Retention Of Hematopoietic Stem/progenitor Cells Research Articles

Novel Evidence That Hematopoietic-Specific PLC-β2 Is Required for Normal Homing and Engraftment of Hematopoietic Stem Cells

Background. We recently identified phospholipase C-β2 (PLC-β2) as the first lipolytic enzyme known to be involved in the mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB) (Leukemia 2016, 4:919-28). The phospholipase C (PLC) family of enzymes consists of 13 members in six subfamilies, including the PLC-δ (1, 3, 4), -β (1-4), -γ (1, 2), -ε, -ζ, and -η isoforms. PLC enzymes are associated with cell surface receptors that convert phosphatidyloinositol-4,5-biphosphate into two important second messengers, diacylglycerol (DAG) and inositol-1,4.5-triphosphate (IP3). Among these isoforms, PLC-β2 is somewhat unique in being a hematopoietic-specific enzyme. Moreover, PLC-β2, when released extracellularly from granulocytes and HSPCs upon stimulation, cleaves glycolipid glycosylphosphatidylinositol anchor (GPI-A) in cell membranes and thus disrupts the structure of membrane lipid rafts, which are important in the retention of HSPCs in BM niches. It is well known that the major BM-retention receptors for HSPCs, CXCR4 and VLA-4, are membrane lipid raft-associated receptors. What is important for the current study, while performing mobilization studies, we previously found that BM cells from these animals show somewhat reduced chemotaxis in response to several chemoattractants involved in BM homing.Aim of the Study. Based on this latter observation, we became interested in the potential role of PLC-β2 in regulating the migration of HSPCs, as this enzyme could be potentially involved in BM homing and engraftment of HSPCs after transplantation. We also explored the potential relationship between PLC-β2 and heme oxygenase 1 (HO-1), which is, as we recently demonstrated, a negative regulator of HSPC trafficking (Cell Transplant. 2016 epub).Materials and Methods. We performed i) homing, ii) short-term engraftment, and iii) long-term competitive engraftment studies in WT mice transplanted with BM cells derived from PLC-β2-/- mice and control (WT) animals. Chemotaxis and adhesion of PLC-β2-/- HSPCs was evaluated in Transwell dishes in response to SDF-1, S1P, C1P, and ATP. The expression of HO-1 was evaluated by real-time PCR and western blotting. Finally, both HSPCs and Gr-1+ cells were evaluated for release of Ca2+ upon stimulation by homing factors.Results. We report here for first time that, besides its role in the release of HSPCs from BM niches, PLC-β2 also regulates the migration of HSPCs in response to chemotactic gradients of BM homing factors, including stromal derived factor-1 (SDF-1), sphingosine-1 phosphate (S1P), ceramide-1 phosphate C1P, and adenosine triphosphate (ATP). Specifically, HSPCs from PLC-β2-KO mice show impaired homing and engraftment in vivo after transplantation into lethally irradiated mice. This decrease in migration of HSPCs can be explained by impaired calcium release and calcium signaling in cells from PLC-β2-KO mice and a high baseline level of HO-1, an enzyme that negatively regulates cell migration, in PLC-β2-KO mouse cells. Downregulation of HO-1 activity in HSPCs improved homing of PLC-β2-/- BM cells.Conclusions. PLC-β2 plays an important role in homing and engraftment of HSPCs. The decrease in migration of HSPCs in PLC-β2-KO mice can be explained by impaired calcium release and a high baseline level of HO-1, an enzyme that negatively regulates cell migration and our data provides evidence that downregulation of HO-1 activity in HSPCs by small molecular inhibitors could become a new strategy to improve homing and engraftment after transplantation. DisclosuresNo relevant conflicts of interest to declare.

Novel Evidence That a Lipolytic Enzyme - Hematopoietic-Specific Phospholipase C Beta 2 - Promotes Mobilization of Hematopoietic Stem Cells By Decreasing Their Lipid Raft-Mediated Bone Marrow Retention and Increasing the Pro-Mobilizing Effects of Granulocytes

Background. Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4β1 integrin VLA-4 receptor, whose specific ligands, stromal-derived factor 1 (SDF-1) and vascular cell adhesion molecule 1 (VCAM-1), respectively, are expressed in BM stem cell niches. The integrity of the membrane lipid rafts containing these BM-retention receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, plays an important role in mobilization of HSPCs into peripheral blood (PB) by i) inducing degranulation of BM-residing granulocytes and ii) promoting their egress from BM into blood so that they permeabilize the endothelial barrier for egress of HSPCs (Leukemia 2010, 24, 976-985). It is known that a hematopoietic-specific lipolytic enzyme, phospholipase C-β2 (PLC-β2), is an intracellular enzyme involved in signaling through the C5a receptor and the chemotactic response of leucocytes to a C5a gradient. On the other hand, if released from the cells, PLC-β2 affects GPI-A by enzymatic digestion and disrupts lipid raft integrity and may affect cell-surface expression of proteins that contain GPI-A, including a truncated form of VCAM-1, the complement cascade inhibitors CD55 and CD59, and uPAR.Hypothesis.Based on the foregoing, we asked whether, in addition to proteolytic enzymes, the lipolytic enzyme PLC-β2 is released from granulocytes during mobilization andplays a role in pharmacological mobilization by disintegrating lipid raft-mediated CXCR4 and VLA-4-dependent BM retention mechanisms and by enhancing the C5a-mediated pro-mobilizing effects of granulocytes.Materials and Methods. To address this novel concept, we evaluated the level of PLC-β2 in BM during mobilization, the effect of this enzyme on adhesion and migration of HSPCs, as well as the status of lipid raft integrity on HSPCs and performed G-CSF- and AMD 3100-induced mobilization studies in PLC-β2-KO and WT animals.Results. We found that PLC-β2, as an intracellular enzyme in granulocytes, mediates the pro-mobilizing responses of these cells in a C5a receptor-dependent manner, specifically, by increasing degranulation of granulocytes and enhancing their C5a-dependent egress from BM into PB. PLC-β2 is also released from granulocytes during their degranulation, and thus the level of this enzyme increases in the BM microenvironment. After its release from granulocytes, PLC-β2 digests GPI-A and thus disintegrates membrane lipid rafts and impairs CXCR4-SDF-1- and VLA-VCAM-1-mediated retention of HSPCs in BM niches. It is also responsible for digestion of a shorter truncated isoform of VCAM-1 in stem cell niches and generation of soluble uPAR. In support of this dual intracellular and extracellular role of PLC-β2, we found that PLC-β2-KO mice are poor mobilizers. These novel and pleotropic effects of this lipolytic enzyme are shown in Figure 1.Conclusions. PLC-β2 is the first lipolytic enzyme identified so far that plays a crucial role in pharmacological mobilization of HSPCs, and modification of its activity may lead to better mobilization strategies, which is currently being tested in our laboratories. Moreover, we propose that, in addition to PLC-β2, other lipolytic enzymes are involved in the mobilization process, and this is also currently being investigated in our laboratories. DisclosuresNo relevant conflicts of interest to declare.

Evidence that a lipolytic enzyme—hematopoietic-specific phospholipase C-β2—promotes mobilization of hematopoietic stem cells by decreasing their lipid raft-mediated bone marrow retention and increasing the promobilizing effects of granulocytes

Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4β1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-β2 (PLC-β2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-β2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs.

Novel Evidence That, in Addition to Proteolytic Enzymes, Lipolytic Enzymes Are Involved in Mobilization of Hematopoietic Stem/Progenitor Cells (HSPCs) - an Important Pro-Mobilizing Role Identified for Hematopoietic-Specific Phospholipase C (PLCβ2)

Background. Hematopoietic stem/progenitor cells (HSPCs) express the chemokine receptor CXCR4 and the very late antigen 4 receptor (VLA-4, also known as alpha4beta1 integrin) on their cell surface and are retained in bone marrow (BM) niches by interaction of these receptors with their respective ligands, α-chemokine stromal-derived growth factor 1 (SDF-1) and vascular adhesion molecule 1 (VCAM-1, also known as CD106), which are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). Mobilization studies employing small molecule antagonists of CXCR4 or VLA-4 indicate the importance of both axes in retention of HSPCs in the BM microenvironment. Furthermore, it has been postulated that a crucial role in the mobilization process plays activation of a proteolytic microenvironment (J Clin Invest. 2003;111:187-96) and complement cascade (ComC) (Blood 2004;103:2071-8) in the BM microenvironment. However, to our surprise no attention has been paid so far to the role of lipolytic enzymes. Phospholipase C (PLC) is an enzyme released by neutrophils that cleaves the phosphoglycerol bond in glycosylphospahtidylinositol (GPI anchor), a glycolipid that is attached to the C-termini of several important proteins during posttranslational modification. GPI-linked proteins are thought to be preferentially located in lipid rafts. The most important GPI-anchored proteins include VCAM-1, complement inhibitors CD55 and CD59, and uPAR. We have reported that for retention of HSPCs in BM, both CXCR4 and VLA-4 have to be incorporated into membrane lipid rafts (Blood 2005;105:40-48).Hypothesis. Based on our previous observation that the HSPCs of paroxysmal nocturnal hemoglobinuria patients show defective retention in BM due to lack of functional GPI anchor (Leukemia 2012; 26:1722—5) we have hypothesized that PLC in normal BM may facilitate mobilization of HSPCs by perturbing expression of GPI anchor, which is so crucial for proper retention of HSPCs in BM.Materials and Methods. PLC activity has been measured by ELISA in the BM of mobilized mice as well in conditioned media from neutrophils exposed to several pro-mobilizing factors (G-CSF, AMD3100, C3a, C5a, desArgC5a, and S1P). We also tested the effect of PLC on the expression of VCAM-1 on BM-derived stroma and CD55 and CD59 antigens on BM mononuclear cells. The effect of PLC on incorporation of CXCR4 and VLA-4 into membrane lipid rafts has been studied by confocal microscopy employing murine Sca-1+ and human CD34+ cells. Here, to test our hypothesis, mobilization studies using AMD3100 and G-CSF have been performed in PLCβ2-KO (PLCβ2–/–) mice and their wild type (WT) littermates.Results. Our data indicate that the PLC level increases in BM during mobilization and is released from neutrophils in response to several pro-mobilization factors (G-CSF, AMD3100, C3a, C5a, desArgC5a, and S1P). PLC efficiently cleaves VCAM-1 expressed on BM stromal cells and thus perturbs the VCAM-1–VLA4 interaction as well as removes CD55 and CD59 from BM mononuclear cells, which enhances the pro-mobilizing effects of the ComC. PLC also inhibits lipid raft formation on HSPCs and by this means impairs the normal BM-retention function of CXCR4 and VLA-4. Finally, what is most important, we observed a mobilization defect in PLCβ2–/– mice, as evaluated by the number of mobilized leucocytes, SKL cells, and CFU-GM.Conclusions. We have established for the first time that, in addition to proteolytic enzymes, lipolytic enzymes, including LPC, are upregulated in the BM microenvironment, and that PLC promotes mobilization of HSPCs by perturbing the BM-retention function of GPI-anchored proteins. These data support an important role for GPI anchor-dependent proteins in the retention of HSPCs in BM niches. DisclosuresNo relevant conflicts of interest to declare.

Identification of heme oxygenase 1 (HO-1) as a novel negative regulator of mobilization of hematopoietic stem/progenitor cells.

Activation of complement cascade (ComC) play and important role in mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB). While there are vast experimental data on the mechanisms and factors that induce or promote mobilization of HSPCs, there is relatively less data on negative regulators of this process. We demonstrate for the first time that heme oxygenase-1 (HO-1) that has a well-documented anti-inflammatory potential plays an important and heretofore unrecognized role in retention of HSPCs in BM niches by i) modulating negatively activation of mobilization promoting ComC, ii) maintaining stromal derived factor-1 (SDF-1) level in the BM microenvironment and iii) attenuating chemotactic responsiveness of HSPCs to SDF-1 and sphingosine-1 phosphate (S1P) gradients in PB. Furthermore, our data showing a positive mobilizing effect by a non-toxic small-molecule inhibitor of HO-1 (SnPP) suggest that blockade of HO-1 would be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate better the molecular mechanisms responsible for the potential effect of HO-1 in homing of HSPCs after transplantation.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-014-9547-7) contains supplementary material, which is available to authorized users.

Human CD8+TCR- Facilitating Cells (FC) Upregulate Priming Factors That Enhance Proliferation of HSPC and Increase Responsiveness of HSPC to an SDF-1 Homing Gradient.

CD8+TCR- graft facilitating cells (FC) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPC) in syngeneic and allogeneic recipients. We report for the first time the phenotypic and functional characterization of the predominant human CD8+TCR- FC subpopulations. Half of FC are CD56dim/- and half are CD56bright. The majority of CD56dim/- FC coexpress CD3ε+, while CD56bright FC coexpress CD11c+ and CD11b+. Both FC subpopulations significantly promote clonogenicity of HSPC in vitro and enhance their homing to BM after transplantation into NSG mice. Recipients of HSPC combined with CD56dim/- or CD56bright FC showed durable donor chimerism in BM. To test if co-culture of the FC subpopulations with HSPC induces growth factors that promote expansion of HSPC, both CD56dim/- and CD56bright FC were sorted from mobilized PBMC and each subpopulation co-cultured with HSPC for 18 hours. Real time quantitative PCR showed significant upregulation of transcripts for stem cell factor (SCF), fibroblast growth factor (FGF)-2, and FLT-3 ligand (FL) by both FC subpopulations. Upregulation was more pronounced in the CD56bright FC cocultures (3000 fold for FL). To address the effect of FC on homing and engraftment in vivo, we examined the effect of FC on the SDF-1 and CXCR4 axis that plays an important role in homing and retention of HSPC in the hematopoietic niche. Several small innate immunity-related peptides such cathelicidin (LL-37) or β2-defensin (BD-2) enhance responsiveness of HSPC to low SDF-1 gradients. To test if FC subpopulations cultured with HSPC express LL-37 and BD-2, CD56dim/- FC and CD56bright FC were sorted from mobilized PBMC and cultured for 18 hours with HSPC. There was a significant increase in mRNA expression for LL-37 and BD-2 in the group of CD56bright FC cocultured with HSPC. In conclusion, we report here for the first time that FC upregulate several factors that promote expansion of HSPC (SCF, FGF-2, FL) and enhance their homing responsiveness to SDF-1 gradients (LL-37, BD-2) after contact with HSPC. Taken together these data suggest that FC exert an effect on HSPC to promote homing and migration of HSPC. DISCLOSURES:Bozulic, L.: Employee, Regenerex, LLC, a start-up biotech company. Ildstad, S.: Other, Regenerex, LLC, a start-up biotech company, CEO.

Novel Evidence That Sphingosine-1-Phosphate-Mediated Mobilization Of Hematopoietic Stem/Progenitor Cells (HSPCs) During Intravascular Hemolysis Requires Attenuation Of The SDF-1–CXCR4 Retention Axis Of HSPCs In Bone Marrow Niches – Implications For Paroxysmal Nocturnal Hemoglobinuria-Induced Mobilization of HSPCs

BackgroundWe have recently reported that hematopoietic stem/progenitor cells (HSPCs) that harbor mutations of the PIG-A gene are preferentially mobilized into peripheral blood (PB) during hemolytic events in paroxysmal nocturnal hemoglobinuria (PNH) patients (Leukemia 2012;26:1722). This effect has been explained by i) an increase in the plasma level of sphingosine-1-phosphate (S1P), which at physiological doses is a major chemoattractant for HSPCs and is released from lysed erythrocytes (Leukemia 2010;24:976), and ii) the fact that PNH-cloned HSPCs, in contrast to normal HSPCs, show defective SDF-1–CXCR4-mediated retention in BM niches. It is known that under steady-state conditions the concentration of S1P in PB is already 25x higher than its concentration in the BM microenvironment and increases additionally during hemolysis. Aim of the studySince erythrocytes are a major source of plasma S1P, we asked whether massive hemolysis of erythrocytes leading to an additional increase in plasma S1P level would trigger mobilization of HSPCs. Furthermore, to shed more mechanistic light on the mobilization of HSPCs in PNH patients and to distinguish the effect of an increase in the S1P chemotactic gradient in PB plasma from the effect of defective retention of HSPCs in the BM microenvironment, we performed mobilization studies in mice exposed to the hemolysis-inducing agent phenhlhydrazine (PHZ) ± blockade of the BM-retaining SDF-1–CXCR4 axis by AMD3100. Experimental approachNormal C57Bl6 mice were injected with i) PHZ to induce hemolysis and S1P release from erythrocytes, ii) AMD3100 to perturb the SDF-1–CXCR4-mediated retention of HSPCs in BM niches, or iii) both PHZ and AMD3100. Subsequently, we evaluated the number of circulating Sca-1+Kit+Lin– (SKL) HSPCs as well as the number of clonogenic CFU-GM progenitors in PB. In parallel, we evaluated the S1P blood plasma levels by liquid chromatography electrospray ionization tandem mass spectrometry (HPLC ESI MS/MS) and the SDF-1 level by ELISA. In addition, we measured complement cascade (CC) activation by measuring the C5b-C9 (membrane attack complex, MAC) levels. ResultsWe found that hemolysis doubles the PB plasma level of S1P (from 1 to 2 mM). To assess the effect of plasma S1P versus plasma SDF-1 as chemoattractants mediating egress of HSPCs from BM, we employed plasma derived from control and PHZ-treated mice, W146, a receptor-specific inhibitor for the S1P receptor type 1 (S1P1), and the CXCR4 antagonist AMD3100 in Transwell migration assays. We observed that chemotaxis of BM-purified HSPCs was inhibited by blocking the S1P–S1P1 but not the SDF-1–CXCR4 axes, which demonstrates that the S1P level in plasma is a crucial chemoattractant for HSPCs present under normal steady-state conditions and in PHZ-treated mouse plasma. This observation also clearly shows that the S1P gradient, even under steady-state conditions, is already high enough to promote egress of HSPCs from BM into PB and supports our previous observations that, while the SDF-1–CXCR4 axis plays an important role in retention of HSPCs in BM niches, the SDF-1 plasma level is too low to induce egress of HSPCs (Leukemia 2010;24:976). In our in vivo mobilization studies, we observed that, in contrast to AMD3100 administration, PHZ-induced hemolysis alone had a negligible effect on mobilization of HSPCs, with a peak at 6 h after infusion of this hemolysis-inducing agent (Figure 1). However, when we combined PHZ with AMD3100, mice mobilized twice as many HSPCs as with administration of AMD3100 alone. The degree of mobilization of HSPCs correlated with the free Hb level in plasma and with activation of the complement cascade. ConclusionsAt the steady-state conditions S1P level in PB is already a strong chemotactic factros for BM-residing HSPCs. More importantly, to explain the differential mobilization of PNH-affected HSPCs versus normal HSPCs (Leukemia 2012;26:1722), we show here for the first time that hemolysis alone, even if it doubles the S1P level in PB, requires attenuation of CXCR4–SDF-1-mediated retention in BM niches. Thus, PNH-affected HSPCs, due to defective lipid raft formation, have impaired CXCR4-mediated retention in BM niches and are preferentially mobilized into PB. Finally, our data explain why, compared with PNH, HSPCs are mobilized to a much lesser degree in other hemolytic syndromes (e.g., sickle cell anemia).▪ Disclosures:No relevant conflicts of interest to declare.

Further Evidence That HO-1 Regulates SDF-1 Expression in the Bone Marrow Microenvironment and That HO-1-Deficient Mice Show a Defect in the SDF-1–CXCR4 Retention Axis of Hematopoietic Stem/Progenitor Cells in Bone Marrow and Thus Are Easy Mobilizers - Studies in HO-1 Mutant Mice, Irradiation Chimeras, and the Effect of in Vivo Pharmacological Inhibition of HO-1

Abstract 344 Background:Stromal derived factor-1 (SDF-1), which binds to the CXCR4 receptor expressed on the surface of hematopoietic stem/progenitor cells (HSPCs), plays an important role in the retention of HSPCs in BM niches. Heme oxygenase (HO-1) is a stress-responsive enzyme that catalyzes the degradation of heme and plays an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury, atherosclerosis, and cancer. Interestingly, it has also been reported that HO-1 regulates the expression of SDF-1 in myocardium (J Mol Cell Cardiol. 2008;45:44–55). Aim of study:Since SDF-1 plays a crucial role in retention and survival of HSPCs in BM, we become interested in whether HO-1 is expressed by BM stromal cells and whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach:To address this issue, we employed several complementary strategies to investigate HO-1–/–, HO-1+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) the chemotactic responsiveness of HSPCs to an SDF-1 gradient as well as to other chemoattractants, including sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and extracellular nucleotiodes (ATP, UTP), iv) the adhesiveness of clonogenic progenitors to immobilized SDF-1 and stroma, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100, assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. We also exposed mice to the small HO-1 molecular inhibitor tin protoporphyrin IX (SnPP) and studied the effect of this treatment on G-CSF- or AMD3100-induced mobilization of HSPCs. Finally, to prove an environmental HSPC retention defect in HO-1-deficient mice, we created radiation chimeras, wild type mice transplanted with HO-1-deficient BM cells, and, vice versa, HO-1-deficient mice reconstituted with wild type BM cells. Results:Our data indicate that under normal, steady-state conditions, HO-1–/– and HO+/– mice have normal PB cell counts and numbers of circulating CFU-GM, while a lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. However, while BMMNCs from HO-1–/– have normal expression of the SDF-1-binding receptor, CXCR4, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1–/– animals respond more robustly to an SDF-1 gradient, and HO-1–/– animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into PB in response to G-CSF and AMD3100. Both G-CSF and AMD3100 mobilization were also significantly enhanced in normal wild type mice after in vivo administration of HO-1 inhibitor. Finally, mobilization studies in irradiation chimeras confirmed the crucial role of the microenvironmental SDF-1-based retention mechanism of HSPCs in BM niches. Conclusions:Our data demonstrate for the first time that HO-1 plays an important and underappreciated role in modulating the SDF-1 level in the BM microenvironment and thus plays a role in retention of HSPCs in BM niches. Furthermore, our recent data showing a mobilization effect by a small non-toxic molecular inhibitor of HO-1 (SnPP), suggest that blockage of HO-1 could be a promising strategy to facilitate mobilization of HSPCs. Further studies are also needed to evaluate the role of HO-1 in homing of HSPCs after transplantation to BM stem cell niches. Disclosures:No relevant conflicts of interest to declare.

Novel Evidence That Hematopoietic Stem/Progenitor Cells (HSPCs) Are Mobilized During Hemolysis in an Erythrocyte Lysis-Derived, Sphingosine-1-Phosphate (S1P)-Dependent manner—the Crucial Involvement of Complement Cascade (CC) Activation and Attenuation of CXCR4 Retention Signaling

Abstract 3189 Background.Hemolytic syndromes, such as sickle cell anemia and paroxysmal nocturnal hemoglobinuria, are characterized by an increased number of hematopoietic stem/progenitor cells (HSPCs) circulating in peripheral blood (PB). However, the molecular mechanisms responsible for this effect are unclear. In our previous work we have demonstrated that sphingosine-1-phosphate (S1P) released from lysed erythrocytes and activated platelets is a strong chemottractant for bone marrow (BM)-residing HSPCs (Leukemia 2010;24:976–85). Hypothesis.We hypothesized that S1P released from lysed erythrocytes is a major factor responsible for egress of HSPCs from BM into PB in hemolytic syndromes. Experimental approach.To test this hypothesis, normal mice were injected with phenylhydrazine (PHZ), a compound known to induce hemolysis, and we evaluated the number of Sca-1+Kit+Lin– (SKL) HSPCs circulating in PB as well as the number of clonogenic CFU-GM progenitors mobilized into PB. In parallel, we evaluated the blood plasma levels of S1P and stromal derived factor-1 (SDF-1) by sensitive ELISA, the free hemoglobin (Hb) level, as well as complement cascade (CC) activation by measuring the C5b-C9 (membrane attack complex, MAC) level. In some of the experiments, we combined PZH treatment with injection of the CXCR4 antagonist AMD3100. To better assess the role of CC activation, we also performed mobilization in C5-deficient mice, which do not activate the distal part of the CC and thus do not generate C5b-C9/MAC. Results.We found that hemolysis increases the PB plasma level of S1P but does not affect the SDF-1 level. Furthermore, while PHZ-induced hemolysis mobilizes HSPCs into PB with a peak at 6 h after infusion, this mobilization effect is significantly potentiated by administration of AMD3100, which attenuates CXCR4–SDF-1-mediated retention of HSPCs in the BM microenvironment. Of note, PHZ-induced hemolysis together with AMD3100 mobilized twice as many HSPCs as AMD3100 alone. The degree of mobilization of HSPCs correlated with the free Hb level in plasma and activation of the CC (by an increase in MAC level), and, more importantly, mobilization was not seen in C5-deficient mice. Conclusions.We confirmed our previous observation that with the steady-state S1P level, the PB has already established a strong chemotactic gradient for BM-residing HSPCs (Leukemia 2010;24:976–85), which are actively retained in BM niches in a CXCR4–SDF-1-dependent manner. Hemolysis alone, even if it elevates the S1P level in PB significantly, requires two important events i) attenuation of CXCR4–SDF-1 axis-mediated retention in BM niches and ii) simultaneous activation of the CC, which is crucial for induction of permeabilization of the BM–PB barrier. Furthermore, our data also support the notion that the S1P but not the SDF-1 level in PB establishes the critical chemotactic gradient for HSPCs and is responsible for egress of these cells. Based on the observation that C5-deficient mice are poor mobilizers, inactivation of the distal part of the CC should be considered as a therapeutic approach, not only in paroxysmal nocturnal hemoglobinuria but also in other hemolytic syndromes. Disclosures:No relevant conflicts of interest to declare.

Abstract 239: Stem Cell Therapy Improves Critical Limb Ischemia

Introduction: The journey from single cell to complex being is attributable to stem cells role. Adult stem cells originate during ontogeny & persist in specialized niches within organs. Asymmetric division of each stem cell during differentiation produces : one daughter stem cell & one daughter transit amplifying/intermediate cell having migratory properties. Forced migration of hematopoietic stem/progenitor cells (HSPC) from bone marrow into peripheral blood is called mobilization. Accumulating evidence suggests that attenuation of the chemokine stromal derived factor-1(SDF-1)-CXCR4 axis that plays a pivotal role in retention of HSPC in bone marrow (BM) results in the release of these cells from the BM into peripheral blood. Recently, adult cells have been genetically reprogrammed to an embryonic stem cell like state. Induced pluripotent stem cells (IPSCs) were similar to human embryonic stem cells in morphology, proliferative capacity, expression of cell surface antigens, & gene expression. Treatment of ischemic vascular disease of lower limbs remains a significant challenge. Unfortunately, if medical & surgical salvage procedures fail, amputation is an unavoidable result for those patients. Aim of Work: (Hypothesis) To assess the application of implantation of autologous stem/progenitor cell in the treatment of chronic limb ischemia & to evaluate the safety, efficacy & feasibility of this novel therapeutic approach. Methods: A total of 24 patients with chronic limb ischemia not eligible for arterial reconstruction or endovascular procedures were enrolled & randomized (1:1) to either the implanted group or the control group. Control group: Conventional medical therapy in the form of anti platelet therapy & vasodilators. Implanted group: Subcutaneous injection of 300μ g/day of recombinant human granulocyte colony stimulating factor (G-CSF) for 5 days to mobilize stem/progenitor cells from BM. Total leucocytic count is measured daily to follow up successful mobilization of bone marrow mononuclear cells (BMMNCs). Stem cell Harvesting After 5 days peripheral blood mononuclear cells (PBMNCs) were harvested using a cell separator. Samples from apheresis products are subjected to TLC measurement & immunophenotypic characterization of CD34+ cells by flow cytometry. The collected PBMNCs were implanted by multiple intramuscular injections into ischemic limbs. Results: There was significant increase in pain free walking distance & ankle/brachial index (ABI) & significant decreased rest pain. Effectiveness was documented by : reduced number of amputation, increase ABI & improvement of the quality of life in therapeutic group compared to control group. Conclusion: The novel therapeutic approach of PBMNCs implantation in patients with chronic limb ischemia is safe, feasible & effective in decreasing co-morbidity & rate of amputation. Safety was manifested by absence of complications during G-CSF therapy or during harvesting & injection of the stem cells. Recommendations: 1- Future studies on larger number of patients & longer follow up. 2- Controlled studies using different methods & different cell population (PBMNCs, BMMNCs or MSCs) to compare the outcome of each. 3-Studing the role of endothelial progenitor cell dysfunction in different ischemic diseases to develop successful gene therapy.

A Novel Observation That Heme Oxygenase-1 (HO-1) Deficient Mice Are Easy Mobilizers and That HO-1 Plays An Important Role in Maintaining Expression of SDF-1 in Bone Marrow (BM) Stroma and Promotes Retention of Hematopoietic Stem/Progenitor Cells (HSPCs) in the Bone Marrow Microenvironment

Abstract 315Background. Heme oxygenase (HO) is an enzyme that catalyzes the degradation of heme. Two distinct HO isoforms have been identified: HO-2, which is constitutively expressed, and HO-1, which is stress-responsive and plays an important function in various physiological and pathophysiological states associated with cellular stress. HO-1 plays a role in ischemic/reperfusion injury, atherosclerosis, and cancer. It has also been reported that HO-1 regulates expression of a-chemokine stromal derived factor-1 (SDF-1) in myocardium (J Mol Cell Cardiol.2008;45:44–55). Aim of study. Since SDF-1 plays a crucial role in retention and survival of hematopoietic stem cell/progenitor cells (HSPCs) in BM, we become interested in whether deficiency of HO-1 affects normal hematopoiesis and retention of HSPCs in BM. Experimental approach. To address this issue, we employed several complementary strategies to investigate HO-1−/−, HO+/–, and wild type (wt) mouse littermates for i) the expression level of SDF-1 in BM, ii) the number of clonogenic progenitors from major hematopoietic lineages in BM, iii) peripheral blood (PB) cell counts, iv) chemotactic responsiveness of HSPCs to an SDF-1 gradient, iv) adhesiveness of clonogenic progenitors, v) the number of circulating HSPCs in PB, and vi) the degree of mobilization in response to granulocyte-colony stimulating factor (G-CSF) or AMD3100 assessed by enumerating the number of CD34–SKL cells and clonogeneic progenitors (CFU-GM) circulating in PB. Results: Our data indicate that under normal, steady-state conditions, HO-1−/− and HO+/– mice have normal peripheral blood cell counts and numbers of circulating CFU-GM. Interestingly, lack of HO-1 leads to an increase in the number of erythroid (BFU-E) and megakaryocytic (CFU-GM) progenitors in BM. Next, BMMNCs from HO-1−/−have normal expression of the SDF-1-binding receptor, CXCR4, but a 5-times lower level of CXCR7, which is another SDF-1-binding receptor. Of note, we observed that the mRNA level for SDF-1 in BM-derived fibroblasts was ∼4 times lower. This corresponded with the observation in vitro that HSPCs from HO-1−/−animals responded more robustly to an SDF-1 gradient, and HO-1−/−animals mobilized a higher number of CD34–SKL cells and CFU-GM progenitors into peripheral blood in response to G-CSF and AMD3100. Conclusions: Our data demonstrate for the first time that heme oxygenase plays an important and underappreciated role in BM retention of HSPCs and may affect their trafficking. Since small non-toxic molecular inhibitors of HO-1 have been developed for clinical use (e.g., metaloporhirins), blockage of HO-1 could be a novel strategy for mobilizing HSPCs. Our recent in vivo mobilization studies lend support to this hypothesis. Disclosures:No relevant conflicts of interest to declare.

A novel perspective on stem cell homing and mobilization: review on bioactive lipids as potent chemoattractants and cationic peptides as underappreciated modulators of responsiveness to SDF-1 gradients

Hematopoietic stem progenitor cells (HSPCs) respond robustly to α-chemokine stromal-derived factor-1 (SDF-1) gradients, and blockage of CXCR4, a seven-transmembrane-spanning G(αI)-protein-coupled SDF-1 receptor, mobilizes HSPCs into peripheral blood. Although the SDF-1-CXCR4 axis has an unquestionably important role in the retention of HSPCs in bone marrow (BM), new evidence shows that, in addition to SDF-1, the migration of HSPCs is directed by gradients of the bioactive lipids sphingosine-1 phosphate and ceramide-1 phosphate. Furthermore, the SDF-1 gradient may be positively primed/modulated by cationic peptides (C3a anaphylatoxin and cathelicidin) and, as previously demonstrated, HSPCs respond robustly even to very low SDF-1 gradients in the presence of priming factors. In this review, we discuss the role of bioactive lipids in stem cell trafficking and the consequences of HSPC priming by cationic peptides. Together, these phenomena support a picture in which the SDF-1-CXCR4 axis modulates homing, BM retention and mobilization of HSPCs in a more complex way than previously envisioned.

Mobilization of hematopoietic stem and progenitor cells using inhibitors of CXCR4 and VLA-4.

Successful hematopoietic stem cell transplant requires the infusion of a sufficient number of hematopoietic stem/progenitor cells (HSPCs) that are capable of homing to the bone marrow cavity and regenerating durable trilineage hematopoiesis in a timely manner. Stem cells harvested from peripheral blood are the most commonly used graft source in HSCT. Although granulocyte colony-stimulating factor (G-CSF) is the most frequently used agent for stem cell mobilization, the use of G-CSF alone results in suboptimal stem cell yields in a significant proportion of patients. Both the chemokine receptor CXCR4 and the integrin α(4)β(1) (very late antigen 4 (VLA-4)) have important roles in the homing and retention of HSPCs within the bone marrow microenvironment. Preclinical and/or clinical studies have shown that targeted disruption of the interaction of CXCR4 or VLA-4 with their ligands results in the rapid and reversible mobilization of hematopoietic stem cells into the peripheral circulation and is synergistic when combined with G-CSF. In this review, we discuss the development of small-molecule CXCR4 and VLA-4 inhibitors and how they may improve the utility and convenience of peripheral blood stem cell transplantation.

Evidence That a Bioactive Lipid, Ceramide-1 Phosphate (C1P), Is Upregulated In Bone Marrow Microenvironment After Myeloablative Therapy and Is a Potential Novel Homing Factor for Hematopoietic Stem Cells

AbstractAbstract 401Stromal derived factor-1 (SDF-1), when employed at supra-physiological concentrations, is a potent in vitro chemoattractant for hematopoietic stem progenitor cells (HSPCs). However, because this chemokine is extremely sensitive to degradation by proteolytic enzymes (e.g., MMP-2) and as we have observed myeloablative conditioning for hematopoietic transplantation induces a highly proteolytic microenvironment in bone marrow (BM), SDF-1 secreted by stromal cells and osteoblasts must be rapidly degraded under these conditions. While a role for the SDF-1–CXCR4 axis in retention of HSPCs in BM is undisputed, the role of SDF-1 in the homing of HSPCs in a highly proteolytic microenvironment is somewhat less certain and some redundant homing mechanisms may exist. This latter notion is supported by several observations, such as that i) CXCR4-/- fetal liver HSPCs may home to BM in an SDF-1- independent manner, ii) homing of murine HSPCs made refractory to SDF-1 by incubation and co-injection with a CXCR4 receptor antagonist is normal or only mildly reduced, and iii) HSPCs in which CXCR4 has been knocked down by means of an SDF-1 intrakine strategy are able to engraft even in lethally irradiated recipients. To reappraise the role of SDF-1 and other new potential factors in homing of HSPCs, we employed several complementary strategies. First we measured expression of SDF-1 mRNA in BM at 24 and 48 hours after lethal irradiation and observed a ∼3-fold increase. By contrast, the SDF-1 protein level in BM, evaluated by ELISA, surprisingly decreased as compared to non-irradiated mice. Next, we found that after blocking SDF-1 with AMD3100 treatment, conditioned media (CM) from irradiated BM cells still chemoattracted HSPCs. This SDF-1- independent chemotactic activity was resistant to heat inactivation, but was eliminated after stripping by activated charcoal, suggesting the possible involvement of bioactive lipids. Therefore, we began a search for unknown chemoattractants that could direct trafficking of HSPCs, with bioactive lipids as strong candidates, because, as small molecules, they are resistant to proteases. We focused especially on ceramide-1 phosphate (C1P) and sphingosine-1 phosphate (S1P), which are products of membrane-lipids metabolism. It is known that C1P, in contrast to S1P, is retained intracellularly and can be released mostly from damaged cells. Mass spectrometry (MS) analysis revealed that the major isoforms of C1P were detected at higher concentration in supernatant from irradiated BM when compared to supernatant from non-irradiated BM, which suggests that this bioactive lipid and chemoattractant is released from “leaky” BM cells damaged by myeloablative irradiation. We report here for the first time that C1P i) is a strong chemoatttractant for murine and human HSPCs, ii) activates phosphorylation of MAPKp42/44 and AKT in these cells, iii) induces expression of matrix metallopeptidases (MMPs), and iv) modulates adhesion of HSPCs to stroma and endothelium. Furthermore, in direct clonogenic studies, we did not observe any toxic effect of C1P on proliferation of murine and human clonogenic progenitors. We therefore propose a novel paradigm in which C1P is a chemoattractant for HSPCs that, in contrast to SDF-1, is highly resistant to proteolysis. In the proteolytic microenvironment induced in BM after myeloablative radio/chemotherapy, it could play along with SDF-1 an important and, until now, unrecognized role in the homing of HSPCs after transplantation. Furthermore, C1P secreted by damaged cells in other organs (e.g., infarcted myocardium) may in these highly proteolytic or necrotic microenvironments play a similar role in the homing of circulating stem cells involved in regeneration. Disclosures:No relevant conflicts of interest to declare.

Fifth complement cascade protein (C5) cleavage fragments disrupt the SDF-1/CXCR4 axis: Further evidence that innate immunity orchestrates the mobilization of hematopoietic stem/progenitor cells

Having previously demonstrated that the complement system modulates mobilization of hematopoietic stem/progenitor cells (HSPC) in mice, we investigated the involvement of C5 cleavage fragments (C5a/(desArg)C5a) in human HSPC mobilization. C5 cleavage fragments in the plasma were evaluated by enzyme-linked immunosorbent assay using human anti-(desArg)C5a antibody, and expression of the C5a/(desArg)C5a receptor (CD88) in hematopoietic cells by flow cytometry. We also examined the chemotactic responses of hematopoietic cells to C5 cleavage fragments and expression of stromal cell-derived factor-1 (SDF-1)-degrading proteases that perturb retention of HSPC in bone marrow, namely matrix metalloproteinase (MMP)-9, membrane type (MT) 1-MMP, and carboxypeptidase M. We found that plasma levels of (desArg)C5a are significantly higher in patients who are good mobilizers and correlate with CD34(+) cell and white blood cell counts in mobilized peripheral blood. C5 cleavage fragments did not chemoattract myeloid progenitors (colony-forming unit granulocyte-macrophage), but (desArg)C5a did strongly chemoattract mature nucleated cells. Consistently, CD88 was not detected on CD34(+) cells, but appeared on more mature myeloid precursors, monocytes, and granulocytes. Moreover, granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells and polymorphonuclear cells had a significantly higher percentage of cells expressing CD88 than nonmobilized peripheral blood. Furthermore, C5a stimulation of granulocytes and monocytes decreased CXCR4 expression and chemotaxis toward an SDF-1 gradient and increased secretion of MMP-9 and expression of MT1-MMP and carboxypeptidase M. C5 cleavage fragments not only induce a highly proteolytic microenvironment in human bone marrow, which perturbs retention through the CXCR4/SDF-1 axis, but also strongly chemoattracts granulocytes, promoting their egress into mobilized peripheral blood, which is crucial for subsequent mobilization of HSPC.

C-Cbl Regulates Interaction of Immature Hematopoietic Cells with the Bone Marrow Microenvironment by Rac GTPase-Mediated Cytoskeletal Signals.

Abstract 3643Poster Board III-579<Background and purpose>c-Cbl is a ubiquitin E3 ligase and functions as a negative regulator for signals induced by various activated tyrosine kinases, by promoting ubiquitination and proteasomal degradation of these kinases. This inhibitory action is mainly mediated by the tyrosine kinase binding (TKB) and RING finger domains located in the NH3-terminal part of the molecule and these domains are evolutionally well-conserved from nematodes. But mammalian c-Cbl has a stretched structure toward the COOH-terminal end with putative tyrosine residues that can interact with p85 subunit of PI3-kinase, and several reports have denoted that this interaction may be important for cytoskeletal regulation. Recently, mutations of the c-cbl gene have been reported in hematopoietic malignancies, and regulation of hematopoietic stem/progenitor cells (HSPCs) by Cbl is attracting attention. Since it is well-known that cytoskeletal dysregulation is often associated with malignant transformation, in this study, we investigated cytoskeletal regulatory mechanisms mediated by Cbl in hematopoietic cells, using Cbl deficient mice. <Methods and results>To examine the migratory capacity of the HSPCs, lineage negative (Lin(-)) bone marrow cells were set in the Boyden Chamber assay for SDF-1 and fibronectin (FN) were performed. We found that Cbl deficient Lin(-) cells showed significantly decreased migration to these chemoattractants; the migration capacity of the Cbl deficient cells was one eighth for SDF-1 (p = 0.01) and one third for FN (p = 0.007), respectively, compared with the wild-type counterparts. Then, to evaluate in vivo homing ability to the bone marrow microenvironment, transplantation assays were performed. We transplanted 2.5 × 10e6 of Lin(-) HSPCs from Cbl deficient or wild-type Ly5.2 mice into sublethally irradiated wild-type Ly5.1 mice. Three hours after transplantation, chimerism of the transplanted Ly5.2 cells in the bone marrow was examined, and we found that the number of transplanted cells was significantly smaller in Cbl deficient cells, showing that homing capacity of HSPCs in Cbl deficient mice was impaired. Moreover, when we administered G-CSF to Cbl deficient mice, a significantly larger number of Lin(-) Sca1(+) c-Kit(+) cells were mobilized from the bone marrow (p = 0.01), indicating that in Cbl deficient mice, mobilization of HSPCs by G-CSF was also affected. In bone marrow cells or Lin(-) HPSCs of Cbl deficient mice, activity of Rac, a member of small G-protein GTPases, was significantly decreased (p = 0.002). These data ware supported by the experiments with embryonic fibroblast; Rac activities induced by FN stimulation were weaker in Cbl deficient fibroblasts than wild-type ones, and Cbl deficient fibroblasts showed impaired actin rearrangement. <Discussion>We found that Cbl deficient HSPCs showed impaired migration activities to chemoattractants and altered homing and mobilization to and from the bone marrow. Furthermore, Cbl deficient cells had impaired activation of Rac. Currently, little is known about the relationship between Cbl-mediated signals and cytoskeletal regulator Rac, and in this study, we found that Cbl is a positive regulator of Rac activity in the bone marrow hematopoietic cells. Rac activity is reported to be necessary for homing and retention of HSPCs in the bone marrow microenvironment, and our results indicate that interaction of HSPCs with the bone marrow microenvironment, e.g. trafficking of these cells, is regulated by Cbl via Rac GTPase signals. Disclosures:No relevant conflicts of interest to declare.

MicroRNA Mir-23a Regulates SDF-1alpha Expression in Human Bone Marrow-Derived Mesenchymal Stem Cells

MicroRNAs (miRNAs) can regulate hematopoietic stem/progenitor cells (HSPC) by modulation of intrinsic cell components such as transcription factors and receptors. In addition, miRNAs could play a role in the microenvironment were HSPC host and consequently affect HSPC via extrinsic factors, which to our knowledge is an hypothesis that has not been tested. Since the chemokine stromal derived factor 1alpha (SDF-1alpha) is essential for both homing and retention of HSPC in the bone marrow, we tested if miRNAs expressed by human bone marrow-derived mesenchymal stem cells (MSC; a key source of SDF-1alpha), could potentially inhibit SDF-1alpha expression. In deed, using luciferase reporter-systems we show specific binding of miR-23a to the 3′UTR of SDF-1alpha. Consequently, transfection of MSCs with a precursor miR-23a (pre-miR-23a) leads to a 30% reduction of SDF-1alpha at both mRNA and protein levels. In contrast, inhibition of endogenous miR-23a with anti-sense oligonucleotides (anti-miR-23a), leads to a significant increase of SDF-1alpha also at both mRNA (30%) and protein (10%) levels as compared to controls (scramble pre-/anti-miR). As a result, migration of CD34+ HSPC in transwell assays is strongly affected upon overexpression or inhibition of miR-23a in MSCs (with pre-miR-23a 35% less migration; with anti-miR-23a 20% more migration, as compared to respective controls). Interestingly, transforming growth factor beta 1 (TGF-beta1) inhibits SDF-1alpha expression in MSCs in a concentration dependent manner, while miR-23a is increased under same experimental settings. Even more, silencing endogenous miR-23a significantly reduces the effect of TGF-beta1 on SDF-1alpha mRNA and protein levels, suggesting that at least in part TGF-beta1 inhibits SDF-1alpha expression via increasing miR-23a levels. This is to our knowledge the first established connection between miRNA biology and HSPC-niche related factors.

C1q Complement Cascade Protein as a Novel Modulator of the SDF-1-CXCR4 Axis and Hematopoietic Stem/Progenitor Cell Trafficking.

The first component of the complement (C) system C1q has multiple immune functions including acting as a chemoattractant for mature blood cells, and it has recently been reported that hematopoietic stem/progenitor cells (HSPC) express the C1q receptor, C1qRp. We have reported that the complement components C3 and C5 play an important role in the mobilization of HSPC and that G-CSF mobilization activates C by the classical Ig-dependent pathway (Blood 2004; 103:2071; Stem Cells 2007, in press). While C3 plays an important role in the retention of HSPC in BM, C5 is pivotal for their egress into peripheral blood. The aim of this study was to examine a possible role for C1q in the mobilization/homing of HSPC. We found (using RT-PCR and Western blotting) that the C1q gene and protein are strongly expressed in human mobilized peripheral blood (mPB) CD34+ cells, but not in steady-state bone marrow (BM) CD34+ cells. Importantly, we found that G-CSF stimulation of BM cells induces the expression of C1q in CD34+ cells, as detected by flow cytometry. Moreover, we demonstrated the presence of the receptor for C1q, C1qRp, in BM, mPB and cord blood (CB) CD34+ cells as well as in myeloid, megakaryocytic and erythroid progenitors ex vivo-expanded from CD34+ cells. The stromal derived factor (SDF)-1-CXCR4 axis plays a critical role in HSPC homing to BM and we showed using confocal microscopy that pre-incubation (for 1 h) of CB CD34+ cells on C1q-coated slides increased incorporation of the SDF-1 receptor, CXCR4, into membrane lipid rafts while C1q-coated filters increased chemoattraction (compared to bare filters) to an SDF-1 gradient. Although C1q itself is not a chemoattractant for HSPC, it primed the chemotactic response of CD34+ cells to a low SDF-1 gradient (10 ng/mL) to the extent that it approximated response to a high SDF-1 gradient (200 ng/mL), as well as the chemoinvasion of CB CD34+ cells across reconstituted basement membrane (Matrigel) to a low gradient of SDF-1, which was inhibited by the monoclonal anti-C1q antibody. In addition, C1q stimulated matrix-degrading metalloproteinase-9 secretion by CB CD34+ cells. In conclusion, we demonstrated for the first time that G-CSF mobilization strongly upregulates C1q in human HSPC which express the functional C1q receptor and that the first component of the complement cascade is involved in HSPC trafficking. This supports our previous data that C as an important modulator of the SDF-1-CXCR4 axis plays a pivotal role in the mobilization of HSPC.

Role of stromal‐derived factor‐1 in the hematopoietic‐supporting activity of human mesenchymal stem cells

Mesenchymal stem cells (MSC) have the ability to support and maintain hematopoiesis in vitro. However, mechanisms implicated in this support are not fully characterized. In the present study, the role of stromal-derived factor-1 (SDF-1)/CXCR4 axis in the interactions between MSC and hematopoietic stem/progenitor cells (HSPC) was studied. Human bone marrow MSC were plated as feeder layers in Dexter-type long-term cultures (LTC) with human cord blood CD34(+) HSPC. Cultures were supplemented weekly with neutralizing antibodies against CXCR4 or SDF-1 for 5 wk. LTC-initiating cell (IC) activity was strongly dependent on the SDF-1/CXCR4 axis, as both antibodies significantly decreased secondary colony-forming cell production. To assess the effect of SDF-1/CXCR4 axis on progenitor cell proliferation, LTC-IC killing assays were carried out: in LTC of CD34(+) cells in contact with MSC, treatment with anti-CXCR4 antibody significantly reduced the number of cycling progenitors. These results indicate that the SDF-1/CXCR4 axis promotes HSPC proliferation in contact with MSC. Interestingly, when HSPC were separated from MSC by a semipermeable membrane, LTC-IC activity became CXCR4 independent. Multiplex analysis of MSC-conditioned medium revealed that in addition to SDF-1, MSC produced stimulatory and inhibitory factors, such as interleukin (IL)-6, IL-11, granulocyte macrophage-colony stimulating factor as well as monocyte-chemoattractant protein-1. Altogether, human MSC support hematopoiesis in Dexter-type cultures through the activation of the SDF-1/CXCR4 axis. Our data further suggest that SDF-1 stimulates retention of HSPC in MSC niches which expose them to stimulatory and inhibitory factors in a paracrine manner.

Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow

AbstractThe mechanisms regulating the homing/mobilization of hematopoietic stem/progenitor cells (HSPCs) are not fully understood. In our previous studies we showed that the complement C3 activation peptide, C3a, sensitizes responses of HSPCs to stromal-derived factor 1 (SDF-1). In this study, mobilization was induced with granulocyte colony-stimulating factor (G-CSF) in both C3-deficient (C3–/–) and C3a receptor–deficient (C3aR–/–) mice as well as in wild-type (wt) mice in the presence or absence of a C3aR antagonist, SB 290157. The data indicated (1) significantly increased G-CSF–induced mobilization in C3–/– and C3aR–/– mice compared with wt mice, (2) significantly accelerated and enhanced G-CSF–induced mobilization in wt, but not in C3–/– or C3aR–/–, mice treated with SB 290157, and (3) deposition of C3b/iC3b fragments onto the viable bone marrow (BM) cells of G-CSF–treated animals. Furthermore, mobilization studies performed in chimeric mice revealed that wt mice reconstituted with C3aR–/– BM cells, but not C3aR–/– mice reconstituted with wt BM cells, are more sensitive to G-CSF–induced mobilization, suggesting that C3aR deficiency on graft-derived cells is responsible for this increased mobilization. Hence we suggest that C3 is activated in mobilized BM into C3a and C3b, and that the C3a-C3aR axis plays an important and novel role in retention of HSPCs (by counteracting mobilization) by increasing their responsiveness to SDF-1, the concentration of which is reduced in BM during mobilization. The C3a-C3aR axis may prevent an uncontrolled release of HSPCs into peripheral blood. These data further suggest that the C3aR antagonist SB 290157 could be developed as a drug to mobilize HSPCs for transplantation.