Kfir Lapid

Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells

Here we investigated the potential role of bone-resorbing osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Different stress situations induced activity of osteoclasts (OCLs) along the stem cell–rich endosteum region of bone, secretion of proteolytic enzymes and mobilization of progenitors. Specific stimulation of OCLs with RANKL recruited mainly immature progenitors to the circulation in a CXCR4- and MMP-9–dependent manner; however, RANKL did not induce mobilization in young female PTPε-knockout mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress in homeostasis, G-CSF mobilization and stress situations. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF) and osteopontin along the endosteum, which was associated with progenitor mobilization. Finally, the major bone-resorbing proteinase, cathepsin K, also cleaved SDF-1 and SCF. Our findings indicate involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense, linking bone remodeling with regulation of hematopoiesis.

Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells

Steady-state egress of hematopoietic progenitor cells can be rapidly amplified by mobilizing agents such as AMD3100, the mechanism, however, is poorly understood. We report that AMD3100 increased the homeostatic release of the chemokine stromal cell derived factor-1 (SDF-1) to the circulation in mice and non-human primates. Neutralizing antibodies against CXCR4 or SDF-1 inhibited both steady state and AMD3100-induced SDF-1 release and reduced egress of murine progenitor cells over mature leukocytes. Intra-bone injection of biotinylated SDF-1 also enhanced release of this chemokine and murine progenitor cell mobilization. AMD3100 directly induced SDF-1 release from CXCR4+ human bone marrow osteoblasts and endothelial cells and activated uPA in a CXCR4/JNK-dependent manner. Additionally, ROS inhibition reduced AMD3100-induced SDF-1 release, activation of circulating uPA and mobilization of progenitor cells. Norepinephrine treatment, mimicking acute stress, rapidly increased SDF-1 release and progenitor cell mobilization, whereas β2-adrenergic antagonist inhibited both steady state and AMD3100-induced SDF-1 release and progenitor cell mobilization in mice. In conclusion, this study reveals that SDF-1 release from bone marrow stromal cells to the circulation emerges as a pivotal mechanism essential for steady-state egress and rapid mobilization of hematopoietic progenitor cells, but not mature leukocytes.

S1P promotes murine progenitor cell egress and mobilization via S1P1-mediated ROS signaling and SDF-1 release

The mechanisms of hematopoietic progenitor cell egress and clinical mobilization are not fully understood. Herein, we report that in vivo desensitization of Sphingosine-1-phosphate (S1P) receptors by FTY720 as well as disruption of S1P gradient toward the blood, reduced steady state egress of immature progenitors and primitive Sca-1(+)/c-Kit(+)/Lin(-) (SKL) cells via inhibition of SDF-1 release. Administration of AMD3100 or G-CSF to mice with deficiencies in either S1P production or its receptor S1P(1), or pretreated with FTY720, also resulted in reduced stem and progenitor cell mobilization. Mice injected with AMD3100 or G-CSF demonstrated transient increased S1P levels in the blood mediated via mTOR signaling, as well as an elevated rate of immature c-Kit(+)/Lin(-) cells expressing surface S1P(1) in the bone marrow (BM). Importantly, we found that S1P induced SDF-1 secretion from BM stromal cells including Nestin(+) mesenchymal stem cells via reactive oxygen species (ROS) signaling. Moreover, elevated ROS production by hematopoietic progenitor cells is also regulated by S1P. Our findings reveal that the S1P/S1P(1) axis regulates progenitor cell egress and mobilization via activation of ROS signaling on both hematopoietic progenitors and BM stromal cells, and SDF-1 release. The dynamic cross-talk between S1P and SDF-1 integrates BM stromal cells and hematopoeitic progenitor cell motility.

CD45 regulates retention, motility, and numbers of hematopoietic progenitors, and affects osteoclast remodeling of metaphyseal trabecules

The CD45 phosphatase is uniquely expressed by all leukocytes, but its role in regulating hematopoietic progenitors is poorly understood. We show that enhanced CD45 expression on bone marrow (BM) leukocytes correlates with increased cell motility in response to stress signals. Moreover, immature CD45 knockout (KO) cells showed defective motility, including reduced homing (both steady state and in response to stromal-derived factor 1) and reduced granulocyte colony-stimulating factor mobilization. These defects were associated with increased cell adhesion mediated by reduced matrix metalloproteinase 9 secretion and imbalanced Src kinase activity. Poor mobilization of CD45KO progenitors by the receptor activator of nuclear factor κB ligand, and impaired modulation of the endosteal components osteopontin and stem cell factor, suggested defective osteoclast function. Indeed, CD45KO osteoclasts exhibited impaired bone remodeling and abnormal morphology, which we attributed to defective cell fusion and Src function. This led to irregular distribution of metaphyseal bone trabecules, a region enriched with stem cell niches. Consequently, CD45KO mice had less primitive cells in the BM and increased numbers of these cells in the spleen, yet with reduced homing and repopulation potential. Uncoupling environmental and intrinsic defects in chimeric mice, we demonstrated that CD45 regulates progenitor movement and retention by influencing both the hematopoietic and nonhematopoietic compartments.

Pathways Implicated in Stem Cell Migration: The SDF-1/CXCR4 Axis

The hallmark of hematopoietic stem and progenitor cells (HSPCs) is their motility, which is essential for their function, such as recruitment upon demand. Stromal Derived Factor-1 (SDF-1, CXCL12) and its major receptor CXCR4 play major roles in stem cell motility and development. In vitro migration assays, implicating either gradients or cell surface-bound forms of SDF-1, are easy to perform and provide vital information regarding directional and random stem cell motility, which correlate with their repopulation potential in clinical and experimental transplantations. In vivo stem cell homing to the bone marrow, their retention, engraftment, and egress to the circulation, all involve SDF-1/CXCR4 interactions. Finally, other stem cell features such as stem cell survival and proliferation, are also dependent on the SDF-1/CXCR4 axis.

Physiologic corticosterone oscillations regulate murine hematopoietic stem/progenitor cell proliferation and CXCL12 expression by bone marrow stromal progenitors.

The role of corticosterone (Cort), the immune system’s major stress hormone, in the regulation of hematopoietic stem and progenitor cells (HSPCs) and their dynamic bone marrow (BM) microenvironment is currently unknown. We report that corticotropin-releasing factor receptor 1 (CRFR1) mutant mice with chronically low Cort levels showed aberrant HSPC regulation, having higher HSPC numbers and upregulation of the chemokine CXCL12, phenotypes that were restored by Cort supplementation. Expanded stromal progenitors known to support HSPCs were also observed in these low-Cort-containing mice. A similar phenotype was induced in wild-type (WT) mice by Metyrapone, a Cort synthesis inhibitor. Conversely, high Cort exposure induced HSPC apoptosis, reduced long-term BM repopulation and decreased stromal progenitor cell numbers. We documented circadian oscillations of Cort in WT BM but not in CRFR1 mutant mice, leading to diminished circadian BM CXCL12 fluctuations and increased number of circulating HSPCs in these mice. Finally, low Cort induced expansion of stromal progenitors, CXCL12 expression, HSPC proliferation and BM repopulation capacity, involving Notch1 signaling. This was associated with upregulation of the Notch ligand, Jagged1, in BM myeloid cells. Our results suggest that daily physiologic Cort oscillations are critical for balanced HSPC proliferation and function involving Notch1 signaling and their supportive BM microenvironment.

GSK3β regulates physiological migration of stem/progenitor cells via cytoskeletal rearrangement

Regulation of hematopoietic stem and progenitor cell (HSPC) steady-state egress from the bone marrow (BM) to the circulation is poorly understood. While glycogen synthase kinase-3β (GSK3β) is known to participate in HSPC proliferation, we revealed an unexpected role in the preferential regulation of CXCL12-induced migration and steady-state egress of murine HSPCs, including long-term repopulating HSCs, over mature leukocytes. HSPC egress, regulated by circadian rhythms of CXCL12 and CXCR4 levels, correlated with dynamic expression of GSK3β in the BM. Nevertheless, GSK3β signaling was CXCL12/CXCR4 independent, suggesting that synchronization of both pathways is required for HSPC motility. Chemotaxis of HSPCs expressing higher levels of GSK3β compared with mature cells was selectively enhanced by stem cell factor-induced activation of GSK3β. Moreover, HSPC motility was regulated by norepinephrine and insulin-like growth factor-1 (IGF-1), which increased or reduced, respectively, GSK3β expression in BM HSPCs and their subsequent egress. Mechanistically, GSK3β signaling promoted preferential HSPC migration by regulating actin rearrangement and microtubuli turnover, including CXCL12-induced actin polarization and polymerization. Our study identifies a previously unknown role for GSK3β in physiological HSPC motility, dictating an active, rather than a passive, nature for homeostatic egress from the BM reservoir to the blood circulation.

Erratum: Rapid mobilization of hematopoietic progenitors by AMD3100 and catecholamines is mediated by CXCR4-dependent SDF-1 release from bone marrow stromal cells (Leukemia (April 2011) DOI:10.1038/leu.2011.62)

Figure 5 Neurotransmitter stimulation induces functional SDF-1 release and rapid progenitor mobilization. (a, b) SDF-1 levels in the plasma and BM sup. (a) and circulating WBC and progenitor cells (b) in mice treated with NE or the b2 adrenergic antagonist ICI, 1 h after administration. Control mice received injections of PBS, n1⁄4 6 mice/group. Values of plasma SDF-1 levels: 1.1±0.17, 1.8±0.5, 0.6±0.06, 2±0.3, 2.8±0.5 and 1.2±0.1 ng/ml, respectively, *Po0.05 compared with control mice, Po0.05 compared with AMD3100-treated mice. (c) RT-PCR analysis (top) for mRNA expression and flow-cytometry analysis (bottom) for cell surface expression of b2 adrenergic receptor on cultured primary human BMEC. –RT1⁄4 cDNA was prepared without reverse transcriptase as a control. (d) SDF-1 release from primary human BMEC in response to stimulation with ICI (10 ng/ml), n1⁄4 3. (e) AMD3100-induced mobilization of progenitors in control and sympathectomized (6OHDA) of either neonate or adult mice. *Po0.005. n1⁄410–15 mice/group. Leukemia (2011) 25, 1378 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11

Quantifying hematopoietic stem and progenitor cell mobilization.

Allogeneic donor blood cells and autologous peripheral blood leukocytes (PBL), obtained following -clinical mobilization procedures, are routinely used as a major source of hematopoietic stem and progenitor cells (HSPC) for transplantation protocols. It is, therefore, essential to evaluate and to quantify the extent by which the HSPC are mobilized and enriched in the circulation in correlation with their long-term hematopoietic reconstitution capacity. In this chapter, we describe quantitative methods that measure the number of mobilized HSPC according to specific criteria, as well as their functional properties in vitro and in vivo. The described assays are useful for assessment of progenitor cell mobilization as applied to both human and murine HSPC.