An<Emphasis Type="Italic">in vitro</Emphasis> method to study the effects of hematopoietic regulators during immune and blood cell development

In adults, hematopoiesis occurs in bone marrow (BM) through a complex process with differentiation of hematopoietic stem cells (HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34. Methods on hematopoietic regulation are presented to show the effects of the chemokine, stromal-derived growth factor (SDF)-1α and the neuropeptide, substance P (SP). SDF-1α production in BM stroma causes interactions with HSCs, thereby retaining the HSCs in regions close to the endosteum, at low oxygen. Small changes in SDF-1α levels stimulate HSC functions through direct and indirect mechanisms. The indirect method occurs by SP production, which stimulates CD34⁺ cells, supported by ligand-binding studies, long-term culture-initiating cell assays for HSC functions, and clonogenic assays for myeloid progenitors. These methods can be applied to study other hematopoietic regulators.     

A non-redundant function of cyclin E1 in hematopoietic stem cells

A precise balance between quiescence and proliferation is crucial for the lifelong function of hematopoietic stem cells (HSCs). Cyclins E1 and E2 regulate exit from quiescence in fibroblasts, but their role in HSCs remains unknown. Here, we report a non-redundant role for cyclin E1 in mouse HSCs. A long-term culture-initiating cell (LTC-IC) assay indicated that the loss of cyclin E1, but not E2, compromised the colony-forming activity of primitive hematopoietic progenitors. Ccne1^−/− mice showed normal hematopoiesis in vivo under homeostatic conditions but a severe impairment following myeloablative stress induced by 5-fluorouracil (5-FU). Under these conditions, Ccne1^−/− HSCs were less efficient in entering the cell cycle, resulting in decreased hematopoiesis and reduced survival of mutant mice upon weekly 5-FU treatment. The role of cyclin E1 in homeostatic conditions became apparent in aged mice, where HSC quiescence was increased in Ccne1^−/− animals. On the other hand, loss of cyclin E1 provided HSCs with a competitive advantage in bone marrow serial transplantation assays, suggesting that a partial impairment of cell cycle entry may exert a protective role by preventing premature depletion of the HSC compartment. Our data support a role for cyclin E1 in controlling the exit from quiescence in HSCs. This activity, depending on the physiological context, can either jeopardize or protect the maintenance of hematopoiesis.     

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time¹. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).Both trabecular osteoblasts^2,3 and sinusoidal endothelium⁴ constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin⁺ osteoblasts³ and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-1⁵ concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity⁶. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands⁷ and interferon-α⁶ can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin^- c-Kit⁺ Sca-1⁺ CD150⁺ CD48^- CD34^- population has been described⁸. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs⁹. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described¹⁰. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.     

Unique and independent roles for MLL in adult hematopoietic stem cells and progenitors

The Mixed Lineage Leukemia (MLL) gene is essential for embryonic hematopoietic stem cell (HSC) development, but its role during adult hematopoiesis is unknown. Using an inducible knockout model, we demonstrate that Mll is essential for the maintenance of adult HSCs and progenitors, with fatal bone marrow failure occurring within 3 weeks of Mll deletion. Mll-deficient cells are selectively lost from mixed bone marrow chimeras, demonstrating their failure to self-renew even in an intact bone marrow environment. Surprisingly, HSCs lacking Mll exhibit ectopic cell-cycle entry, resulting in the depletion of quiescent HSCs. In contrast, Mll deletion in myelo-erythroid progenitors results in reduced proliferation and reduced response to cytokine-induced cell-cycle entry. Committed lymphoid and myeloid cells no longer require Mll, defining the early multipotent stages of hematopoiesis as Mll dependent. These studies demonstrate that Mll plays selective and independent roles within the hematopoietic system, maintaining quiescence in HSCs and promoting proliferation in progenitors.     

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34⁺CD38⁺CD33⁺ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3⁺ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.     

Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines

Extracellular nucleotides are emerging as important regulators of inflammation, cell proliferation and differentiation in a variety of tissues, including the hematopoietic system. In this study, the role of ATP was investigated during murine hematopoiesis. ATP was able to reduce the percentage of hematopoietic stem cells (HSCs), common myeloid progenitors and granulocyte–macrophage progenitors (GMPs), whereas differentiation into megakaryocyte–erythroid progenitors was not affected. In addition, in vivo administration of ATP to mice reduced the number of GMPs, but increased the number of Gr-1⁺Mac-1⁺ myeloid cells. ATP also induced an increased proliferation rate and reduced Notch expression in HSCs and impaired HSC-mediated bone marrow reconstitution in sublethally irradiated mice. Moreover, the effects elicited by ATP were inhibited by suramin, a P2 receptor antagonist, and BAPTA, an intracellular Ca²⁺ chelator. We further investigated whether the presence of cytokines might modulate the observed ATP-induced differentiation. Treatment of cells with cytokines (stem cell factor, interleukin-3 and granulocyte–monocyte colony stimulator factor) before ATP stimulation led to reduced ATP-dependent differentiation in long-term bone marrow cultures, thereby restoring the ability of HSCs to reconstitute hematopoiesis. Thus, our data suggest that ATP induces the differentiation of murine HSCs into the myeloid lineage and that this effect can be modulated by cytokines.     

Vinculin Is Indispensable for Repopulation by Hematopoietic Stem Cells,Independent of Integrin Function

Vinculin is a highly conserved actin-binding protein that is localized in integrin-mediated focal adhesion complexes. Although critical roles have been proposed for integrins in hematopoietic stem cell (HSC) function, little is known about the involvement of intracellular focal adhesion proteins in HSC functions. This study showed that the ability of c-Kit⁺Sca1⁺Lin⁻ HSCs to support reconstitution of hematopoiesis after competitive transplantation was severely impaired by lentiviral transduction with short hairpin RNA sequences for vinculin. The potential of these HSCs to differentiate into granulocytic and monocytic lineages, to migrate toward stromal cell-derived factor 1α, and to home to the bone marrow in vivo were not inhibited by the loss of vinculin. However, the capacities to form long term culture-initiating cells and cobblestone-like areas were abolished in vinculin-silenced c-Kit⁺Sca1⁺Lin⁻ HSCs. In contrast, adhesion to the extracellular matrix was inhibited by silencing of talin-1, but not of vinculin. Whole body in vivo luminescence analyses to detect transduced HSCs confirmed the role of vinculin in long term HSC reconstitution. Our results suggest that vinculin is an indispensable factor determining HSC repopulation capacity, independent of integrin functions.     

Dysfunction of Bone Marrow Vascular Niche in Acute Graft-Versus-Host Disease after MHC-Haploidentical Bone Marrow Transplantation

Acute graft-versus-host disease (aGvHD) is the most common complication of allogeneic hematopoietic stem cell transplantation (HSCT), which is often accompanied by impaired hematopoietic reconstitution. Sinusoidal endothelial cells (SECs) constitute bone marrow (BM) vascular niche that plays an important role in supporting self-renewal capacity and maintaining the stability of HSC pool. Here we provide evidences that vascular niche is a target of aGvHD in a major histocompatibility complex (MHC)–haploidentical matched murine HSCT model. The results demonstrated that hematopoietic cells derived from GvHD mice had the capacity to reconstitute hematopoiesis in healthy recipient mice. However, hematopoietic cells from healthy donor mice failed to reconstitute hematopoiesis in GvHD recipient mice, indicating that the BM niche was impaired by aGvHD in this model. We further demonstrated that SECs were markedly reduced in the BM of aGvHD mice. High level of Fas and caspase-3 expression and high rate of apoptosis were identified in SECs, indicating that SECs were destroyed by aGvHD in this murine HSCT model. Furthermore, high Fas ligand expression on engrafted donor CD4⁺, but not CD8⁺ T cells, and high level MHC-II but not MHC-I expression on SECs, suggested that SECs apoptosis was mediated by CD4⁺ donor T cells through the Fas/FasL pathway.     

Rac GTPases differentially integrate signals regulating hematopoietic stem cell localization

The molecular events that regulate engraftment and mobilization of hematopoietic stem cells and progenitors (HSC/Ps) are still incompletely defined. We have examined the role of the Rho GTPases Rac1 and Rac2 in HSC engraftment and mobilization. Rac1, but not the hematopoietic-specific Rac2, is required for the engraftment phase of hematopoietic reconstitution, because Rac1(-/-) HSCs did not rescue in vivo hematopoiesis after transplantation, but deletion of Rac1 after engraftment did not impair steady-state hematopoiesis. Rac1(-/-) HSC/Ps showed impaired spatial localization to the endosteum but near-normal homing to the medullary cavity in vivo. Interaction with the bone marrow microenvironment in vitro was markedly altered. Whereas post-engraftment deletion of Rac1 alone did not impair hematopoiesis, deficiency of both Rac1 and Rac2 led to massive mobilization of HSCs from the marrow associated with ineffective hematopoiesis and intense selection for Rac-expressing HSCs. This mobilization was reversible by re-expression of Rac1. In addition, a rationally designed, reversible small-molecule inhibitor of Rac activation led to transient mobilization of engraftable HSC/Ps. Rac proteins thus differentially regulate engraftment and mobilization phenotypes, suggesting that these biological processes and steady-state hematopoiesis are biochemically separable and that Rac proteins may be important molecular targets for stem cell modification.     

Bone marrow progenitor cells induce a regulatory autologous proliferative T lymphocyte response

The proliferative response of human T lymphocytes to autologous bone marrow progenitor cells was studied by in vitro coculture in autologous serum. Irradiated enriched bone marrow progenitor cells induced the proliferation of cocultured peripheral blood T cells, with maximal proliferation at 8 days and stimulator:proliferator ratios of 1/1. This autologous proliferative T lymphocyte response was completely abrogated by the inclusion of anti-HLA-DR, anti-CD2, or anti LFA-3 antibodies into the coculture, and partially inhibited by anti-CD4. Repetitive stimulation with autologous progenitors at days 14 and 28 expanded and further enriched the autoreactive T cells, which proliferated specifically in the presence of autologous progenitors. When incubated for 12 h with bone marrow before short term hematopoietic culture, these autoreactive T cells inhibited hematopoiesis 60 to 100%. These data indicate that a subset of T lymphocytes recognize proliferating hematopoietic progenitors and regulate the growth and differentiation of normal bone marrow cells.     

Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood

Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC activity have been identified, as have downstream lineage-committed progenitors, but multipotent progenitor activity has not been uniquely isolated. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. Utilizing in vivo transplantation studies and complementary in vitro assays, we demonstrate that the Lin-CD34+CD38-CD90+CD45RA- cord blood fraction contains HSC and isolate this activity to as few as 10 purified cells. Furthermore, we report the first prospective isolation of a population of candidate human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.     

Piwi Genes Are Dispensable for Normal Hematopoiesis in Mice

Hematopoietic stem cells (HSC) must engage in a life-long balance between self-renewal and differentiation to sustain hematopoiesis. The highly conserved PIWI protein family regulates proliferative states of stem cells and their progeny in diverse organisms. A Human piwi gene (for clarity, the non-italicized “piwi” refers to the gene subfamily), HIWI (PIWIL1), is expressed in CD34⁺ stem/progenitor cells and transient expression of HIWI in a human leukemia cell line drastically reduces cell proliferation, implying the potential function of these proteins in hematopoiesis. Here, we report that one of the three piwi genes in mice, Miwi2 (Piwil4), is expressed in primitive hematopoetic cell types within the bone marrow. Mice with a global deletion of all three piwi genes, Miwi, Mili, and Miwi2, are able to maintain long-term hematopoiesis with no observable effect on the homeostatic HSC compartment in adult mice. The PIWI-deficient hematopoetic cells are capable of normal lineage reconstitution after competitive transplantation. We further show that the three piwi genes are dispensable during hematopoietic recovery after myeloablative stress by 5-FU. Collectively, our data suggest that the function of the piwi gene subfamily is not required for normal adult hematopoiesis.     

Erythroid differentiation is augmented in Reelin-deficient K562 cells and homozygous reeler mice

Reelin is an extracellular glycoprotein that is highly conserved in mammals. In addition to its expression in the nervous system, Reelin is present in erythroid cells but its function there is unknown. We report in this study that Reelin is up-regulated during erythroid differentiation of human erythroleukemic K562 cells and is expressed in the erythroid progenitors of murine bone marrow. Reelin deficiency promotes erythroid differentiation of K562 cells and augments erythroid production in murine bone marrow. In accordance with these findings, Reelin deficiency attenuates AKT phosphorylation of the Ter119⁺CD71⁺ erythroid progenitors and alters the cell number and frequency of the progenitors at different erythroid differentiation stages. A regulatory role of Reelin in erythroid differentiation is thus defined.     

Blood loses it when nerves go bad

Myeloproliferative neoplasms are diseases that arise in the stem cells of the blood. In a recent paper published in Nature, Arranz et al. demonstrated that abrogation of sympathetic nerve fibers reduced bone marrow Nestin⁺ mesenchymal cells, which in turn led to an expansion of hematopoietic stem cells and progression of myeloproliferative neoplasms.The stromal cell compartment, or non-hematopoietic cells, of the bone marrow has emerged as an important driver of cell state in hematopoietic stem cells (HSCs) and hematopoietic stem/progenitor cells (HSPCs) in a non-autonomous manner. The HSC niche is not well defined and a number of studies suggest that there are specialized niches for the unique regulation of HSCs and HSPCs¹. Several studies suggest that HSCs reside in a perivascular niche in which a heterogenic population of perivascular mesenchymal stromal cells (MSCs) with overlapping expression of Nestin, LepR, Prx1, or Mx1 each synthesize multiple factors (e.g., CXCL12 and SCF) that promote maintenance and/or localization of HSCs^1,2. Endothelial cells (Tie2⁺)³, and MSCs (NG2⁺/LepR⁻) surrounding arterial vessels⁴, are other units of the niche reported to regulate HSC numbers and quiescence, respectively. Furthermore, osteoprogenitors lining the endosteal surface have a more indirect role in the regulation of HSCs⁶. Thus, it appears that hematopoietic regulation and differentiation in the bone marrow microenvironment is governed at multiple levels⁵.Increasing evidence suggests that dysregulation of the bone marrow microenvironment may participate in blood malignancies. For instance, perturbations of the miRNA processing or ribosomal components through Dicer1 deletion in immature osteolineage cells induced myelodysplasia in mice, followed by the rare emergence of acute myeloid leukemia (AML)⁶. Others reported that β-catenin stabilization in mature osteolineage cells resulted in Notch pathway activation, myelodysplastic syndrome, and highly penetrant AML in mice⁷. In humans, ∼5% of post-transplant AML patients relapse with a leukemia of donor cell origin, suggesting that some patients may have a microenvironmental driver of leukemogenesis⁸. Together, these studies are consistent with a role of the bone marrow microenvironment in maintaining the integrity of hematopoiesis and restricting oncogenesis. When the well-orchestrated regulation of hematopoiesis is disrupted, blood malignancies might occur.The study by Arranz et al.⁹ is a continuation of prior work identifying perivascular bone marrow Nestin⁺ MSCs affected by sympathetic nerve fibers to regulate HSCs¹⁰. Previous studies that a perturbed bone marrow microenvironment modulates myeloproliferative neoplasms (MPNs)^6,7 prompted the authors to further investigate the role of Nestin⁺ MSCs in MPN, specifically MPN associated with Janus kinase 2 (JAK2) mutations^11,12.The authors first analyzed Nestin expression in bone marrow samples from MPN patients and discovered that despite elevated blood-vessel density, Nestin⁺ cell numbers and mRNA expression were reduced. Similar findings were observed in genetically engineered mice that recapitulate human MPNs (e.g., Mx1-cre; JAK2^V617F), indicating that Nestin⁺ MSCs might play a role in MPN. Arranz et al. proceeded to investigate whether a selective depletion of Nestin⁺ MSCs mimics the MPN mouse model. Mice depleted of Nestin⁺ MSCs showed an expansion of HSCs, due to a drop in CXCL12 expression, accompanied by increased hematopoietic progenitors in bone marrow, peripheral blood and spleen, indicative of MPN. Extensive genome-wide RNA-sequencing studies revealed enrichment of Schwann cell- and neural-related genes in Nestin⁺ MSCs derived from MPN mice. This result prompted the authors to explore the role of sympathetic nerve fibers and nonmyelinating Schwann cells in MPN patients and the MPN mouse model. Strikingly, both MPN patients and MPN mice had reduced sympathetic nerve fibers and nonmyelinating Schwann cells adjacent to Nestin⁺ cells in the bone marrow. Multiplex ELISA experiments identified that mutant HSCs secrete IL-1β, which induced apoptosis in bone marrow Schwann cells by Caspase-1 activation followed by neuronal damage. Neural-glial damage in turn compromised Nestin⁺ MSCs survival and led to MPN. Finally, the authors rescued the MPN phenotype partially in MPN mice by treating the mutant mice with IL-1R antagonist, a neuroglial protection agent (4-methylcatechol), or a β3-adrenergic agonist (BRL37344) which compensated for deficient sympathetic stimulation. This treatment was selective against mutant hematopoietic progenitors and preserved normal HSCs, and this effect could only be observed in vivo. Therefore, the authors concluded that the effect was niche-dependent (Figure 1).Open in a separate windowFigure 1Bone marrow neuropathy leads to mutant HSC expansion in MPN. (1) IL-1β is released by mutant HSCs, which induces Caspase-1-dependent apoptosis in sympathetic nerve fibers, ensheathed by nonmyelinating Schwann cells. (2) Neural-glial damage leads to a reduced noradrenergic sympathetic stimulation of Nestin⁺ MSCs and loss of MSCs. (3) Aberrant neural regulation sensitizes Nestin⁺ MSCs to IL-1β-induced apoptosis with a subsequent drop in CXCL12 expression. (4) HSC and progenitor cell proliferation is increased, followed by MPN pathogenesis. (5) Nestin⁺ MSCs survival and function can be restored by the neuroglial protective agent 4-methylcatechol, the β3-adrenergic agonist BRL37344, or by blocking IL-1R. MPN, myeloproliferative neoplasm; HSC, hematopoietic stem cell; MSC, mesenchymal stem cell; NA, noradrenaline; AR, adrenergic receptor; IL, Interleukin; IL-1R, Interleukin-1 receptor.While the prevailing understanding of cancer as a disease in which changes in the cell of origin drive oncogenic transformation, these studies point to the potential for the microenvironment as a critical cooperator in the malignant process for at least some neoplasms. This study emphasizes that there may be a two-way perturbation process required for MPN. HSCs acquire a mutation (e.g., JAK2-V617F mutation) that leads to cell expansion and the mutant HSC perturbs the bone marrow niche, which further drive HSCs into neoplasia. Based on these findings, the authors postulated that neural-glial protective agents and β3-adrenergic agonists may subvert the process and be therapeutically useful. Therefore, this model provides insight into how the neural compartment of the bone marrow microenvironment can serve as a modulator of malignancy and offers a novel, testable approach for treating MPNs — by not only targeting the malignant cell, but also by selectively targeting the unhealthy niche.     

Robo4 Plays a Role in Bone Marrow Homing and Mobilization,but Is Not Essential in the Long-Term Repopulating Capacity of Hematopoietic Stem Cells

Roundabout (Robo) family proteins are immunoglobulin-type surface receptors critical for cellular migration and pathway finding of neuronal axons. We have previously shown that Robo4 was specifically expressed in hematopoietic stem and progenitor cells and its high expression correlated with long-term repopulating (LTR) capacity. To reveal the physiological role of Robo4 in hematopoiesis, we examined the effects of Robo4 disruption on the function of hematopoietic stem cells (HSCs) and progenitors. In Robo4-deficient mice, basic hematological parameters including complete blood cell count and differentiation profile were not affected. In contrast to the previous report, HSC/hematopoietic progenitor (HPC) frequencies in the bone marrow (BM) were perfectly normal in Robo4^−/− mice. Moreover, Robo4^−/− HSCs were equally competitive as wild-type HSCs in transplantation assays and had normal long-term repopulating (LTR) capacity. Of note, the initial engraftment at 4-weeks after transplantation was slightly impaired by Robo4 ablation, suggesting a marginal defect in BM homing of Robo4^−/− HSCs. In fact, homing efficiencies of HSCs/HPCs to the BM was significantly impaired in Robo4-deficient mice. On the other hand, granulocyte-colony stimulating factor-induced peripheral mobilization of HSCs was also impaired by Robo4 disruption. Lastly, marrow recovery from myelosuppressive stress was equally efficient in WT- and Robo4-mutant mice. These results clearly indicate that Robo4 plays a role in HSC trafficking such as BM homing and peripheral mobilization, but is not essential in the LTR and self-renewal capacity of HSCs.