u0094 A 0 200 400 600 800 1000 0 200 400 600 800 1X0 PI PI 260 / 200- 70% 150- I 100 60-0-10 week-old Lin- BM 60 100 160 200 260 Hoechst 33342 Figure 2.2: F A C S profiles of the distribution o f different lin\" populations in G 0 , G i and S / G 2 / M . A . The left panel shows a representative F A C S contour plot for E l 4 . 5 Ter l 19\" fetal liver (FL) cells after staining with Hst and Py. Right panel shows the profile for the same cells after staining for K i67 . B. The left panel shows a representative F A C S contour plot for lin\" bone marrow ( B M ) cells from 3 week-old mice after staining the cells with Hst and Py. The middle panel shows the profile for the sorted Go cells after staining for K i 6 7 (>90% of the Go cells showed no K i 6 7 expression). The right panel shows the profile for the sorted G1/S/G2/M cells after staining for K i 6 7 (>99% of the G i / S / G 2 / M cells expressed Ki67). C . and D. Representative F A C S contour plots for lin\" B M cells from 4 week-old and 10 week-old mice after staining the cells with Hst and Py. 100 o TJ Q) C O a> a a E14.5FL 3 wk BM 4 wk BM 10wkBM o o S o cT o M Figure 2.3: The cycling activity of C R U s is down-regulated between 3 and 4 weeks o f age. Results shown are the number of C R U s per 10 5 initial total viable cells. For each tissue source, the difference in the yields of C R U s in the 2 subsets compared was significantly different (P<.001). For fetal liver (FL), these were depleted of Ter l 19 + cells; for the 3 and 4 week-old bone marrow ( B M ) cells, all l i n + cells except M a c l + cells had been removed and for, the 10 week-old B M cells, all l i n + cells including M a c l + cells were removed. Values shown are the mean \u00C2\u00B1 S E M from data pooled from at least 3 experiments per tissue. 101 A Before culture G 1 + 6hrs S/G2/M + 6hrs Before culture (3 O I t CM ' o 35 G 1 + 6hrs S/G2/M + 6hrs == o \u00C2\u00A7 Bi J-Sorted ted S/G2 Bi s-Sorted ted S/G2 \u00C2\u00A3 O 65-fold increase (P<.001) in the number of C R U s detected when C R U s in S/G2/M were cultured and a >128-fold increase (P<.00T) when the cultured cells were sorted for Gicells. Values shown are the mean \u00C2\u00B1 S E M o f results from at least 3 experiments. 102 A In vivo treatments Figure 2.5: The engraftment defect o f H S C s in S/G2/M is corrected by treatment of the host, but not the cells, with SDF-1 G2. A . Effect of injecting prospective recipients, 2 hours post-irradiation and 2 hours prior to transplant with 10 ng/ml SDF-1 G2(+) or P B S (-). Starting equivalents of 4,000 G] cells per recipient mouse or 12,000 S/G2/M cells per recipient mouse were similarly tested. Results show a new ability of fetal liver (FL) H S C s in S/G2/M and 3 week bone marrow ( B M ) H S C s in S / G 2 / M to engraft only when they are transplanted into SDF-1 G2 treated recipients, whereas treated recipients were no more likely to be engrafted long term by H S C s in G i than were untreated recipients. Results are combined from 3 independent experiments. B. Effect o f in vitro treatment of sorted Ter l 19' F L cells in G i or S/G2/M for 30 minutes at 37\u00C2\u00B0C in serum-free medium plus various additives, as shown, on C R U detection. When present, SF was used at a concentration of 50 ng/mL, SDF-1 at either 100 ng/ml or 300ng/m and SDF-1G2 at 300 ng/ml. In vitro treatment had no significant effect on the number o f mice that subsequently showed multi-lineage repopulation from starting cells in either G i or S/G2/M. Results are combined from 3 independent experiments. 103 A G, cells into SDF-1 G2-treated recipient 10\" O 103 Q. < . 10* 10\" 44 t 3 o ^ ^ ^ 4 1 (^Hi)12 Po((, 10J 10* Gr-1/Mac-1 - PE 10\u00C2\u00B0 101 102 10; B220 - PE B S/G2/M cells into SDF-1 G2-treated recipient 10' 10\" 10* 10' 10\u00C2\u00B0 101 102 10: 27 32 w ft\" B220 - PE Gr-1/Mac-1 - PE C S/G2/M cells into PBS-treated recipient 10\" i \u00C2\u00A3 1 0 -< ^ 1 0 * 10\" 10\" 10\u00C2\u00B0 10* 10\" 10\u00C2\u00B0 101 102 103 104 Gr-1/Mac-1 - PE 10u 1 ob\" 10\u00C2\u00B0 101 102 103 10* B220 - PE 10* 10\" 10\" 101 10\" | 28 17 # 8 10\u00C2\u00B0 101 102 103 104 Ly1 - PE 10\u00C2\u00B0 101 102 103 104 Ly1 - PE -[Jjfj CO 10\u00C2\u00B0 101 102 103 104 Ly1 - PE Figure 2.6: Donor-derived repopulation o f SDF-1 G2-treated mice. Shown are representative F A C S profiles of donor-specific cells detected after dual staining for the donor-type Ly5 allotype and various lineage-specific markers. A . Example of a positively engrafted PBS-treated recipient of fetal liver (FL) cells in G | . B. Example o f a positively engrafted SDF-lG2-treated recipient o f F L cells in S / G 2 / M . C . Example of a PBS-treated recipient o f F L cells in S / G 2 / M that does not show donor-derived hematopoiesis. 104 10 \u00E2\u0080\u00A2 F L G 1 \u00E2\u0080\u00A2 F L S / G 2 / M H 3 w k G 1 \u00E2\u0080\u00A2 3 wk S / G 2 / M CD44 SDF-1 CXCR4 a4-lnt c-kit c-mpl VCAM-1 Figure 2.7: Gene expression analysis of the G i and S / G 2 / M subsets of highly purified lin\" Seal C D 4 3 + M a c l + HSCs from fetal liver and 3-week bone marrow. Gene expression in G i was set equal to 1 and the fold change in transcript levels in the corresponding S / G 2 / M fraction is shown. Results shown are the mean \u00C2\u00B1 S E M of data from 2-3 biological replicates measured in triplicate. The difference between the level of SDF-1 transcripts between the 2 pairs of G i and S / G 2 / M samples is significant, P<.05) 105 Table 2.1: Limit ing dilution data for C R U frequency determinations for E l 8 . 5 fetal liver and bone marrow cells (data pooled from 5 experiments). fetal liver bone marrow No. of cells injected No . of negative mice No. of cells injected No. of negative mice per mouse (xlO 3 ) per total mice injected per mouse (xlO 2 ) per total mice injected 2800 1 /9 4725 0 / 4 250 0 / 6 2800 1/9 80 5 / 2 0 2500 0 / 3 20 11 /14 1575 1/4 800 6 / 1 2 200 9 / 1 5 C R U frequency 1 / 73,000 C R U frequency 1 / 95,000 (range defined + 1 / 59,000 (range defined + 1 / 75,400 b y \u00C2\u00B1 S E M ) -1/91,000 by \u00C2\u00B1 S E M ) , - 1 /119 .000 106 Table 2.2. Limit ing dilution data for C R U frequency determinations for l in bone marrow cells from 3 and 4 week-old mice (pooled data from 2 experiments). 3-week bone marrow 4-week bone marrow N o . of cells injected No. of negative mice per mouse (x 10 3) per total mice injected No. of cells injected No. of negative mice per mouse (x 103) per total mice injected 12 0 / 3 4 4 / 6 11 0 / 3 4 4 / 6 C R U frequency 1 / 6,500 (range defined + 1 / 4,000 b y \u00C2\u00B1 S E M ) - 1 / 1 0 , 0 0 0 1 \u00E2\u0080\u00A2 . C R U frequency (range defined by \u00C2\u00B1 S E M ) 1 / 6,300 + 1 / 4,000 - 1 / 10,000 107 R E F E R E N C E S 1. Szilvassy, S.J., Humphries, R.K., Lansdorp, P.M., Eaves, A.C., and Eaves, C.J. 1990. Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc. Natl. Acad. Sci. USA. 87:8736-8740. 2. Harrison, D.E., Astle, C M . , and Lerner, C. 1984. 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Methods. 25:402-408. 119 CHAPTER 3 HEMATOPOIETIC STEM CELLS TRANSITION ABRUPTLY AND PROGRAMMATICALLY FROM A FETAL TO AN ADULT STATE. The work presented in this Chapter was submitted for publication Michelle B. Bowie, David G. Kent, Brad Dykstra,, Kristen D. McKnight, Lindsay McCaffrey Pamela A. Hoodless and Connie J. Eaves David Kent isolated the R N A and generated the cDNA preparations, Brad Dykstra assisted with the analysis of stage-specific GM-contribution, Kristen McKnight isolated E l 8.5 fetal bones and Lindsay McCaffrey assisted with C R U analysis. 120 INTRODUCTION The identification in the 1950's of cells in adult mouse bone marrow that can individually regenerate all lineages of the blood and lymphoid systems provided the first direct evidence of a pluripotent hematopoietic stem cell (HSC) (1 ;2). Since that time much effort has been devoted to the development of quantitative assays for enumerating, characterizing and purifying HSCs . Currently there is general agreement that HSCs can be most specifically identified by endpoints that detect competitively and permanently (>4 months) repopulated immunologically compatible recipients whose endogenous blood-forming system has been seriously compromised (e.g., by irradiation and/or genetic modification). H S C quantitation can then be achieved using a limiting dilution approach coupled with a strategy for keeping the recipients alive with a minimally competitive source and dose of H S C s (3). This has allowed many shared biological properties and associated molecular features of H S C s from different stages of ontogeny to be identified (4). However, in spite of these common properties, extensive heterogeneity in their individual behaviour is a hallmark of every source of H S C thus far studied (5-8). Changes in the behaviour of H S C s from different stages of development have also been recognized for many years. O f particular note is the finding that H S C s from midgestation mouse fetal livers (embryonic day (E)14.5) regenerate HSCs in irradiated recipients at a faster rate than is seen with transplants of adult bone marrow HSCs (9-11). This, in turn, results in an increased rate of regeneration of derivative myeloid progenitor cell types detected by short term colony assays both in vivo (colony-forming unit-spleen, CFU-S) and in vitro (colony-forming cells, CFCs) and their more mature progeny (6; 11; 12). The size, content and longevity of clones produced in vivo by individual H S C s likely reflects their immediate self-renewal history; that is, the frequency with which they have generated one or 121 more daughter HSC(s) during their expansion post-transplant. However, changes in other H S C properties during development could also affect their clonal outputs post-transplantation: for example, changes that control their proliferative activity, cell cycle time, survival, or lineage-restriction. Comparisons of the responses of genetically-altered H S C s and their wi ld type counterparts stimulated in identical environments have led to a growing list of intrinsic determinants of H S C self-renewal activity (13-20), and a few of these have been reported to affect fetal and adult HSCs differentially (13;20). Definitive evidence that the self-renewal of H S C s can be modulated by the types and concentrations of growth factors to which the H S C s are exposed both in vivo (21) and in vitro (14;22-24) has also been demonstrated. Some of these latter effects differ on fetal and adult HSCs , such as in vitro conditions that support adult but not fetal H S C self-renewal (25). Interestingly, the timing of changes in the influence of specific determinants of H S C regenerative properties during development has not been previously explored. Recently, we showed that fetal mouse H S C s are maintained as a wholly cycling population until 3 weeks after birth, at which point they switch within one week to a largely quiescent population (as shown in Chapter 2). Here we show that this rapid change in H S C proliferative activity is accompanied by a similarly abrupt switch in the regenerative and differentiation properties these cells display after transplantation into irradiated recipients. In addition, we show that this switch occurs independently of the age of the host in which the H S C s are amplifying and is accompanied by pronounced changes in their transcriptional control. 122 RESULTS Kinetic analysis of the different rates of HSC production by transplants from fetal and adult sources Previous reports have provided some limited, albeit key information about the different kinetics of H S C regeneration in irradiated recipients of fetal and adult H S C s (6; 18), including effects of the dose as well as the source of the H S C s (CRUs) initially transplanted (26). Therefore, to maximize the differences displayed by HSCs from different sources, while also restricting other possible contributing variables, the number of C R U s transplanted into each primary W41 recipient was set at 10, regardless of the tissue from which they had been obtained (i.e., the contents of either 10 5 Ter l 19\" E l 4 . 5 fetal liver cells (27) or 10 s lin\" adult bone marrow cells (28)). Groups of recipients were then sacrificed from one to 24 weeks later and the changing bone marrow content of in vivo amplified donor-derived H S C s determined by performing limiting dilution transplantation assays in secondary W41 recipients. This general experimental design is shown schematically in Figure 3.1 A . The pooled results from 4 such experiments revealed 2 distinct and highly consistent patterns of C R U amplification in primary recipients of fetal liver and adult bone marrow C R U s (Figure 3.IB). Initially (during the first week), there was no net change in the number of donor-derived H S C s detected in the bone marrow of the transplanted mice. Then, between the first and second week, the number of fetal liver-derived C R U s expanded rapidly (8-fold), whereas during the same interval, bone marrow-derived C R U s increased their numbers very little (1.5-fold). Thereafter, the rates of expansion of the 2 populations of C R U s in vivo became similar, doubling approximately every week, until saturation of the regenerated C R U content. 123 To determine whether the different kinetics of C R U regeneration obtained from transplants of fetal liver and adult bone marrow cells might be due to the co-transplantation of accessory cells unique to one of these tissues, the rates of regeneration of the progeny of each was re-examined in mice injected with both (i.e., transplants of Ly5.1 fetal liver cells containing 10 C R U s were co-injected with Ly5.2 adult bone marrow cells, also containing 10 C R U s , into both Ly5.1 and Ly5.2 W41 recipients) and the C R U output from each donor C R U type was then measured 2, 4 and 8 weeks later in secondary recipients. The results of this experiment with mixed primary transplants showed that the kinetics of C R U regeneration from the co-injected fetal liver and adult bone marrow C R U s were the same as when these cells were transplanted separately (data not shown). Taken together, these results show that fetal and adult H S C s expand at markedly different rates in irradiated mice during the second week after transplantation and that these differing rates of expansion are cell autonomous. Identification of an abrupt and programmed switch in the regenerative properties of HSCs between 3 and 4 weeks after birth We next sought to investigate the timing and pace of change in H S C (CRU) regenerative properties from those typical of the C R U s present in the E l 4.5 fetal liver to those typical of the C R U s present in adult bone marrow. We therefore repeated the experiment shown in Figure 3.1 A using bone marrow cells from mice at intermediate stages of development (i.e. E l 8.5 fetal bone marrow, and 3 and 4-week post-natal bone marrow) as the cells initially transplanted. In each case, preliminary experiments were undertaken to define the number of cells that contained 10 C R U s . This dose was then injected into each primary recipient and secondary limiting dilution transplants were performed from one to 4 weeks later to determine 124 the kinetics of expansion of H S C s derived from the injected cells initially transplanted into the primary recipients. These experiments focused on the initial 4-week period post-transplant because the greatest difference between the rate of expansion of fetal liver and adult bone marrow C R U s in vivo had been seen at this time (Figure 3.IB). A s shown in Figure 3.2A and B , the results of the experiments with C R U s from donors at intermediate stages of development revealed that the E l 8 . 5 fetal bone marrow and week 3 post-natal bone marrow C R U s expanded with the same kinetics as the E l 4 . 5 fetal liver C R U s , whereas the week 4 post-natal bone marrow C R U s behaved just like the transplants of adult bone marrow C R U s . We next asked whether this abrupt transition in the regenerative properties of C R U s was unique to the weanling mouse entering puberty or whether a similarly timed switch might also be seen during the expansion of fetal liver-derived C R U s in adult recipients. To discriminate between these possibilities, we transplanted secondary mice with 10 C R U s that had been regenerated in primary recipients of 10 fetal liver C R U s transplanted 6 weeks previously. We then assessed the kinetics of expansion of the progeny of these in vivo expanded fetal liver C R U s during their further expansion in the secondary recipients by performing limiting dilution C R U transplants in tertiary recipients (Figure 3.1 A ) . This experiment showed that within 6 weeks, the C R U s derived from fetal liver C R U s had acquired the regenerative properties of adult bone marrow C R U s (Figure 3.2C). Thus, regardless of the age or physiological conditions of the host in which fetal liver C R U s proliferate, they generate a cohort of C R U s with \"adult\" C R U regeneration properties within the same time frame. 125 Identification of an abrupt and programmed switch in the differentiation behaviour of HSCs between 3 and 4 weeks after birth To investigate whether the changes in H S C (CRU) regenerative activity were accompanied by parallel changes in their differentiation properties, we examined the different types of W B C s they produced at 16 weeks (Figure 3.3). The percentage of all donor W B C s that expressed L y 6 G and/or M a c l ( G M ) was more than 2-fold higher in recipients of fetal liver C R U s than in recipients of adult bone marrow C R U s . A fetal liver-like G M output was obtained in primary recipients of E l 8.5 bone marrow and 3 week-old bone marrow cells, as well as in secondary recipients of C R U s from mice transplanted with fetal liver C R U s 1 -2 weeks earlier. In contrast, an adult-like output change of G M was seen in secondary recipients of C R U s from mice transplanted with 3 week-old bone marrow cells, 2 weeks earlier. Similarly, this adult-like output was seen in primary recipients of 4 week-old bone marrow cells, secondary recipients of adult bone marrow cells, and tertiary recipients of C R U s from secondary mice that had been transplanted 1 -2 weeks earlier with bone marrow from primary recipients of fetal liver C R U s transplanted 6 weeks previously (refer to Figure 3.1 A schematic - indicated by asterisks on the 3\u00C2\u00B0 line). Interestingly, a G M output intermediate between that exhibited by fetal liver and that by adult bone marrow C R U s was seen in secondary recipients of C R U s from primary mice that had been transplanted with 3 week-old bone marrow cells for just 1 week (chequered bar), suggesting that the C R U s ultimately tested were comprised of a mixture of fetal-like and adult-like C R U s . Collectively, these findings point to the operation of an intrinsically controlled switch that alters the differentiation properties of C R U between 4.5 and 5.5 weeks after E14.5 (i.e. approximately 3-4 weeks after birth), regardless of whether the fetal liver C R U s are left in situ in their endogenous developmental setting or are transplanted directly into a myeloablated adult recipient. 126 The cell cycle transit time of self-renewing HSC does not change during ontogeny The different rates of H S C (CRU) expansion measured in recipients of fetal and adult C R U s could be influenced by several parameters: differences in the frequency of symmetric self-renewal divisions undertaken, differences in H S C cell cycle transit times and differences in H S C death. These are difficult parameters to measure in vivo but can be assessed in single cell cultures of highly purified C R U populations maintained under conditions that optimize each of these parameters. We have previously reported the kinetics of division of approximately 40% pure populations of adult bone marrow C R U s cultured under conditions that allow a 2- to 4-fold net expansion of their numbers (14;24). Experiments showed that E l 4.5 fetal liver C R U s could be purified to purity of approximately 10% by isolation of the l i n \" S c a - l + M a c l + C D 4 3 + subset as shown by both limiting dilution transplants and transplants of single purified cells (Figure 3.4A). In preliminary experiments (for a detailed description please refer to Chapter 4), we found that 50 ng/ml SF gave a full net maintenance of fetal liver C R U numbers after 48 hours in culture and this growth factor condition was superior in this regard to any of a variety of other growth factor cocktails tested, including that found to be optimal for adult bone marrow C R U s (i.e., 300 ng/ml SF + 20 ng/ml IL-11 \u00C2\u00B1 1 ng/ml Flt3-ligand (14;24)). The kinetics of cell division of fetal liver H S C s were then monitored visually in single cell cultures containing 50 ng/ml SF and the results compared to previously published data for adult bone marrow HSCs (14;24). A s shown in Figure 3.4B, under these conditions, fetal liver HSCs divided with the same 14-hour cell cycle transit time as adult bone marrow HSCs , but without any initial delay, due to the fact that all of the fetal liver H S C s are in cycle whereas all of the purified adult bone marrow H S C s are quiescent (see Chapter 2). In addition, it can be seen that there was essentially no cell death in these cultures as previously shown for optimally stimulated adult 127 bone marrow H S C s (24). Thus, under optimally mitogenic conditions that also preserve H S C activity, all o f the fetal liver HSCs completed a first division within 28 hours whereas adult bone marrow H S C s would be just starting to divide at this time. More prolonged monitoring of the fetal liver H S C cultures showed that the fetal liver cells continued to proliferate in the same semi-synchronous fashion as previously shown for adult bone marrow HSCs and were thus completing their second and third divisions at the same times as adult bone marrow cells were found to be completing a first and second division, respectively (Figure 3.4C). Based on these findings, it seems likely that the expansion of fetal liver and adult bone marrow H S C s stimulated in the bone marrow of myeloablated mice also occurs with minimal H S C death, with the same cell cycle transit times for both sources of HSCs . Accordingly, differences in the frequency of symmetric self-renewal divisions would be inferred. Interestingly, the same kinetics of division were seen when these highly purified fetal liver cells were cultured in the cocktail used for adult bone marrow H S C s (i.e., 300 ng/ml SF + 20 ng/ml IL-11), even though this was found to lead to a rapid loss of H S C activity (see Chapter 3), suggesting that the cell cycle transit times of H S C s undergoing an initial differentiation step is the same as that of H S C s maintaining self. Identification of differences in gene expression between purified fetal liver and adult bone marrow HSCs that also distinguish the same phenotypes of cells in the bone marrow of 3 and 4 week-old mice Identification of an abrupt transition between 3 and 4 weeks after birth of a number of properties in H S C s that distinguish these cells in the fetal liver and adult bone marrow (see Figure 3.5) suggested that changes in these properties might be accompanied by corresponding changes in gene expression. To test this hypothesis, highly enriched E14.5 fetal liver ( l inSca-128 1 + M a c l + C D 4 3 + , Figure 3.4A) and adult bone marrow (lin\"Rho d u l l SP) (24) H S C s populations were isolated (-20% and 40% pure, respectively), R N A extracts obtained and c D N A preparations made. Similar isolates were obtained from the l i n \" S c a - l + M a c l + C D 4 3 + and lin\" R h o d u l l S P cells isolated from 3 and 4-week old bone marrow cells, respectively, based on the assumption that the phenotype of their HSCs , like their cycling (Chapter 2), self-renewal and differentiation properties (Chapter 3) would be similar to the phenotype of fetal liver HSCs (Chapter 3) and adult bone marrow H S C s (24), respectively. We then looked for changes in the levels of transcripts for a number of candidate genes. Several genes previously reported to play a role in H S C expansion (cyclinD2, Ikaros, rae-28 and MEF) were found to be more highly expressed (P<.05) in fetal liver and 3-week bone marrow H S C s as compared to H S C s in 4 and 10 week bone marrow (Figure 3.6). Bmi-1, c-Kit, Gfi-1, Notchl and Ship showed no difference in expression in any of the HSC-enriched samples. However, a marked and permanent upregulation of expression of ATM, Ezh2, Gata-2 and Runxl was noted in the purified H S C s of 4 and 10 week-old bone marrow as compared to their earlier counterparts (P<.05). Increased expression of Sci in adult vs. fetal H S C s was also noted, but expression of this gene was not tested in the 3 and 4-week bone marrow HSCs . DISCUSSION Here we have identified a unique time point in the expansion of H S C s when these cells undergo an abrupt and tightly regulated change in properties that control their ability to execute symmetric self-renewal divisions. After this point, the H S C s show a decreased ability to rapidly generate large outputs of progeny in long-term in vivo reconstitution assays. Concomitant with this change in self-renewal control is an alteration in the relative proportions of terminally differentiated cells they generate, as evidenced by a changed average 129 output of granulocytes and monocytes. O f note, the timing and unexpectedly abrupt kinetics of these co-ordinated changes in H S C properties mirror precisely the change in cycling status of H S C s in vivo (Chapter 2). Unt i l 3 weeks after birth, the H S C s remain a wholly cycling population, but between 3 and 4 weeks after birth they are converted to a largely quiescent population. Taken together, these findings point to the existence of a novel and rapidly executed master switch that controls a spectrum of key H S C properties. Interestingly, although there is no previous report of such a switch at this time, several studies have described changes in other H S C properties around this time (expression of CD34, CD38, and podocalyxin and Hoechst33342 and Rhodamine-123 efflux properties) (29-35). We also show here that when optimally stimulated, fetal liver H S C s (El4.5) and adult bone marrow H S C s have negligible apoptotic frequencies and identical cell cycle times (14 hours). In this latter respect, they differ from the more mature progeny that are generated from these cells which, in the fetal liver, clearly cycle more rapidly (36;37). Thus the enhanced rate of expansion of fetal liver H S C s after transplantation into irradiated adult hosts is likely due to differences in the intrinsic mechanisms that control their self-renewal ability, as opposed to differences in regulators of their cell cycle transit times. The concept of an intrinsically determined switch regulating H S C functions was further supported by experiments indicating that the rate of H S C regeneration post-transplant changes after a fixed period of H S C expansion, independent of the particular in vivo environment in which the expansion took place (Figure 3.2C). To gain further insight into genes that might be involved in these developmentally programmed changes in H S C behaviour, we looked for correlated patterns of change in the expression of a number of candidates previously implicated in H S C control (reviewed in Chapter 1). This survey included the following specific genes: ATM, which has been shown to 130 play a role in mediating the stress-response of H S C s (38); Bmi-1, mel-18, rae-28, and Ezh2, P c G proteins thought to be important in the initiation (Ezh2) and maintenance (Bmi-1, mel-18, rae-28) of gene repression and involved in the regulation of H S C self-renewal (39-41); cyclinD2, a gene that is upregulated in proliferating cells and may contribute to H S C expansion when overexpressed (42); 2 genes that play critical roles in adult but not fetal HSCs ; i.e. c-Kit, the receptor for SF, a ligand that promotes self-renewal signalling (13) and Gfi-1, a zinc finger repressor thought to restrict the proliferation of H S C s (43); Gata-2, whose expression in fetal and adult HSCs is driven by different promoters (44;45); Ikaros, a D N A -binding subunit critical for H S C s expansion (46); MEF, a gene whose absence in HSCs results in their increased quiescence (47); Notchl (48-50), Runxl (51-53) and Sci (54-57) 3 genes critical to the generation of H S C s in the fetus but not in the adult (Chapter 1); and Ship; of interest due to a possible role in HSCs (58) and of major interest to the lab of Dr. Gerry Krystal, also in the Terry Fox Laboratories. These studies revealed a remarkably consistent pattern of gene expression in the 4 populations analyzed (E14.5 fetal liver HSCs and 3, 4 and 10 week-old bone marrow HSCs) . Although changes in the expression of Gata-2 were consistent with the fact that the promoter used by this gene changes during development (45), none of the results obtained for other categories of genes were predicted. For example: within the P c G genes: expression of Bmi-1 was unchanged whereas expression of rae-28 was down-regulated after 3 weeks and the opposite was true for Ezh2 and mel-18. Previous evidence of differential roles in fetal liver and adult bone marrow H S C s (e.g., Runxl, Sci and Notchl) was similarly unpredictive. Nevertheless, for those genes whose expression did change, a remarkably consistent pattern was identified, as might be expected for a major switch in programming, in which genes involved in the expansion of H S C s through promotion of cell cycle or self-renewal (CyclinD2, 131 Ikaros, MEF and rae-28) were more highly expressed in H S C s with fetal liver-like properties and genes involved in H S C maintenance {ATM, Ezh2, Gata-2, and mel-18) were more highly expressed in H S C s with adult properties. It is interesting to note that the levels of expression of ATM, Ezh2, and Gata-2 were particularly elevated in the H S C s from 4-week bone marrow. It is thus conceivable that the products of these genes, or the regulators of their expression, may play an important role in regulating the actual transition that takes place in these cells between 3 and 4 weeks of age. It w i l l also be important to confirm the H S C purities in the 3 and 4-week bone marrow phenotypes analyzed and to investigate their potential functional effects on influencing the developmental switch herein described. M A T E R I A L S A N D M E T H O D S Mice. C57Bl/6-Pep3B-Ly5.1 (Pep3B) and congenic C5TBM6-W4l/W4,-lsf52 (W41) mice were bred, maintained and used in experiments as donors and recipients, respectively, in the Animal Resource Centre of the B C Cancer Agency according to protocols approved by the University o f British Columbia in accordance with Canadian Council o f Animal Care guidelines. A l l mice were kept under microisolation conditions and supplied with sterile food and water. Cell preparation. Livers were removed from E14.5 Pep3b fetuses and fetal femurs from E l 8.5 Pep3b fetuses. Both tissues were placed in Hank's balanced salt solution (HF: StemCell Technologies) containing 2% fetal bovine serum (FBS) (HF/2), and a cell suspension obtained by forcing the tissue through a sieve using the plunger of a 3 ml syringe. Ce l l aggregates were removed using a 70 pm filter and, where indicated, Ter l 19 + (erythroid) cells were removed 132 immunomagnetically (using EasySep reagents and equipment; StemCell Technologies) as recommended by the supplier. Bone marrow cells were harvested from 3 week, 4 week and 3-4 month-old Pep3B mice by flushing excised femurs and tibiae with Dulbecco's Min imum Essential medium ( D M E M ) containing 2% F B S . Where indicated, L i n + (B220 + , Ter l 19 +, L y 6 G + , M a c l + , L y l + ) cells were removed immunomagnetically (using EasySep). Purification of HSCs. E14.5 Ter l 19\" fetal liver and 3 week-old bone marrow cells were first incubated with biotinylated anti-Grl (RB6-8C5, granulocytes), anti-B220 (RA3-6B2, B lymphocytes), and ant i -Lyl (53-7.3, T lymphocytes), all of which were prepared in the Terry Fox Laboratory, TER-119, anti-CD4 and a n t i - N K l . l (all from Becton Dickenson [BD]), and phycoerythrin (PE)-labelled anti-Sca-1 (BD) and various combinations of the following: fluorescein isothiocyanate (FITC)-conjugated anti-CD43, or anti-CD34 and allophycocyanin (APC)-conjugated anti-Mac 1 or c-Kit (all from B D ) . 4 week-old bone marrow or 10 week-old bone marrow cells were similarly stained for lineage-antibodies, including M a c l , selecting those that are negative for these, and enriched for cells that did not retain the dyes Rhodamine and Hoechst 33342, as previously described (24). Staining was carried out with cells in ice-cold H F plus 5% rat serum (Sigma Chemicals) and 3 ug/ml Fc receptor blocking antibody (2.4G2) at 10 7 cells/ml for at least 30 minutes in the dark. Cells were then washed in H F and incubated for an additional 30 minutes on ice with streptavidin-PE-TexasRed. Cells were washed again in HF/2 and then resuspended in HF/2 plus 2 ug/ml propidium iodide (PI, Sigma). Cells were kept cold and protected from light during the analysis and sorting on a F A C S Vantage or F A C S Ar i a (BD). 133 Transplantation and HSC quantification. W41 mice were irradiated with 360 cGy of 250 k V p X-rays and then varying numbers of Pep3b cells injected as indicated. H S C s were identified retrospectively by their ability to produced B , T and granulopoietic W B C s in the W41 mice for >16 weeks after staining peripheral blood (PB) samples with antibodies for donor (Ly5.1) and recipient (Ly5.2) CD45 allotypes plus lymphoid (B220 and L y l ) and myeloid ( G r l and M a c l ) cell surface markers, as previously described (Chapter 2 ) . Mice were considered to be repopulated by >HSC when >1% of the total P B W B C s were L y 5 . 1 + and these included a contribution of >1% L y 5 . 1 + cells to the L y l + , ( L y 6 G / M a c l ) + and B 2 2 0 + populations. Donor-type H S C frequencies in the injected test populations were then determined from the proportions of recipients scored using Poisson statistics and the method of maximum likelihood (L-Calc software, StemCell Technologies) (28). To evaluate the frequency of H S C s from transplants of single purified cells, the cells were sorted using the F A C S into the individual wells of a 96-well round-bottom plate containing 200 ul of serum-free medium (SFM) and visually confirmed to contain only one cell per well . S F M consisted of Iscove's M E M supplemented with a serum substitute (BIT\u00E2\u0084\u00A2, from StemCell Technologies) and 10\"4 M 2-mercaptoethanol. The entire contents of each well (1 cell + S F M ) were then taken up into a l m l syringe and injected intravenously into individual irradiated W41 recipients. In this case, the frequency of H S C s was calculated directly from the proportion of injected mice that showed multi-lineage Ly5.1 repopulation for 16 weeks. Values for total H S C s regenerated per transplanted mouse were calculated by multiplying H S C frequencies by 4x the number of cells recovered from 2 femurs and 2 tibias, assuming 2 femurs and 2 tibias contain - 2 5 % of the total bone marrow cells in an adult mouse (59). 134 Short term cultures. Single l i n ~ S c a - l + C D 4 3 + M a c l + E14.5 fetal liver cells were deposited using the automatic cell deposition unit of a F A C S Vantage directly into the individual round bottom wells of a 96-well plate containing S F M with either 300 ng/ml Steel factor (SF) and 20 ng/ml IL-11 or 50 ng/ml SF only. Wells were then maintained at 37 C for up to 50 hours and monitored by direct visualization at 4-hour intervals using an inverted microscope to determine the timing of the first and second cell divisions (the interval during which first 2, and later 3 or 4 cells were first seen in each well). Real-time PCR. Cells were sorted into 1 ml HF/10 and R N A was isolated using the PicoPure\u00E2\u0084\u00A2 R N A Isolation K i t (Arcturus Biosciences Inc.) as recommended by the supplier including a 15 minute D N A s e l treatment (Qiagen). R N A was eluted into an 11 pi volume and stored at -80\u00C2\u00B0C. A c D N A preparation was then generated using the Superscr ip t\u00E2\u0084\u00A2 III First-Strand Synthesis System for R T - P C R (18080093, Invitrogen) again as recommended by the manufacturer, with the reaction scaled up to use 25ul. Quantitative real-time P C R was performed using the following primer pairs (5' to 3'): ^ rM(NM_007499.1) forward G C A G A G T G T C T G A G G G T T T G T and reverse A A C T T C C A G C A A C C T T C A C C ; Bmi-1 ( N M 007552.3) forward A A A C C A G A C C A C T C C T G A A C A and reverse T C T T C T T C T C T T C A T C T C A T T T T T G A ; c-Kit ( N M 021099.2) forward G A T C T G C T C T G C G T C C T G T T and reverse C T G A T T G T G C T G G A T G G A T G ; CyclinD2 (NM_009829.2) forward G G C C A A G A T C A C C C A C A C T and reverse A T G C T G C T C T T G A C G G A A C T ; Ezh2 (NM_007971.1) forward C A T C G A A G G C A G T G G A G T C and reverse G T C T G G C C C A T G A T T A T T C T T C ; Gata-2 ( N M 008090.3) forward T G A C T A T G G C A G C A G T C T C T T C and reverse A C A C A C T C C C G G C C T T C T ; Gfi-1 ( N M 010278.1) forward 135 C T G C T C A T T C A C T C G G A C A C and reverse A T T T G T G G G G C T T C T C A C C T ; Ikaros (NM_001025597) forward C C T G A G G A C C T G T C C A C T A C C and reverse A C G C C C A T T C T C T T C A T C A C ; MEF (NM_019680) forward T C T G T G G A T G A G G A G G T T C C and reverse G G G T G C T G G A G A A G A A C T C A ; mel-18 (NM_009545.1) forward T T C C C C C T C T T A A C G A T T T G and reverse G A T C C T G G A G G C T G T T T C C T ; Notch-1 ( N M 008714.2) forward G C A C A A C T C C A C T G A T C C T G and reverse G C A A A G C C G A C T T G C C T A ; rae-28 ( N M 007905.1) forward G T C C C A G G C C C A G A T G T A T and reverse C C C C A T T A G G C A T C A G G A ; Sci (NM_011527.1) forward T G A G A T G G A G A T T T C T G A T G G T C and reverse C A A A T G C C C C A T T C A C A T T ; Gapdh ( N M 008084) was used as an endogenous control: forward A A C T T T G G C A T T G T G G A A G G and reverse A T G C A G G G A T G A T G T T C T G G . Statistical Analyses. Comparisons were made using the Wald test except in Figure 3.3, where the students T-test was used. 136 B Weeks post-transplant Figure 3.1: Fetal liver H S C s self-renew to a greater extent than bone marrow H S C s in a transplant. A. Schematic of the experimental design followed to measure the rate of self-renewal of 10 HSCs in vivo or 10 HSCs previously expanded for 6 weeks in an adult recipient from 10 starting fetal liver (FL) HSCs . B. Results shown are the number of donor C R U per recipient, as determined by secondary C R U assays, from the transplant of E14.5 F L (black circles) or 10 week-old bone marrow ( B M ) (open squares) after x weeks in a primary recipient. Values shown are the mean \u00C2\u00B1 S E M of results from 4 experiments. 137 Figure 3.2: H S C s switch from fetal liver-like SR to adult bone marrow-like SR abruptly and intrinsically. Results shown are the number of donor C R U per recipient, as determined by secondary C R U assays, from the transplant of 10 donor HSCs , assayed after weeks 1, 2, 3 and 4. For comparison, the similar results from 10 fetal liver (FL) (grey circles) and 10 bone marrow ( B M ) (grey squares) are shown. Values shown are the mean \u00C2\u00B1 S E M of results from 2 experiments. A . E l 8.5 fetal bone marrow H S C s self-renew at the same rate as E14.5 fetal liver C R U . Open circles denote the E l 8 . 5 fetal bone marrow (FBM)-generated C R U per recipient. B. A clear transition in self-renewal potential from fetal liver-like to bone marrow (BM)-l ike is revealed between 3 and 4 week-old B M HSCs . Open diamonds denote the 3-week-old BM-generated C R U per recipient and closed diamonds denote the 4-week-old B M -generated C R U per recipient. C . After 6 weeks in a transplant, FL-derived HSCs self-renew at the same rate as B M C R U . Closed triangles denote the donor C R U . The experimental design is shown in Figure 3.IB. 138 Figure 3.3: Fetal liver-like and adult bone marrow-like H S C s generate distinct P B lineage contribution patterns when transplanted into irradiated recipients. Bars indicate the mean % G M of donor W B C at 16 weeks post-transplant of 4 to 24 recipients of multiple (-3-6) H S C s of the source indicated. Error bars indicate the standard error of the mean. The percentage of all donor W B C s that express L y 6 G and/or M a c l ( G M ) is more than two-fold higher in recipients of fetal liver (FL) H S C s than recipients of adult bone marrow ( A B M ) H S C s (leftmost bars). With one exception, H S C s from other developmental stages and/or transplanted into secondary or tertiary recipients show distinctly FL- l ike or A B M - l i k e repopulation patterns (remaining bars). Black bars indicate a significantly (p<0.05) greater G M contribution than primary recipients of A B M . White bars indicate a significantly lower (p<0.05) G M contribution than primary recipients of F L . Checkered bar indicates both a significantly greater % G M than A B M recipients and a significantly lower % G M than F L recipients. 139 B Single Iin-Sca1+Mac1+CD43+ cells deposited Into wells \u00C2\u00A9 Q 0 2hr Single cells visually confirmed Timing of each * ufpiaSI 1st division IQSKSIS9' (2 ceils) Timing of each 2nd division (4 cells) e o UJ \u00E2\u0080\u00A2> > 100 80 60 ~ 40 E o 20 0 1\" division FL \u00C2\u00B0 3 r d division FL j 20 30 40 Incubation time (hr) Figure 3.4: Purification and culture of fetal liver HSCs reveals a similar cell cycle length as adult bone marrow HSCs . A . L i n \" S c a - l + C D 4 3 + M a c l + E14.5 fetal liver (FL) cells are enriched for H S C activity. Cells were isolated based on the indicated phenotypes and assayed for C R U content by either limiting dilution, or where appropriate, by single-cell injection. Values shown are the mean \u00C2\u00B1 S E M of results from at least 2 experiments. L S C D 4 3 M a c l + cells were assayed in 4 different experiments by limiting dilution and 5 different experiments by single cell injection. B. Schematic of the experimental set-up followed to measure the in vitro division kinetics of purified HSCs . C . In vitro division kinetics of single, pure (20%) F L H S C s compared to single, pure (40%) bone marrow ( B M ) HSCs reveals a same cell cycle transit time. The percentage of cumulative divisions is shown with respect to time in culture. F L H S C s are shown as open circles when cultured in self-renewal conditions of 50 ng/ml SF and closed circles when cultured in differentiating-conditions of 300 ng/mL SF and 20 ng/mL IL-11. 140 E14.5 birth 3 wk 4 wk 10 wk Pre-switch HSCs: Post-switch HSCs: Cycling Quiescent High self-renewal Lower self-renewal High GM output Reduced GM output Figure 3.5: N e w H S C developmental switch discovered. A distinct and abrupt transition in H S C behaviour occurs both endogenously and post-transplantation, after the same length of time. HSCs switch from fetal liver-like to adult bone marrow-like behavioural characteristics. Pre-switch H S C s are characterized by actively cycling, high rates self-renewal in transplants and high GM-regeneration per donor-derived W B C s . Post-switch HSCs are characterized by a large percentage in quiescence, low rates of self-renewal in transplants and low GM-regeneration per donor-derived W B C s . 141 A Figure 3.6: Gene expression analysis of purified fetal liver, 3-wk, 4-wk and 10-wk-old bone marrow HSCs . Gene expression in the adult bone marrow ( A B M ) or 4 week bone marrow ( B M ) H S C s was calculated as a fold-change relative to gene expression in the fetal liver (FL) or 3 week B M HSCs , respectively, with values within each comparison normalized to levels of Gapdh expression from each. A. 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Boggs,S.S., Chervenick,P.A., and Boggs,D.R. 1972. The effect of postirradiation bleeding or endotoxin on proliferation and differentiation of hematopoietic stem cells. Blood 40:375-389. 152 CHAPTER 4 STEEL FACTOR RESPONSIVENESS REGULATES THE HIGH SELF-RENEWAL PHENOTYPE OF FETAL HEMATOPOIETIC STEM CELLS The work presented in this Chapter was submitted for publication Michelle B . Bowie and Connie J. Eaves David Kent isolated the R N A and generated c D N A . 153 I N T R O D U C T I O N In the studies described in Chapters 2 and 3, a developmental switch that appears to simultaneously alter two key H S C properties - proliferative status and self-renewal potential -was identified. To investigate the molecular basis of this switch, with respect to the change in self-renewal potential, an in vitro model that supports fetal H S C self-renewal divisions was sought. Much effort has been directed to analyzing the growth factor requirements for maintaining bone marrow H S C s in culture and cocktails that support moderate expansion of these cells have been identified (1-11). Similar efforts with fetal liver H S C s are more limited and have shown that their growth factor responses are quite different from those of adult H S C s (12). The disparity between the high self-renewal capacity of fetal liver H S C s in vivo (13-15) and the inability to achieve their maintenance in vitro has led to a series of experiments testing the potential of various cell types to substitute for soluble growth factors in this regard (1;16;17), followed by the report that IGF-2, a product of some supportive cell types, might be effective (17). The W41 mouse has a single point mutation in the tyrosine kinase domain of the SF receptor, c-Kit , resulting in attenuated signalling following ligand binding as compared to the wild-type (+/+) receptor (18). The importance of c-Kit activation for stimulating adult H S C self-renewal has been well documented (4;9;19;20). The effect of the W41 mutation on H S C generation in vivo is much less severe in the fetus than in the adult (19). Evidence of a minimal requirement of fetal liver cells with a H S C phenotype to be stimulated by SF in order to expand their numbers has also been reported (21). These findings led us to hypothesize that the SF responsiveness of H S C s might also be programmed to switch; to a decreased state. This decrease in SF responsiveness in H S C s might thereby reduce both their ability to execute symmetric self renewal divisions and their ability to be mitogenically activated and therefore 154 be an important regulator of the switch in H S C properties described in Chapters 2 and 3. The studies described in this Chapter were designed to test this possibility that a change in SF responsiveness can account for the change in H S C cycling status and/or self-renewal activity as shown in Chapters 2 and 3. RESULTS Fetal liver CRUs are more sensitive to SF than adult bone marrow HSCs In a first series of experiments, 11 cocktails of 5 different growth factors were assessed in 12 experiments for their ability to maintain fetal liver C R U s in culture for at least 48 hours. The results are shown in Figure 4.1. Consistent with previous reports, most cocktails failed to sustain a significant level of C R U activity over a 48 hour period in culture. However, an important exception to this result was obtained when only SF was present at a concentration of 50 ng/ml. Under this condition, all the fetal liver H S C s divided (see Figure 3.4C), but the number o f C R U s present after 2 days was maintained. Intriguingly, 50 ng/ml o f SF in the presence of 20 ng/ml IL-11 was not able to maintain fetal liver C R U s for 48 hours in culture. This suggests that 20 ng/mL IL-11 is inhibitory to fetal liver C R U self-renewal, in the presence of 50 ng/ml SF alone. To more fully define the SF sensitivity of fetal liver HSCs , a full dose response curve was then generated using the same experimental design. A s shown in Figure 4.2, the generation of daughter C R U s in these cultures was optimal when SF was present at a concentration of 50 ng/ml SF, with significantly reduced C R U outputs at 2x higher or 5-fold lower SF concentrations. B y comparison, optimal self-renewal of cultured C R U s from adult bone marrow requires 300 ng/ml SF (4). Thus the maintenance of in vivo repopulating activity by fetal liver and adult bone marrow C R U s stimulated to divide is highly 155 dependent on SF activation but, in this regard, fetal liver C R U s are 6-fold more sensitive to SF than their adult counterparts. Fetal liver and adult bone marrow HSCs express the same levels of c-Kit. Next, a series of experiments were undertaken to determine whether the different responses exhibited by fetal liver and adult bone marrow H S C s could be attributed to differences in their expression of the SF receptor, c-Kit. Accordingly, the l i n \" S c a l + C D 4 3 + M a c l + a n d l in\"Rho d u l l SP subpopulations of fetal liver and adult bone marrow cells, respectively, from Pep3b (+/+) mice (-20% and - 3 0 % pure HSCs , as described in Chapter 3 and (22), respectively) were isolated and the levels of c-Kit m R N A and cell surface protein then determined (Figure 4.3). Quantitative R T - P C R measurements of c-Kit transcript showed no difference between the 2 highly enriched H S C populations compared. F low cytometric analysis of the level of c-Kit protein expressed on the cell surface in the same highly purified C R U populations yielded similar results to the m R N A data, as expected from historical data obtained from less purified populations (Figure 4.3 and (21)). W41 fetal liver CRUs mimic +/+ adult bone marrow CRUs in their SF requirement for self-maintenance in vitro The W41 mutation encodes a form of c-Kit that confers a reduced signalling capacity of this receptor following SF binding; W41 C R U s would thus be characterized by a reduced SF sensitivity than their +/+ counterparts. Given that W41 mice have normal numbers of H S C s in the fetus but not in the adult, this reduced SF sensitivity might only affect H S C expansion once these mice age past 3 weeks, the same time point at which self-renewal potential, cycling activity and multilineage potential decrease. To directly assess the SF responsiveness of W41 156 C R U self-renewal at different stages of development, we used the same type of dose-response analysis of 2-day cultured cells performed on +/+ C R U s (Figure 4.2). These experiments showed that the E14.5 fetal liver C R U s from W41 mice have a SF sensitivity very similar to adult bone marrow C R U s from +/+ mice, with a peak response in terms of W41 C R U self-maintenance in cultures that contained 500 ng/ml SF. When adult bone marrow C R U s from W41 mice were tested in the same way, their SF sensitivity was found to be at least 10-fold further reduced. Thus, in spite of a greatly reduced ability to activate normal pathways downstream of c-Kit , the C R U s in W41 mice appear to undergo the same switch in SF responsiveness that characterizes the development of adult C R U s from +/+ mice. To determine whether the decreased SF responsiveness of C R U s in the W41 fetal liver relative to +/+ fetal liver C R U might nevertheless be sufficient to maintain these cells in cycle in vivo, l i n \" S c a l + C D 4 3 + M a c l + fetal liver cells were isolated from W41 E14.5 fetal livers and then stained with Hst to determine the distribution of G0/G1 and S/G2/M cells within this fraction of +/+-CRU-phenotype cells. Although the C R U content of this phenotype in the W41 fetal liver was not assessed, the frequency of these cells among the Ter l 19-depleted cells was the same as in the corresponding +/+ Ter l 19-depleted cells (1/4115 \u00C2\u00B1 913 W41 fetal liver cells, n=2 vs. 1/4944 \u00C2\u00B1 834 +/+ fetal liver cells, respectively), consistent with a similar frequency of C R U s (19). The Hst staining revealed an approximately equal proportion of H S C s between the Go/Gi (2N D N A ) and S / G 2 / M (>2N D N A ) fractions (Figure 4.4), indicating that the W41 fetal liver H S C s are a cycling population, despite lower SF responsiveness. 157 The proliferative activity of W41 CRUs is not impaired in the fetus but the self-renewal in vivo mimics the characteristic reduced activity of adult +/+ CRUs W41 H S C s are hyposensitive to soluble SF in vitro, like +/+ adult bone marrow HSCs , but their sensitivity to the more potent, membrane-bound SF (23) under transplantation conditions in vivo might be similar to +/+ fetal liver. We therefore examined the self-renewal activity of W41 fetal liver C R U s in vivo, using the same serial transplant design outlined in Chapter 3. Accordingly, the 10 4 fetal liver cells (estimated to contain 10 W41 fetal liver C R U s (19)) were transplanted into lethally irradiated Pep3b mice and then at weekly intervals up to 4 weeks later, the number of W41 C R U s regenerated in the bone marrow of these hosts was measured by performing limiting dilution C R U assays in secondary Pep3b recipients. The results, shown in Figure 4.5, indicate that the rate of W41 fetal liver C R U self-renewal in vivo was the same as previously characterized for C R U s from +/+ adult bone marrow (Chapter 3), not +/+ fetal liver. DISCUSSION Based on observations in Chapter 3, suggesting that fetal liver and adult bone marrow HSCs self-renew using different mechanisms, an initial objective was to identify critical signalling pathways for fetal liver H S C self-renewal. In vitro analysis of potentially supportive growth factor conditions revealed that 50 ng/mL of SF, in the absence of other growth factors, can maintain fetal liver H S C s for 48 hours. This condition obviously does not recapitulate the HSC-supportive environment of the E14.5 fetal liver and suggests that a variety of other parameters may be required for optimal regenerative potential, such as cell-cell interactions. It did, however, draw attention to the fact that 50 ng/mL is a lower dose than that previously described as optimal for adult HSCs (4). 158 The objective of the experiments was then to determine whether a change in SF signalling might explain the reduced proliferative activity and self-renewal potential acquired by H S C s during development. Here we demonstrate that a change in SF signalling alone is not sufficient to alter the proliferative activity of HSCs , as W41 E14.5 fetal liver HSCs are a cycling population, but we provide evidence that the reduced self-renewal potential of HSCs , both in vitro and in vivo, can be caused by a reduced sensitivity to SF. Dose response curves for SF-dependent H S C self-renewal in vitro revealed that the sensitivity of fetal liver and adult H S C s differed by a factor of approximately 6 (50 ng/mL vs. 300 ng/mL), occurred independently of the amount of c-Kit expressed by these cells and correlated with their self-renewal behaviour in vivo as assessed quantitatively in a transplant-based regeneration assay. A s predicted by earlier studies, this change in SF sensitivity was not mediated by a change in c-Kit expression. Therefore, it seems more likely that these differential effects of SF are due either to a developmentally regulated change in the surface organization of c-Kit , or in the coordinated integration of c-Kit activation within additional signalling pathways, or in downstream events that mediate effects of c-Kit activation on self-renewal decisions. For example, in the latter case, a downstream effector of c-Kit might be envisaged to become limiting as a result of the fetal-to-adult switch. Previous studies have shown that the concentration of SF required to maximize the mitogenesis of adult H S C s is much lower than what is required to maximize their self-renewal in vitro (4). This differential requirement for c-Kit activation might explain why the reduced signalling capacity of W41 fetal liver HSCs would be sufficient to sustain their proliferation at a high rate in the fetus, in spite of an impaired self-renewal activity when stimulated to grow in the microenvironment of the irradiated adult bone marrow. The findings of Iscove and Nawa that the self-renewal of adult 159 bone marrow H S C s from +/+ mice could be enhanced by in vivo administration of SF (and IL-11) is consistent with this hypothesis (20). It is important to acknowledge that soluble SF is not as potent as membrane-bound SF in c-Kit signalling and in vivo, membrane-bound SF is critical to adequately sustain hematopoiesis (23). Therefore, the SF concentration requirements documented here and in previous studies (4) presumably mirror those required to match the signalling achievable from cell-bound SF in vivo. Although not pinpointed here, we propose that the change in SF responsiveness is likely to occur abruptly between 3 week and 4 weeks after birth, similarly to the observed change in self-renewal potential at this time point (Chapter 3). This could easily be determined by future comparison of the SF dependence of H S C self-renewal in vitro from 3 and 4 week-old mice. M A T E R I A L S A N D M E T H O D S Animals. C57Bl/6:Pep3B (Ly5.1) and W41 (Ly5.2) mice were used as donors and recipients, respectively, or vice versa, as indicated. Cel l suspensions were prepared from E l 4 . 5 fetal liver and the bone marrow of 10 week-old adult mice as described in Chapter 2. CRU assay. When Pep3B mice were used as recipients, they were irradiated with 2 doses of 400 cGy X-rays, separated by 4 hours. W41 recipients were irradiated once with 360 cGy X -rays. A l l transplants were injected intravenously. Recipients were analyzed for multi-lineage ( G r l , M a c l , B220, L y l ) donor-derived repopulation after 16 weeks and C R U frequencies calculated as described in Chapter 2. 160 HSC cultures. Fetal liver cells depleted of Ter l 19 + cells and W41 bone marrow cells depleted of l i n + cells (EasySep, StemCell Technologies) were cultured at a concentration of 10 6 cells/ml, for 48 hours in serum-free medium with various growth factors as indicated. After 48 hours, cells were harvested and C R U assays were performed. RNA analysis. The l i n \" S c a - l + C D 4 3 + M a c l + fraction of E14.5 fetal liver cells and the lin'Rho-d u l l S P C D 4 5 m i d fraction of adult bone marrow cells were isolated by F A C S as described in Chapter 2 and (22), respectively. R N A was isolated, transcribed into c D N A and quantitative-R T - P C R was then performed (as described in Chapter 2). Primers for c-Kit (NM_021099.2) were as follows: forward A C A A G A G G A G A T C C G C A A G A and reverse G A A G C T C A G C A A A T C A T C C A G , for Gapdh (NM_008084) were as follows: forward primer A A C T T T G G C A T T G T G G A A G G , reverse primer A T G C A G G G A T G A T G T T C T G G . Protein expression. E14.5 fetal liver and week 10 bone marrow cells were stained with lin\" (no M a c l ) S c a l + C D 4 3 + and lin\" SP R h o d u \" as described in Chapter 2 and (22), analyzed by F A C S and the geometric mean of their c-Kit fluorescence intensities determined and compared. Cell cycle analysis. The l i n \" S c a l + C D 4 3 + M a c l + fraction of E l 4.5 Ter l 19\" fetal liver cells from were co-stained with Hoechst 33342 and their distribution in Go/Gi vs. S/G2/M assessed using gates to distinguish cells with 2n vs. >2n D N A , respectively, as described in Chapter 2. Assays of HSC self-renewal in vivo. 10 5 unfractionated fresh Vf^'/W41 fetal liver cells, containing an estimated 10 C R U s (19), were injected intravenously into irradiated primary Pep3b hosts and then 1,2,3 and 4 weeks later, groups of these were sacrificed and the bone 161 marrow cells from these primary recipients was harvested from all four leg bones. Single cell suspensions were prepared and tested for regenerated ff^'/W41 C R U content by limiting dilution assays performed in secondary irradiated Pep3b recipients, as described above and in Chapter 3. Statistical Analysis. Comparisons were made using the Wald test 1 6 2 F I G U R E S 20 40 60 80 100 CRU ouput (% of input) Figure 4.1: Comparison of the effects of different growth factor cocktails on fetal liver C R U self-maintenance in vitro The number of C R U s recovered after 48 hours from each culture is expressed as a percent of the input number., C R U numbers were determined by 16-week limiting dilution transplantation assays as described in Chapter 2. Values shown are the mean \u00C2\u00B1 S E M of results pooled from 2 to 5 experiments. Growth factor concentrations are in ng/ml. 163 Figure 4.2: Steel factor dose response curves for the in vitro self-renewal of +/+ and W41 C R U s from fetal liver and adult bone marrow. Data shown for each source of C R U s are expressed as a percent of the number of C R U s recovered in the culture that yielded the maximum C R U output: +/+ fetal liver (FL) (open circles); W41 F L (solid circles); W41 bone marrow ( B M ) (solid squares). Data for +/+ B M is redrawn from results presented in (4). 164 A B Rhodamine Hst Red cKit-APC Figure 4.3: Comparison of c-Kit expression on fetal liver and adult bone marrow HSCs . A. Representative profile of E14.5 Ter l 19- fetal liver (FL) viable, lin\" cells assessed for Seal and CD43 expression, further analyzed for intensity of c-Kit protein expression. B. Shown are the results of fold-change in cKit gene expression relative to gapdh between purified Lin\" S c a l + C D 4 3 + M a c l + F L H S C s (as shown in A ) and Lin\"Rho d u \" SP bone marrow ( B M ) H S C s (as shown in C) on the left, and the geometric mean fluorescence intensity (M.F.I) of c-Kit protein expression in each of these same populations. C. Representative profile of adult B M viable cells, selected within the Lin\"Rho d u l fraction, further enriched as SP cells and assessed for intensity of c-Kit protein expression. 165 ( CD43-FITC Hoechst 33342 Figure 4.4: Cel l cycle analysis of W41 fetal liver HSCs . 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Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc.Natl.Acad.Sci. U.S.A 89:1502-1506. 170 22. Uchida,N., Dykstra,B-, Lyons,K.J . , Leung,F.Y., and Eaves,C.J. 2003. Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics. Exp.Hematol. 31:1338-1347. 23. Broudy,V.C. 1997. Stem cell factor and hematopoiesis. Blood 90:1345-1364. 171 C H A P T E R 5 C O N C L U S I O N S A N D F U T U R E D I R E C T I O N S In this thesis, I have presented the results of experiments that, for the first time, define the timing of changes in H S C proliferative activity, self-renewal and differentiation behaviour that occur during development. These have revealed the unexpected discovery of an abrupt and coordinated change in all o f these properties between 3 and 4 weeks after birth. For each of these parameters, a quantitative assay/endpoint was devised and then H S C s from fetal, weanling and young adult mice were assessed with respect to each parameter. The first evidence of this \"switch\" came from studies of the proliferative activity of the H S C s present at each of these stages of development, as assessed using 3 different methodologies (as described in Chapter 2), and subsequently confirmed by gene expression analyses of highly purified HSCs populations isolated from these different sources. These studies indicated that all H S C s are maintained in a state of constant turnover until 3 weeks after birth regardless of their location (in the liver or bone marrow) but then, within a period of one week >80% have transitioned into a quiescent state without further change in this level of proliferative activity through to adulthood. Interestingly, when the self-regenerative properties of H S C s from the same stages of development were subsequently examined (Chapter 3), these were also found to transition from a fetal phenotype to an adult phenotype at exactly the same time. Thus, it was possible to show that the H S C s in transplanted bone marrow from 3 week-old mice displayed the rapid HSC-regenerating activity characteristic of fetal HSCs , whereas the results for similarly assessed H S C s from the bone marrow of 4 week-old mice were superimposable on the data for 172 H S C s from adult bone marrow, which display a much slower HSC-regenerating activity. In addition, I undertook a series of experiments with highly purified fetal liver H S C s in vitro which demonstrated that, unlike their differentiating progeny (1 ;2), the cell cycle transit time of optimally stimulated fetal and adult H S C s is identical. These experiments are important as they strongly suggest that the different rates of H S C regeneration obtained by fetal and adult H S C s in the bone marrow of irradiated adult mice is determined by differences in their self-renewal response to the conditions activated in these hosts. A s also shown in Chapter 3, the spectrum of differentiated progeny generated by transplanted H S C s also changes at precisely the same time during development (resulting in a reduced output of circulating granulocytes and monocytes by the HSCs present in mice that are 4 weeks-old or more). Both the distinctive high self-renewal behaviour and higher output of granulocytes and/or monocytes were also found to change after the same duration of fetal H S C proliferation in transplanted adult hosts. Thus the switch itself, as well as its precise timing, does not appear to be dependent on extrinsic conditions that are determined either by the site or the developmental status of the host, favouring the view that they are intrinsically programmed. The unexpected speed and synchrony of these changes suggests the operation of some master \"switch\" mechanism. And , i f the mechanism that regulates the intrinsic responsiveness of H S C s to factors that control their cycling status proves to be independent of the environment in which the HSCs are located, as found for their self-renewal potential and distribution of mature progeny output, this would imply that this putative switch may, itself be intrinsically regulated and pre-programmed - perhaps determined either by time and/or the number of divisions HSCs are stimulated to execute. 173 Because of the evidence indicating that all H S C s are cycling up until 3 weeks after birth in the normal mouse, genetic strategies to promote H S C quiescence might be anticipated to cause a delay in the timing of the 'switch' seen here to occur between 3 and 4 weeks of age. Recently, the loss of the transcription factor M E F was shown to promote the quiescence of adult H S C s (3). Therefore, it would be interesting in the future to examine i f this might also be true of fetal HSCs , and i f so, whether the predicted delay in altered self-renewal activity would also be seen. This is of particular interest, given that Chapter 3 also shows that M E F is up-regulated in pre-switch HSCs . The other genes found to be higher in expression, such as Ikaros (4-6) or rae-28 (7), both genes shown to be critical to H S C expansion, would also be candidates of interest for this strategy. M E F _ / \" bone marrow H S C s have also been found to respond differently when maintained under homeostatic conditions as compared to conditions activated in response to hematologic injury. Conversely, forced overexpression in pre-switch H S C s of genes found to be higher expressed in post-switch H S C s might also facilitate inducing a premature post-switch state. Particularly, A T M , Ezh2 and Gata-2, the genes with the highest fold change in the 4 week versus 3 week bone marrow H S C s would be strong candidates to study. These findings point to the likelihood that a small set of genes, including MEF, rae-28, Ikaros, Ezh2, Gata-2 and ATM, may be part of a program that co-regulates H S C proliferative activity and self-renewal function. Other candidates are the cyclin-dependent kinase inhibitor (CKI) p 2 1 C i p l / W a f l which when deleted, has been shown to increase specifically the proportion of cycling H S C s (but not their derivative progenitors) and under these stress conditions self-renewal activity appeared to be compromised (8), the p l S 1 1 ^ 4 0 C K I , shown to limit the potential of adult H S C self-renewal in vivo (9), or genes within the specific clusters on chromosome 11, shown to be disproportionately distinctly expressed in 174 D B A / 2 versus C57BL/6 mice (10), specifically related to their demonstrated differences in H S C proliferation (11). One can also envisage various mechanisms that might mediate such effects; for example, a very long-lived (or functionally sequestered) protein or m R N A that decayed at a slow rate to finally reach a critical threshold level. This type of cell division-related threshold has been well studied in the case of telomeres, and the regulation of telomere length and telomerase activity is tightly linked to cell cycle regulation (12). Yet telomeres seem unlikely to be a playing this type of critical role in murine H S C s as it has been proposed that telomere shortening evolved as a tumor suppressor mechanism not found in short-lived mammals, such as mice (13). In fact, it has been demonstrated that significant numbers of generations of mice must be followed before defects due to telomere shortening become evident, as mouse telomeres are extremely long (14; 15). However, this does not preclude that a particular length of telomeres, long as it may be, is a threshold signal for a cascade of 'switch' responses to begin. The demonstration that adult bone marrow H S C s have shorter telomeres than those from the fetal liver or cord blood seems to suggest a continued decline in telomere length occurs with age (16). The rapid switch that takes place in the type of globin expressed in maturing erythroblasts has also been well-studied. There, a role for the chromatin-remodeling complex ( P Y R complex) and its TF binding partner, Ikaros, has been well demonstrated. Mice null for Ikaros not only show a significant loss of HSCs (4), but display a delay in the murine embryonic to adult P-globin switch (17). c D N A array analysis on these mice indicated that several hematopoietic-specific genes across all lineages were changed in the day 14 embryos. Therefore Ikaros, PYR and a number of other chromatin-remodelling genes, such as members 175 of the polycomb-group (Bmi-1, mel-18, rae28, Ezh2) (18-20) continue to be strong candidates to test for playing a role in the H S C switch demonstrated in this Thesis. It is worth noting that i f such a switch was determined to be cell division-dependent, it would not necessarily imply that one final H S C division was required for a transition from the fetal to the adult state. This could instead be quickly and easily regulated without the need of a change-of-state cell division, by changing any number of factors within the same cell: a loss or gain in a transcription factor, or set of transcription factors, again past a given threshold; any epigenetic changes, etc. Complicating the matter further is the heterogeneity of the adult H S C , for example with obvious subtypes related to differences in their patterns of repopulation in vivo (21). Further investigations of H S C properties that are similarly altered during development may also offer new clues to the mechanisms that regulate the changes observed here. A s described in the Introduction to this Thesis (Chapter 1), there are a number of cell surface markers whose expression is different between adult and fetal H S C s and in some cases, evidence of a post-natal change between 3. and 10 weeks of age has been reported (22-28). More carefully timed analyses w i l l be useful to determine how closely these changes track relative to the functional changes described in this thesis. If coordinated, common transcription factor binding sites in the promoters of genes encoding for these cell surface markers, along with those of genes found in Chapter 3 to change in coordination, could then be sought in silico. Particularly, to look for activators of expression of genes found to be up-regulated in post-switch HSCs and repressors of those down-regulated in this population, as playing a role in promoting the switch itself. 176 A corresponding mechanistic change in HSCs has been described in Chapter 4, which points to a role for c-Kit signalling, responsible, at least, for to the change in self-renewal potential. Chapter 4 describes a change in the H S C responsiveness to SF signalling as H S C s progress through development, to explain the reduced self-renewal potential that HSCs acquire during development, as described in Chapter 3, but not their reduced proliferative potential. A full dose-response curve showed that the sensitivity of fetal liver and adult HSCs differ, but that W41 fetal liver H S C s were similar to +/+ adult HSCs . However, purified fetal liver H S C s were shown to express the same level of c-Kit m R N A as adult bone marrow HSCs and even the same mean fluorescence intensity of c-Kit expressed on the cell surface of pure fetal liver and adult bone marrow HSCs , suggesting that there is likely to be a developmentally regulated change in the surface organization of c-Kit or in a downstream or cooperative signalling component. W41 fetal liver H S C s were demonstrated to also share the same in vivo self-renewal kinetics as adult bone marrow HSCs , indicating that H S C responsiveness to SF signalling was directly related to the rate of self-renewal. These results suggest that the levels of SF expressed in the bone marrow of an irradiated recipient is limiting to adult bone marrow, but not +/+ fetal liver HSCs , and treatment of recipients of bone marrow H S C s with SF could therefore be a means by which to increase the rate of regeneration following bone marrow transplantation. These findings raise the question of which effectors of SF signalling, potentially transcription factors, might be regulating the intensity of c-Kit downstream signalling, as these may be critical points of control for the potential 'master switch'. A set of transcription factors acting as negative regulators of expression of key components to c-Kit signalling would be presumed to be up-regulated at a point in time between 3 and 4 weeks post-birth. 177 Alternatively, complexes involved in the repression of promoter accessibility of genes mediated c-Kit signalling may be up-regulated, either as direct mediators of chromatin condensation or by enhancing rapid methylation of the promoter region; each of which would result in a decreased amount of expression of such key genes in H S C s after 3 weeks post-birth. In Chapter 2, an examination of the differences in cycling status of H S C s through development highlighted one HSC-cycl ing property that did not change through development: H S C s in S / G 2 / M phase of the cell cycle have a perpetual inability to engraft irradiated recipients. This has obvious clinical ramifications, as expanded H S C s are routinely used when there is a need for HSCs in the clinic and therefore a significant proportion of these are useless under current procedures. Chapter 2 provides evidence that this defect can be overcome when the recipient environment specifically (not the test cells) is pre-treated with an antagonist to SDF-1 . However, the mechanism by which the host interacts with a cell autonomous property such as the cell cycle status is still unclear, specifically after treatment with the SDF-1 antagonist. We have proposed a logical model based on the additional evidence that SDF-1 is up-regulated in S / G 2 / M H S C s compared to H S C s in G i , suggesting that the high levels of SDF-1 in these cells saturates the C X C R 4 receptors expressed on these same cells, preventing their ability to respond to SDF-1 gradients, unless these gradients were very strong. In Chapter 2, we hypothesized that SDF-1 G2 treatment can generate a stronger gradient of SDF-1 in the host, allowing S / G 2 / M HSCs to now engraft. It is of great interest to determine whether SDF-1 G2 treatment can result in increased levels of SDF-1 expressed at niche cells. Since the SDF-1 antagonist acts on C X C R 4 in the niche, but not cell-autonomously on stem cells, the population of cells in the recipient's environment that do express C X C R 4 and mediate the observed effects are of great interest to identify and characterize. Various candidates, such as stromal and epithelial cells could be isolated and tested in vitro for their 178 differential ability to bind to S / G 2 / M and G 0 / G i HSCs . A s well , the ability of S / G 2 / M and Go/Gi HSCs to migrate through these cells could be tested, both before and after SDF-1 G2 treatment of the niche cells under investigation. Such experiments w i l l provide evidence of the mechanism that explains these exciting observations. There are potential clinical applications of the findings presented in this Thesis. For example, in Chapter 2, the results of experiments on the S / G 2 / M engraftment defect predict that intrafemoral injection of transplants is unlikely to be a useful strategy for improving the therapeutic effectiveness of HSCs induced to expand in vitro. Particularly exciting is the idea that pre-treatment of recipients of cycling cells with an SDF-1 antagonist could potentially improve the therapeutic effectiveness of these cells by as much as 100% (assuming that half of the H S C in a cycling population are in S / G 2 / M ) . The induction of adult bone marrow H S C cycling is a consequence of culturing to expand these prior to transplantation or to genetically modify these; currently readily used and increasingly attempted strategies, respectively. The information presented in Chapter 4 could also provide new strategies for expanding HSCs , particularly as one growth factor condition, 50 ng/ml SF, has now been shown to maintain fetal liver H S C s in culture. Again, the ability to maintain fetal liver H S C s in culture is a necessity for successful attempts at genetic modification. 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"Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Analysis of developmentally programmed changes in hematopoietic stem cells"@en . "Text"@en . "http://hdl.handle.net/2429/18465"@en .