Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche

Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1(hi) monocytes (MOs), Gr-1(lo) MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin(+) niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169(+) MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin(+) niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.     

The bone marrow stem cell niche grows up: mesenchymal stem cells and macrophages move in

Stem cell niches are defined as the cellular and molecular microenvironments that regulate stem cell function together with stem cell autonomous mechanisms. This includes control of the balance between quiescence, self-renewal, and differentiation, as well as the engagement of specific programs in response to stress. In mammals, the best understood niche is that harboring bone marrow hematopoietic stem cells (HSCs). Recent studies have expanded the number of cell types contributing to the HSC niche. Perivascular mesenchymal stem cells and macrophages now join the previously identified sinusoidal endothelial cells, sympathetic nerve fibers, and cells of the osteoblastic lineage to form similar, but distinct, niches that harbor dormant and self-renewing HSCs during homeostasis and mediate stem cell mobilization in response to granulocyte colony-stimulating factor.     

Rapid mobilization of murine and human hematopoietic stem and progenitor cells with AMD3100, a CXCR4 antagonist

Improving approaches for hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) mobilization is clinically important because increased numbers of these cells are needed for enhanced transplantation. Chemokine stromal cell derived factor-1 (also known as CXCL12) is believed to be involved in retention of HSCs and HPCs in bone marrow. AMD3100, a selective antagonist of CXCL12 that binds to its receptor, CXCR4, was evaluated in murine and human systems for mobilizing capacity, alone and in combination with granulocyte colony-stimulating factor (G-CSF). AMD3100 induced rapid mobilization of mouse and human HPCs and synergistically augmented G-CSF-induced mobilization of HPCs. AMD3100 also mobilized murine long-term repopulating (LTR) cells that engrafted primary and secondary lethally-irradiated mice, and human CD34(+) cells that can repopulate nonobese diabetic-severe combined immunodeficiency (SCID) mice. AMD3100 synergized with G-CSF to mobilize murine LTR cells and human SCID repopulating cells (SRCs). Human CD34(+) cells isolated after treatment with G-CSF plus AMD3100 expressed a phenotype that was characteristic of highly engrafting mouse HSCs. Synergy of AMD3100 and G-CSF in mobilization was due to enhanced numbers and perhaps other characteristics of the mobilized cells. These results support the hypothesis that the CXCL12-CXCR4 axis is involved in marrow retention of HSCs and HPCs, and demonstrate the clinical potential of AMD3100 for HSC mobilization.     

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.     

SDF-1/CXCR4信号通路在造血干细胞归巢中作用的研究进展

造血干细胞移植(HSCT)是治疗白血病、淋巴瘤、再生障碍性贫血等恶性血液病的一种有效治疗手段.但是,HSCT后造血功能的恢复与归巢至骨髓造血微环境中的造血干细胞(HSC)数目有关.HSCT输注入受者体内的HSC不会立刻发挥作用,而是先经历一系列复杂的过程归巢至骨髓造血微环境后,才能继续增殖并分化为相应的效应细胞发挥作用.其中,供者HSC与众多细胞及细胞因子间的相互作用是决定HSC归巢、增殖及分化的关键因素.基质细胞衍生因子(SDF)-1及其唯一的受体CXC趋化因子受体(CXCR)4所构成的SDF-1/CXCR4信号通路,可能在HSC归巢中发挥重要作用.为了更深入地研究HSC归巢的具体过程和SDF-1/CXCR4信号通路在该过程中发挥的作用,现对SDF-1、CXCR4的结构、作用机制,以及SDF1/CXCR4信号通路在HSC归巢中作用的研究进展进行综述.     

基质细胞衍生因子SDF及其受体CXCR4在造血干/祖细胞动员及归巢过程中的作用

基质细胞衍生因子(SDF)属CXC型趋化因子，主要在骨髓基质细胞和骨髓内皮等细胞表达，特异性地引起表达CXCR4的造血干细胞的趋化反应，因此在造血干细胞的迁移和归巢中起着重要作用。本文对SDF及其受体CXCR4在造血干细胞动员以及归巢过程中的机理进行简要综述。     

Microenvironmental targeting of Wnt/β-catenin signals for hematopoietic stem cell regulation

Importance of the field: Microenvironment has emerged as crucial element in the regulation of hematopoietic stem cells (HSCs) in the stem cell niche. Recent studies have demonstrated that Wnt/β-catenin signals are clearly involved in the stem cell niche, raising the possibility that the hematopoietic microenvironment is a potential target for stem cell therapy.

Areas covered in this review: In this review, the biological outcomes of Wnt/β-catenin signals on HSC activity are summarized in various study models and potential reasons for discrepancies are discussed. Evidence for the microenvironmental targeting of Wnt/β-catenin signals and studies that verify this concept is summarized.

What the reader will gain: We show that distinct effects of Wnt/β-catenin on HSCs can be caused depending on their target of activation in the hematopoietic microenvironment. We show stromal targeting of Wnt/β-catenin signals and cross-talk with notch signals in the niche during bone marrow regeneration enhancing the self-renewal of HSCs.

Take home message: The identification of stroma-mediated Wnt/β-catenin effects now allows the integration of discrepancies in previously reported findings and suggests that the stromal targeting of wnt/β-catenin signals could serve as a potential therapeutic strategy for activating a stem cell niche for the efficient regeneration of HSCs.     

Endothelium and NOTCH specify and amplify aorta-gonad-mesonephros–derived hematopoietic stem cells

Hematopoietic stem cells (HSCs) first emerge during embryonic development within vessels such as the dorsal aorta of the aorta-gonad-mesonephros (AGM) region, suggesting that signals from the vascular microenvironment are critical for HSC development. Here, we demonstrated that AGM-derived endothelial cells (ECs) engineered to constitutively express AKT (AGM AKT-ECs) can provide an in vitro niche that recapitulates embryonic HSC specification and amplification. Specifically, nonengrafting embryonic precursors, including the VE-cadherin–expressing population that lacks hematopoietic surface markers, cocultured with AGM AKT-ECs specified into long-term, adult-engrafting HSCs, establishing that a vascular niche is sufficient to induce the endothelial-to-HSC transition in vitro. Subsequent to hematopoietic induction, coculture with AGM AKT-ECs also substantially increased the numbers of HSCs derived from VE-cadherin⁺CD45⁺ AGM hematopoietic cells, consistent with a role in supporting further HSC maturation and self-renewal. We also identified conditions that included NOTCH activation with an immobilized NOTCH ligand that were sufficient to amplify AGM-derived HSCs following their specification in the absence of AGM AKT-ECs. Together, these studies begin to define the critical niche components and resident signals required for HSC induction and self-renewal ex vivo, and thus provide insight for development of defined in vitro systems targeted toward HSC generation for therapeutic applications.     

The stem cell niches in bone

The stem cell niche is composed of a specialized population of cells that plays an essential role in regulating adult stem cell self-renewal and differentiation. In adults, osteoblasts, responsible for osteogenesis, and hematopoietic cells, responsible for hematopoiesis, are closely associated in the bone marrow, suggesting a reciprocal relationship between the two. It was recently discovered that a subset of osteoblasts functions as a key component of the HSC niche (namely, the osteoblastic niche), controlling HSC numbers. HSCs interact not only with osteoblasts but also with other stromal cells, including endothelial cells. Sinusoidal endothelial cells in bone marrow have been revealed as an alternative HSC niche called the vascular niche. In this Review we compare the architecture of these 2 HSC niches in bone marrow. We also highlight the function of osteoblasts in maintaining a quiescent HSC microenvironment and the likely role of the vascular niche in regulating stem cell proliferation, differentiation, and mobilization. In addition, we focus on studies of animal models and in vitro assays that have provided direct insights into the actions of these osteoblastic and vascular niches, revealing central roles for numerous signaling and adhesion molecules. Many of the discoveries described herein may contribute to future clinical treatments for hematopoietic and bone-related disorders, including cancer.     

Children are not little adults: just ask their hematopoietic stem cells

HSCs differ during ontogeny in some important parameters, including anatomic site of residence and cell cycling characteristics. In this issue of the JCI, Bowie et al. show that postnatal HSCs as well as fetal liver HSCs in mice are active in the cell cycle at much higher rates than that of adult HSCs; however, this increased frequency of cycling abruptly ceases 4 weeks after birth (see the related article beginning on page 2808). The cycling postnatal HSCs expressed high levels of CXC chemokine ligand 12 (CXCL12, also known as stromal cell-derived factor 1 [SDF-1]), a chemokine previously implicated in stem cell trafficking to the marrow cavity and shown to be expressed by cells within the hematopoietic microenvironment. These cells also possessed an engraftment defect impeding reconstitution in irradiated recipient mice, which was reversible by pretransplant administration of antagonists of the CXCL12 receptor, CXCR4. Such agents are currently clinically available, suggesting that this approach could be used to improve stem cell transplantation and engraftment.     

Hematopoietic stem cells from poor and good mobilizers are qualitatively equivalent

BACKGROUND: Marrow damage from chemo‐ and radiation therapies has been suggested to affect quality and quantity of hematopoietic stem cell (HSC) products. We tested the hypothesis that CD34+ cells (HSCs) from low mobilizers are qualitatively inferior to HSCs from high mobilizers. STUDY DESIGN AND METHODS: HSC quality was defined by proportion of primitive HSC subsets (CD34+CD38?, CD34+HLA‐DR?, and CD34+ in G0 stage of cell cycle), the proportion of HSCs that express CXCR4 and CD26 homing proteins, and days to neutrophil and platelet (PLT) engraftments after transplant. HSC content and CD34 subsets analyses were performed using flow cytometry following the ISHAGE protocol. We evaluated the HSC quantity and quality of 139 autologous filgrastim‐mobilized HSC products. Patients were categorized into low, moderate, and high mobilizers if their total HSC collection was less than 3 × 10⁶, 3 × 10⁶ or more and less than 5 × 10⁶, and 5 × 10⁶/kg or more, respectively. RESULTS: The median number of primitive CD34 subsets increases with increasing HSC numbers and this association was significant (p = 0.001). However, when the ratios of the primitive CD34 subsets to total HSC counts were compared among the mobilization groups, the ratios were not significantly different. Coexpression of neither CD26 nor CXCR4 with CD34 antigen correlated with HSC mobilization. Evaluation of days to neutrophil engraftment among the mobilization groups did not show a significant difference (p = 0.1). However, days to PLT engraftment among the mobilization groups was significantly different (p = 0.05). CONCLUSION: The quality of HSCs from low mobilizers was comparable to HSCs from high mobilizers.     

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.     

Recombinant IL-7/HGFβ efficiently induces transplantable murine hematopoietic stem cells

Difficulty obtaining sufficient hematopoietic stem cells (HSCs) directly from the donor has limited the clinical use of HSC transplantation. Numerous attempts to stimulate the ex vivo growth of purified HSCs with cytokines and growth factors generally have induced only modest increases in HSC numbers while decreasing their in vivo reconstituting ability. We previously developed a recombinant single-chain form of a naturally occurring murine hybrid cytokine of IL-7 and the β chain of hepatocyte growth factor (rIL-7/HGFβ) that stimulates the in vitro proliferation and/or differentiation of common lymphoid progenitors, pre-pro-B cells, and hematopoietic progenitor cells (day 12 spleen colony-forming units) in cultures of mouse BM. Here we used the rIL-7/HGFβ in culture to induce large numbers of HSCs from multiple cell sources, including unseparated BM cells, purified HSCs, CD45^– BM cells, and embryonic stem cells. In each instance, most of the HSCs were in the G₀ phase of the cell cycle and exhibited reduced oxidative stress, decreased apoptosis, and increased CXCR4 expression. Furthermore, when injected i.v., these HSCs migrated to BM, self-replicated, provided radioprotection, and established long-term hematopoietic reconstitution. These properties were amplified by injection of rIL-7/HGFβ directly into the BM cavity but not by treatment with rIL-7, rHGF, and/or rHGFβ.     

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.     

New agents for mobilizing peripheral blood stem cells

Transplantation with bone marrow (BM) hematopoietic stem cells (HSC) has been used for curative therapy of hematologic diseases and inborn errors of metabolism for decades. More recently, alternative sources of HSC, particularly those induced to exit marrow and traffic to peripheral blood in response to external stimuli, have become the most widely used hematopoietic graft and show significant superiority to marrow HSC. Although a variety of agents can mobilize stem cells with different kinetics and efficiencies and these agents can be additive or synergistic when used in combination, currently G-CSF is the predominant stem cell mobilizer used clinically based upon potency, predictability and safety. Recent studies have demonstrated that the interaction between the chemokine stromal-derived factor 1 (SDF-1/CXCL12) and its receptor CXCR4 serves as a key regulator of HSC trafficking. AMD3100, a novel bicyclam CXCR4 antagonist, induces the rapid mobilization of HSC with both short- and long-term repopulation capacity. Mobilization with G-CSF and AMD3100 in clinical trials resulted in more patients achieving sufficient PBSC for transplantation than with G-CSF alone. Thus, chemokine axis-mobilization could allow rapid PBSC harvests with increased cell yields in difficult-to mobilize patients. Studies of autologous and allogeneic transplantation of AMD3100 mobilized grafts demonstrated prompt and stable engraftment.Enhanced homing properties of chemokine axis-mobilized PBSC suggest that these cells may have greater therapeutic utility in other areas including tissue repair and regeneration.     

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.     

Hematopoietic stem cells proliferate until after birth and show a reversible phase-specific engraftment defect

The regulation of HSC proliferation and engraftment of the BM is an important but poorly understood process, particularly during ontogeny. Here we show that in mice, all HSCs are cycling until 3 weeks after birth. Then, within 1 week, most became quiescent. Prior to 4 weeks of age, the proliferating HSCs with long-term multilineage repopulating activity displayed an engraftment defect when transiting S/G2/M. During these cell cycle phases, their expression of CXC chemokine ligand 12 (CXCL12; also referred to as stromal cell-derived factor 1 [SDF-1]) transiently increased. The defective engrafting activity of HSCs in S/G2/M was reversed when cells were allowed to progress into G1 prior to injection or when the hosts (but not the cells) were pretreated with a CXCL12 antagonist. Interestingly, the enhancing effect of CXCL12 antagonist pretreatment was exclusive to transplants of long-term multilineage repopulating HSCs in S/G2/M. These results demonstrate what we believe to be a new HSC regulatory checkpoint during development. They also suggest an ability of HSCs to express CXCL12 in a fashion that changes with cell cycle progression and is associated with a defective engraftment that can be overcome by in vivo administration of a CXCL12 antagonist.