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Novel methods for high level ex vivo expansion of hematopoietic stem cells Sekulovic, Sanja 2005

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NOVEL METHODS FOR HIGH LEVEL EX VIVO EXPANSION OF HEMATOPOIETIC STEM CELLS By S A N J A S E K U L O V I C B . S c . Molecular Biology and Physiology, University of Belgrade, 2001 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I 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 O F M A S T E R O F S C I E N C E In T H E F A C U L T Y O F G R A D U A T E S T U D I E S ( Medica l G e n e t i c s ) T H E U N I V E R S I T Y O F BRIT ISH C O L U M B I A September , 2005 © San ja Sekulov ic , 2005 ABSTRACT The development of strategies to extensively expand HSCs ex vivo could greatly improve the utility of hematopoietic stem cell (HSC) - based therapies. In addition to potential clinical applications, such an advance would provide an invaluable tool for studying the mechanisms underlying H S C self-renewal. Engineered overexpression of the homeobox transcription factor HOXB4 has emerged as a powerful stimulator of hematopoietic stem cell (HSC) expansion ex vivo (>40-fold net increase in 2 weeks). More recent studies of the properties of natural and engineered NUP98-HOX fusion genes, initially of interest to us for their role in human AML, suggested these molecules might have similar effects on HSCs. To examine whether specific NUP98 and HOX fusion genes stimulate murine H S C expansion in short term liquid cultures, 3x10 6 marrow cells from mice given 5-fluorouracil 4 days previously were prestimulated with IL-3, IL-6 and S F , retrovirally transduced with MSCV- IRES-GFP retroviral vectors also encoding NUP98-HOXB4, NUP98-HOXA10, or HOXB4 (only) or nothing as controls and then cultured for another 6 days with the same growth factors. Limiting dilution assays were used to determine the frequency and hence number of Competitive long-term (>4months) lympho-myeloid Repopulating Units (CRU) present before and after culture. The results of these experiments showed that the C R U content of the cultures of NUP98-HOXB4-, and NUP98-HOXA10-transduced cells increased 290-fold and >2000-fold, respectively, i.e. ~4 and i i >25x the effect obtained with HOXB4 and >104 and >105x the yield of C R U in the control cultures. Similar results were obtained in cultures of NUP98-HOXA10-transduced cells that were initiated with limiting numbers of C R U s (1-2), demonstrating that the cells targeted were not a rare subset of HSCs . Additional studies of the same design showed that the effect of NUP98-HOXA10 on H S C expansion was preserved when sequences flanking the homeodomain were removed, thus identifying the homeodomain as the key HOX gene sequence required in concert with the N-terminal region of NUP98. These findings demonstrate a greater potency of NUP98-HOX fusions as novel agents for H S C expansion ex vivo, reveal the essential contribution of the DNA-binding homeodomain to achieve this effect and set the stage for the design of minimal HOX-based fusion proteins for future studies. iii TABLE OF CONTENTS TITLE PAGE i ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii ABBREVIATIONS x ACKNOWLEDGEMENTS xiii CHAPTER 1 INTRODUCTION 1.1 Hierarchical Mode l of Hematopoies is and the Crit ical Ro le of Hematopoiet ic S t e m Ce l l s ( H S C s ) 1 1.2 Hematopoiet ic S tem Ce l ls ( H S C s ) 5 1.2.1 Ex is tence of H S C s 5 1.2.2 Detect ion and quantification of H S C s 6 1.2.3 Detect ion and quantification of H S C s 10 1.2.4 Funct ional properties of H S C s 12 1.2.5 Phenotyp ic characterizat ion of H S C s 12 1.2.6 Sel f - renewal of H S C s 15 1.2.7 Potential cl inical appl icat ions of ex wVo expanded H S C s 16 1.2.8 Extr insic factors regulating H S C functions 18 1.2.9 Intrinsic factors regulating H S C functions 20 1.3 HOX G e n e s and H S C function 24 iv 1.3.1 HOX gene organizat ion and express ion 24 1.3.2 HOX gene express ion and roles in hematopoies is 27 1.3.3 HOXB4 - a potent stimulator of H S C expans ion 28 1.3.4 Other H O X genes that have an ability to promote H S C expans ion 30 1.4 Thes i s Object ives 33 C H A P T E R 2 MATERIALS AND METHODS 2.1 Retroviral Vec to rs . . 34 2.2 Mice 35 2.3 Infection of Primary Murine B M Ce l ls 36 2.4 C R U A s s a y 37 2.5 F low Cytometry 37 2.6 Purif ication of S c a 1 + L i n " Ce l l s 38 2.7 Proviral Integration Ana lys is 38 C H A P T E R 3 R E S U L T S 3.1 NUP98-HOX Fus ion G e n e s Stimulate a Very Large Expans ion of H S C s in Culture 39 3.2 A NUP98-HOX Fus ion G e n e Containing Only the HD of HOXA10 Reta ins the Full H S C Ex Vivo Expans ion Activity of the Parent Fus ion G e n e 44 3.3 M J P 9 8 - H O X - t r a n s d u c e d Ce l l s Reta in Mult i l ineage Repopulat ion Ability 48 3.4 Polyc lonal Recovery of A / l /P98-HOX- t ransduced H S C s 51 3.5 Ex vivo Expans ion of A/L/P98-HOX,470-t ransduced B M Ce l ls in Cul tures Initiated with Smal l Numbers of Input C R U s - Direct Ev idence of High Level C lona l H S C Expans ion 53 CHAPTER 4 DISCUSSION 59 CHAPTER 5 REFERENCES 63 vi LIST OF TABLES Table 3.1. - Summary of l ineage distribution of GFP+ cel ls in P B of t ransplanted recipients 49 vii LIST OF FIGURES Figure 1.1. - The hematopoiet ic hierarchy 4 Figure 1.2. - HOX chromosomal organization 26 Figure 2.1. - Structures of retroviruses used in the study 35 Figure 3.1. - Genera l experimental design 40 Figure 3.2. - Long-term reconstitution of recipients transplanted with GFP-, HOXB4-, NUP98-HOXB4- or NUP98-HOXA70-transduced cel ls 41 Figure 3.3. - Limiting dilution analysis (LDA) for estimation of C R U f requencies in 10-day cultures of H S C s t ransduced with var ious HOX fusion genes 42 Figure 3.4. - Ex vivo expans ion of t ransduced H S C s after 10 days of cul ture. . .43 Figure 3.5. - Long-term reconstitution of recipients transplanted with GFP-, HOXB4- or NUP98-HOXA10hd-transduced cel ls 46 Figure 3.6. - Ex vivo expans ion of t ransduced H S C s after 10 days of culture....47 Figure 3.7. - Representat ive peripheral blood F A C S profiles of recipients transplanted with NUP98-HOXB4-, NUP98-HOXA10- or NUP98-HOXA 10hd-t ransduced cel ls 48 Figure 3.8. - Southern blots showing common integration patterns of vector D N A in reconstituted myeloid and lymphoid t issues of representative recipients of NUP98-HOX expanded cel ls 50 Figure 3.9. - Polyc lonal recovery of A/L /P98-HOX-t ransduced H S C s 52 Figure 3.10. - Exper imental protocol for examining the ex vivo expans ion of / V I / P 9 8 - H O X A 70-transduced B M cel ls in cultures initiated with 1-2 C R U s 54 Vll l F i g u r e 3.11. - Long-term reconstitution of recipients by GFP- or NUP98-HOXA 70-transduced cel ls expanded ex vivo in cultures initiated with 1-2 C R U s 56 F i g u r e 3.12. - Southern blots of D N A from representative mice transplanted with A/ l /P98-HOX/470- t ransduced B M cel ls expanded ex vivo from cultures initiated with 1-2 C R U s 58 ix A B B R E V I A T I O N S 5-FU 5-fluorouracil A M L acute myelogenous leukemia Antp Antennaped ia A N T - C antennapedia complex B M bone marrow B-cel ls B- lymphocytes C B cord blood C D cluster of differentiation C F C colony forming cell C F U colony forming unit C F U - E colony forming unit - erythroid C F U - G colony forming unit - granulocyte C F U - G M colony forming unit - granulocyte macrophage C F U - G E M M colony forming unit granulocyte, erythroid, macrophage, megakaryocyte C F U - M colony forming unit - macrophage C F U - S colony forming unit - sp leen c G y cent iGray C R U competit ive repopulating unit C R U s competit ive repopulating units D M E M Du lbecco 's modified Eag le 's medium D N A deoxyr ibonucleic acid F A C S f luorescence-act ivated cell sorting F B S fetal bovine serum F L fetal liver Flt3-L Flt3-l igand G - C S F granulocyte-colony stimulating factor G F P green f luorescence protein G V H D graft versus host d i sease H D homeodomain H F Hank 's balanced salt solution with 2 % F B S H O M - C homeot ic complex H S C hematopoiet ic stem cell H S C s hematopoiet ic stem cel ls IL interleukin I R E S internal r ibosomal entry site L C liquid culture Lin l ineage markers L T C - I C ling term culture-initiating cell L T R long terminal repeat LTR-ce l l s long-term repopulating cel ls N A 1 0 N U P 9 8 - H O X A 1 0 NA IOhd N U P 9 8 - H O X A 1 0 h d N B 4 N U P 9 8 - H O X B 4 N K cel ls natural killer cel ls N O D / S C I D non-obese diabetes / severe combined immunodef ic iency N U P 9 8 nucleoporin-98 P B peripheral blood P B X 1 pre-B-cel l leukemia transcription factor 1 PI propidium iodide P S prestimulation R B C red blood cell R B C s red blood cel ls R U repopulation unit 1 Sca-1 stem cell antigen S F steel factor S L cel ls S e a l + L i n " cel ls S P s ide population S T R - c e l l s short-term repopulating cel ls T-cel ls T- lymphocytes W B C white blood cell W B C s white blood cel ls xii A C K N O W L E D G E M E N T S I am deeply grateful to my supervisors, Dr. Keith Humphr ies and Dr. Conn ie E a v e s , for the opportunity to do graduate training at Terry Fox Laboratory and for their fervent support and sel f less guidance throughout this project. I a lso truly appreciate their encouragement to present the results of this research at the International Soc iety for Exper imental Hematology 34th annual scientif ic meet ing. I would like to thank Dr. Hideaki Ohta for providing invaluable initial leads pointing us towards the high potency of engineered HOX fusion genes to stimulate murine H S C expans ion in vitro and Si lv ia Bakov ic for p leasant and productive team work during the project. They were both very helpful in teaching me the a s s a y s for hematopoiet ic cel ls. I would a lso like to thank Dr. S u z a n Imren for her constant encouragement in both academic and personal endeavors throughout my time in the lab. Dr. Pame la Hood less served on my graduate committee and I am thankful for her ass is tance in project d iscuss ions. Finally, I send my thanks to members of the Humphr ies and the E a v e s lab (Bob, Er ic, Caro l ina , Nick, Lars, Rhonna , Fredrick, Patty, Adr ian, Christy, Brad and Andrea) for making T F L such a fun place to work. Lastly, but far from the least, I would like to thank my parents, my sister and my husband for their end less love and encouragement. Xll l CHAPTER 1 INTRODUCTION 1.1 Hierarchical Model of Hematopoiesis and the Critical Role of Hematopoietic Stem Cells (HSCs) Hematopoies is is the life-long process of blood cell development. B e c a u s e the l i fespan of most mature blood cel ls is relatively short (days), their replacement and the mechan isms that control this process are essent ia l for survival. The establ ishment and permanent maintenance of hematopoiesis relies on the presence of a smal l subset of pluripotent hematopoiet ic stem cel ls ( H S C s ) that can amplify their own numbers and/or differentiate as needed. A pool of these cel ls is retained in the bone marrow (BM) throughout adult life, although they are est imated to compr ise on ly 0.01% of the total B M compartment. The hematopoiet ic sys tem of mice and humans is organized as a hierarchy of cell types with differing capaci t ies for self-renewal, proliferation, and differentiation. Hematopoies is is thought to proceed irreversibly through a ser ies of l ineage commitment steps, during which the most primitive pluripotent H S C s give rise to multipotent myeloid- or lymphoid-restricted progenitors that in turn give rise to committed progenitors - the final output being the mature functional circulating blood cel ls. The myeloid l ineage includes those cel ls responsib le for carbon dioxide and oxygen transport (erythrocytes), blood clotting (platelets) and those involved in mounting a phagocyt ic response to foreign organ isms (granylocytes, monocytes, macrophages) . The lymphoid l ineage includes cel ls involved in humoral (B- lymphocytes) and cellular immunity (T cel ls and natural killer cells) (Figure1.1). Despite their common origin, cel ls belonging to the myeloid l lineages are produced in the B M , whereas many cells of the lymphoid lineages undergo further development in the spleen, thymus and lymph nodes. HSCs are defined by their functional attributes, the potential to generate and maintain a lifetime output of all of the terminally differentiated lymphoid and myeloid cell types that comprise the blood, BM, spleen, and thymus (Jordan and Lemischka, 1990, Szilvassy and Cory, 1994). These cells include the eight major hematopoietic lineages: B and T lymphocytes; erythrocytes; megakaryocytes/platelets; basophils/mast cells; eosinophils; neutrophils, and monocytes/macrophages. To support this potential, HSCs possess an extremely high proliferative potential. It is estimated that in normal humans there are approximately 50 million HSCs, some of which can generate up to 10 1 3 mature blood cells over a normal lifespan (reviewed in Szilvassy, 2003). In mice, it has been shown that a single stem cell can regenerate and maintain a significant proportion of the lymphohematopoietic system following transplantation into an irradiated or immunocompromised host (Dick et al., 1985; Keller et al., 1985, Lemischka et al., 1986). Proliferation and differentiation are not necessarily tightly coupled, and in the most primitive HSCs this renders a variable capacity for self-renewal, the cardinal property of all stem cell types. Self-renewal is critical for HSCs because they are constantly subjected to physiological stresses that stimulate their recruitment into maturational pathways. HSC self-renewal, at least at the population level, thus ensures that sufficient numbers of HSCs are available to meet the demands of hematopoiesis over a normal adult lifespan. Furthermore, their potential for reconstituting the hematopoietic system has allowed 2 the development of powerful clinical therapies such as for leukemias and genetic blood disorders based on HSC transplantation. Given the pivotal role of HSCs, much effort has been directed at developing tools for their detection and in understanding and ultimately exploiting the molecular mechanisms that control their self-renewal. A longstanding major goal has been to develop methods that would enable significant levels of HSC self-renewal to occur in vitro. Such a development would enhance efforts to understand the mechanisms controlling self-renewal and enable broader and safer application of HSC-based therapies. In this thesis work, I have focused on a recently developed strategy for achieving HSC expansion ex vivo, which is based on the forced expression in primitive hematopoietic cells of HOX transcription factors and related molecules. In the following sections, the key concepts, assays and regulatory mechanisms of HSC function that guided and enabled the research undertaken, are briefly reviewed. 3 HEMATOPOIETIC ASSAYS CRU LTC-IC CFU-S CFC £ ® LTR-HSC STR-HSC I 1 Hematopoietic f stem cells J FACS QMP Multipotent progenitors f § ) M E P "1 ( & ) GMP 1 * • f BFU-E CFU-Meg CFU-M CFU-G CFU-Eo CFU-Baso DMI CFll-E Megakaryocyte I CO ® ® w V + + i ft ® Pro-T Pro-B f> Ci) Fre-B • i Neutrophil Eosinophil Basophil T Ceil B Cell NKCell Erythrocyte Platelets Macrophage Figure 1.1: The hematopoietic hierarchy. Pluripotent H S C s are shown at the top of the hierarchy (the circular arrow indicates H S C self-renewal). Longterm repopulating (LTR) H S C s give rise to short term repopulating (STR) HSCs , which give rise to multipotent progenitors. Commitment to either myeloid or lymphoid lineages produces common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs), which give rise to more lineage-restricted megakaryocytic-erythroid progenitors (MEP) and granulocyte-macrophage (GMP) progenitors. Further commitment events produce unilineage progenitors, which mature into functional end cells. Some hematopoietic assays are aligned with the developmental level of the cell type they detect. Potentials for proliferation and self-renewal both decrease with differentiation. 4 1.2 Hematopoietic Stem Cells (HSCs) 1.2.1 Existence of HSCs Early indications that hematopoiesis is sustained by the cont inuous differentiation of cel ls from a common pluripotent stem cell population and that such cel ls may exist came from studies in which the transplantation of chromosomal ly marked cel ls into lethally irradiated mice resulted in the long-term recovery of both lymphoid and myeloid cel ls that shared the same unique chromosomal markers (Wu et a l . , 1968). Later, these findings were also confirmed by studies that demonstrated common sites of retroviral integration in lymphoid and myeloid cel ls regenerated following bone marrow transplantation (Dick et a l . , 1985; Kel ler et al . , 1985, C a p e l et a l . , 1989). Addit ional direct ev idence for H S C s has most recently derived from methods to enrich for rare hematopoiet ic subpopulat ions and the demonstrat ion of the capaci ty of multi l ineage repopulation capacity of single transplanted cel ls (Osawa et a l . 1996, E m a et a l . , 2000; Uch ida et a l . , 2003). Moreover, plating of such single cel ls into t issue culture wells, revealed repopulating cel ls within the first division progeny - demonstrat ing self-renewal capacity. W h e n the two daughter cel ls were separated, however, retention of long-term repopulating ability could somet imes be found in only one of the two wel ls, indicating that asymmetr ic self-renewal div is ions occur in vitro. 5 1.2.2 Detection and quantif ication of H S C s One of the first methods for detecting and quantifying primitive hematopoietic cells in vivo was the colony-forming unit - spleen (CFU-S) assay (Till and McCulloch, 1961). In this assay, hematopoietic cells obtained from the bone marrow or spleen of donor mice are transplanted intravenously into syngeneic recipients, previously exposed to a lethal dose of irradiation. Approximately, 10% of the injected cells become lodged in the spleen (Lord and Hendry, 1973), where they proliferate and generate clonal macroscopic colonies within 1-2 weeks. Counts of the colonies obtained then provide a measure of the CFU-S frequency in the initial cell suspension injected. The number and types of cells comprising each colony can also be determined to assess the proliferation and differentiation potential of the founder cell. Self-renewal potential can be evaluated by re-transplanting cells from individually excised colonies into secondary irradiated hosts to determine whether CFU-S were regenerated during the formation of the primary colony (Siminovitch et al., 1963). Originally, this assay was thought to detect HSCs with longterm regenerative potential. However, subsequent studies showed that this was not the case, although some HSCs may form spleen colonies (Spangrude and Johnson, 1990). The strongest evidence that many CFU-S are different from HSCs has come from cell separation experiments where it was possible to physically separate CFU-S from HSCs detected by endpoints of long-term repopulation of transplanted recipients (Jones etal., 1990; Ploemacherand Brons, 1989). 6 The results of the early studies of C F U - S nevertheless led to recognit ion of the importance of quantitative a s s a y s specif ic for cel ls with long-term in vivo lympho-myeloid reconstitution ability and catalyzed many important concepts and mode ls concern ing the properties and regulation of H S C s . Currently, the abundance and properties of H S C s are best determined using t ransplantat ion-based assays . H S C s are identified by their unique ability to give long-term lymphomyeloid reconstitution of intravenously transplanted myeloablated recipients pretreated by exposure to lethal or near-lethal doses of ionizing radiation. The injected H S C s home to the B M and re-establ ish multi l ineage hematopoiesis. However, heterogeneity exists amongst cel ls that are capable of some multi-l ineage reconstitution and the most primitive subsets can be further defined by the durability of their capacity to sustain donor-der ived hematopoies is . The repopulation unit (RU) assay developed by Harr ison (1980) measures the ability of a cell sample to repopulate irradiated hosts relative to another reference source of repopulating cel ls (e.g. 1 0 5 normal B M cells) contained in the transplant innoculum. The test sample is injected, along with a standard dose of freshly isolated and genotypical ly dist inguishable competitor cel ls, into a congenic irradiated murine recipient. After 3-4 months, the relative repopulation of myeloid and lymphoid cel ls by the 2 donor sources is calculated, and an R U value calculated by the formula: RU=(P*C) / (100-P) , where P is the measured percentage of test cel l-derived hematopoies is , and C is the number of competitor cel ls used. The magnitude of the R U value reflects both H S C number (quantity) and its cell output capaci ty. This a s s a y thus compares the stem cell activity of a cell population relative to some 7 reference population, but does not measure H S C frequency directly. Addit ional statistical methods can be applied to the var iance data obtained from such exper iments to calculate the frequencies of the input H S C s if certain assumpt ions are made about their output potential, which we now know is not an invariant feature of different H S C populations (Rebel et a l . , 1996). A method for measur ing H S C frequency based on limiting dilution a s s a y of cel ls with longterm competit ive lympho-myeloid repopulation activity is the Compet i t ive Repopulat ing Unit or C R U a s s a y (Sz i lvassy et a l . , 1990, Sz i l vassy et a l . , 2002). Th is procedure uses the principles of limiting dilution analys is to measure the f requency of cel ls in a given suspens ion that have transplantable long-term repopulating ability and can individually generate both lymphoid and myeloid progeny (Sz i lvassy et a l . , 2002). A s recipients, normal mice are pretreated with a lethal dose of radiation (myeloablative treatment), or c-kit mutant mice whose H S C s are defective (Miller et a l . , 1996) are treated with a sublethal dose of irradiation. Th is treatment of the hosts maximizes the sensitivity of the assay and reduces the compet ing endogenous H S C population to a minimum, creating an environment in which the engrafting H S C s will be optimally st imulated. In order for a limiting dilution analys is of the H S C content of the test cell suspens ion to be performed, the recipients must be able to survive regardless of whether they receive any H S C s in the test cel ls injected. Survival of normal recipients is assured by co transplanting them with hematopoiet ic cel ls of the same genotype that contain sufficient numbers of short-term repopulating cel ls but minimal numbers of long-term repopulating cel ls (e.g. 1 0 5 normal bone marrow cells). Survival of c-kit mutant hosts is similarly 8 assured by pretreating them with a dose of radiation that al lows significant numbers of endogenous cel ls to survive and avoids the requirement of transplanting addit ional cel ls. The differentiated blood cell progeny of the test cel ls and the recipients must be genetically dist inguishable and a s s e s s e d at a time when the regenerated cel ls can be assumed to represent the exclusive output of cel ls with life-long H S C potential. Strains of mice congenic with the C 5 7 B 1 / 6 mouse are typically used to al low the blood cell progeny of the test cel ls to be uniquely identified by C D 4 5 (Ly5) allotype markers (Sz i lvassy and Cory, 1993). Quantif ication of H S C s is achieved by application of Po i sson statistical analys is on the proportion of animals that test negative for the test cel l-derived repopulation at each cell dose transplanted, where the dose at which 3 7 % of an imals are negative is estimated to contain 1 C R U . In practice, a threshold of >1% test cel l-derived myeloid and lymphoid peripheral blood (PB) cel ls detected >4 months post-transplant has been shown to rigorously detect a long term lympho-myeloid repopulating cel l . Us ing this assay , the f requency of H S C s in the B M of a mouse has been est imated to be about 1 in 10,000 nucleated cel ls. Methods for detecting and assay ing human H S C s have also been deve loped based on the remarkable observat ion that primitive human hematopoiet ic cel ls can engraft and contribute to long-term lympho-myeloid hematopoies is in certain xenogene ic recipients that are adequately immunocompromised (e.g. fetal sheep and immunodeficient mouse strains (Zanjani et al . , 1994; Dick, 1996). Immunocompromised mice bearing non-obese diabetes (NOD) and severe combined immunodef ic iency (SCID) genotypes can tolerate hematopoiet ic grafts 9 from human sources (Cashman et al . , 1977). This has al lowed for the development of a quantitative in vivo functional assay for human H S C s (Bhatia et a l . , 1997; Connea l ly et al . , 1997). Limiting doses of the test sample are injected into semi -lethally irradiated N O D - S C I D hosts, and transplanted mice are scored as posit ive (engrafted) if they contain a threshold level of human lymphoid and myeloid cel ls. A s in the murine C R U assay , Po isson statistics are then employed to quantitate the H S C frequency. 1.2.3 Detection and quantification of progenitor cells The discovery of l ineage-restricted progenitor cel ls in the late 1960s led to a model whereby cel ls become irreversibly committed to l ineages, with progressive restrictions in potentiality, as they progress through hematopoiet ic differentiation. In vitro sys tems for supporting both murine and human hematopoies is have also been deve loped and adapted in various ways to al low speci f ic subpopulat ions of hematopoiet ic progenitor cel ls to be detected and quantif ied. The colony-forming cell ( C F C ) assay is a functional assay to test for the presence of progenitor cel ls that can form colonies of mature progeny in semi-sol id medium containing appropriate growth factors (Bradley et a l . , 1967; Ichikawa et al . , 1966). C F C s can produce from one (uni) to many (multi) l ineages and the greater the colony s ize and content of the different l ineages, the more primitive the cell from which the colony is thought to be der ived. For example, a multi-l ineage progenitor, C F U - G E M M (colony forming unit -granulocyte, erythroid, macrophage, megakaryocyte) gives rise to a very large colony composed of granulocytes, macrophanges, erythrocytes and megakarocytes, 10 and is more primitive than a uni-l ineage progenitor, C F U - E (colony forming unit -erythroid) which gives rise to smal ler colonies of only erythroid cel ls (Metcalf, 1984). Al though colonies have been shown to be clonal (Metcalf and Moore, 1971), most C F C s display a limited or no capacity for self-renewal as no or very few co lon ies are normally formed when primary colonies are re-plated into secondary a s s a y s (Siminovitch et al . , 1963). Ce l l purification techniques further revealed that most C F C s could be separated from more primitive cel ls with repopulating activity. C F C s are a lso more numerous than C F U - S and could be detected in spleen colonies as the progeny of C F U - S (Metcalf, 1984). These observat ions formed the basis of ass igning these cel ls to different steps in a hierarchy of hematopoiet ic cell differentiation (Metcalf, 2001). A n in vitro assay that has shown greater specificity for quantifying hematopoiet ic cel ls that appear to be more primitive than most C F C s is the long-term culture initiating cell (LTC- IC) assay . This assay detects a cell that can initiate sustained myelopoies is when co-cultured on stromal feeder layers (Sutherland et al . , 1989). Th is assay w a s developed from the observat ion that mature granulocytes and macrophages can be produced for several months when unseparated suspens ions of B M cel ls are cultured at high density in media containing horse serum and cort icosteroids. Subsequent studies showed that the primary need for serum was to generate a competent feeder layer of stromal cel ls that then stimulate the proliferation and differentiation of very primitive hematopoiet ic cel ls in the absence of exogenously suppl ied growth factors. In order to enable the latter to be 11 quantif ied independently of the ability of cel ls in the test suspens ion to form a competent feeder layer, irradiated pre-establ ished marrow feeders or irradiated monolayers of a number of human or murine fibroblast cell l ines can be used (reviewed in E a v e s and E a v e s , 2004). 1.2.4 Functional properties of HSCs A s s a y s for H S C s are based on 3 critical, and in large part unique functional H S C properties: capacity for self-renewal, generating at least one identical daughter cel l ; and capacity for long-term multi-l ineage hematopiet ic reconstitution. Us ing these criteria and avai lable assays a number of key properties and regulatory mechan isms of H S C s have emerged. 1.2.5 Phenotypic characterization of HSCs Currently, H S C s cannot yet be directly and consistently identified on the bas is of any unique morphological , physical or cell surface characterist ics, although a number of different phenotypic markers can now be used in combinat ion to obtain H S C s in nearly pure form from adult mouse B M . The most commonly ana lyzed markers are cell surface antigens against which specif ical ly reactive monoclonal ant ibodies have been made. These antibodies can then be labeled either directly or indirectly (via a secondary antibody) with a unique f luorochrome and used to dist inguish cel ls as positive or negative on the basis of their acquired f luorescence. Mult i -parameter flow cytometry has a great power to dist inguish cel ls based on these molecular ly-determined features, in an objective and quantitative way, with a high 12 degree of specificity. In addition, this technology can be used not only for cell analys is but also for their separat ion into viable subsets defined by the analys is . T h e s e isolated cel ls can then be assayed for their functional attributes. In this way, the phenotype of different functionally defined cell populations can be identified. Many stem cell markers have been descr ibed over the past 20 years (reviewed in V i sse r and Bekkum, 1990, Civ in and Gore , 1993, Uch ida et al . , 1993, Sz i l vassy and Hofman, 1995) . Murine H S C s are character ized by their high express ion of s tem cell antigen (Sca)-1 and low levels of Thy-1 . They are a lso character ized by the absence of l ineage (Lin) ant igens expressed predominantly on terminally differentiated lymphocytes ( C D 4 5 R / B 2 2 0 , C D 3 , C D 4 , C D 8 ) , myeloid ( C D 1 1 b / M a c - 1 , Ly -6G/Gr-1) , and erythroid (TER-119) cel ls. Notably, however, low levels of C D 4 and Mac-1 can be induced on some H S C s , particularly following activation by 5 -FU treatment of donor mice (Sz i lvassy and Cory, 1993, W e i s s m a n et a l . , 1997). S c a 1 + T h y - 1 l 0 L i n " cel ls have the capacity for long term reconstitution of lethally irradiated mice (Uchida and W e i s s m a n , 1992). The co-express ion of c-kit further enr iches for H S C s and c-kit + murine B M progenitors and therefore the c-k i t + Sca1 + L in " cell population is often used as a source of murine stem cel ls (Orlic et a l . , 1993). Human H S C s are character ized by high express ion of C D 3 4 , intermediate express ion of c-kit and Thy-1 , and low or no expression of C D 3 8 . Most strategies to isolate H S C s rely on utilizing the cell sur face ant igens descr ibed above, however, other properties of H S C s can be used in addit ion when isolating these primitive cel ls. The majority of H S C s in adult B M are bel ieved to be in a quiescent, non-cycl ing state (Ogawa et al . , 1993), although more recent studies 13 indicating that up to 10% of L T R - H S C randomly enter cell cycle per day, with all H S C entering the cell cycle in 1-3 months (Bradford et al . , 1997, Chesh ie r et a l . , 1999). B a s e d on the qu iescence theory, sorting for H S C residing in G 0 / G 1 cell cyc le phase with low levels of R N A , can enrich for these cel ls. V iab le dyes, such as the D N A dye Hoechst 33342 and the R N A dye Pyronin Y , are often used for this purpose (Gothot et al . , 1997). Another dye commonly used in stem and progenitor cell enr ichment is the mitochondria dye Rhodamine-123 (Rho), which does not stain primitive hematopoiet ic cel ls but does stain most other cel ls in the B M (Li et a l . , 1992, Spangrude and Johnson , 1990). Recent ly, another method based on the propensity of H S C s to actively pump out the Hoecht 33342 dye has emerged. A unique side population ( S P ) of cel ls is observed when the f luorescent properties of cel ls stained with this dye are s imul taneously observed at 2 wavelengths. S P cel ls from mouse B M were found to be highly enr iched for C F U - S and multi l ineage repopulating cel ls (Goodel l et a l . , 1996). Hoechst is actively exc luded by the A B C transporter protein A B C G 2 in H S C s , and overexpress ion of A B C G 2 leads to an expans ion of cel ls with an S P phenotype (Zhou et a l . , 2001). The S P phenotype of the H S C s in adult mouse B M has been useful to dev ise relatively simple strategies for isolating populations that are at least 4 0 % pure C R U s (Uchida et al . , 2003). The S P phenotype initially attracted considerable interest as a potential marker for H S C s from many spec ies because a smal l S P population can be demonstrated in hematopoiet ic cel ls from many spec ies including humans (Goodel l et al . , 1997). Moreover, H S C s in human fetal liver were 14 found to have an S P phenotype (Uchida et al . , 2001) but this is not the c a s e for H S C s in human cord blood (Fischer et al . , 2005). Unfortunately, many of the avai lable markers for discriminating the most primitive hematopoiet ic cell types cannot be used to enumerate changes in their numbers reliably, particularly under c i rcumstances where their activation or cycl ing status may have been altered, because expression of these markers is labile under these condit ions (Zanjani et al . , 2003; Uchida et al . , 2004). Thus , in order to measure levels of H S C expans ion in culture is still necessary to rely on functional a s s a y s rather than phenotype. However, the increasing refinement in methodologies for phenotype discrimination, when validated by functional assays , do offer great promise for obtaining highly purified populations that can then be used for gene express ion and proteomic studies. 1.2.6 Self-renewal of HSCs H S C s appear early in embryogenes is and subsequent ly amplify their numbers to levels that are maintained for the l i fespan of the individual. During ontogeny, there is a great expans ion of all hematopietic cel ls, including H S C s , to meet the growing needs of the body. The murine fetal liver (FL) at 12 days post-conceptus (dpc) contains approximately 40 H S C s , as detected by the C R U assay . By 16 dpc this number has expanded 30-fold to 1500 H S C s (Ema and Nakauch i , 2000) and by adulthood a further 13-fold expansion brings the total H S C cnotent up to 20,000 (Sz i lvassy et a l . , 1990). From this expans ion we can infer that extensive H S C self-renewal occurs during ontogeny. 15 H S C self-renewal a lso occurs in myeloablated hosts undergoing hematologic recovery, as for example, following a B M transplant. This has been formally demonstrated by recovery of cel ls capable of giving donor-der ived long-term repopulation of multiple secondary recipients from mice previously transplanted with only a single H S C (Dick et al . , 1985; Kel ler et al . , 1985; Lemischka et a l . , 1986; Brecher et a l . , 1993; O s a w a et al.,1996). Quantitative ana lyses have shown that H S C s recovered from transplant recipients can be up to 100-fold higher than the number transplanted (Pawlliuk et a l . , 1996). 1.2.7 Potential clinical applications of ex vivo expanded HSCs The ability to activate H S C s into division without causing their differentiation would be an immensely useful tool for both experimental and clinical appl icat ions. The capaci ty for sustained self-renewal is fundamental for the increasing appl icat ion of H S C - b a s e d therapies in a wide range of malignant and genetic disorders. From that perspect ive, the development of strategies to extensively expand H S C s ex vivo could greatly enhance the safety and application of H S C - based therapies in treatment of mal ignancies, gene therapy and other areas. H S C transplantation following a myeloablat ive condit ioning regimen is the only potenially curative treatment for aplast ic anemia, hemoglobinophat ies and leukemias, as it results in a full or partial replacement of the d iseased hematopoiet ic t issue with normal cel ls. However, there are significant risks associated with H S C transplantation, including toxicity of condit ioning regimens, graft versus host d i sease ( G V H D ) and mortality (reviewed in Anasett i et al . , 2001). Whi le H S C s obtained directly from the patient 16 (autologous H S C s ) are used for rescuing patients from the effects of high dose of chemotherapy or used as the target for gene therapy vectors, H S C s obtained from another person (al logeneic H S C s ) are used to treat hematological mal ignancies by replacing the malignant hematopoiet ic system with normal cel ls. The number of H S C s injected is bel ieved to be critical to ensure the eff icacy of B M transplantation procedures (Mavroudis et a l . , 1996, Sierra et al . , 2000). Therefore, amplif ication of H S C numbers would be useful in severa l contexts, both to overcome existing limitations and to develop new transplantation approaches . A success fu l H S C expans ion strategy could also potentially al low the use of much smal ler harvests of sources of H S C (e.g., mobi l ized peripheral b lood, bone marrow) thereby reducing the cost and the risk of H S C collection. It might a lso accelerate the rate of H S C recovery following B M transplantation and allow use of T-cel l depleted donor grafts, which reduce the incidence of and severity of G V H D . W h e n such grafts were used in pediatric transplantation, the rate of T cell recovery w a s dependent on the dose of the C D 3 4 + cel ls that were administered. High doses of C D 3 4 + cel ls (>20x10 6 cel ls per kg) led to a marked decrease in the time required for T cell reconstitution (Handgretinger et al . , 2001, Lang et al . , 2004). However, in many c a s e s , this high number of CD34+ H S C s could not be col lected from the donor. Another promising new source of H S C s for transplantation is cord blood (CB) , which gives a reduced incidence of G V H D due to the naive immunological state of the cel ls (Rocha et al . , 2001). However, there is also a higher rate of mortality because of failed or delayed engraftment. This latter issue relates to the smal l s ize of C B grafts and their consequent ly low stem cell content. Higher H S C d o s e s have 17 consistently been found to correlate with improved d isease- f ree survival and reduced transplant-related mortality (Mavroudis et a l . , 1996, Sierra et a l . , 2000). Until H S C expans ion methods are dramatically improved, the use of C B material will remain limited in adults. H S C - b a s e d gene therapy is a growing treatment option for patients with hematological defects (reviewed in C a v a z z a n a - C a l v o and Hace in -Bey-Ab ina , 2001). It involves collecting H S C s from the patient and genetical ly modifying them with a therapeutic t ransgene. These procedures require ex vivo culture of hematopoiet ic cel ls and usually result in significant H S C losses, s ince in most culture condit ions differentiation is favored over expansion. Moreover, genomic integration of retrovirus-based vectors requires target cel ls to be proliferating (Sadela in et al . , 2000). Activation of H S C s into the S / G 2 / M phase of the cell cycle a lso results in the transient loss of engraftment potential (Habibian et a l . , 1998 G l imm et al . , 2000). Therefore, an ability to expand H S C s ex vivo could greatly improve the cl inical ou tcomes of H S C - b a s e d gene therapies. In addition to these potential cl inical appl icat ions, an ability to increase the number of H S C s in culture would provide a useful tool and source for studying the molecular mechan isms underlying H S C self-renewal. 1.2.8 Extrinsic regulators of HSC function Whi le the clinical imperative is high, harnessing and enhancing H S C self-renewal potential remains a formidable chal lenge (see Sauvageau et a l . , 2004. for recent review). A variety of in vitro condit ions have now been descr ibed that permit 18 enormous expans ion of C F C s and substantial expans ion even of L T C - I C s (Petzer et al . , 1997; Zandst ra et a l . , 1997; Hoffman, 1999). However, the in vitro expans ion of rigorously defined H S C s has not been achieved under the same condit ions. The largest reprodicble in vitro expans ion of murine H S C s to date, using exogenous growth factors, is a 4-fold net increase of C R U s that is obtained in serum-free medium containing a combinat ion of interleukin-11 (IL-11), flt3-ligand (flt3-L) and Steel factor (SF) (Miller and E a v e s 1997). In a similar fashion, human C B H S C s were found to undergo a 2-4-fold net increase after 4-8 days culture in serum-free medium containing a rich cocktail of growth factors: flt3-L, S F , granulocyte colony-st imulating factor ( G - C S F ) , IL-3 and IL-6 (Bahtia et al . , 1997, Connea l ly et a l . , 1997). Intrinsic dif ferences in cytokine requirements from different sources of H S C s were demonstrated, s ince human H S C s have different optimal cytokine combinat ions from murine H S C s . C lues to extrinsic mediators of H S C self-renewal are also emerging from a broader understanding of the key receptor signaling pathways involved in the development and maintenance of the hematopoiet ic sys tem. Perhaps , some of the most compel l ing ev idence of early developmental growth factors impacting of H S C self-renewal have emerged from studies where presentation of the No tch l l igand as an engineered immobil ized form (Del ta l ) , together with a cocktai l of growth factors ( S F , flt3-L, IL-6 and IL-11), resulted in a several log increase in the number of cel ls capab le of short term lymphoid and myeloid repopulation after 28-day culture (Varnum-Finney et. a l . , 2003). Furthermore, addition of the soluble form of Son i c Hedgehog protein along with a cocktail of hematopoiet ic growth factors (flt3-L, S F , 19 G - C S F , IL-3 and IL-6) to a liquid cultures of human B M cel ls, a lso enhanced recovery of human H S C s over 7 day culture period (Bhardwai et al . , 2001). Purif ied Wnt3A was shown to synergize with low doses of S F to induce proliferation of H S C -enr iched cel ls p laced in single-cel l cultures (Willert et al . , 2003). S u c h f indings raise opt imism that further refinement of culture condit ions, growth factors etc. may enable striking increases in H S C numbers. 1.2.9 Intrinsic regulators of H S C function The decis ion made by individual H S C s to self-renew (or not) has long been thought to be largely determined by stochastical ly regulated intrinsic mechan isms. Th is concept was first developed from studies showing a large variability in the numbers of C F U - S generated in individually a s s e s s e d sp leen colonies (Siminovitch et a l . , 1963) and the demonstrat ion that this variability is predicted by a probabil istic model (Till et a l . , 1964) in which the likelihood of each C F U - S producing at least one progeny C F U - S throughout the formation of a spleen colony is only slightly higher than 0.5 (Vogel et al . , 1968). It is important to note that the concept of s tem cell self-renewal ou tcomes being descr ibed as probabilistic at a population level does not mean that the probability of stem cell self-renewal cannot be inf luenced. The limitations to this model were that they did not accommodate the possibil ity of pre-existent C F U - S hetergeneity in self-renewal potential or variations in the micro-environment in which each spleen colony develops (reviewed in Till and McCu l l och , 1980). Later, the role of the environmental variability as a determining factor w a s largely ruled out by replicating these findings in vitro (Humphries et a l . , 1981). 20 However, even when self-renewal responses appear to be optimally supported by external factors, intrinsic mediators may be limiting resulting in a stochast ic picture of response outcome at the population level. Through the use of forward-genetic approaches, particularly loss-of-function or gain-of-function mouse models, some of the genes and related signal ing pathways that are important in these outcomes have been identified. However, the identification of important H S C regulatory genes is likely incomplete. Thus decipher ing the way in which these various regulatory pathways interact and identifying key common molecular target(s) remains a major chal lenge. Whi le much has been learned with regard to extrinsic factors that can promote the survival and proliferation of H S C s , growth factor-stimulated pathways that might promote self-renewal have proven more difficult to elucidate. A hematopoiet ic cel l 's developmental state is reflected by its complement of expressed genes . However, specif ic regulatory programs that control the gene express ion repertoire to maintain the H S C state or trigger their differentiation remain unknown. Never the less, analys is of genes involved in leukemia and/or early development have begun to provide some important c lues and, in particular, have drawn attention to various regulators of gene express ion such as transcription factors, cell cycle regulators and chromatin modifiers. Interestingly, gene expression experiments have shown that multipotent progenitor cel ls express many l ineage-associated genes at low levels (Hu et a l . , 1997). Development along a given l ineage thus appears to involve both activation of l ineage-speci f ic maturation factors and repression of genes assoc ia ted with alternate 21 l ineages. Transcript ion factors act on both processes , thereby promoting l ineage cho ices . They act at all hematopoiet ic branch points, including specif icat ion to the hematopoiet ic fate, branching of the major lymphoid and myeloid l ineages and commitment of bipotent progenitors to single l ineages (reviewed in Orkin, 2000, Sh ivdasan i and Orkin, 1996). G e n e s required for the specif ication of hematopoiet ic potential include SCL (also cal led tal-1) and LM02 (also cal led rbtn2), which encode basic helix-loop-helix and LIM-domain type transcription factors, respectively. Mice lacking either of these genes have a complete absence of primitive hematopoiesis and die at approximately 10 dpc (Porcher et a l . , 1996, Warren et al . , 1994). S C L and L M 0 2 proteins interact physical ly (Larson et a l . , 1996, W a d m a n et al . , 1994) and their similar loss-of-function phenotypes suggest co-operat ive transcriptional control of hematopoiet ic-speci f ic genes . Both are further required for erythroid differentiation, where they form a complex together with the transcription factors G A T A 1 , E 2 A and Ldb1 (Wadman et a l . , 1997). Ectopic express ion of S C L or L M 0 2 in T-cel ls leads to the generat ion of T cell acute lymphoid leukemia (Brown et al . , 1990, Larson et al . , 1996). Ikaros is an intrinsic factor, which acts at the lympho-myeloid branching point, promoting specif icat ion to the lymphoid l ineage. Mice lacking Ikaros lack all B-lymphocytes and precursors, as well as fetal T lymphocytes, although a few C D 4 + T cel ls are aberrantly produced (Wang et a l . , 1996). Ikaros represses transcription of non-lymphoid genes via recruitment of histone deacety lase complexes to speci f ic promoters (Kim et a l . , 1999), thereby altering chromatin accessibi l i ty. 2 2 Further specif icat ion within the lymphoid l ineage comes from factors such as P a x 5 , which directs cel ls along the B-lymphiod l ineage while repressing differentiation along alternate l ineages. Pax5 knockout mice lack mature B-cel ls and precursors (Urbanek et al . , 1994) and pro-B cel ls from these mice will reconstitute T but not B lymphoid cel ls in transplanted mice (Rolnik et al . , 1999). L ineage specif ication within the myeloid system comes in part from c ross-antagonism between G A T A - 1 and P U . 1 . G A T A - 1 promotes erythroid and megakaryocyte differentiation, while PU.1 acts on disparate lymphoid and myeloid l ineages. G A T A - 1 and PU.1 directly interact with and inhibit one another (Nerlov et a l . , 2000, Rekhtman et al . , 1999). Thus the ultimate l ineage choice will be dec ided and then reinforced by the relative levels of these 2 transcription factors. Recent ly, H O X family transcription factors have emerged as important regulators of hematopoiesis, acting at various levels of the hematopoiet ic hierarchy. The following sect ion will d i scuss the roles of these proteins in hematopoies is and their potency to expand H S C s ex vivo. 23 1.3 HOX Genes in HSC Function 1.3.1 Hox gene organization and expression The Homeobox (HOX) genes were first d iscovered in Drosophila melanogaster (Lewis, 1978.). The term "homeobox" takes its origin from an old genet ic term, the homeotic mutation, which descr ibes Drosophila mutations in which the identity of one body segment was transformed into that of another: for example , the development of legs in the position where antennae are normally located. D. melanogaster has 8 homeobox genes divided in 2 clusters, Antennapedia (ANT-C) and Bithorax (BX-C) complexes. Together the 2 groups of clustered genes make up the Drosophila homeotic complex (HOM-C). A large number of genes involved in pattern formation and morphogenes is during fruit fly development are homeobox genes. It was quickly recognized that these genes are present in all animal genomes, including man and mouse and play crucial roles in pattern formation and t issue identity throughout the animal k ingdom (Akam 1989). In mammals there are 2 main groups of HOX genes : c lass I, or the clustered Hox genes that have high homology to Antennipedia and c lass II, a diverged group of homeobox genes that have low homology to Antennipedia (Krumlauf, 1994). There are 39 c lass I H O X genes known in mammals , organized in four clusters, A - D , each containing 9-11 genes on 4 different ch romosomes (Boncicel l i et a l . , 1989, Scott, 1992, Zel tser et al . , 1996). Based on homology, HOX genes in separate clusters can be al igned in groups, resulting in 13 paralogs (i.e. HOXA4, HOXB4, 24 H0XC4 and H0XD4). The high homology within paralogs suggests that a quadrupl icat ion o f a single gene cluster has occurred during evolution (Hol land et a l . , 1994, Kappen et al . , 1993, Schughart et a l . , 1989). Figure 1.2 shows the organizat ion of the clusters and compar ison with the corresponding Drosophila homeobox genes . HOX genes are expressed during embryonic development co- l inear with chromosomal order. In Drosophila, genes at the 3' ends of clusters were found to be expressed earliest in development, with more 5' genes expressed later. Co-l ineari ty a lso extends to the spatial domains of HOM-C gene express ion, with 3' genes expressed in more anterior structures and more 5' genes having sequential ly more posterior express ion domains. This temporal and spatial co-linearity is a lso true for mammal ian HOX gene expression during development (Dolle et al . , 1989, G r a h a m et a l . , 1989, Izpisua-Belmonte and Duboule, 1992), suggest ing that gene order might have been conserved in order to maintain this tightly linked express ion pattern. Regulat ion of HOX gene express ion, which is not fully understood, involves most likely complex c ross- and auto-regulatory mechan isms s ince it is known that HOX genes can affect the expression of each other (Zappavigna et al . , 1991). H O X proteins are DNA-binding transcription factors. They have domains for D N A binding and for protein-protein interactions. The most prominent structure of all H O X proteins is their homeodomain (reviewed in Gehr ing et a l . , 1994). The structure of homeodomain , 60-amino acid D N A binding domain, has been deduced by N M R (Billatar et a l . , 1990, Q ian et al . , 1989) and X-ray crystal lography (Kiss inger et a l . , 1990, Li et al . , 1995). At the N-terminal end is flexible arm, fol lowed by a lpha helix I, 25 which is connected by a loop to alpha helix II. The helix-turn-helix sequence connect ing hel ices II and III forms a highly conserved structure common to many D N A binding proteins. Footprinting, E M S A and trans-activation a s s a y s have shown that H O X proteins bind D N A as monomers to 5 ' -TAAT-3 ' core motif (Kal ionis and O'Farrel l , 1993). Helix III acts to recognize this sequence and binds D N A in the major groove. The flexible N-terminal arm binds to bases in the minor groove and the loop between hel ices II and III binds to the D N A backbone (Otting et a l . , 1990). Drosophila ANT-C BX-C a: O i r u.i I 2 HoxC I HoxA I A l HoxB [ B T < CO "O ~o < < Human 1 1 I I I I I C H R O M O S O M E A2 A3 A4 A 5 A6 A? A9 A10 A l l B2 B 3 B4 B5 B6 B7 B8 B9 • • • • 813 C4 C6 CIO ;11 C12 C13 HoxD D l D3 D4 D8 D9 D10 D111| D121| D13 human mouse a: 7 6 O ir. 17 11 1.4.1 1-w 12 15 CL 2 2 Direction of HOX gene transcription Early - 5' Late F i g u r e 1.2 HOX c h r o m o s o m a l o r g a n i z a t i o n . The 4 mammal ian H O X clusters (A-D) are shown, with al ignments to the Drosophila ANT-C and BX-C. G roups 1-13 are cal led paralog groups sharing high sequence homology in the homeodomain . B lack boxes indicate lack of gene. G e n e s at the 3' ends of the clusters are expressed earl iest and most anterior, with sequential ly later and more posterior express ion of more 5' genes . 26 1.3.2 HOX gene expression and roles in hematopoiesis A role for H O X genes in hematopoiesis was first demonstrated in human and murine hematopoiet ic cell l ines. The first reports indicated l ineage specif icity of the Hox clusters, e.g. , HOXB genes predominantly expressed in erythroid cell l ines while HOXA genes were active in myeloid cel ls and HOXC in lymphoid cell l ines (Lowney et a l . , 1991, Mathews et a l . , 1991, Lawrence et al . , 1993). This l ineage specificity is, however, not true for all HOX genes , where some show a much broader express ion pattern. Members of the HOXD cluster were found not to be expressed in hematopoiet ic cel ls (Thompson et al . , 2003). Express ion analys is of primitive human hematopoiet ic CD34+ subpopulat ions revealed that nine of 11 HOXA genes ; 8 of 9 HOXB genes and 4 of 9 HOXC genes tested, were expressed in this population (Giampaolo et a l . , 1994, Moretti et al . , 1994, Sauvageau et al . , 1994). Express ion of the HOXA genes was strongest, fol lowed by the HOXB genes and thereafter the HOXC genes . HOX genes located 3 ' in the cluster, such as HOXB3 and HOXB4, were mainly expressed in the primitive subset of C D 3 4 + cel ls and then down-regulated as the primitive cel ls start to mature. In contrast, 5 ' located HOX genes , such as HOXA9 and HOXA10, had prolonged express ion and were also found in more differentiated populations (Sauvageau et al . , 1994). S u c h findings suggested that HOX genes play functional roles in early s tages of hematopoiet ic growth and differentiation. Further express ion analysis and gain- or loss- function studies in mouse models indeed confirmed that H O X genes have the ability to specif ical ly regulate different s tages of hematopoietic development, including the self-27 renewal/proliferation of H S C s (Thorsteinsdottir et al . , 2002; Antonchuk et a l . , 2001 ; Buske et a l . , 2002) and the differentiation of myeloid and lymphoid l ineages (Owens and Hawley, 2002; Buske and Humphries, 2000). In recent years, H O X genes have been strongly l inked to human leukemia (Lawrence et al . , 1999; Rozovska ia et a l . , 2001 ; Kawagoe et a l . , 1999Afonja et al . , 2000; Imamura et al . , 2002) by their observed aberrant express ion and by translocat ions involving their cofactor P B X 1 (Kamos et a l . , 1993) and upstream regulators (Ayton et al . , 2001). Moreover, the d iscovery of chromosomal translocations involving a growing list of A b d - B H O X genes provided support for the direct involvement of H O X genes in the pathobiology of human leukemia (Slape and Ap ian 2004). Further elucidation of the cel lular and molecular p rocesses that are involved in normal and/or leukemic hematopoies is and controlled by the complex H O X - b a s e d regulatory network, holds promise for developing new tools to expand H S C s and for providing a deeper understanding of mechan isms underlying H S C self-renewal. 1.3.3 HOXB4 - a potent stimulator of HSC expansion The effects of HOXB4 gene on hematopoiet ic cel ls were initially d iscovered by using a retroviral-expression vector (Sauvageau , G . et al., 1995). The most dramatic effect observed in recipients of / - /OXB4-transduced cel ls was an enhanced regenerat ion of donor-derived H S C s . Thus when lethally irradiated mice were reconstituted with B M cel ls that were t ransduced with a control vector, H S C numbers in the B M regenerated to only 5 - 1 0 % of normal levels, whereas B M cel ls t ransduced the HOXB4 vector regenerated normal numbers of H S C s . However, further 28 expans ion of the H S C compartment did not occur (Thorsteinsdottir et a l . , 1999), showing that the effects of HOXB4 are still subject to normal homeostat ic controls. Stud ies indicating an ability of H O X B 4 to enhance the self-renewal of H S C s in vivo provided a bas is for investigating its potential to promote an expans ion of H S C s in vitro (Antonchuk et a l . , 2002; Kros l et a l . , 2003 ; Kros l et a l . , 2003). T h e s e studies demonstrated that HOXB4 can stimulate the ex vivo expans ion of H S C s , when suppl ied either as a t ransduced c D N A or as externally del ivered protein and the H S C s produced retain their normal differentiation and long-term repopulation potential. Adult mouse H S C s engineered to overexpress HOXB4 expand 40-fold after 2 weeks of culture in media containing IL-3, IL-6 and S F , while the number of untransduced of GFP- t ransduced H S C s decreased by 30-60-fold (Antonchuk et a l . , 2002) . The rapid ex vivo H S C expans ion induced by H O X B 4 has further been exploited with the development of H O X B 4 fusion proteins with the protein transduction domain of HIV T A T protein, that can be del ivered directly to t issue culture medium to achieve H S C expans ion in short term liquid culture (Krosl et a l . , 2003) . T A T fusion proteins moved freely between the medium and intracellular compartments. However, the majority of T A T - H O X B 4 protein is lost after a 4 hour incubation in serum-containing media and the half-life of intracellular H O X B 4 is only approximately 1 hour. H S C s exposed to T A T - H O X B 4 for 4 days expanded by about 4 to 6-fold and were 8-20 t imes more numerous than H S C s in control cultures. T h e s e f indings indicate that H S C expansion induced by T A T - H O X B 4 is comparab le to that induced by HOXB4 retrovirus during a similar period of observat ion and 29 encourage further development of more potent Hox-based molecules that could be adapted to delivery as proteins rather than by gene transfer. Stimulatory effects of HOXB4 have also been demonstrated on human H S C s following retrovirally-engineered overexpress ion. Studies by Buske et al showed 5-fold expans ion of C B cel ls that repopulate N O D / S C I D mice after just 1-2 days in culture (Buske et a l . , 2002). In addition, studies by Baum 's group showed increased levels of HOXB4- t ransduced CD34+ cel ls in S C I D recipients compared to controls (Sch ied lme ie re t a l . , 2003). The mechan ism of HOXB4-med ia ted expans ion of H S C s is not well understood. It is known that HOXB4 can co-operatively dimerize with P B X 1 (pre-B-cell leukaemia transcription factor 1) (Krosl et a l . , 1998) which is encoded by a proto-oncogene that is required for the maintenance of definitive (adult) hematopoies is (Dimartino et al . , 2001). Downregulat ion of P B X 1 express ion using a vector that co -expressed both a P B X 1 ant isense sequence and H O X B 4 resulted in a further increase in H S C expans ion relative to that observed using HOXB4 a lone (Krosl et a l . , 2003). 1.3.4 Other HOX genes that have an ability to promote HSC expansion Although HOXB4 has been the most extensively studied H O X gene for its ability to increase H S C self-renewal, this potential may not be unique to HOXB4, and may extend to other intact or variant H O X genes . For example, H O X 4 9 w a s initially studied for its strong involvement in acute myeloid leukemia. However , when compared with controls, recipients of HOX/49- t ransduced cel ls had about a 15-fold 30 increase in transplantable lymphomyeloid long-term repopulating cel ls, during the preleukemic phase of the d isease (Thorsteinsdottir et al . , 2002). In the s a m e study, it was demonstrated that overexpression of HOXA9 greatly enhances H S C regenerat ion in transplantation chimeras, leading to an expans ion of myeloid C F C s and accompan ied by a partial block in B lymphopoiesis. T h e s e data, together with the preferential express ion of HOXA9 seen in primitive hematopoiet ic cel ls (Sauvageau et a l . , 1994) and the reduction in H S C numbers seen in Hoxa9 homozygous mutant mice (Lawrence et al . , 1998), suggest that this gene might qualify as a regulator of primitive hematopoiet ic cel ls and be capable a lso of promoting H S C expans ion ex vivo. Another major way in which HOX genes have been implicated in leukemia is through their involvement in translocations with nucleoporin 98 {NUP98). Abdominal-B HOX genes are the most common fusion partners of NUP98, identified in patients with myeloid leukemia (Lam and Ap ian , 2001). The N U P 9 8 protein is a component of the nuclear pore complex, which regulates nucleocytoplasmic transport of protein and R N A (Radu et al . , 1995). All NUP98-HOX fusions reported to date include the N-terminus of N U P 9 8 which contains a region of multiple phenylalanine-glycine repeats that may act as a transcriptional co-activator through binding to C B P / p 3 0 0 (Kasper et a l . , 1999). They also contain the C-terminus of the HOX gene product (A9, A 1 1 , A 1 3 , C 1 1 , C 1 3 or D13), including the intact homeodomain and a variable portion of the f lanking amino ac ids (Lam and Ap ian , 2001). A s detai led below, f indings from studies of properties of natural and engineered fusions have provided intriguing new leads to potent molecules for H S C expans ion. 31 Initially these NUP98-HOX fusion genes were analyzed for their effects on t ransduced murine B M transplants, which demonstrated the leukemogenic activity of fus ions containing Abdominal-B members (i.e., NUP98-HOXA10), but not Antennapedia members (i.e., NUP98-HOXB4 or NUP98-HOXB3)(P\neau\{ et a l . , 2004). It was also found that these NUP98-HOXfusion genes have a potent ability in vitro to block hematopoiet ic differentiation and to promote the sel f-renewal of primitive progenitors, as indicated by serial replating of C F C s or mass ive expans ion of C F U - S numbers in short term (7 day) cultures. Interestingly, NUP98-HOXA10 had a much more potent activity in this regard than did NUP98-HOXB4. 32 1.4 Thesis objectives The exper iments in this study were driven by a primary goal to develop new strategies for achieving higher levels of H S C expans ion ex vivo than can be obtained with H O X B 4 . B a s e d on the work showing that N U P 9 8 - H O X proteins have a more powerful ability to suppress early hematopoiet ic cell differentiation than H O X B 4 , my work focused on the potential use of NUP98-HOX fusion genes as potential stimulators of H S C expans ion in an ex vivo sys tem. The major a ims of these studies were as follows: 1) To del ineate the potency of NUP98-HOX fusion genes for H S C expans ion ex vivo; 2) To determine if the HOXA10 homeodomain sequence was sufficient as part of the NUP98-HOXA10 fusion gene to stimulate H S C expans ion ; 3) To a s s e s s the quality of A/L/P98-/- /OX-transduced and expanded H S C in regard to long-term lympho-myeloid repopulation ability; 4) To demonstrate clonal ex-vivo expans ion of NUP98-HOX t ransduced H S C s . 33 CHAPTER 2 MATERIALS AND METHODS 2.1 Retroviral Vectors Vectors were based on the murine stem cell virus ( M S C V ) vector originally descr ibed by Hawley which has virtues of expressing well in H S C and later cel ls and is not prone to expression si lencing (Hawley et al . , 1994). All vectors used an internal r ibosomal entry site ( IRES) to enable efficient translation of 2 proteins from a single L T R driven transcript. All vectors a lso contained a GFP c D N A in the second posit ion to provide a convenient marker to identify and select t ransduced cel ls and serve as a surrogate marker of the co-expressed HOX gene. Al l vectors (Figure 2.1.) have been descr ibed previously (Antonchuk et al . , 2001; Pineault et a l . , 2004). Constructs were val idated by sequenc ing and correct express ion and t ransmiss ion were conf irmed by Western blot and Sothern blot analys is. Product ion of high-titre helper-free retroviruses was carried out by standard procedures (Pawliuk et a l . , 1994), using virus-containing supernatants from transfected amphotropic Phoen ix packaging cel ls (Kinsel la and Nolan, 1996) to t ransducer the ecotropic packaging cell line G P + E 8 6 (Markowitz et al . , 1988). 34 Figure 2.1. Structures of retroviruses used in the study Three new NUP98-HOX fusion genes were engineered b y fusing the c D N A sequence corresponding to the homeobox-conta in ing exon of HOXA10 (NA10) or HOXB4 (NB4), or just homeodomain (hd) of HOXA10 {NAIOhd) to that of NUP98. Only NA 10 retained its Pbx-interacting motif, which is indicated as a black rectangle. GFP HOXB4 NUP98HOXB4 NUP98HOXA10 LTR IRES eGFP LTR LTR hd IRES eGFP LTR LTR NUPS8 hd IRES eGFP LTR LTR NUP38 1 hd IRES eGFP LTR LTR IMUP38 hd IRES eGFP LTR 2.2 Mice Mice were bred and maintained at the British Co lumb ia C a n c e r R e s e a r c h Cent re animal facility accord ing to the guidel ines of the Canad ian Counc i l on An imal C a r e . Al l B M donors and recipients were chosen based on their C D 4 5 cell sur face marker genotype. Transplant donor/recipient pairs were either C57BI /6Ly -Pep3b 35 (Pep3b) mice that express Ly5.1 and C57BI /6 -W* W ( W 4 1 ) mice that express Ly5.2, or Pep3b mice that express Ly5.1 and C 5 7 B L / 6 J (B6) mice that express Ly5.2, or B6 mice that express Ly5.2 and Pep3b mice that express Ly5.1 . 2.3 Infection of Primary Murine BM Cells Primary mouse B M cel ls were t ransduced as previously descr ibed (Antonchuk et al . , 2002). Briefly, B M cel ls were extracted from mice injected intravenously 4 days previously with 150 mg/kg 5-fluorouracil (5-FU) (Fauld ing, Underdaler, Austral ia) and the cel ls were then cultured for 2 days in Du lbecco 's modif ied Eag le ' s medium ( D M E M ) supplemented with 1 5 % fetal bovine serum ( F B S ) , 10 ng/ml human IL-6, 6 ng/ml murine IL-3, and 100 ng/ml murine S F . Med ia , serum and growth factors were purchased from StemCel l Techno log ies (Vancouver , B C , Canada ) . After stimulation, the cel ls were harvested and infected by co -cultivation on irradiated (4,000 c G y X-rays) G P + E - 8 6 viral producer cel ls with the addit ion of 5 ug/ml protamine sulfate (S igma, Oakvi l le, O N , Canada) . Loose ly adherent and nonadherent cel ls were recovered from these co-cultures after 2 days and cultured a further 6 days in the same medium without protamine sulfate. For bulk culture experiments, 3 x 1 0 6 cel ls were seeded in a 10 cm dish at day 0, and the equivalent of 3 x 1 0 5 starting cel ls (day 0) were replated into the s a m e s ize dish on days 6 or 7. For cultures initiated with small numbers of bulk B M cel ls (5000) or sorted S c a - 1 + Lin" cel ls (500), cel ls were cultured in a 96-well at day 0, and replated into a 24-well plate at day 7. 36 2.4 CRU Assay H S C s were detected and evaluated using a limiting dilution transplantation-based assay for cel ls with competit ive, long-term, lympho-myeloid repopulating function. The basic procedure (Sz i lvassy et a l . , 1990) and a modification employing sublethally irradiated W 4 1 recipients (450 c G y 1 3 7 C s gamma radiation) with an endogenous source of competitor cel ls, have been descr ibed in detail previously (Miller et. al . 1997, Antonchuk et a l . , 2002; Antonchuk et. al . , 2001;). Briefly, lethally (810 c G y of X-ray) or sublethally (450 c G y of 1 3 7 C s - g a m m a radiation) mice were injected with the cultured cel ls in varying dilutions, and their P B cel ls were col lected and ana lyzed by flow cytometry > 16 weeks post-transplant to look for ev idence of regenerated lymphoid and myeloid cel ls derived from the t ransduced (GFP+) cel ls injected. Mice that had >1% donor-derived ( G F P + ) cel ls in all subpopulat ions of myeloid cel ls ( G r - 1 + and/or Mac -1 + ) , B cel ls (B220 + ) , and T cel ls ( C D 4 + and/or C D 8 + ) were cons idered to be repopulated with t ransduced cel ls. C R U f requencies were calculated by applying Po i sson statistics to the proportion of negative recipients at different dilutions using Limit Dilution Analys is software (StemCel l Technolog ies) . 2.5 Flow Cytometry For analys is of transplant recipients, 100 u.l of blood was extracted from the tail vain, and the erythrocytes were lysed with ammonium chloride (S temCel l Technol ig ies) . Leukocyte samp les suspended in H F were incubated sequential ly on ice with the following monoclonal antibodies: a combinat ion of biotinylated anti-Ly5.1 37 (anti-Ly5.2) and either phycoerythrin (PE)- labeled B220 or combinat ion of P E -labeled Gr-1 and Mac-1 or combinat ion of PE- labe led C D 4 and C D 8 and then a l lophycocyanin (APC)- labe led streptavidin. Al l antibodies were purchased from Pharmingen (San Diego, Cal i fornia, U S A ) . Al l samples were washed with H F and 1ucj/ml PI prior to analysis. 2.6 Purification of Sca-1+Lin' Cells B M cel ls were stained with f luorescein isothiocyanate (FITC)- labeled anti-S c a - 1 , a l lophycocyanin (APC)- labe led anti-c-kit, and PE- labe led ant i -Gr-1, anti-B220 , ant i -CD4, ant i -CD8 and Ter119 antibodies. Sort ing was performed on a F A C S A r i a system (Becton Dickinson, S a n J o s e , C A ) . Sorted cel ls were counted using a hemocytometer and plated into a 96-well plate. 2.7 Proviral Integration Analysis G e n o m i c D N A was isolated with D N A z o l reagent (Invitrogen, Car l sbad , C A ) , as recommended by the manufacturer, and Southern blot analys is was performed as previously descr ibed (Sauvageau et. a l . , 1994). Unique proviral integrations were identified by digestion of D N A with EcoRI, which c leaves once within the provirus and at var ious d is tances outside in the host genome. Digested D N A w a s then separated in 0 .8% agarose gel by electrophoresis and transferred to zeta-probe membranes (B io -Rad , M iss i ssauga , ON) . Membranes were probed with a [ 3 2 P] d C T P GFP sequence . 3 8 CHAPTER 3 RESULTS 3.1 NUP98-HOX Fusion Genes Stimulate A Very Large Expansion of HSCs in Culture A first ser ies of experiments were des igned to test the possibil ity that NUP98-HOX fusion genes have a similar or even greater potency to stimulate H S C expans ion in vitro than HOXB4, which was previously documented to expand H S C s more than 40-fold in 2-week cultures (Antonchuk et al . , 2002). Therefore, H S C numbers were measured in cultures of GFP-, HOXB4-, NUP98-HOXB4- and /Vl /P98-/- /OX,470-transduced mouse B M cel ls by performing limiting dilution C R U a s s a y s before and after 10 days in vitro without select ion of G F P - e x p r e s s i n g cel ls. A s shown schemat ical ly in Figure 3.1, B M cel ls were harvested 4 days after intravenous injection of donor mice with 5 -FU and individual cultures were initiated (Day 0) with 3 x 1 0 6 cel ls per culture. Ce l l s were pre-stimulated for 2 days with IL-6, IL-3 and S F prior to being retrovirally t ransduced with G F P control or HOXB4, NUP98-HOXB4 or NUP98-HOXA10 vectors (2 additional days of co-culture with virus producers) and were then cultured for another 6 days in suspens ion with the s a m e growth factors. O n day 10, more than 7 5 % of the cel ls in each culture were G F P + and various doses of starting cell equivalents were transplanted into irradiated recipients. 39 Figure 3.1. - General experimental design DayO Day4 5-FU mouse BM cells } 2 days , y Prestimulatjon 3xiu«cel ls 2 days 6 days CM M3 axit^ cens 3x10* cells 3 x 10* cells 3 x 10* cells DayO CRU Assay By limiting dilution analysis of CRU Infection GFP HOXB4 NUP9&HOXB4 NUPQ8MOXA10 ?JUP9frHOXA10h<i', Liquid culture EXPANSION PHASE DMEM, 15% FCS, IL-3, IL-6, SF DayO CRU Assay By limiting dilution analysis of CFtU The contribution of transduced (GFP+) cells to the lymphoid and myeloid reconstitution of the transplanted recipients was determined by flow cytometry at 16 weeks after transplantation. Recipients having at least 1% of transduced cells in both lymphoid and myeloid compartments were considered to be positively reconstituted (Figure 3.2.). 40 _ „ „ 8 8 8 Tx. D o s e - » Starting o^l -equivalent : O i j3 § 10 i £ 2 | *J *? in c in o O u fi. 1 # £ o E 0.1 8 8 O D O CO Expt. I 8 8 8 8 o o <s> oi oo to Expt. I Expt. I $tt! • • o o o 8 8 8 o o o 8 8 8 V 8 8 S R D m r-u • • • • 8 8 8 H GFP NB4 NA10 GFP H0XB4 NB4 NA10 GFP H0XB4 NB4 NA10 Assay of starting (Day 0) cells Assay of transduced and cultured (Day 10) cells Figure 3.2. - Long-term reconstitution of recipients transplanted with GFP-, H0XB4-, NUP98-HOXB4- or NUP98-HOXA70-transduced cells. S h o w n is donor-derived (Day 0) or dono r -de r i ved /GFP + (Day 10) reconstitution of P B at 16 weeks post transplantation. Black d iamonds represent reconstituted recipients having > 1% of dono r -de r i ved /GFP + cel ls in myeloid (Gr -1 + /Mac -1 + ) and lymphoid ( B 2 2 0 + and C D 4 + / C D 8 + ) subpopulat ions. White d iamonds represent non-reconstituted recipients having < 1% of dono r -de r i ved /GFP + cel ls and/or lacking ability to reconstitute myeloid or lymphoid compartment. Transplantat ion (Tx) dose is expressed in starting cell equivalents. (Data obtained by Dr. Hideaki Ohta.) 41 The proportion of "negative" recipients as a function of the number of cel ls injected was analyzed by Po isson statistics to calculate G F P + C R U f requencies. At the start of the pre-stimulation culture period, this value was -1 in 5000 cel ls. Thus - 6 0 0 C R U s were used to initiate each culture. Figure 3.3. shows the C R U frequencies at the end of the 10 days of culture but expressed on the bas is of starting cell equivalents for a representative experiment. T h e s e show that the yield of C R U dramatical ly increased in the cultures containing the HOXB4 and NUP98-HOX t ransduced cel ls, in contrast to the marked dec rease in C R U frequency documented in the control culture (Figure 3.3). 0 50 100 150 200 250 4k 20k 40k 60k 80k 100k 120k Cell dose (starting cell equivalents) Figure 3.3. - Limiting dilution analysis (LDA) for estimation of CRU frequencies in 10-day cultures of HSCs transduced with various HOX fusion genes. If - 3 7 % of recipients transplanted with the given dose are not repopulated or negative, that exact dose should contain 1 C R U . Transplantat ion dose is expressed in starting cell equivalents. (Data obtained by Dr. Hideaki Ohta.) 42 These findings were consistent in multiple experiments. Pooled data (at least 3 for each gene tested) are presented in Figure 3.4. The results show an overall net amplification of C R U numbers of 80-fold, 290-fold and >2000-fold in the cultures of HOXB4-, NUP98-HOXB4- and NUP98-HOXA1 (Mransduced ceils and an overall net decline in C R U numbers of 50-fold in the control cultures of GFP-transduced cells. The expanded C R U populations were restricted to HOXB4-, NUP98-HOXB4- or NUP98-HOXA f Otransduced cells, as there was complete concordance between the presence of regenerated donor-derived cells (identified by CD45 allotype markers) and transduced (GFP+) cells in the reconstituted recipients. Figure 3.4. - Ex vivo expansion of transduced HSCs after 10 dys of culture For each expanding agent tested, results of at least three independent experiments were pooled and expressed as the mean +/- standard error mean (SEM) of the C R U numbers per culture of 3 x 10 6 starting cells. In some experiments, for culture containing NUP98-HOXA10-transduced cells, limiting dilution was not reached, thus the level of H S C expansion was estimated to be at least 2000-fold. (Data obtained by Dr. Hideaki Ohta.) GFP HOXB4 NB4 NA10 4 3 Together, these results establ ish the ability of the NUP98-HOXB4 and NUP98-HOXA10 fusion genes to mimic the ability of HOXB4 to amplify H S C s ex vivo thus extending this activity to fusion genes of NUP98 and HOXB4 and the Abdominal-B c lass H O X gene, HOXA10. Moreover, the potency of HOXB4 appears enhanced as part of a fusion with NUP98 with even greater potency obtained with the NUP98-HOXA10 fusion gene. 3.2 A NUP98-HOX Fusion Gene Containing Only the Homeodomain of HOXA10 Retains the Full HSC Ex Vivo Expansion Activity of the Parent Fusion Gene Fol lowing the finding that very marked expansion of H S C s can be ach ieved in vitro by forced express ion of an engineered fusion between NUP98 and the second exon of HOXA10, we further analyzed the HOXA10 sequences required to ach ieve this effect. The second exon of NUP98-HOXA10 encodes the homeodomain plus another 16 N terminal amino acids that provide a PBX-b ind ing motif (Chang et a l . , 1996) and another 15 C terminal amino ac ids of unknown function. Recent studies of the leukemogenic properties of various NUP98-HOX fusion genes , including NUP98-HOXA10, have revealed that the homeodomain is essent ia l for blocking hematopoiet ic differentiation (Pineault et al . , 2004) and P B X 1 knock down studies have shown that the in vivo compet i t iveness of HOX64-ove rexp ress ing cel ls is enhanced >20-fold (Krosl et al . , 2003). I therefore hypothesized that the homeodomain might be necessary and perhaps sufficient to provide the H S C expanding properties of NUP98-HOX fusion proteins. To test this hypothesis, a NUP98-HOX fusion gene retaining only the H O X sequence encoding the 61 amino 44 acid homeodomain of HOXA10 was constructed, placed in an M S C V vector (hereafter referred to as the NUP98-HOXA1Ohd vector, Figure 2.1), and then tested for its ability to expand H S C s in culture by compar ison to the parental fusion gene using the same protocol as outlined above. O n the 1 0 t h day of culture, the percentage of GFP+ cel ls in each culture was again above 75%. C R U a s s a y s performed on the cel ls before and after the 10-day prestimulation-transduction-expans ion culture period showed that the C R U content of the culture containing NUP98-HOXA10hd-Xransduced cel ls had increased -1500- fo ld-essent ia l ly identical to the levels obtained previously using the NUP98-HOXA10 vector (Figures 3.5 and 3.6). T h e s e f indings indicate that the DNA-bind ing homeodomain alone as part of a NUP98-HOX fusion gene is sufficient to promote the high levels of H S C expans ion in culture ach ieved with the full length fusion gene and further demonstrate that the PBX-b ind ing motif to be d ispensable for this activity. 45 Expt. IV Expt. V Expt. VI Tx. D o s e -> Starting eel equivalent 100 I I 0.1 8 3 8 O O O o ur> T-'M o o H H o o o O O O O lO 8 8 8 P " o o o o o o o 8 8 8 ? " " o <N o 000 ov 000 001 o o o o o io 8 8 8 9 « O >N O O O O 8 8 8 O O O o « ^ O Q N M o « ,-: <N ° O O O <N 8 R " •N • 1 • • • • • • 1 • X V • • 0 O /-} k w 0 (> <> 0 o 0 ° 0<> GFP NA10t)d GFP NAWttd GFP NA10 NAIOhd Assay of startiny (Day 0) cells Assay of transduced an d cult ured (Day 10) cells Figure 3.5. - Long-term reconstitution of recipients transplanted with GFP-, HOXB4- or /Vl/P98-rYOX470 /7a ' - transduced cel ls. Shown is donor-derived (Day 0) or dono r -de r i ved /GFP + (Day 10) reconstitution of P B at 6 months post-transplantation. B lack d iamonds represent reconstituted recipients having > 1% of dono r -de r i ved /GFP + cel ls in myeloid (Gr -1 + /Mac -1 + ) and lymphoid ( B 2 2 0 + and C D 4 + / C D 8 + ) subpopulat ions. White d iamonds represent non-reconstituted recipients having < 1% of dono r -de r i ved /GFP + cel ls and/or lacking ability to reconstitute myeloid or lymphoid compartment. Transplantat ion (Tx) dose is expressed in starting cell equivalents. 46 Figure 3.6. - Ex vivo expansion of transduced HSCs after 10 days of culture For each expanding agent tested, results of three independent experiments were pooled and expressed as the mean +/- standard error mean (SEM) of the C R U numbers per culture of 3 x 1 0 6 starting cells. In some experiments, for culture containing NUP98-HOXAIOhd-iransduceti cells, limiting dilution was not reached, thus the level of H S C expansion was estimated to be at least 1000-fold. 47 3.3 A/l/P98-HOX-transduced Cells Retain Multi-lineage Repopulating Ability To confirm that the expanded HSCs retained full multi-lineage repopulating ability, detailed immunophenotypic flow cytometric analysis was performed on blood samples from mice transplanted > 16 weeks previously. The presence of transduced (GFP+) cells in the myeloid, B-lymphoid, T-lymphoid and red blood cell compartments from representative recipients indicated that the expanded HSCs were not compromised in their capacity to differentiate along all myeloid and lymphoid lineages examined (Figure 3.7). NUP9&4SOXA10hd NUPmHOXB4 NUP98MOXA10 R B C s f CD o C M W B C s | C O Q O a o • 1 • 14 I m '4 5 9 | 4 42 .. § * . :: 1 1 3 11 1 .. i s 6 3 ... ... ... ... 3 14 1 3 1 1 0 dp 3 4 i l l 5 # 2 2 I 2 5 2 5 9 21 4 5 16 1 3 3 6 1 H 3 3 GFP Figure 3.7. - Representative peripheral blood FACS profiles of recipients transplanted with NUP98-HOXB4-, NUP98-HOXA10- or NUP98-HOXA10hd-transduced cells. Representative recipients received equivalent of 200, 200 or 250 starting cells, respectively. GFP-expressing cells were present in myeloid (Gr-1+Mac-1+), B-lymphoid (B220+) and T-lymphoid (CD4 +CD8 +) compartments as well as red blood compartment (RBC) of transplanted recipients. 48 Recip ients transplanted with H0XB4- or A/ i /P98-HOXe4-transduced cel ls demonstrated normal l ineage distributions, similar to those seen in normal, unmanipulated mice. O n the other hand, although recipients of NUP98-HOXA10-t ransduced cel ls d isplayed substantial contributions to all l ineages, there was a modest increase in proportion of myeloid as compared to lymphoid cel ls. However, these recipients remained healthy, not showing any s igns of an incipient myeloproliferative disorder and/or leukemia during the period of at least one year post transplantation that the mice were fol lowed. Simi lar detai led ana lyses carr ied out on recipients of expanded NUP98-HOXA10hd4ransduce<i cel ls a lso showed normal lympho-myeloid distributions and high-level contributions to the red blood cell compartment (Table 3.1.). Table 3.1. - Summary of lineage distribution of GFP+ cells in PB of transplanted recipients Expanding aaent % of G M cells in G F P + compartment ± SD % of B cells in G F P + compartment ± SD % of T cells in G F P + compartment ± SD (-) -» CTL 16 .27±7 .35 5 2 . 6 7 ± 1 5 . 2 8 17.99 + 9.64 HOXB4 4 0 . 6 8 ± 1 5 . 7 0 46.60 ± 1 5 . 6 2 5 . 8 5 ± 2 . 2 3 NB4 4 5 . 3 8 ± 1 6 . 2 2 5 1 . 4 4 ± 15.69 8.31 ± 1 . 5 5 NA10 7 0 . 9 4 ± 3 . 6 5 3 2 . 8 7 ± 7 . 7 1 5 . 5 2 ± 1 . 9 3 NAIOhd 2 7 . 4 6 ± 8 . 3 0 4 1 . 6 6 ± 8 . 2 2 2 9 . 5 2 ± 4 . 1 1 49 To further examine the lympho-myeloid repopulating capacity of HSCs recovered after expansion ex-vivo, Southern blot analysis of proviral integrations was performed on DNA extracts from enriched populations of BM myeloid cells, splenic B cells and thymic T cells from representative recipients of NUP98-HOXA10-or NUP98-HOXA1 Ohd-transduced cells. The identical patterns of proviral integration sites observed confirmed the pluripotent nature of NUP98-HOXA10- or NUP98-HOXA10hd-\ransduced HSC clones (Figure 3.8). NA10 NAIOhd BM Sp Thy BM Sp Thy ^ •j jjgj j * nm0 **>0* 'ItsiK*' Figure 3.8. - Southern blots, showing common integration patterns of vector DNA in reconstituted myeloid (BM) and lymphoid (Spleen and Thymus) tissues of representative recipients of NUP98-HOX expanded HSCs. Representative recipients reconstituted by NUP98-HOXA10- or /VI/P98-HOXA 7 Ofrd-transduced cells, received equivalent of 500 or 200 starting cells, respectively. (Data obtained by Dr. Hideaki Ohta) 50 3.4 Polyclonal Recovery of A/l/P98-HOX-transduced HSCs Further ev idence of the polyclonal composit ion of the H S C s obtained in cultures of NUP98-HOXA10- or /VcJP98-HOX/470/7d-transduced cel ls w a s obtained by Southern blot analys is of proviral integrations in B M D N A isolated from recipients of these cel ls at late time points (>6 months post-transplant). For this purpose, multiple recipients transplanted with various doses of cel ls harvested from a single culture of GFP, NUP98-HOXA10 or NUP98-HOXA1 Ohd-transduced cel ls were ana lyzed . G iven the marked decl ine in H S C content in G F P control cultures, recipients of these cel ls even at the highest transplant dose (250,000 starting cell equivalents) showed a smal l number of proviral integrations with the same proviral integration pattern in all positive mice (data not shown). In contrast, recipients of cel ls from the cultures of NUP98-HOXA10 and NUP98-HOXA1 Ohd-transduced cel ls showed highly complex and distinct proviral integration patterns at much lower transplant doses (e.g. 200 or 20 starting cell equivalents) and the extent of polyclonality was clearly related to the dose of t ransduced cel ls transplanted (Figure 3.9). Thus , only in recipients of the lowest dose of NUP98-HOXA10 and NUP98-HOXA1 Ohd cel ls (~2 starting cell equivalents) was a simple proviral integration pattern apparent consistent with the injection of these mice with a near limiting dilution of t ransduced H S C s . At these lowest transplant doses , unique patterns were apparent in different recipients, indicating that the >2000-fold H S C expans ion ex-vivo measured by C R U a s s a y s reflected a highly polyclonal population of H S C s in the expans ion cultures from which they were obtained. Variat ions in the autoradiographic intensities of bands that represent these integrations suggest that each recipient of the lowest cell dose 51 was reconstituted by one or two unique c lones with up to 3 proviral integrations per clone. This result would also agree with the C R U frequencies measured (<1 in 2 starting cell equivalents) for the cultures of NUP98-HOXA10- or N U P 9 8 - H O X A 1 0 h d -t ransduced cel ls. NUP98-HOXAW NUP98-HOXA10hd r Tx. Case 4 Starting cell equivalent D O CM O o o o in in o M M M M M Mouse « > 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 13 m,.m-'HI I • * . %# f I • M I • • Figure 3.9. - Polyc lonal recovery of A/L/P98-HOX-transduced H S C s . Southern blot analys is of proviral integrations in D N A isolated from B M of multiple recipients transplanted with var ious doses of NUP98-HOXA10- and NUP98-HOXA10hd-t ransduced cel ls, 16 weeks post transplantation. Transplantat ion (Tx.) dose is expressed in starting cell equivalents. (NUP98-HOXA10 data obtained by Dr. Hideaki Ohta.) 52 3.5 Ex Vivo Expans ion of NUP98-HOXA10-transduced BM cel ls in Cultures Initiated with Smal l numbers of Input C R U s - Direct Evidence of High Level Clonal H S C Expans ion A s a further test of the ability of NUP98-HOXA10 to stimulate high level c lonal expans ion of H S C s , additional experiments were carried out in which individual cultures were initiated with smal l numbers of 5FU-pretreated B M cel ls per well , est imated to contain 1 or 2 C R U s . E a c h well was then subjected to transduct ion, culture and assayed individually for C R U content at the end of the 10 day culture period as for the larger cultures (Figure 3.10). 53 dO d2 d4 d10 Input 25,000 cells/well Input 5,000 cells/well I n f e c t i o n ] GFP NA10 CRU assay/well 1,'2 well 1/10 well 1150 well 1(2 well 1(10 well 1(50 well 1(250 well Input 1,000 J SL cells/well SORT GFP 1/2.5 well 1/25 well Input 500 J SL cells/well [ NA10 1/2,500 well 1/25,000 well 1/250,000 well Figure 3.10. - Experimental protocol for examining the ex vivo expansion of NUP98-HOXA70-transduced BM cel ls in cultures initiated with 1-2 C R U s . Cultures were initiated with 25,000 or 5,000 (-1-2 CRUs) 5-FU pre-treated mouse B M cells per well, prestimulated with IL-3, IL-6 and S F and retrovirally transduced with GFP control or NA10 vectors, respectively. 6 days after infection, individual wells were harvested and various fractions of each/single well were transplanted into irradiated recipients. By day 10, percentage of G F P + cells in wells containing GFP-transduced or /WW0-transduced cells was above 65%. Also at day 0 5-FU pre-treated mouse BM cells were sorted for Sca1 +Lin" stem cell enriched population and cultures were initiated with 1,000 or 500 (~2 CRUs) per well. Cells were again prestimulated with IL-3, IL-6 and SF and retrovirally transduced with GFP control or NA10 vectors, respectively. 6 days after infection, individual wells were harvested and various fractions of each/single well were transplanted into irradiated recipients. By day 10, percentage of G F P + cells in wells containing GFP-transduced or NA10-transduced cells was above 75%. 54 In these exper iments none of the recipients transplanted with GFP- t ransduced cel ls showed G F P + cel ls in their reconstituted blood cel ls, indicating the expected loss of H S C s in these cultures whereas up to 6 recipients transplanted with var ious fractions of the single well containing NUP98-HOXA 70-transduced cel ls demonstrated long-term lympho-myeloid reconstitution. T h e s e results confirm the extensive C R U expans ion obtainable with these vectors even when starting with limiting numbers of C R U s . The fact that recipients of as little as 1/250 t h of one of these cultures were highly reconstituted points to expans ions of 125-fold as suggested by the bulk cultures (Figure 3.11). Moreover, in experiment carried out with phenotypical ly def ined, CRU-en r i ched S c a 1 + L i n " cel ls that al lowed even smal ler numbers of starting cel ls to be used (i.e., as low as 500 cel ls, est imated to contain 2 C R U s ) , measurement of C R U frequencies before and after the culture period again showed a high degree of reconstitution of recipients transplanted with extremely low (1 /2500 t h or even 1/25000 t h) fractions of single cultures of NUP98-HOXA10-t ransduced cel ls whereas no reconstitution was obtained with the GFP- t ransduced cel ls (Figure 3.11). T h e s e findings further document the very high levels of c lonal expans ion of C R U s obtainable with NUP98-HOXA10 and suggest that the responsive cel ls have a S e a l + L i n " phenotype. 55 c o c o u 4> 10 Bulk BM cells A . Sea l 'L in - BM cells 1/10 1/50 1/10 1/50 1/10 1/50 1)250 1/10 1/50 1/250 1 i'in 1)50 1/10 1/50 1/10 1/50 1/2.5 1/25 o o o in in CJ CJ .— a o o o o m in ! <^  CJ : j • • * • • ! : • ! u • • ! • ; • i . * ] * : : i • ! ! ! • ; i i » [ ! o i I ! - V I i 0 0 0 <} * 0 Ut -> #1 ; #2 GFP #1 #2 | #3 | NA10 #4 #5 #1 GAP #1 ! #2 NA10 Figure 3.11. - Long-term reconstitution of recipients by GFP- or NUP98-HOXA70-transduced cells expanded ex vivo in cultures initiated with 1-2 CRUs. S h o w n is dono r -de r i ved /GFP + reconstitution of P B at > 16 weeks post transplantation. Whi le the control cultures exper ienced significant H S C dec rease , which was confirmed by inability of GFP- t ransduced cel ls to reconstitute any of transplanted recipients, the NUP98-HOXA10 cultures ach ieved at least 125-fold (bulk B M cells) or 1250-fold ( S c a 1 + L i n " B M cells) net H S C increase. B lack d iamonds represent reconstituted recipients having > 1% of dono r -de r i ved /GFP + cel ls in myeloid (G r -1 + /Mac -1 + ) and lymphoid ( B 2 2 0 + and C D 4 + / C D 8 + ) subpopulat ions. White d iamonds represent non-reconstituted recipients having < 1% of donor-d e r i v e d / G F P + cel ls and/or lacking ability to repopulate myeloid or lymphoid compartment. Transplantat ion (Tx.) dose indicates proportion of each/s ingle well . 56 Proviral integration analysis of B M D N A from representative recipients that received various cell doses from single wells also displayed either a single proviral integration pattern (well #1 - bulk B M cells) or not more than two patterns (well #1 -S c a U n B M cel ls, #2 - bulk B M cel ls and #3 - bulk B M cells) (Figure 3.12.), confirming that the likelihood that the expans ions obtained in these cultures were c lonal . Moreover, toward the end of the culture period and just before the transplantation, a portion of a well#1, initially containing S c a + L i n " B M cel ls, was resorted for this cell population and transplanted into several recipients (Figure 3.12.). Proviral integration analysis of B M D N A from these mice showed the s a m e integration pattern, providing ev idence that the expanded C R U s retain these phenotypic features during their generat ion in vitro. 57 Well #1 Well U2 Well #3 Well #1 Unsorted Resorted SL _ o o o O O O O o i n a o o o o o w i n i n i n Proportion of each well >^ C J S C B i c ^ B ^ i e i S C C C C! o i n o *** - • HI*1' mm •P Bulk BM cells 5,000fwell Sca +Lin BM cells 500fwell Figure 3.12. - Southern blots of DNA from representative recipients transplanted with M7P98-HOX/170-transduced BM cells expanded ex vivo from cultures initiated with 1-2 CRUs. 58 C H A P T E R 4 D I S C U S S I O N Recent studies have provided intriguing evidence that the ability to expand H S C s might not be unique to HOXB4, but may extend to other intact HOX genes, notably HOXA9 (Thorsteinsdottir et al., 2002), or engineered NUP98-HOX fusion genes, initially studied for their role in leukemogenesis. The recent studies indicating that these fusion genes can promote C F C self-renewal in vitro and block their differentiation (Pineault et. al., 2004) suggested that these activities extend to H S C s and allow the enhancement of HSC expansion in vitro. My results reveal a striking potency of multiple fusion genes of NUP98 and HOX, to stimulate multi-log expansion of murine HSCs in short term liquid culture. Interestingly, the NUP98-HOXB4 fusion gene showed a 7 to 8-fold greater potency than that previously documented for HOXB4 (average H S C expansion of 300-fold versus 40-fold). Moreover, even higher levels of H S C expansion were achieved with the NUP98-HOXA10 fusion gene, which stimulated a >2000-fold expansion of HSCs ; i.e., a potency 7- and 50-fold greater than that exhibited by NUP98-HOXB4 or HOXB4, respectively. Of further interest, the H S C expansion effect of NUP98-HOXA10 was preserved when sequences flanking the homeodomain were removed, thus identifying the homeodomain as the key HOX gene sequence required in concert with the N-terminal region of NUP98. Analysis of proviral integrations and of cultures initiated with limiting numbers of enriched H S C populations provided strong support for the view that the remarkable output of H S C s achieved after transducing mouse BM cells with NUP98-HOXA10 is due to clonal expansions of all pre-existing H S C s transduced. However, regardless of the precise nature of the cellular target, 59 these findings point to NUP98-HOXA10-based molecules as promising new tools for stimulating high level ex vivo expansion of HSCs. The underlying molecular mechanism governing this striking increase in potency of NUP98-HOXA10 and even NUP98-HOXB4 for HSC expansion compared to HOXB4 is currently unclear. In part, it may be due to dominant transcriptional activation properties attributable to NUP98 rather than intrinsic properties of intact HOX protein. Alternatively, a potentially increased stability of the HOX component within the fusion protein could be important. The ability of NUP98-HOXA10 to stimulate a greater H S C expansion than NUP98-HOXB4 is consistent with previous observations that NUP98-HOXA10 is a much stronger inhibitor of hematopoietic differentiation in vitro compared to NUP98-HOXB4 (Pineault et al., 2004). This property of NUP98-HOXA10 may reflect its ability to affect overlapping but not identical target genes. Given that the fundamental mechanism of HOX function is modulation of gene expression, the identification of NUP98-HOXB4 and NUP98-HOXA10 target genes would be anticipated to help elucidate the mechanisms involved in NUP98-HOX-mediated H S C expansion. In addition, the cultured NUP98-HOXB4- and NUP98-HOXA10-transduced cells would be expected to contain large numbers of actively self-renewing H S C s and should thus serve as ideal populations for global gene expression approaches to analyze their unique properties and dissect the complex regulatory networks that control HSC fate decisions. We have clearly shown that homeodomain alone of HOXA10 is sufficient for H S C expansion effect in context of a NUP98 fusion protein. Interestingly, the P B X 60 binding motif was not required for achieving high levels of H S C expans ion . Moreover , with homeodomain alone, no s igns of abnormali t ies in l ineage distributions were evident, indicating perhaps that the P B X domain may be responsib le for some deleterious effects. Interestingly, HOXA10 was reported to bind the p21(Cip1/Waf1) promoter and activate p21 transcription in the presence of the cofactors, P B X 1 and MEIS1 (Bromleigh, 2000). Combined with the observat ion that the P B X binding motif is d ispensable for NUP98-HOXA10-pronio\ed H S C expans ion and the H S C expanding potential of HOXB4 is augmented by ant isense P B X 1 , it might be speculated that NUP98-HOX fusions somehow inhibit p21, facilitating H S C entry into the cell cyc le. Evaluat ion of the effect of NUP98-HOX fus ions on cell cycle or cell division would allow this idea to be further explored. Finally, it is remarkable that multi-log clonal H S C expans ion w a s ach ieved within a period of 6 days, indicating at least 10 self-renewal doubl ings. T h e s e must therefore have been symmetr ical self-renewal divisions, assuming a cell cyc le time of 12-16 hours. Interestingly, upon transplantation, the cel ls behaved "normally" in terms of their ability to repopulate all l ineages with no evident increase in primitive subpopulat ions. Indeed, preliminary measurements of the number of C R U s regenerated in primary mice suggest that the t ransduced C R U s reach normal levels but do not exceed these (data not shown). Therefore, for H S C expans ion to occur, appropriate extrinsic condit ions (e.g., growth factors, absence of inhibitors) are required. In conc lus ion, a strategy for consistently achieving very high expans ion of H S C s ex vivo using a single agent has been deve loped. These f indings raise 61 optimism that further prolongation of the time in culture or modification of the growth factors used may enable even greater levels of H S C expansion ex vivo to be obtained. The approach described here also appears ideally suited to the type of protein delivery system afforded using TAT-fusion protein technology (Krosl, 2003) which might allow H S C expansion without gene manipulation and be of great interest for human applications. 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