Combination of low O(2) concentration and mesenchymal stromal cells during culture of cord blood CD34(+) cells improves the maintenance and proliferative capacity of hematopoietic stem cells

The physiological approach suggests that an environment associating the mesenchymal stromal cells (MSC) and low O(2) concentration would be most favorable for the maintenance of hematopoietic stem cells (HSCs) in course of ex vivo expansion of hematopoietic grafts. To test this hypothesis, we performed a co-culture of cord blood CD34(+) cells with or without MSC in presence of cytokines for 10 days at 20%, 5%, and 1.5% O(2) and assessed the impact on total cells, CD34(+) cells, committed progenitors (colony-forming cells-CFC) and stem cells activity (pre-CFC and Scid repopulating cells-SRC). Not surprisingly, the expansion of total cells, CD34(+) cells, and CFC was higher in co-culture and at 20% O(2) compared to simple culture and low O(2) concentrations, respectively. However, co-culture at low O(2) concentrations provided CD34(+) cell and CFC amplification similar to classical culture at 20% O(2) . Interestingly, low O(2) concentrations ensured a better pre-CFC and SRC preservation/expansion in co-culture. Indeed, SRC activity in co-culture at 1.5% O(2) was higher than in freshly isolated CD34(+) cells. Interleukin-6 production by MSC at physiologically low O(2) concentrations might be one of the factors mediating this effect. Our data demonstrate that association of co-culture and low O(2) concentration not only induces sufficient expansion of committed progenitors (with respect to the classical culture), but also ensures a better maintenance/expansion of hematopoietic stem cells (HSCs), pointing to the oxygenation as a physiological regulatory factor but also as a cell engineering tool.     

Transient disruption of autocrine TGF-beta signaling leads to enhanced survival and proliferation potential in single primitive human hemopoietic progenitor cells.

Hemopoietic stem cells (HSCs) are maintained at relative quiescence by the balance between the positive and negative regulatory factors that stimulate or inhibit their proliferation. Blocking the action of negative regulatory factors may provide a new approach for inducing HSCs into proliferation. A variety of studies have suggested that TGF-beta negatively regulates cell cycle progression of HSCs. In this study, a dominant negatively acting mutant of TGF-beta type II receptor (TbetaRIIDN) was transiently expressed in HSCs by using adenoviral vector-mediated gene delivery, such that the effects of disrupting the autocrine TGF-beta signaling in HSCs can be directly examined at a single cell level. Adenoviral vectors allowing the expression of TbetaRIIDN and green fluorescence protein in the same CD34(+)CD38(-)Lin(-) cells were constructed. Overexpression of TbetaRIIDN specifically disrupted TGF-beta-mediated signaling. Autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells was studied in single cell assays under serum-free conditions. Transient blockage of autocrine TGF-beta signaling in CD34(+)CD38(-)Lin(-) cells enhanced their survival. Furthermore, the overall proliferation potential and proliferation kinetics in these cells were significantly enhanced compared with the CD34(+)CD38(-)Lin(-) cells expressing green fluorescence protein alone. Therefore, we have successfully blocked the autocrine TGF-beta-negative regulatory loop of primitive hemopoietic progenitor cells.     

Role of Flk-1 in mouse hematopoietic stem cells.

It was reported that human hematopoietic stem cells in bone marrow were restricted to the CD34(+)KDR(+) cell fraction. We found that expression levels of Flk-1, a mouse homologue of KDR, were low or undetectable in mouse Lin(-)c-Kit(+)Sca-1(+)CD34(low/-) cells as well as Hoechst33342(-) cells (side population), which have long-term reconstitution capacity. Furthermore, neither Flk-1(+)CD34(low/-) cells nor Flk-1(+)CD34(+) cells had long-term reconstitution capacity in mouse. Taken together with other observations using Flk-1-deficient mice, these results indicate that Flk-1 is essential for the development of hematopoietic stem cells in embryo but not for the function of hematopoietic stem cells in adult mouse bone marrow.     

Detection of murine adult bone marrow stroma-initiating cells in Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells

We attempted to characterize the phenotype of cells which initiate fibroblastic stromal cell formation (stroma-initiating cells: SICs), precursor cells for fibroblastic stromal cells, based on the expression of cell surface antigens. First, we stained adult murine bone marrow cells with several monoclonal antibodies and separated them by magnetic cell sorting. SICs were abundant in the c-kit(+), Sca-1(+), CD34(+), VCAM-1(+), c-fms(+), and Mac-1(-) populations. SICs were recovered in the lineage-negative (Lin(-)) cells but not the Lin(+) cells. When macrophage colony-stimulating factor (M-CSF) was absent from the culture medium, no stromal colony appeared among the populations enriched in SICs. Based on these findings, the cells negative for lineage markers and positive for c-fms (M-CSF receptor) were further divided on the basis of the expression of c-kit, VCAM-1, Sca-1 or CD34 with a fluorescence-activated cell sorter. SICs were found to be enriched in the Lin(-)c-fms(+)c-kit(low) cells and Lin(-)c-fms(+)VCAM-1(+) cells but not in Lin(-)c-fms(+)Sca-1(+) cells and Lin(-)c-fms(+)CD34(low) cells. As a result, the SICs were found to be present at highest frequency in Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells: a mean of 64% of the SICs in the Lin(-) cells were recovered in the population. In morphology and several characteristics, the stromal cells derived from Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells resembled fibroblastic cells. The number of Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells in bone marrow of mice injected with M-CSF was higher than that in control mice. In this study, we identified SICs as Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells and demonstrated that M-CSF had the ability to increase the cell population in vivo.     

Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1(+) stem cells from bone-marrow microenvironment

The mechanism by which angiogenic factors recruit bone marrow (BM)-derived quiescent endothelial and hematopoietic stem cells (HSCs) is not known. Here, we report that functional vascular endothelial growth factor receptor-1 (VEGFR1) is expressed on human CD34(+) and mouse Lin(-)Sca-1(+)c-Kit(+) BM-repopulating stem cells, conveying signals for recruitment of HSCs and reconstitution of hematopoiesis. Inhibition of VEGFR1, but not VEGFR2, blocked HSC cell cycling, differentiation and hematopoietic recovery after BM suppression, resulting in the demise of the treated mice. Placental growth factor (PlGF), which signals through VEGFR1, restored early and late phases of hematopoiesis following BM suppression. PlGF enhanced early phases of BM recovery directly through rapid chemotaxis of VEGFR1(+) BM-repopulating and progenitor cells. The late phase of hematopoietic recovery was driven by PlGF-induced upregulation of matrix metalloproteinase-9, mediating the release of soluble Kit ligand. Thus, PlGF promotes recruitment of VEGFR1(+) HSCs from a quiescent to a proliferative BM microenvironment, favoring differentiation, mobilization and reconstitution of hematopoiesis.     

A sensitive and quantitative polymerase chain reaction-based cell free in vitro non-homologous end joining assay for hematopoietic stem cells

Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative cell free in vitro non-homologous end joining (NHEJ) assay using linearized plasmids as the substrates and quantitative polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via NHEJ in less than 1 μg 293T cell nuclear proteins or nuclear extracts from about 5,000 to 10,000 human BM CD34(+) hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34(+)CD38(-) cells) are less proficient in the repair of DSBs by NHEJ than HPCs (CD34(+)CD38(+) cells). In contrast, mouse quiescent HSCs (Pyronin-Y(low) LKS(+) cells) and cycling HSCs (Pyronin-Y(hi) LKS(+) cells) repaired the damage more efficiently than HPCs (LKS(-) cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as LIG4. These findings suggest that the qPCR-based cell free in vitro NHEJ assay can be used to sensitively measure the ability of human and mouse HSCs to repair DSBs.     

Co-culture of cord blood CD34(+) cells with human BM mesenchymal stromal cells enhances short-term engraftment of cord blood cells in NOD/SCID mice

BACKGROUND: The major challenge for cord blood transplantation (CBT) is higher rates of delayed and failed engraftment. In an attempt to broaden the application of CBT to more candidates, ex vivo expansion of hematopoietic stem/progenitor cells in CB is a major area of investigation. The purpose of this study was to employ human BM mesenchymal stromal cells (hBM-MSC) as the feeding-layer to expand CB cells ex vivo. METHODS: In this study, hBM-MSC were isolated and characterized by morphologic, mmunophenotypic and RT-PCR analysis. The hBM-MSC at passage 3 were employed as the feeding-layer to expand CB CD34(+) cells in vivo in the presence of thrombopoietin, flt3/flk2 ligand, stem cell factor and G-CSF. The repopulating capacity of the ex vivo-expanded CB cells was also evaluated in a NOD/SCID mice transplant experiment. RESULTS: After 1 or 2 weeks of in vitro expansion, hBM-MSC supported more increasing folds of CB in total nucleated cells, CD34(+) cells and colony-forming units (CFU) compared with CB without hBM-MSC. Furthermore, although NOD/SCID mice transplanted with CB cells expanded only in the presence of cytokines showed a higher percentage of human cell engraftment in BM than those with unexpanded CB CD34(+) cells, expanded CB cells co-cultured with hBM-MSC were revealed to enhance short-term engraftment further in recipient mice. DISCUSSION: Our study suggests that hBM-MSC enhance in vitro expansion of CB CD34(+) cells and short-term engraftment of expanded CB cells in NOD/SCID mice, which may be valuable in a clinical setting.     

Human marrow stromal precursors are alpha 1 integrin subunit-positive

In this work we studied the expression of adhesion molecules on primate human and non-human marrow stromal cells (primary cultures and lines) and on human CD34(+) hematopoietic normal and leukemic precursors. Differential expression of alpha1 integrin subunit was observed, since this molecule was intensely expressed by marrow stroma but not detected on CD34(+) cells. We used this difference to select, in fresh bone marrow samples, alpha 1-positive cells. We found that all stromal precursors giving rise to colony-forming units-fibroblasts (CFU-F) were present in the alpha 1-positive fraction. No colonies were detected in the alpha 1-negative fraction even after 2 weeks of culture. Phenotypic studies of stromal cells derived from alpha1-positive cells and grown in long-term marrow culture indicated that these cells were similar to stromal cells from primary cultures. We also observed early upregulation of alpha 4 and alpha 2 integrin subunits in cultures derived from alpha1-positive cells with maximal expression by day 10 (26 and 51%, respectively) preceding a gradual decline to low to nil values at day 30 (4.5 and 12%). These data indicate that alpha 1 integrin subunit is a marker for both mature stromal cells and stromal precursors, while alpha 2 and alpha 4 integrin subunits are expressed primarily by immature cells.     

Human CD34+ CD133+ hematopoietic stem cells cultured with growth factors including Angptl5 efficiently engraft adult NOD-SCID Il2rγ-/- (NSG) mice

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34(+) CD133(+) cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg(-/-) (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34(+) CD133(+) fraction of expanded cells and that CD34(+) CD133(+) cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.     

Promotive effect of macrophage colony-stimulating factor on long-term engraftment of murine hematopoietic stem cells

Large ex vivo expansion of hematopoietic stem cells (HSCs) sufficient for use in clinical applications has not been achieved, although the influence of some cytokines including SCF, IL-11, Flt3-L, and TPO for this purpose has been reported. We present evidence for an indirect effect of macrophage colony-stimulating factor (M-CSF) on expansion of murine HSCs. Fresh Lin(-/low) cells were isolated from Ly5.1 mouse bone marrow and cultured with or without M-CSF in the presence of SCF + IL-11 + Flt3-L or SCF + IL-11 + TPO for 6 days. The expanded cells were harvested and transplanted into lethally irradiated Ly5.2 recipients with competitor cells. Culture of Lin(-/low) cells with M-CSF significantly enhanced long-term engraftment. When the more enriched HSC populations of Lin(-/low) c-Kit(+) Sca-1(+) cells were used as a source of HSCs, such a promotive effect was not observed, in agreement with negative expression of the M-CSF receptor (c-Fms). However, co-culture with Lin(-/low) c-Fms(+) resulted in a significant increase of long-term engraftment. These results suggested that M-CSF is an indirect stimulator for ex vivo expansion of HSCs in the presence of SCF, IL-11, Flt3-L, and TPO. These observations provide new directions for ex vivo expansion and insight into new engraftment regulation through M-CSF signaling.     

Mesenchymal stem/stromal cells induce the generation of novel IL-10--dependent regulatory dendritic cells by SOCS3 activation

Suppression of immune response by mesenchymal stem/stromal cells (MSCs) is well documented. However, their regulatory effects on immune cells, especially regulatory dendritic cells, are not fully understood. We have identified a novel Sca-1(+)Lin(-)CD117(-) MSC population isolated from mouse embryonic fibroblasts (MEF) that suppressed lymphocyte proliferation in vitro. Moreover, the Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced hematopoietic stem/progenitor cells to differentiate into novel regulatory dendritic cells (DCs) (Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs) when cocultured in the absence of exogenous cytokines. Small interfering RNA silencing showed that Sca-1(+)Lin(-)CD117(-) MEF-MSCs induced the generation of Sca-1(+)Lin(-)CD117(-) MEF-MSC-induced DCs via IL-10-activated SOCS3, whose expression was regulated by the JAK-STAT pathway. We observed a high degree of H3K4me3 modification mediated by MLL1 and a relatively low degree of H3K27me3 modification regulated by SUZ12 on the promoter of SOCS3 during SOCS3 activation. Importantly, infusion of Sca-1(+)CD117(-)Lin(-) MEF-MSCs suppressed the inflammatory response by increasing DCs with a regulatory phenotype. Thus, our results shed new light on the role of MSCs in modulating regulatory DC production and support the clinical application of MSCs to reduce the inflammatory response in numerous disease states.     

Molecular cloning of murine STAP-1, the stem-cell-specific adaptor protein containing PH and SH2 domains

To identify the novel substrate of c-kit which is important for hematopoietic stem cell self-renewal or differentiation, CD34-low/negative, Sca-1-positive, c-kit-positive, and lineage marker-negative (CD34(low/-)Sca-1(+)c-kit(+)Lin(-)) cells were sorted by a fluorescence-activated cell sorter from mouse bone marrow cells and a yeast two-hybrid cDNA library was constructed. By screening with c-kit as bait, we cloned a novel cDNA, designed STAP-1, encoding an adaptor protein with a Pleckstrin homology domain, the Src homology 2 (SH2) domain, and a number of tyrosine phosphorylation sites. RT-PCR analysis revealed that STAP-1 expression is restricted in the bone marrow cell fraction expressing c-kit. The highest expression was observed in the CD34(low/-)Sca-1(+)c-kit(+)Lin(-) stem cell-enriched fraction. The murine myeloid cell line, M1, expressed a high level of STAP-1. However, the expression was strongly repressed in response to leukemia inhibitory factor (LIF) which induced monocytic differentiation of M1 cells, suggesting that STAP-1 is associated with the undifferentiated cell type. A two-hybrid assay indicated that STAP-1 bound not only to c-kit but also to c-fms but not to JAK2 or Pyk2. In 293 cells, STAP-1 was tyrosine-phosphorylated by activated c-kit. An in vitro binding assay suggested that the STAP-1 SH2 domain interacted with several tyrosine-phosphorylated proteins including c-kit and STAT5. These suggest that STAP-1 functions as an adaptor molecule downstream of c-kit in hematopoietic stem cells.     

Stirred culture of peripheral and cord blood hematopoietic cells offers advantages over traditional static systems for clinically relevant applications

The ability to culture hematopoietic cells in readily characterizable and scalable stirred systems, combined with the capability to utilize serum-free medium, will aid the development of clinically attractive bioreactor systems for transplantation therapies. We thus examined the proliferation and differentiation characteristics of peripheral blood (PB) mononuclear cells (MNC), cord blood (CB) MNC, and PB CD34(+) cells in spinner flasks and (control) T-flask cultures in both serum-containing and serum-free media. Hematopoietic cultures initiated from all sources examined (PB MNC, CB MNC, and PB CD34(+) cells) grew well in spinner vessels with either serum-containing or serum-free medium. Culture proliferation in spinner flasks was dependent on both agitator design and RPM as well as on the establishment of critical inoculum densities (ID) in both serum-containing (2 x 10(5) MNC/mL) and serum-free (3 x 10(5) MNC/mL) media. Spinner flask culture of PB MNC in serum-containing medium provided superior expansion of total cells and colony-forming cells (CFC) at high ID (1.2 x 10(6) cells/mL) as compared to T-flask controls. Serum-free spinner culture was comparable, if not superior, to that observed in serum-containing medium. This is the first report of stirred culture of PB or CB MNC, as well as the first report of stirred CD34(+) cell culture. Additionally, this is the first account of serum-free stirred culture of hematopoietic cells from any source.     

Immature hematopoietic stem cells undergo maturation in the fetal liver

Hematopoietic stem cells (HSCs), which are defined by their capacity to reconstitute adult conventional mice, are first found in the dorsal aorta after 10.5 days post coitus (dpc) and in the fetal liver at 11 dpc. However, lympho-myeloid hematopoietic progenitors are detected in the dorsal aorta from 9 dpc, raising the issue of their role in establishing adult hematopoiesis. Here, we show that these progenitors are endowed with long-term reconstitution capacity, but only engraft natural killer (NK)-deficient Rag2γc(-/-) mice. This novel population, called here immature HSCs, evolves in culture with thrombopoietin and stromal cells, into HSCs, defined by acquisition of CD45 and MHC-1 expression and by the capacity to reconstitute NK-competent mice. This evolution occurs during ontogeny, as early colonization of fetal liver by immature HSCs precedes that of HSCs. Moreover, organ culture experiments show that immature HSCs acquire, in this environment, the features of HSCs.     

The direct profibrotic and indirect immune antifibrotic balance of blocking the cannabinoid 2 receptor

Cannabinoid 2 (CB2) receptors expressed on immune cells are considered to be antifibrogenic. Hepatic stellate cells (HSCs) directly interact with phagocytosis lymphocytes, but the nature of this interaction is obscure. We aimed to study the effects of CB2 receptors on hepatic fibrosis via their role in mediating immunity. Hepatic fibrosis was induced by carbon-tetrachloride (CCl(4)) administration in C57BL/6 wild-type (WT) and CB2 knockout (CB2(-/-)) mice. Irradiated animals were reconstituted with WT or CB2(-/-) lymphocytes. Lymphocytes from na?ve/fibrotic WT animals and healthy/cirrhotic hepatitis C virus were preincubated in vitro with or without CB2 antagonist, evaluated for proliferation and apoptosis, and then cocultured with primary mouse HSCs or a human HSC line (LX2), respectively. Lymphocyte phagocytosis was then evaluated. Following CCl(4)-administration, CB2(-/-) mice developed significant hepatic fibrosis but less necroinflammation. WT mice harbored decreased liver CD4(+) and NK(+) cells but increased CD8(+) subsets. Na?ve CB2(-/-) mice had significantly decreased T cell subsets. Adoptive transfer of CB2(-/-) lymphocytes led to decreased fibrosis in the irradiated WT recipient compared with animals receiving WT lymphocytes. Moreover, necroinflammation also tended to decrease. In vitro, a CB2-antagonist directly increased human HSC activation and increased apoptosis and decreased proliferation of mice/human T cells (healthy/fibrotic) and their phagocytosis. We concluded that CB2(-/-) lymphocytes exert an antifibrotic activity, whereas lack of CB2 receptor in HSCs promotes fibrosis. These findings broaden our understanding of cannabinoid signaling in hepatic fibrosis beyond their activity solely in HSCs.     

Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45(low) c-Kit(+) cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45(low) c-Kit(-) cells that showed a granulocyte morphology; CD45(high) c-Kit(low/-) that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45(low) c-Kit(+) cells from the AGM culture had the abilities to reproduce CD45(low) c-Kit(+) cells and differentiate into CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) cells, whereas CD45(low) c-Kit(-) and CD45(high) c-Kit(low/-) did not produce CD45(low) c-Kit(+) cells. Furthermore, CD45(low) c-Kit(+) cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45(low) c-Kit(+) cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells.     

Specific marking of hESCs-derived hematopoietic lineage by WAS-promoter driven lentiviral vectors

Genetic manipulation of human embryonic stem cells (hESCs) is instrumental for tracing lineage commitment and to studying human development. Here we used hematopoietic-specific Wiskott-Aldrich syndrome gene (WAS)-promoter driven lentiviral vectors (LVs) to achieve highly specific gene expression in hESCs-derived hematopoietic cells. We first demonstrated that endogenous WAS gene was not expressed in undifferentiated hESCs but was evident in hemogenic progenitors (CD45(-)CD31(+)CD34(+)) and hematopoietic cells (CD45(+)). Accordingly, WAS-promoter driven LVs were unable to express the eGFP transgene in undifferentiated hESCs. eGFP(+) cells only appeared after embryoid body (EB) hematopoietic differentiation. The phenotypic analysis of the eGFP(+) cells showed marking of different subpopulations at different days of differentiation. At days 10-15, AWE LVs tag hemogenic and hematopoietic progenitors cells (CD45(-)CD31(+)CD34(dim) and CD45(+)CD31(+)CD34(dim)) emerging from hESCs and at day 22 its expression became restricted to mature hematopoietic cells (CD45(+)CD33(+)). Surprisingly, at day 10 of differentiation, the AWE vector also marked CD45(-)CD31(low/-)CD34(-) cells, a population that disappeared at later stages of differentiation. We showed that the eGFP(+)CD45(-)CD31(+) population generate 5 times more CD45(+) cells than the eGFP(-)CD45(-)CD31(+) indicating that the AWE vector was identifying a subpopulation inside the CD45(-)CD31(+) cells with higher hemogenic capacity. We also showed generation of CD45(+) cells from the eGFP(+)CD45(-)CD31(low/-)CD34(-) population but not from the eGFP(-)CD45(-)CD31(low/-)CD34(-) cells. This is, to our knowledge, the first report of a gene transfer vector which specifically labels hemogenic progenitors and hematopoietic cells emerging from hESCs. We propose the use of WAS-promoter driven LVs as a novel tool to studying human hematopoietic development.     

SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells

To improve our ability to identify hematopoietic stem cells (HSCs) and their localization in vivo, we compared the gene expression profiles of highly purified HSCs and non-self-renewing multipotent hematopoietic progenitors (MPPs). Cell surface receptors of the SLAM family, including CD150, CD244, and CD48, were differentially expressed among functionally distinct progenitors. HSCs were highly purified as CD150(+)CD244(-)CD48(-) cells while MPPs were CD244(+)CD150(-)CD48(-) and most restricted progenitors were CD48(+)CD244(+)CD150(-). The primitiveness of hematopoietic progenitors could thus be predicted based on the combination of SLAM family members they expressed. This is the first family of receptors whose combinatorial expression precisely distinguishes stem and progenitor cells. The ability to purify HSCs based on a simple combination of SLAM receptors allowed us to identify HSCs in tissue sections. Many HSCs were associated with sinusoidal endothelium in spleen and bone marrow, though some HSCs were associated with endosteum. HSCs thus occupy multiple niches, including sinusoidal endothelium in diverse tissues.