International Journal of

Progress in Hematology

HEMATOLOGY

Biology of Normal and Acute Myeloid Leukemia Stem Cells John E. Dick,a Tsvee Lapidotb a Department of Molecular and Medical Genetics, University of Toronto, and Division of Cell and Molecular Biology, University Health Network, Toronto, Ontario, Canada; bDepartment of Immunology, Weizmann Institute, Rehovot, Israel

Received September 29, 2005; accepted October 2, 2005

Abstract The substantial understanding that has been gained over the past 5 decades of the biology of blood formation is largely due to the development of functional quantitative assays for cells at all stages of differentiation, from multipotential stem cells to mature cells. The majority of studies have involved the mouse because the ease with which repopulation studies can be carried out with this animal model allows the assay of complete lineage development from stem cells. In the past decade, advances in repopulation assays for human stem cells using xenotransplantation have greatly enhanced our understanding of human stem cell biology. Importantly, the xenotransplantation methodology has also been used to identify the cancer stem cell that initiates and sustains leukemic proliferation, providing key evidence for the cancer stem cell hypothesis. This hypothesis argues that cancer cells are functionally heterogeneous and hierarchically organized such that only specific cells are capable of sustaining tumor growth and continuously producing the cells that make up the bulk of the tumor. Recent studies have also brought into focus the importance of the intimate relationship between the stem cell (normal or leukemic) and its microenvironment. Coming into view are the molecular players involved in stem cell homing, migration, and adhesion, as well as the cellular components of the microenvironmental niche. Here we review recent studies that have begun to elucidate the interplay between normal and leukemic human stem cells and their microenvironment. Int J Hematol. 2005;82:389-396. doi: 10.1532/IJH97.05144 ©2005 The Japanese Society of Hematology Key words: Cancer stem cells; NOD/SCID; Xenotransplantation; Cancer; Leukemia

blood development was developed over many years of studies and was based on the analysis of fractionated cell populations. Recent data indicate that the developmental pathway emerging from any specific single HSC may be more complex. Experiments in which the differentiation outcome from a single HSC or from highly purified fractions has been assessed have suggested that extensive maturation can occur even within one of the daughter cells that arise after a mitotic event, rather than having to pass through all of the intermediate steps of lineage commitment that are outlined in the classic representations of blood development [4-8]. Unique to the most primitive HSC is self-renewal potential, a property that enables life-long blood production because the stem cell is replaced with one of the daughter cells when the normally quiescent HSC undergoes a mitosis [9]. Although the molecular machinery for self-renewal is poorly understood, this process appears to be carefully regulated such that, in the steady state, HSC numbers are maintained [10,11]. However, under conditions of high demand, such as those occurring after transplantation, the HSC expand exponentially for a period of time before lapsing into steady-state maintenance. It is becoming clear that dysregulation of self-renewal lies at the heart of the neoplastic

1. Existence of Different Classes of Human Stem Cells The traditional view of the hematopoietic system is that of a highly regulated cellular hierarchy. Mature cells are continuously generated from progenitors detected with clonogenic assays (colony-forming cells [CFC]). Primitive long-term culture–initiating cells (LTC-IC) and extended LTC-IC (ELTC-IC) can differentiate into CFC of all myeloid lineages in stroma-based cultures and proliferate up to 10 weeks, but they possess little capacity for self-renewal. A subclass of LTC-IC with multilineage B-cell and myeloid developmental potential also has been identified with a variety of stromabased assays [1-3]. Ultimately, the hierarchy originates from hematopoietic stem cells (HSC), which possess extensive proliferation and differentiation potential. This picture of

Correspondence and reprint requests: John E. Dick, PhD, Division of Cell and Molecular Biology, University Health Network, Suite 7-700, 620 University Ave, Toronto, Ontario, Canada M5G 2C1 (e-mail: [email protected]).

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process. In neoplastic diseases, the cancer stem cells that sustain neoplastic cell growth apparently have lost the tight control of self-renewal, resulting in expanded numbers of cancer stem cells (discussed in more detail below). Thus, understanding the molecular regulation of the stem cell selfrenewal program and how perturbation in this regulation initiates proliferative diseases such as leukemia represent a major challenge of biological research. HSC are rare and difficult to assay because they can be reliably studied only by in vivo repopulation experiments. Thus, our understanding of HSC biology gained over more than 40 years is derived from murine studies because of the availability of quantitative assays for individual HSC in mice. In contrast, progress in the study of human HSC lagged until the development of the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) xenotransplantation assay, a method that has revolutionized the study of both normal and leukemic human hematopoiesis (reviewed in [12,13]). The quantitative assay for human HSC (termed SCIDrepopulating cells [SRC] or competitive repopulating units) [14-17] is based on the lymphomyeloid repopulation of immune-deficient mice. A detailed insight into the biology of SRC is emerging in terms of their frequency, cell surface phenotype, cytokine responsiveness, cell cycle kinetics, homing, and relationship to CFC and LTC-IC (reviewed in [12,13]). Of course, it is important to reiterate that the SRC assay is only a surrogate and that a certain level of uncertainty still exists as to whether the results with this assay are identical to those of HSC assayed by transplantation into humans. Recently, insight was gained into the composition of the human HSC pool with the identification of different classes of HSC with short-term and long-term repopulating capacities (termed short-term SRC and long-term SRC) [18]. The in vivo fates of individual SRC were analyzed during the repopulation of NOD/SCID mice by clonal analysis of retrovector integration sites. Through analysis of serial bone marrow (BM) aspirates, we demonstrated that the repopulation was oligoclonal with extensive variability in self-renewal capacity as well as in the lifespan and proliferative capacity of individual SRC. The existence of multiple distinct classes of human HSC is consistent with clonal analysis data from studies in mice [19] and primates [20]. Thus, the existence of HSC classes that differ only in self-renewal capacity suggests that self-renewal is a regulated cellular process and that this regulation is the basis for HSC heterogeneity. This idea needs to be proved. The availability of an assay to detect different SRC classes forms the basis for purifying and then identifying the molecular factors that regulate the developmental fate of each class and for ultimately determining how HSC decide, upon cell division, to generate daughter cells that selfrenew (ie, retain parental HSC properties) or commit to differentiation pathways.

2. Purification of HSC Combining an assessment of cell surface expression with assays of stem and progenitor function provides a powerful tool to uncover lineage relationships and to purify each HSC class. Because absolute purity is the ultimate goal of purification and is testable, as elegantly demonstrated for murine

HSC by Osawa et al [21], Benveniste et al [22], and others, only by single-cell transplantation, the development of an assay for the transplantation of a single SRC represents an elusive goal. A high proportion of CD34+CD38– cells give rise to lymphomyeloid LTC-IC and ELTC-IC clones after 5 and 10 weeks of culturing (up to 48% and 16%, respectively) on MS-5 stroma [2,3,23]. Most SRC transplanted into NOD/ SCID mice (SRC are typically assayed between 4 weeks and >12 weeks after intravenous injection) are Lin–CD34+CD38– [15,24], although very rare CD34– SRC exist in the Lin–CD34–CD38– fraction [25-27]. The estimated frequency of SRC in the CD34+CD38– cell fraction is 1 in 617 [15,24], although accounting for xenogenic seeding efficiency suggests that the actual SRC frequency may be as low as 1 in 34 [28,29]. The Lin–CD34+CD38+ fraction engrafts poorly in NOD/SCID mice but does engraft in natural killer (NK) activity–depleted NOD/SCID mice [30-32]. The graft size is relatively small, often myeloerythroid restricted, and not sustained beyond 8 to 12 weeks.Thus, purified Lin–CD34+CD38+ cells contain a short-term repopulating stem cell. The sensitivity to residual NK activity distinguishes these cells from the short-term SRC identified by clonal tracking in NOD/ SCID mice. Collectively, the clonal-tracking and cell purification studies provide compelling evidence that the HSC compartment as assayed by repopulation is heterogeneous and that purification enriches subclasses.

3. Novel SRC Classes Detected by an Intrafemoral Transplantation System All HSC repopulation assays are based on intravenous injection, a complex process that requires circulation through the blood, recognition and extravasation through the BM vasculature, and migration to a supportive microenvironment. Thus, the presence of residual host factors that resist engraftment (see above; short-term repopulating stem cells are detected only in NK activity–deficient mice) and the theoretical possibility that some classes of HSC may remain undetected because they are incapable of homing demonstrate the need for an improved SRC assay system [33-35]. By using direct intrafemoral injection, we identified a rapid SRC (R-SRC) within the Lin–CD34+CD38+(low)CD36– subpopulation that is poorly detected by the method of intravenous injection [36]. R-SRC rapidly generate high levels of human myeloid and erythroid cells within the injected femur, migrate to the blood, and colonize individual bones of NOD/ SCID mice within 2 weeks following transplantation. The presence of SRC activity in the Lin–CD34+CD38+(low) fraction but low activity in the Lin–CD34+CD38+(hi) fraction likely explains the inconsistent reports of SRC potential among Lin–CD34+CD38+ cells [24,30]. Unique engraftment properties, a higher frequency, and cell surface phenotype distinguish R-SRC from previously identified SRC classes. NOD/SCID xenograft models employing intrafemoral procedures provide powerful tools to detect novel classes of HSC that are either rare or poorly detected with traditional intravenous injection–based assays [33]. In addition, even known SRC classes are more efficiently detected with assays

Normal and Leukemic Stem Cells

based on intrafemoral injection. For example, the SRC frequency in Lin–CD34+CD38– cells is 1 in 40 with intrafemoral injection (versus 1/600 with intravenous transplantation) [35], a value similar to the seeding fraction–corrected value we referred to earlier. Similarly, NOD/SCID
2-microglobulin–null (NOD/SCID-
2m–/–) mice, in which NK cells are genetically depleted, show a 10-fold increase in the efficiency of SRC engraftment [37], although this result may be due to the enhanced detection of CD34+CD38+ short-term repopulating cells that were undetected in untreated NOD/SCID mice. This result indicates that immune recognition is an important determinant of host resistance to xenotransplantation of this SRC class [30,31]. Several groups have recently transplanted cells either intravenously or intrahepatically into neonatal mice at a time when the innate immune system is not fully developed and the mouse is still growing [38,39]. Again, this model provides robust human cell engraftment from lower numbers of SRC. In contrast, we showed that simple intraperitoneal transplantation into neonatal mice does not lead to HSC-derived engraftment; rather, only mature lymphocytes proliferate and infiltrate murine tissues to create a graft-versus-host–like syndrome [40]. These results show that the microenvironment into which HSC are transplanted is an important parameter (see below). We evaluated human engraftment in various NOD/SCID models in combination with an assessment of the intrafemoral and intravenous transplantation routes to determine the most effective assay for characterizing R-SRC [33]. Because NK cells were previously shown to be an important factor in resisting early engraftment [30,31,41], we used NOD/SCID-
2m–/– mice. Additionally, we injected the anti-CD122 antibody, which is directed against the
chain of the interleukin 2 receptor, into NOD/SCID mice, because this antibody targets several mature hematopoietic cell populations, including NK cells and macrophages [41]. Mice were evaluated for the level of human cell engraftment at 2 weeks and 6 weeks to determine the optimal NOD/SCID xenotransplantation model. These experiments showed that intrafemorally injected anti-CD122–treated NOD/SCID mice generated the highest engraftment (in the injected femur as well as other hematopoietic territories) [33], indicating the superiority of carefully selecting the recipients and the route of injection.

4. CD34– SRC Until recently, all human HSC were believed to be Lin–CD34+. However, this dogma was challenged by the discovery of Lin–CD34– HSC in humans and mice [21,25,26].We found repopulating cells, which we termed CD34neg-SRC, within the Lin–CD34–CD38– fraction [26]. This new HSC class did not express classic HSC-associated markers (CD34, HLA-DR, or Thy-1), lacked CFC and LTC-IC, and exhibited clear differences in cytokine-stimulated growth compared with CD34pos-SRC. This evidence, together with the results of experiments with repopulation in sheep xenografts [25,42] and CD34pos-SRC production on HESS5 stroma [43], suggests that CD34+ HSC originate from CD34– HSC. Murine studies and some human studies suggest developmental or activation modulation of CD34 expression on HSC [44-46].

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However, the human CD34 gene possesses different genetic regulatory elements, and the distinctive growth properties of human CD34– HSC suggest that they may be biologically distinct [47]. CD34neg-SRC are difficult to study because of their rarity and poorly reproducible engraftment properties. Importantly, the intrafemoral transplantation approach detected much higher numbers of CD34neg-SRC, which exhibited slower but sustained engraftment compared with CD34pos-SRC [48]. The CD34neg-SRC are defined by the exclusion of cellular markers rather than by the presence of distinctive markers, making them difficult to purify. However, Hoechst dye–excluding “side population” cells seem to include some of them [49].

5. Leukemic Stem Cells Transplantation of human leukemia into immune-deficient mice generates a disease in the mice that recapitulates the human disease [50]. In acute myeloid leukemia (AML), an impaired differentiation program results in the excess production of leukemic blasts, the vast majority of which have limited proliferative capacity. Morphologically, AML reflects abnormal development in one of the major blood lineages, but the blasts from different patients are heterogeneous with respect to the lineage antigens they express.The human AML-initiating cell, termed the SCID leukemia-initiating cell (SL-IC), was identified and purified by transplantation into SCID mice [51]. The CD34+CD38– cell fraction that represents from 0.1% to 1% of the AML cell population contained all SL-IC, whereas only clonogenic leukemia progenitors were found in other fractions. Upon transplantation of CD34+CD38– cells, the entire cellular diversity was recapitulated, conclusively establishing that leukemia is a hierarchy sustained by rare leukemic stem cells (LSC) in a process closely resembling normal development [51,52]. HSC and SL-IC are quiescent and share similarities in cell surface phenotype [50], although LSC possess higher self-renewal capacities [52]. These data support a model in which the cellular targets for transformation commonly are HSC, as opposed to lineage-committed progenitors or the bulk nonclonogenic blast population (detailed arguments reviewed in [13,50]). According to this model, the heterogeneity of leukemic properties derives from the variable differentiation of LSC due to the direct influence of specific transformation–related or progression-related gene(s) and not from the degree of lineage commitment of targeted progenitors. This model predicts that LSC and HSC share many of the same properties that render them stem cells, especially their self-renewal potential. As is noted below, the stem cell model of leukemogenesis was recently given a strong boost with the discovery that Bmi-1 is a key regulator of self-renewal in both LSC and HSC [9,53].Thus, normal and neoplastic stem cells share the molecular machinery of self-renewal. However, the so-called cell-of-origin problem in human leukemia is complex. One can imagine a scenario in which progenitor cells are endowed with self-renewal capacity because of genetic or epigenetic alterations and thus are converted into LSC [54]. Direct experimental tools are required to determine which stem or progenitor cell in the normal hierarchy needs to be targeted with oncogenic alterations. Progress in creating such models is being made by

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transducing normal human hematopoietic cells with oncogenes, alone or in combination [55,56]. These studies are still in their infancy; however, it is clearly possible to observe the initial steps in the leukemogenic process of how a normal cell alters the properties of proliferation, differentiation, and selfrenewal as a consequence of oncogene expression [57,58]. Moreover, it is possible to determine the rules of how human cells can be made to become neoplastic. More progress has been made to carry cell-of-origin studies into the murine system. Several oncogenes have been transduced into populations of purified murine stem and progenitor cells, with varied consequences. For example, transduction of the MLL-ENL oncogene was found to induce AML in both murine stem cells and progenitor populations, although more progenitors were required for transplantation [59]. Similarly, MOZ-TIF caused leukemia to arise from both populations [60]. In contrast, leukemia arose from the stem cell fraction only when bcr-abl or c-jun was overexpressed [60,61]. Thus, only in some circumstances can progenitors (ie, nonstem cells) represent targets of transformation. Moreover, the marked monoclonality or oligoclonality in these studies suggests that the many thousands of transduced stem or progenitor cells were not all equally able to give rise to leukemia, suggesting the existence of some functional heterogeneity in populations of cells selected on the basis of cell surface markers. Additionally, whether the murine and human systems follow identical rules for transformation is not known; however, the murine studies have set the paradigm for how such human studies need to proceed. We have recently found that individual LSC, which we have detected by clonal marking with lentivectors, differ widely in self-renewal potential, demonstrating that the LSC pool is not homogeneous but still retains aspects of normal HSC organization, in which stem cells of differing selfrenewal and repopulation potentials exist [62]. Only a minority of LSC possessed a high self-renewal capability for enabling the initiation of AML following serial transplantation in NOD/SCID mice. It is surprising that some LSC were undetectable in primary mice and emerged only in secondary or even tertiary recipients, indicating that this novel LSC class possesses extensive self-renewal potential but does not readily generate daughter cells committed to the expansion of leukemic blasts. The discovery of complexity in the LSC pool strongly supports the concept of their derivation from normal HSC and establishes that the initiating leukemogenic processes do not entirely abolish the developmental program of normal HSC [50]. The quiescent properties of the LSC demonstrate the difficulty in developing leukemic therapies, which thus far have relied on eradicating cycling cells [63]. Thus, understanding the oncogenic mechanisms that initiate and sustain neoplastic growth requires knowledge of the molecular regulators for both the normal HSC that are initially targeted and the LSC that result [64].

6. In the Footsteps of the LSC Niche The mechanisms and key regulators of HSC migration and development are not fully understood. Definitive repopulating stem cells require interactions with the BM stromal cells, which regulate their migration, self-renewal, and devel-

opment in order to continuously maintain the production of new blood cells throughout life (discussed in [65]). Similarly, human and murine stromal cells support the proliferation of malignant human cells in vitro and in some cases can also attenuate their uncontrolled growth [66]. Although recent studies have identified the murine HSC niche and the central role in the endosteum region of bone-forming osteoblasts, which support stem cells via notch,
-catenin, and Tie-2 signaling [67-69], little is known about the human stem cell niche in the adult BM, let alone the leukemic niche. Many types of malignant cells, including some human breast cancer and prostate cancer cells, metastasize to the BM (discussed in [65]) as part of their abnormal migration and proliferation, suggesting that BM stromal cells also support the continuous growth of malignant cells. In contrast to metastasizing cancer cells, all human leukemias are hematopoietic and BM derived, and most leukemic cells at diagnosis are confined to the hematopoietic system, ie, the BM, blood, and spleen. However, some leukemias with a poor prognosis are associated with invasion to nonhematopoietic organs. For example, some childhood pre–B-cell acute lymphoblastic leukemia (pre–B ALL) cells infiltrate the spinal central nervous system [70] as well as cells in xenograft models [71], and some AML subtypes, such as M5, can infiltrate peripheral organs such as the liver and can form chloromas in the skin and gum [72]. HSC migration and development (proliferation and differentiation) are sequential events that overlap in their regulation; that is, these events are mostly regulated by the same factors (reviewed in [65]). For example, the chemokine SDF-1 and its receptor, CXCR4, are involved in multiple stem cell checkpoints, which include migration, survival, and development. This chemokine can induce stem cell migration, proliferation, and B-lymphocyte differentiation. Of interest is that CXCR4 signaling is essential for definitive human CD34+CD38–/low SRC function in immune-deficient NOD/SCID and NOD/SCID-
2m–/– mouse transplant recipients (reviewed in [65]). More importantly, the migration of mobilized human CD34+ cells toward a gradient of SDF-1 in vitro correlates with these cells’ in vivo repopulation potential in autologous transplantation patients and in NOD/ SCID mouse transplant recipients (reviewed in [65]). This ligand induces the secretion of metalloproteinases MMP-2 and MMP-9 by different cell types, including migrating human CD34+CXCR4+ progenitors in the circulation but not stationary CD34+ progenitors in the BM. These enzymes can help migrating cells penetrate the physical extracellular matrix barrier and can also inactivate and degrade SDF-1. In the functional preclinical NOD/SCID mouse model, the homing of normal human CD34+ progenitors and CD34+CD38–/low SRC, their retention, and their high-level multilineage repopulation are dependent on interactions between murine SDF-1 (which is cross-reactive) and human CXCR4, which is expressed by the CD34+ progenitors (reviewed in [65]). In the BM, this ligand and its receptor are expressed by multiple stromal and hematopoietic cell types. High levels of SDF-1 expression by human BM endothelial cells and immature bone-forming osteoblasts in the stem cell–rich endosteum region have been documented [73]. These results are of interest because most immature murine progenitors are located in the endosteum region at the bor-

Normal and Leukemic Stem Cells

der zone between the bone and the marrow; these progenitors are located to a lesser degree in periarterial sites (discussed in [73]). SDF-1 and CXCR4 are regulated by HIF-1. The BM is partially hypoxic during steady-state homeostasis because of reduced oxygen levels [74]; therefore, both SDF-1 and CXCR4 are constitutively expressed by BM stromal and hematopoietic cells. Both homing and engraftment/retention of human CD34+ progenitors in the murine BM microenvironment require the activation of adhesion molecules, which are needed for cell migration and adhesion to stromal cells. SDF-1 activates the major adhesion molecules LFA-1, VLA-4, VLA-5, and CD44 on immature human CD34+ cells; these molecules are essential for these cells’ SRC function (homing and engraftment) (reviewed in [65]). For example, SDF-1 stimulation induces cytoskeleton rearrangement and protrusion formation, in which CD44 is relocated to the leading edge of the migrating cells. It is interesting that inhibition of CD44 by neutralizing antibodies vetoes SDF-1 activation, documenting a cross talk between chemokine receptors and the adhesion machinery. Hyaluronic acid is the primary ligand of CD44 and is highly expressed in the stem cell–rich endosteum region of the human BM and in the endothelium of small blood vessels in which migration is thought to take place [75]. Thus, ligands of CD44 and other adhesion molecules (such as VLA-4) expressed by the BM stroma may dictate stem cell quiescence, lack of motility, and the secretion of MMP-2 and MMP-9 in the niche. Importantly, leukemic human AML cells, which adhere to stromal cells, are protected from druginduced apoptosis because of the activation of survival pathways such as BCL2 [76,77]. VLA-4–expressing AML cells can better adhere to fibronectin and stromal cells in response to cytokine stimulation, thereby protecting them from apoptosis in vitro and possibly leading to minimal residual disease in vivo with the potential to trigger leukemic relapse [78]. Both normal human SRC and leukemic SL-IC obtained from AML and pre–B ALL patients require CXCR4 signaling for their homing and engraftment in transplanted NOD/ SCID mice [65,72,79,80]. Of interest is that the same SDF-1–expressing BM endothelium sites were used by both normal murine stem cells and human pre–B ALL NALM-6 cells for their homing and retention within the endothelial niches [79]. However, differences in CXCR4 signaling and retention/adhesion to the murine BM were also documented. Atypical protein kinase C (PKC)- phosphorylation as part of CXCR4 signaling is essential for engraftment (but not homing) of normal CD34+ SRC. Mice treated with PKC pseudosubstrates rapidly mobilized murine progenitors to the circulation, revealing the importance of this signaling for retention of progenitors in the marrow [81]. Different inhibitors only partially overlapped in the inhibition of normal versus leukemic pre–B ALL cell migration and proliferation. For example, Toxin B, an inhibitor of RhoA, Rac, and CDC42, prevents homing of malignant pre–B ALL cells but does not prevent homing of normal human CD34+ cells [80]. In addition, because AML progenitors and maturing cells secrete high levels of proteolytic enzymes such as elastase and because this enzyme and cathepsin G can degrade both SDF-1 and cleave the N terminus of CXCR4, differences in adhesion interactions that are needed to retain normal and

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malignant human progenitors in the murine BM were able to be documented [72,82]. Administering neutralizing antiCXCR4 antibodies to chimeras preengrafted with AML or normal cord blood cells revealed stronger adhesion/retention interactions by the normal cells, which were less affected than the AML progenitors. Similarly, anti-CD44 antibody treatment preferentially prevents homing of malignant SLIC in NOD/SCID chimeric mice and leads to their eradication in preengrafted AML NOD/SCID mice, compared with normal chimeras [83]. Of interest is that this antibody can also lead to the apoptosis and differentiation of AML cells in vitro [84]. In addition, the levels of elastase in the circulation of patients with new AML diagnoses correlated with the levels of malignant blast cells. Furthermore, inhibition of elastase in NOD/SCID mouse transplant recipients significantly reduced in vivo homing, in vitro proliferation, and egress of malignant human AML cells (including immature CD34+) cells from BM to the circulation. Again, human cord blood CD34+ cells and a minority of AML cells were less affected, suggesting that different levels of elastase inhibition are needed for normal cells [82]. Of interest is that human pre–B ALL cells responded better than normal progenitors to lower concentrations of SDF-1 [80]. At present, total body irradiation and chemotherapy are the major clinical treatments for inducing the DNA damage aimed at curing leukemic patients. However, these treatments also induce the secretion of factors that also positively influence human LSC. For example, increases in SDF-1 secretion and CXCR4 expression via HIF-1 activation occur with DNA damage. These changes increase the function of normal SRC [65,73] and could increase the migration, proliferation, and dissemination of leukemic progenitors. In addition, we have documented the involvement of SDF-1 and CXCR4 in human chloromas [72], and CXCR4 levels correlate with extramedullary infiltration and poor prognosis in childhood pre–B ALL [73]. In addition, recent publications have revealed that FLT-3 mutations associated with poor prognosis in AML and increased engraftment of NOD/SCID mice are mediated via CXCR4 expression and signaling [85,86]. These results suggest that specific inhibitors of HIF-1–mediated SDF-1 expression and CXCR4 signaling are needed to complement current clinical protocols. The levels of another HIF-1–regulated cytokine, vascular endothelial growth factor, in the plasma of AML patients also have important prognostic value, and inhibition of this cytokine and/or its receptors is also needed [87]. Finally, the similarities and differences between normal human SRC and leukemic SLIC reveal that the overlap is only partial and that differences in signaling, adhesion, proliferation, and migration can be manipulated to specifically eliminate the malignant clone while only minimally affecting normal hematopoiesis.

7. Summary There has been an explosion of information concerning normal and leukemic human stem cells in the past decade. More recently, we have finally been gaining insight into the microenvironmental influences that act on these stem cells. An understanding of this complex interaction should yield

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novel approaches for manipulating these stem cells for clinical purposes: for the purpose of enhanced engraftment in the case of normal stem cells and for interfering with this process in the case of LSC for the purpose of bringing about their eradication.

Acknowledgments This work was supported by grants to J.E.D. from The Stem Cell Network of National Centres of Excellence, the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society and the Terry Fox Foundation, the Canadian Institutes for Health Research, Genome Canada through the Ontario Genomics Institute, the Leukemia and Lymphoma Society, and a Canada Research Chair.

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