Ash1l controls quiescence and self-renewal potential in hematopoietic stem cells

Rapidly cycling fetal and neonatal hematopoietic stem cells (HSCs) generate a pool of quiescent adult HSCs after establishing hematopoiesis in the bone marrow. We report an essential role for the trithorax group gene absent, small, or homeotic 1-like (Ash1l) at this developmental transition. Emergence and expansion of Ash1l-deficient fetal/neonatal HSCs were preserved; however, in young adult animals, HSCs were profoundly depleted. Ash1l-deficient adult HSCs had markedly decreased quiescence and reduced cyclin-dependent kinase inhibitor 1b/c (Cdkn1b/1c) expression and failed to establish long-term trilineage bone marrow hematopoiesis after transplantation to irradiated recipients. Wild-type HSCs could efficiently engraft when transferred to unirradiated, Ash1l-deficient recipients, indicating increased availability of functional HSC niches in these mice. Ash1l deficiency also decreased expression of multiple Hox genes in hematopoietic progenitors. Ash1l cooperated functionally with mixed-lineage leukemia 1 (Mll1), as combined loss of Ash1l and Mll1, but not isolated Ash1l or Mll1 deficiency, induced overt hematopoietic failure. Our results uncover a trithorax group gene network that controls quiescence, niche occupancy, and self-renewal potential in adult HSCs.     

Clonal expansion capacity defines two consecutive developmental stages of long-term hematopoietic stem cells

Long-term hematopoietic stem cells (HSCs [LT-HSCs]) are well known to display unpredictable differences in their clonal expansion capacities after transplantation. Here, by analyzing the cellular output after transplantation of stem cells differing in surface expression levels of the Kit receptor, we show that LT-HSCs can be systematically subdivided into two subtypes with distinct reconstitution behavior. LT-HSCs expressing intermediate levels of Kit receptor (Kit^int) are quiescent in situ but proliferate extensively after transplantation and therefore repopulate large parts of the recipient’s hematopoietic system. In contrast, metabolically active Kit^hi LT-HSCs display more limited expansion capacities and show reduced but robust levels of repopulation after transfer. Transplantation into secondary and tertiary recipient mice show maintenance of efficient repopulation capacities of Kit^int but not of Kit^hi LT-HSCs. Initiation of differentiation is marked by the transit from Kit^int to Kit^hi HSCs, both of which precede any other known stem cell population.Hematopoietic stem cells (HSCs) replenish millions of mature hematopoietic cell types every second throughout life but also maintain the HSC pool over time. HSC function is assessed by their capacity to repopulate the blood system of lethally irradiated recipient mice in the long term. The most immature HSC pool is functionally heterogeneous, and HSCs vary in their differentiation potential and duration of reconstitution (Copley et al., 2012; Muller-Sieburg et al., 2012). However, the magnitude of repopulation, thus white blood cell output per donor HSC, was only retrospectively associated with specific reconstitution patterns determined by lineage choice (Dykstra et al., 2007). Therefore, it remains unknown whether clonal expansion capacities are predetermined in donor cells or whether the magnitude of repopulation is determined by the microenvironment of the recipient.Kit expression is widely used for the prospective isolation of HSCs, and the stem cell factor (SCF)–Kit signaling axis is pivotal for normal pool size and function of fetal and adult HSCs (Russell, 1979; Ikuta and Weissman, 1992). Consistently, alterations in Kit signaling profoundly affect adult HSC function (Ogawa et al., 1991; Czechowicz et al., 2007; Waskow et al., 2009; Ding et al., 2012; Deshpande et al., 2013). Furthermore, Kit alleles resulting in hypomorphic expression of the receptor are loss of function alleles (Russell, 1979; Thorén et al., 2008; Waskow et al., 2009), suggesting that reduced densities of Kit expression correlate with loss of “stemness.” In contrast, cells expressing low levels of (Doi et al., 1997; Matsuoka et al., 2011) or lacking (Ortiz et al., 1999) Kit receptor expression were suggested to contain quiescent long-term HSCs (LT-HSCs). However, differences in the clonal expansion capacities of HSCs expressing distinct levels of the Kit receptor were not reported.To assess whether expansion capacities are predetermined within donor HSCs and whether this function identifies novel cellular subtypes within the most immature HSC pool, we transplanted LT-HSCs that differed in the density of the expression of the Kit receptor. Donor cells repopulated recipient mice to two significantly different magnitudes: HSCs with intermediate levels of Kit receptor expression (Kit^int) contained greater expansion capacities compared with HSCs expressing high densities of the Kit receptor (Kit^hi), suggesting that HSC clonal growth potential is predetermined in a cell-intrinsic fashion. We further provide evidence that these HSC subtypes are two consecutive developmental stem cell stages within the most immature HSC pool and that transit from Kit^int to Kit^hi LT-HSCs marks the onset of differentiation and is associated with significant loss of expansion capacities. Gene expression profiles ex vivo and after SCF trigger suggest that the inherent differences are based on distinct cycling and adhesive activities.     

Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region

Hematopoietic stem cells (HSCs) emerge during embryogenesis and maintain hematopoiesis in the adult organism. Little is known about the embryonic development of human HSCs. We demonstrate that human HSCs emerge first in the aorta-gonad-mesonephros (AGM) region, specifically in the dorsal aorta, and only later appear in the yolk sac, liver, and placenta. AGM region cells transplanted into immunodeficient mice provide long-term high level multilineage hematopoietic repopulation. Human AGM region HSCs, although present in low numbers, exhibit a very high self-renewal potential. A single HSC derived from the AGM region generates at least 300 daughter HSCs in primary recipients, which disseminate throughout the entire recipient bone marrow and are retransplantable. These findings highlight the vast regenerative potential of the earliest human HSCs and set a new standard for in vitro generation of HSCs from pluripotent stem cells for the purpose of regenerative medicine.     

Thrombopoietin expands hematopoietic stem cells after transplantation

Multiple lines of evidence indicate that thrombopoietin (TPO) contributes to the development of hematopoietic stem cells (HSC), supporting their survival and proliferation in vitro. To determine whether TPO supports the impressive expansion of HSC observed following transplantation, we transplanted normal marrow cells into lethally irradiated Tpo(-/-) and Tpo(+/+) mice and quantified HSC self-renewal and expansion and hematopoietic progenitor cell homing. Although essentially identical numbers of marrow-associated colony forming unit-culture (a surrogate measure of stem cell homing) were observed in each type of recipient 24 hours following transplantation, we found that a minimum of fourfold greater numbers of marrow cells were required to radioprotect Tpo-null mice than to radioprotect controls. To assess whether long-term repopulating (LTR) HSCs self-renew and expand in Tpo(-/-) recipients or controls, we performed limiting-dilution secondary transplants using donor cells from the Tpo(-/-) or Tpo(+/+) recipients 5-7.5 weeks following primary transplantation. We found that LTR HSCs expand to levels 10-20 times greater within this time period in normal recipients than in Tpo-null mice and that physiologically relevant amounts of TPO administered to the Tpo(-/-) recipients could substantially correct this defect. Our results establish that TPO greatly promotes the self-renewal and expansion of HSCs in vivo following marrow transplantation.     

RARgamma is critical for maintaining a balance between hematopoietic stem cell self-renewal and differentiation

Hematopoietic stem cells (HSCs) sustain lifelong production of all blood cell types through finely balanced divisions leading to self-renewal and differentiation. Although several genes influencing HSC self-renewal have been identified, to date no gene has been described that, when activated, enhances HSC self-renewal and, when inactivated [corrected] promotes HSC differentiation. We observe that the retinoic acid receptor (RAR)gamma is selectively expressed in primitive hematopoietic precursors and that the bone marrow of RARgamma knockout mice exhibit markedly reduced numbers of HSCs associated with increased numbers of more mature progenitor cells compared with wild-type mice. In contrast, RARalpha is widely expressed in hematopoietic cells, but RARalpha knockout mice do not exhibit any HSC or progenitor abnormalities. Primitive hematopoietic precursors overexpressing RARalpha differentiate predominantly to granulocytes in short-term culture, whereas those overexpressing RARgamma exhibit a much more undifferentiated phenotype. Furthermore, loss of RARgamma abrogated the potentiating effects of all-trans retinoic acid on the maintenance of HSCs in ex vivo culture. Finally, pharmacological activation of RARgamma ex vivo promotes HSC self-renewal, as demonstrated by serial transplant studies. We conclude that the RARs have distinct roles in hematopoiesis and that RARgamma is a critical physiological and pharmacological regulator of the balance between HSC self-renewal and differentiation.     

Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation

Multipotent self-renewing hematopoietic stem cells (HSCs) are responsible for reconstitution of all blood cell lineages. Whereas growth stimulatory cytokines have been demonstrated to promote HSC self-renewal, the potential role of negative regulators remains elusive. Receptors for tumor necrosis factor (TNF) and Fas ligand have been implicated as regulators of steady-state hematopoiesis, and if overexpressed mediate bone marrow failure. However, it has been proposed that hematopoietic progenitors rather than stem cells might be targeted by Fas activation. Here, murine Lin(-)Sca1(+)c-kit(+) stem cells revealed little or no constitutive expression of Fas and failed to respond to an agonistic anti-Fas antibody. However, if induced to undergo self-renewal in the presence of TNF-alpha, the entire short and long-term repopulating HSC pool acquired Fas expression at high levels and concomitant activation of Fas suppressed in vitro growth of Lin(-)Sca1(+)c-kit(+) cells cultured at the single cell level. Moreover, Lin(-)Sca1(+)c-kit(+) stem cells undergoing self-renewal divisions in vitro were severely and irreversibly compromised in their short- and long-term multilineage reconstituting ability if activated by TNF-alpha or through Fas, providing the first evidence for negative regulators of HSC self-renewal.     

Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression

Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.     

Changes in the proliferative activity of human hematopoietic stem cells in NOD/SCID mice and enhancement of their transplantability after in vivo treatment with cell cycle inhibitors

Human hematopoietic tissue contains rare stem cells with multilineage reconstituting ability demonstrable in receptive xenogeneic hosts. We now show that within 3 wk nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver cells regenerate near maximum levels of daughter human hematopoietic stem cells (HSCs) able to repopulate secondary NOD/SCID mice. At this time, most of the human HSCs (and other primitive progenitors) are actively proliferating as shown by their sensitivity to treatments that kill cycling cells selectively (e.g., exposure to high specific-activity [(3)H]thymidine in vitro or 5-fluorouracil in vivo). Interestingly, the proliferating human HSCs were rapidly forced into quiescence by in vivo administration of stromal-derived factor-1 (SDF-1) and this was accompanied by a marked increase in the numbers of human HSCs detectable. A similar result was obtained when transforming growth factor-beta was injected, consistent with a reversible change in HSCs engrafting potential linked to changes in their cell cycle status. By 12 wk after transplant, most of the human HSCs had already entered G(o) and treatment with SDF-1 had no effect on their engrafting activity. These findings point to the existence of novel mechanisms by which inhibitors of HSC cycling can regulate the engrafting ability of human HSCs executing self-renewal divisions in vivo.     

The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance

The role of autophagy, a lysosomal degradation pathway which prevents cellular damage, in the maintenance of adult mouse hematopoietic stem cells (HSCs) remains unknown. Although normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs leads to leukemia. Therefore, mechanisms protecting HSCs from cellular damage are essential to prevent hematopoietic malignancies. In this study, we crippled autophagy in HSCs by conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system. This resulted in the loss of normal HSC functions, a severe myeloproliferation, and death of the mice within weeks. The hematopoietic stem and progenitor cell compartment displayed an accumulation of mitochondria and reactive oxygen species, as well as increased proliferation and DNA damage. HSCs within the Lin(-)Sca-1(+)c-Kit(+) (LSK) compartment were significantly reduced. Although the overall LSK compartment was expanded, Atg7-deficient LSK cells failed to reconstitute the hematopoietic system of lethally irradiated mice. Consistent with loss of HSC functions, the production of both lymphoid and myeloid progenitors was impaired in the absence of Atg7. Collectively, these data show that Atg7 is an essential regulator of adult HSC maintenance.     

Differential impact of Ink4a and Arf on hematopoietic stem cells and their bone marrow microenvironment in Bmi1-deficient mice

The polycomb group (PcG) protein Bmi1 plays an essential role in the self-renewal of hematopoietic and neural stem cells. Derepression of the Ink4a/Arf gene locus has been largely attributed to Bmi1-deficient phenotypes in the nervous system. However, its role in hematopoietic stem cell (HSC) self-renewal remained undetermined. In this study, we show that derepressed p16(Ink4a) and p19(Arf) in Bmi1-deficient mice were tightly associated with a loss of self-renewing HSCs. The deletion of both Ink4a and Arf genes substantially restored the self-renewal capacity of Bmi1(-/-) HSCs. Thus, Bmi1 regulates HSCs by acting as a critical failsafe against the p16(Ink4a)- and p19(Arf)-dependent premature loss of HSCs. We further identified a novel role for Bmi1 in the organization of a functional bone marrow (BM) microenvironment. The BM microenvironment in Bmi1(-/-) mice appeared severely defective in supporting hematopoiesis. The deletion of both Ink4a and Arf genes did not considerably restore the impaired BM microenvironment, leading to a sustained postnatal HSC depletion in Bmi1(-/-)Ink4a-Arf(-/-) mice. Our findings unveil a differential role of derepressed Ink4a and Arf on HSCs and their BM microenvironment in Bmi1-deficient mice. Collectively, Bmi1 regulates self-renewing HSCs in both cell-autonomous and nonautonomous manners.     

Fgd5 identifies hematopoietic stem cells in the murine bone marrow

Hematopoietic stem cells (HSCs) are the best-characterized tissue-specific stem cells, yet experimental study of HSCs remains challenging, as they are exceedingly rare and methods to purify them are cumbersome. Moreover, genetic tools for specifically investigating HSC biology are lacking. To address this we sought to identify genes uniquely expressed in HSCs within the hematopoietic system and to develop a reporter strain that specifically labels them. Using microarray profiling we identified several genes with HSC-restricted expression. Generation of mice with targeted reporter knock-in/knock-out alleles of one such gene, Fgd5, revealed that though Fgd5 was required for embryonic development, it was not required for definitive hematopoiesis or HSC function. Fgd5 reporter expression near exclusively labeled cells that expressed markers consistent with HSCs. Bone marrow cells isolated based solely on Fgd5 reporter signal showed potent HSC activity that was comparable to stringently purified HSCs. The labeled fraction of the Fgd5 reporter mice contained all HSC activity, and HSC-specific labeling was retained after transplantation. Derivation of next generation mice bearing an Fgd5-CreERT2 allele allowed tamoxifen-inducible deletion of a conditional allele specifically in HSCs. In summary, reporter expression from the Fgd5 locus permits identification and purification of HSCs based on single-color fluorescence.Hematopoietic stem cells (HSCs) function to maintain blood homeostasis throughout life via their unique ability to differentiate into all blood cell types and to self-renew. These properties, along with the robust ability of HSCs to engraft myeloablated recipients in the setting of BM transplantation, have established the clinical paradigm for therapeutic stem cell use (Weissman, 2000).Originally described by Till and McCulloch (1961), HSCs were first experimentally defined by their ability to form macroscopic colonies in the spleens (CFU-S) of irradiated recipients after BM transplantation that histological examination revealed contained multiple blood lineages, and cytological examination revealed were clonally derived (Becker et al., 1963). Together with the demonstration that a subset of CFU-S colonies had the potential to reform colonies when transplanted into secondary recipients (Siminovitch et al., 1963), the defining properties of hematopoietic stem cells—multipotency and self-renewal—were established. In the 50 yr since these seminal studies were conducted, the experimental study of HSCs has flourished, leading to a profound level of understanding of their biology. These efforts were enabled through the development of several in vivo and in vitro assays that permitted evaluation of HSC self-renewal and multilineage potential, and by methods that allowed purification of HSCs by FACS. HSCs were initially reported to be enriched within the Thy1^lowLineage⁻ fraction of the murine BM (Muller-Sieburg et al., 1986), and subsequently cells with a Thy1^lowLineage⁻Sca1⁺ immunophenotype were shown to possess long-term multilineage repopulating activity (Spangrude et al., 1988). The immunophenotype of HSCs was further refined, culminating with the demonstration that single cells purified from the Lineage⁻Sca1⁺c-kit⁺ (LSK)CD34^−/low fraction of the BM of adult mice could function to long-term multilineage reconstitute irradiated recipients at the clonal level (Osawa et al., 1996). Additional cell surface markers that have also been used to enrich for HSC activity include: CD105 (Chen et al., 2002), Flk2/Flt3 (Christensen and Weissman, 2001), CD201/PROCR (Balazs et al., 2006), ESAM (Ooi et al., 2009; Yokota et al., 2009), and CD150, CD48, and CD244 (Kiel et al., 2005a) among others. In addition to immunophenotype, intravital dye efflux activity has also proven to be an effective strategy for enriching for HSC activity (Bertoncello et al., 1985; Wolf et al., 1993; Goodell et al., 1996).Although immunophenotype combined with flow cytometry has become the principle technique used for identifying and studying diverse cells types, genetically engineered reporter strains have also enabled the identification and study of other cell types, including tissue-specific stem cells from other organs. For example, rapidly cycling intestinal stem cells were identified with the use of an Lgr5 reporter (Barker et al., 2007), whereas a population of more slowly cycling stem cells in the intestinal crypt were marked with a reporter for telomerase (Montgomery et al., 2011). In the developing embryo, reporter strains for Isl1 (Laugwitz et al., 2005) and WT1 (Zhou et al., 2008) have been combined with lineage-tracing experiments to identify cardiac progenitors in the developing heart. In the skin, a Tet-inducible H2B-GFP reporter stain was used in conjunction with a keratinocyte-specific driver to isolate label-retaining stem cells in the epidermis (Tumbar et al., 2004). A similar H2B-GFP label retention strategy was later used by two independent groups to explore the turnover of HSCs, showing that a label-retaining population of cells with potent HSCs activity resides in a state of prolonged dormancy during steady-state homeostasis (Wilson et al., 2008; Foudi et al., 2009). Importantly, depending on vector design, introducing reporter cassettes into specific genomic loci (knock-in) can also lead to the disruption of the targeted gene, permitting analysis of the null (knockout) genotype when targeted alleles are crossed to homozygosity.With the goals of identifying novel genes that could be used to specifically report on HSC activity within the murine BM, we performed a system-wide microarray screen of hematopoietic stem, progenitor, and effector cells, and identified a set of genes whose expression was highly restricted to the HSC compartment. Generation of mice with targeted reporter knock-in/knock-out alleles at three of the identified genes, Sult1a1, Clec1a, and Fgd5 revealed that whereas knockout of Sult1a1 and Clec1a were viable and had normal HSC function, nullizygosity of Fgd5 was embryonic lethal at midgestation, though the generation and function of definitive HSCs was not affected by loss of Fgd5. Of the three reporter alleles, only Fgd5 explicitly marked immunophenotypic HSCs in the adult marrow at steady state and after transplantation. BM cells isolated based solely on reporter signal of the Fgd5 reporter mice showed robust HSC activity, with all stem cell activity residing within the labeled fraction. These results demonstrate that HSCs can be identified and purified from the BM of Fgd5 reporter mice by single color fluorescence. Finally, the development of Fgd5-CreERT2 mice permitting inducible deletion of Floxed alleles specifically in HSCs represents an invaluable genetic resource for exploring gene function in HSC biology.     

In vitro self-renewal division of hematopoietic stem cells

Little is known about how hematopoietic stem cells (HSCs) self-renew. We studied the regeneration of HSCs in culture. Effects of various cytokines on cell division of CD34(-/low) c-Kit(+)Sca-1(+) lineage marker-negative (CD34(-)KSL) bone marrow cells of the mouse were first evaluated in serum-free single cell culture. We then performed a competitive repopulation assay on divided cells to ask if such cell division involved self-renewal of HSCs.In the presence of stem cell factor (SCF), thrombopoietin (TPO) induced a first cell division of CD34(-)KSL cells more efficiently than did interleukin (IL)-3 or IL-6. Multilineage repopulating cells were detected in a significant proportion of cells derived from single cells in culture with TPO and SCF, although this culture condition led to a substantial decrease in HSC number. These regenerated repopulating cells could be further transplanted into secondary recipients. When paired daughter cells were separately studied, one of a pair gave rise to repopulating cells with self-renewal potential, suggesting asymmetric self-renewal division. This study provides evidence that one HSC regenerates at least one HSC in culture.     

CXCR4 is required for the quiescence of primitive hematopoietic cells

The quiescence of hematopoietic stem cells (HSCs) is critical for preserving a lifelong steady pool of HSCs to sustain the highly regenerative hematopoietic system. It is thought that specialized niches in which HSCs reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. We report that CXCL12 produced by bone marrow (BM) stromal cells is not only the major chemoattractant for HSCs but also a regulatory factor that controls the quiescence of primitive hematopoietic cells. Addition of CXCL12 into the culture inhibits entry of primitive hematopoietic cells into the cell cycle, and inactivation of its receptor CXCR4 in HSCs causes excessive HSC proliferation. Notably, the hyperproliferative Cxcr4(-/-) HSCs are able to maintain a stable stem cell compartment and sustain hematopoiesis. Thus, we propose that CXCR4/CXCL12 signaling is essential to confine HSCs in the proper niche and controls their proliferation.     

Critical role for Gimap5 in the survival of mouse hematopoietic stem and progenitor cells

Mice and rats lacking the guanosine nucleotide-binding protein Gimap5 exhibit peripheral T cell lymphopenia, and Gimap5 can bind to Bcl-2. We show that Gimap5-deficient mice showed progressive multilineage failure of bone marrow and hematopoiesis. Compared with wild-type counterparts, Gimap5-deficient mice contained more hematopoietic stem cells (HSCs) but fewer lineage-committed hematopoietic progenitors. The reduction of progenitors and differentiated cells in Gimap5-deficient mice resulted in a loss of HSC quiescence. Gimap5-deficient HSCs and progenitors underwent more apoptosis and exhibited defective long-term repopulation capacity. Absence of Gimap5 disrupted interaction between Mcl-1-which is essential for HSC survival-and HSC70, enhanced Mcl-1 degradation, and compromised mitochondrial integrity in progenitor cells. Thus, Gimap5 is an important stabilizer of mouse hematopoietic progenitor cell survival.     

Transduction of long-term-engrafting human hematopoietic stem cells by retroviral vectors

Gene therapy using retroviral vectors to transfer functional exogenous genes into hematopoietic stem cells (HSCs) promises to provide a permanent cure for a wide array of both hematopoietic and nonhematopoietic disorders by virtue of the fact that retroviral vectors permanently integrate into the host cell genome and HSCs are able to self-renew and give rise to differentiated progeny throughout the life span of the patient. However, for transduction and genomic integration to occur, the target cells must undergo cell division and express the appropriate retroviral receptor, requirements that have thus far hindered attempts at inserting exogenous genes into human HSCs in vitro. In the present studies, we used the fetal sheep xenograft model of human hematopoiesis to evaluate whether human long-term engrafting HSCs could be transduced in vivo, within a fetal microenvironment. We transplanted adult human bone marrow-derived CD34(+)Lin(-) cells into preimmune fetal sheep recipients and subsequently (19 days later) administered clinical-grade murine retroviral vector supernatants to these fetal hematopoietic chimeras. Our results demonstrate that this approach successfully transduced adult human HSCs within all seven sheep that survived the procedure, and that these transduced HSCs had the ability to serially engraft primary, secondary, and tertiary fetal sheep recipients. Transgene expression persisted throughout the serial transplantation. The successful in vivo transduction of long-term engrafting human HSCs with the existing generation of murine retroviral vectors has significant implications for developing new approaches to pre- and postnatal gene therapy.     

De novo DNA methyltransferase is essential for self-renewal, but not for differentiation, in hematopoietic stem cells

DNA methylation is an epigenetic modification essential for development. The DNA methyltransferases Dnmt3a and Dnmt3b execute de novo DNA methylation in gastrulating embryos and differentiating germline cells. It has been assumed that these enzymes generally play a role in regulating cell differentiation. To test this hypothesis, we examined the role of Dnmt3a and Dnmt3b in adult stem cells. CD34(-/low), c-Kit(+), Sca-1(+), lineage marker(-) (CD34(-) KSL) cells, a fraction of mouse bone marrow cells highly enriched in hematopoietic stem cells (HSCs), expressed both Dnmt3a and Dnmt3b. Using retroviral Cre gene transduction, we conditionally disrupted Dnmt3a, Dnmt3b, or both Dnmt3a and Dnmt3b (Dnmt3a/Dnmt3b) in CD34(-) KSL cells purified from mice in which the functional domains of these genes are flanked by two loxP sites. We found that Dnmt3a and Dnmt3b function as de novo DNA methyltransferases during differentiation of hematopoietic cells. Unexpectedly, in vitro colony assays and in vivo transplantation assays showed that both myeloid and lymphoid lineage differentiation potentials were maintained in Dnmt3a-, Dnmt3b-, and Dnmt3a/Dnmt3b-deficient HSCs. However, Dnmt3a/Dnmt3b-deficient HSCs, but not Dnmt3a- or Dnmt3b-deficient HSCs, were incapable of long-term reconstitution in transplantation assays. These findings establish a critical role for DNA methylation by Dnmt3a and Dnmt3b in HSC self-renewal.