Hematopoietic Stem/progenitor Cells Migration Research Articles

Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) by Downregulating Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) Activity.

Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and peak at early morning hours. The level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) that drives activation of Nlrp3 inflammasome. To support this, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Migration of HSPCs is also impaired at the intracellular level by the anti-inflammatory enzyme heme oxygenase-1 (HO-1) which is an inhibitor of Nlrp3 inflammasome. It is also well known that circadian rhythm mediates PB level of melatonin released from the pineal gland. Since trafficking of HSPCs is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from BM into PB by inhibiting Nlrp3 inflammasome activation. We provide anevidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs both in vitro migration assays and in vivo during pharmacological mobilization. This correlated with inhibition of cholesterol synthesis required for a proper membrane lipid raft (MLRs) formation and an increase in expression of HO-1-an inhibitor of Nlrp3 inflammasome. Since melatonin is a commonly used drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more mechanistic light on a role of melatonin in the diurnal circulation of HSPCs.

Novel Evidence That NADPH Oxidase 2 (Nox-2) Enhances Trafficking of Hematopoietic Stem Progenitor Cells (HSPCs) By Involving ROS Mediated Activation of Nlrp3 Inflammasome

Background. NADPH oxidase 2 (Nox-2) is a superoxide-generating enzyme that forms reactive oxygen species (ROS) to regulate the redox state involved in the self-renewal of hematopoietic stem/progenitor cells (HSPCs). Moreover, ROS are released from Nox-2 expressed by cell surface and mitochondria membranes and are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates several aspects of HSPCs biology, including their trafficking, proliferation, and metabolism. Our previous research demonstrated a novel role of Nlrp3 inflammasome in the migration, mobilization, homing, and engraftment of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). The role of ROS was extensively studied in the proliferation of hematopoietic cells; however, its role in the migration of HSPCs is not very well understood. Aim of the study.Based on the role of Nox-2 in activating Nlrp3 inflammasome, we hypothesized that activation of Nox-2-ROS-Nlrp3 inflammasome axis in both HSPCs and BM microenvironment orchestrates trafficking of HSPCs.Materials and Methods. First, we studied in Transwells chemotaxis of Nox-2 deficient cells to bone marrow (BM) chemoattractants, including SDF-1, S1P, and eATP. Next, to shed more light on the role of Nox2 in the trafficking of HSPCs, we performed in Nox-2-KO animals G-CSF- and AMD3100-induced pharmacological mobilization. We also studied homing and engraftment of Nox-2-KO cells in BM of wild-type (WT) animals and homing and engraftment o WT BM cells in Nox-2-KO recipients. Nox-2-KO mice were also sublethal irradiated, and we followed the recovery of peripheral blood counts in these animals. Activation of Nlrp3 inflammasome in Nox-2-KO murine Sca-1+c-kit+lineage- HSPCs was evaluated by Glow assay. To ''bypass'' Nox-2-ROS activation in control experiments, nigericin has been employed as a direct activator of Nlrp3 inflammasome. Since membrane lipid rafts (MLRs) play a crucial role optimal migration of HSPCs, we evaluated the formation of membrane lipid rafts by employing confocal analysis in Nox-2-KO cells stimulated with SDF-1. We also studied the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in providing lipid components for MLRs assemble in activated Nox-2-KO cells. Results. We noticed that Nox-2-KO animals have a profound defect in the trafficking of HSPCs as evidenced by impaired chemotaxis to BM chemoattractants, decrease in pharmacological mobilization, and defect in homing and engraftment after transplantation. Nox-2-KO mice have also defect in recovery from sublethal irradiation. At the molecular level, HSPCs from Nox-2-KO mice displayed a defect in activation of Nlrp3 inflammasome and in cholesterol and sphingolipids synthesizing enzymes, which provide lipid components required for optimal assemble of MLRs. These defects were partially ameliorated by using direct activator of Nlrp3 inflammasome that is nigericin. Conclusions. We provide for the first time evidence that Nox-2-ROS-Nlrp3 inflammasome axis operates in normal HSPCs and is crucial for regulating trafficking and post-irradiation recovery of these cells. These effects are mediated by providing components for MLRs required for optimal migration and signaling in HSPCs

Novel Evidence That Pineal Gland Hormone Melatonin Inhibits Migration of Hematopoietic Stem/Progenitor Cells (HSPCs) By Inhibiting Nlrp3 Inflammasome and Upregulating Heme Oxygenase-1 (HO-1) - Implications to Understand Better Diurnal Trafficking of HSPCs and Anti-Inflammatory Effects of Melatonin

Background. Hematopoietic stem progenitor cells (HSPCs) follow the diurnal circulation rhythm in peripheral blood (PB) with nadir during late night and pick at early morning hours. We have reported that the level of these cells in PB correlates with activation of innate immunity pathways, including complement cascade (ComC) (Stem Cell Rev Rep. 2018;14(5):677-685) that drives activation of Nlrp3 inflammasome (Stem Cell Rev Rep. 2020;16(2):335-343). Therefore, mice both in defective ComC activation as well as Nlrp3 inflammasome do not show typical changes in the diurnal level of circulating HSPCs. Cell migration is also inhibited by the anti-inflammatory enzyme heme oxygenase-1 (HO-1), which inhibits Nlrp3 inflammasome (Leukemia. 2022;36(1):23-32). It is well known that circadian rhythm is regulated by the release of melatonin from the pineal gland, which also has some anti-inflammatory effects (J. Pineal Res. 2018;65:e12525). Aim of the study.Since stem cell trafficking is driven by innate immunity-induced sterile inflammation and melatonin has an anti-inflammatory effect, we hypothesized that melatonin could negatively impact the release of HSPCs from bone marrow (BM) into PB by inhibiting Nlrp3 inflammasome activation. Materials and Methods. To test this hypothesis, first, we tested the expression of melatonin receptors in murine and human HSPCs. We employed Transwell system to see the effect of melatonin on the migration of human and murine HSPCs to the SDF-1 gradient. Next, we performed pharmacological mobilization of normal mice with G-CSF or AMD3100 in the presence of melatonin. Finally, employing Glow assay, we evaluated the effect of melatonin on activation of Nlrp3 inflammasome in HSPCs stimulated by BM chemotactic factors and the effect of melatonin on expression of migration inhibitory HO-1. Since membrane lipid rafts (MLRs) are crucial for the proper response of cells to chemoattractants, we also evaluated the effect of melatonin on the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in the synthesis of cholesterol and sphingolipids that are major constituents of MLRs. Results. We noticed that HSPCs express melatonin receptors, and melatonin inhibits in vitro Transwell assays migration of HSPCs and negatively affects mobilization of these cells into PB in response to G-CSF and AMD3100 administration. We also observed that melatonin inhibits Nlrp3 inflammasome activation and the formation of MLRs. This is explained by a decrease in the expression of enzymes such as SREBP2, HMGCs, and HMGCR involved in cholesterol synthesis leading to impaired formation of MLRs. Moreover, melatonin increases in HSPCs expression of heme oxygenase-1 (HO-1), that is a known inhibitor of Nlrp3 inflammasome and a negative regulator of cell migration. Conclusions. We provide for the first-time evidence that melatonin being a ''sleep regulating pineal hormone'' directly inhibits migration of HSPCs in vitro assays and in vivo their release from BM in response to G-CSF and AMD3100. This is correlated with inhibition of cholesterol synthesis, which results in defective assembly of MLRs, as well as a decrease in Nlrp3 inflammasome activity and an increase in expression of HO-1. Since melatonin is a commonly used shelf drug, this should be considered while preparing a patient for the procedure of HSPCs mobilization. More importantly, our studies shed more light on molecular mechanisms of melatonin involvement in the diurnal circulation of HSPCs.

Novel Evidence That Intracellular Complement Network (complosome) Is Expressed in Hematopoietic Stem Cells (HSPCs) and Regulates Their Trafficking, Metabolism, and Proliferation in an Intracrine-Dependent Manner By Engaging Pattern Recognition Receptor Nlrp3 Inflammasome

Background. Complement cascade (ComC) proteins, including C3 and C5 components, are synthesized in the liver; however, recent reports indicate their expression in lymphocytes as well (Immunity 2020;52(3):513-527). Accordingly, intracellular ComC proteins have been reported to be cleaved to active C3a and C5a mediators that interact with corresponding receptors in an intracrine-depended manner. This novel regulatory loop operating in lymphocytes has been named ''complosome''. Aim of the study.Because hematopoiesis and lymphopoiesis have a common stem cell origin and similar mechanisms are involved in regulating the biology of both lineages, we asked if complosome is also expressed and functional in normal murine hematopoietic stem progenitor cells (HSPCs). We also address a challenging question if intracellular ComC mediators could activate pattern recognition receptor Nlrp3 inflammasome that, as we reported, regulates trafficking, proliferation and metabolism of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). Materials and Methods. First, we phenotyped purified murine and human HSPCs for expression of major complement elements, including C3, C5, C3aR, C5aR, and cathepsin L that cleaves intracellular C3 to C3a. Next, we focused on a crucial distal executive part of ComC, protein C5, and evaluated in C5-KO and C5aR-KO mice the number of HSPCs and their clonogeneic potential. We also studied hematopoietic recovery of these animals after sublethal irradiation (650 cGy). Activation of Nlrp3 inflammasome in C5a-KO and C5aR-KO in response to hematopoietic growth factors and cytokines was evaluated by immunofluorescence Glow assay. We also studied homing and engraftment of C5-KO and C5aR-KO mice-derived HSPCs transplanted into normal WT recipients. Finally, we performed a metabolomic analysis of C5-KO, C5aR-KO, and control WT mice in steady-state conditions and after stimulation with selected hematopoietic growth factors and cytokines. Results. We show for the first time that purified murine and human HSPCs express complosome elements, including C3, C5, C3aR, C5aR, and cathepsin L. Next, C5-KO and C5aR-KO mice also have a lower number of HSPCs and clonogenic progenitors in bone marrow (BM), and display defect of hematopoietic recovery from sublethal irradiation. Furthermore, BM cells isolated from these animals had impaired homing and engraftment in normal WT recipients, supported by the observation that HSPCs from complosome deficient mice had a defect in migration to stromal-derived factor-1 (SDF-1), which correlated with decreased activation of Nlrp3 inflammasome. This defect could be alleviated by direct activation of Nlrp3 inflammasome by nigericin. Finally, our metabolic studies revealed a defect in glucose, amino acids, and lipid metabolism in C5-KO and C5aR-KO mice which correlates with complosome deficiency and Nlrp3 inflammasome activation. Conclusions. We provide for the first time evidence that complosome is expressed and functional in normal HSPCs and by engaging Nlrp3 inflammasome regulates metabolism, proliferation, and migration of HSPCs. Thus, our data sheds new intriguing light on innate immunity as a regulator of hematopoiesis that involves intracellular activation of Nlrp3 inflammasome. We currently focus on Nlrp3 inflammasome-released alarmins released from the cells in response to complosome-Nlrp3 inflammasome activation that accounts for observed phenomena. These studies are essential to develop more efficient ex vivo expansion strategies of HSPCs and to understand better intrinsic mechanisms that regulate metabolism of these cells.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

Meteorin links the bone marrow hypoxic state to hematopoietic stem/progenitor cell mobilization.

Hematopoietic stem/progenitor cells (HSPCs) are supported and regulated by niche cells in the bone marrow with an important characterization of physiological hypoxia. However, how hypoxia regulates HSPCs is still unclear. Here, we find that meteorin (Metrn) from hypoxic macrophages restrains HSPC mobilization. Hypoxia-induced factor 1α and Yin Yang 1 induce the high expression of Metrn in macrophages, and macrophage-specific Metrn knockout increases HSPC mobilization through modulating HSPC proliferation and migration. Mechanistically, Metrn interacts with its receptor 5-hydroxytryptamine receptor 2b (Htr2b) to regulate the reactive oxygen species levels in HSPCs through targeting phospholipase C signaling. The reactive oxygen species levels are reduced in HSPCs of macrophage-specific Metrn knockout mice with activated phospholipase C signaling. Targeting the Metrn/Htr2b axis could therefore be a potential strategy to improve HSPC mobilization for stem cell-based therapy.

A Novel View of the Role of Prostaglandin E2 (PGE2) in Facilitating Engraftment of HSPCs By Activating the NOX2-ROS-Nlrp3 Inflammasome Axis to Incorporate the CXCR4 Receptor into Membrane Lipid Rafts

Background . It is known that prostaglandin E2 (PGE2) increases the homing and engraftment of hematopoietic stem/progenitor cells (HSPCs). However, aside from its role in upregulation of CXCR4 receptor expression on the surface of these cells, the exact mechanism has not been proposed. We have demonstrated in the past that an important step enabling the migration of HSPCs is the incorporation of CXCR4 into membrane lipid rafts on the leading surface (leading edge, in two dimensions) of migrating cells, which facilitates its interaction with cell migration signaling pathways (Wysoczynski M et al. Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient. Blood. 2005;105(1):40-48). Recently, we reported that Nlrp3 inflammasome-deficient HSPCs show a defect in lipid raft formation that results in defective migration of these cells in response to an SDF-1 gradient and their defective homing and engraftment after transplantation (Adamiak, M et al. Nlrp3 Inflammasome Signaling Regulates the Homing and Engraftment of Hematopoietic Stem Cells (HSPCs) by Enhancing Incorporation of CXCR4 Receptor into Membrane Lipid Rafts. Stem Cell Rev and Rep (2020). https://doi.org/10.1007/s12015-020-10005-w). An important activator of Nlrp3 inflammasomes is reactive oxygen species (ROS). Importantly, the enzyme that generates ROS, known as NADPH oxidase 2 (NOX2), is also associated with cell membrane lipid rafts. Hypothesis. Given the known roles of PGE2, membrane lipid rafts, and the Nlrp3 inflammasome in migration, homing, and engraftment of HSPCs, we hypothesized that PGE2 signaling promotes Nlrp3 inflammasome activation in a Nox2-ROS-dependent manner that results in incorporation of CXCR4 into membrane lipid rafts, which better explains the role of PGE2 in these phenomena.Materials and Methods. To test this hypothesis, murine SKL and human CD34+ cells enriched for HSPCs were stimulated with PGE2 to evaluate activation of genes of the Nlrp3 inflammasome complex at the mRNA and protein levels. Next, HSPCs from Nox2-KO mice were tested for membrane lipid raft formation in functional chemotaxis assays in response to SDF-1 gradients under conditions promoting membrane lipid raft formation. Formation of membrane lipid rafts in Nox2-KO cells was also evaluated by confocal analysis in the presence or absence of PGE2. Finally, the effect of the PGE2-Nox2-Nlrp3 inflammasome axis on the formation of membrane lipid rafts was evaluated in the presence of the ROS scavenger N-acethyl-cysteine (NAC). Results. We provide for the first time evidence that PGE2 activates Nlrp3 inflammasomes in HSPCs in a Nox2-ROS-dependent manner. This Nlrp3 inflammasome activation increases at the leading surface of migrating HSPCs with incorporation of the CXCR4 receptor into membrane lipid rafts. Formation of membrane lipid rafts was absent in Nox2-KO and Nlrp3-KO mouse HSPCs and in normal wild type cells after their exposure to NAC. Moreover, we also observed that Nox2-KO and Nlrp3-KO mice had a lower basal level of CXCR4 expression. Conclusions. Our results for the first time explain the role of PGE2 in promoting homing and migration of HSPCs, which occurs in response to PGE2 by activation of the Nox2-ROS-Nlrp3 inflammasome axis and thereby promotes incorporation of the CXCR4 receptor into membrane lipid rafts. Moreover, basal expression of the CXCR4 receptor was at a low level on the surface of HSPCs from Nlrp3-KO mice. Thus, our results provide evidence for the importance of the Nox2-ROS-Nlrp3 inflammasome axis in PGE2-mediated homing and engraftment of HSPCs and the role of PGE2-mediated lipid raft formation for optimal responsiveness of CXCR4 to SDF-1 in the BM microenvironment. Disclosures No relevant conflicts of interest to declare.

Paracrine CCL17 and CCL22 signaling regulates hematopoietic stem/progenitor cell migration and retention in mouse fetal liver

Fetal liver (FL) is the major embryonic hematopoietic organ and a site where circulating hematopoietic stem/progenitor cells (HSPCs) reside. However, HSPC migration/retention mechanisms in FL remain unclear. A chemokine screen revealed that the CCR4 ligands CCL17 and CCL22 are highly expressed in mouse embryonic day (E) 12.5 FL. Flow cytometric analysis confirmed CCR4 expression in FL HSPCs. To identify sources of CCL17 and CCL22, we fractionated FL into various cell types and found that Ccl17 and Ccl22 were predominantly expressed in HPCs/matured HCs. In vitro cell migration analysis confirmed enhanced HSPC migration in the presence of HPCs/matured HCs. Furthermore, exo-utero injection of anti-CCR4 neutralizing antibody into pregnant mice significantly reduced the number of FL HSPCs in embryos. These data demonstrate a paracrine mechanism by which HSPC migration/retention is regulated by CCL17 and CCL22 secreted from HPCs or matured HCs in FL.

A Platform for Studying of the Three-Dimensional Migration of Hematopoietic Stem/Progenitor Cells.

Hematopoietic stem/progenitor cells (HSPCs) have the property to return to the bone marrow, which is believed to be critical in situations such as HSPC transplantation. This property plays an important role in the stemness, viability, and proliferation of HSPCs, also. However, most in vitro models so far have not sufficiently simulated the complicate environment. Here, we proposed a three-dimensional experimental platform for the quantitative study of the migration of HSPCs. After encapsulating osteoblasts (OBs) in alginate beads, we quantified the migration of HSPCs into the beads due to the physical environment using digital image processing. Intermittent hydrostatic pressure (IHP) was used to mimic the mechanical environment of human bone marrow without using any biochemical factors. The expression of stromal cell-derived factor 1 (SDF-1) under IHP was measured. The results showed that the presence of OBs in the hydrogel scaffold initiate the movement of HSPCs. Furthermore, the IHP promotes the migration of HSPCs, even without the addition of any biochemical factors, and the results were confirmed by measuring SDF-1 levels. We believe this suggested three-dimensional experimental platform consisting of a simulated in vivo physical environment and encapsulated OBs should contribute to in vitro migration studies used to investigate the effects of other external factors.

Ikzf1 regulates embryonic T lymphopoiesis via Ccr9 and Irf4 in zebrafish

Ikzf1 is a Krüppel-like zinc-finger transcription factor that plays indispensable roles in T and B cell development. Although the function of Ikzf1 has been studied extensively, the molecular mechanism underlying T lymphopoiesis remains incompletely defined during the embryonic stage. Here we report that the genetic ablation of ikzf1 in mutant zebrafish resulted in abrogated embryonic T lymphopoiesis. This was ascribed to impaired thymic migration, proliferation, and differentiation of hematopoietic stem/progenitor cells (HSPCs). Ccr9a and Irf4a, two indispensable factors in T lymphopoiesis, were the direct targets of Ikzf1 and were absent in the ikzf1 mutants. Genetic deletion of either ccr9a or irf4a in the corresponding mutant embryos led to obvious T cell development deficiency, which was mainly caused by disrupted thymic migration of HSPCs. Restoration of ccr9a in ikzf1 mutants obviously promoted HSPC thymus homing. However, the HSPCs then failed to differentiate into T cells. Additional replenishment of irf4a efficiently induced HSPC proliferation and T cell differentiation. Our findings further demonstrate that Ikzf1 regulates embryonic T lymphopoiesis via Ccr9 and Irf4 and provide new insight into the genetic network of T lymphocyte development.

34: Hematopoietic Stem/Progenitor Cell Dependent Participation of Innate Lymphoid Cells in Low-Intensity Sterile Inflammation

Innate lymphoid cells (ILCs) are increasingly acknowledged as important mediators of immune homeostasis and pathology. They act as early orchestrators of immunity and as such they make up interesting therapeutic targets for several diseases and possibly a means to peripheral tolerance. Taking advantage of an animal model of sterile peritonitis, induced by intraperitoneal injection of zymosan, a fungal derived TLR-2 ligand we studied the progenitor–successor relationship between the hematopoietic stem/progenitor cells (HSPC) and ILCs at the site of sterile inflammation. Our results demonstrate that the strength of the inflammatory signal affects the capacity of BM-derived HSPCs to migrate and give rise in situ to ILCs. Both low- and high-dose of zymosan injections triggered the appearance of mature ILCs in the peritoneal cavity. Only in low-dose injected mice, the recovered ILCs seemed to be dependent on an in situ differentiation of BM-derived HSPCs and/or ILC2 precursors (ILC2P) wherein high-dose, the stronger inflammatory environment seemed to be able to induce the emergence of ILCs independently of BM-derived HSPCs. To further elucidate the importance of the inflammatory environment per se for the attraction of mature ILCs, an anti- inflammatory drug was used. Treatment of mice with alpha-1 anti-trypsin (AAT) 15 min after injection of either low- or high- dose of zymosan, led to the blockade of the migration of HSPCs into the peritoneal cavity resulting in a loss of even mature ILCs in mice injected with the low-dose of zymosan. Contrary, in mice injected with high-dose, progenitor cells of type 2 of ILCs could be still recovered, but not mature ILC2 cells. We suggest that the relationship between HSPCs and ILCs seem to be affected by the strength of the inflammatory stimuli and that the use of anti-inflammatory molecules like A1 antitrypsin may attenuate the inflammation and to some extent prevent the migration of HSCPs to the site of inflammation and the emergence of mature ILCs.

Distinct Roles for Matrix Metalloproteinases 2 and 9 in Embryonic Hematopoietic Stem Cell Emergence, Migration, and Niche Colonization.

SummaryHematopoietic stem/progenitor cells (HSPCs) are formed during ontogeny from hemogenic endothelium in the ventral wall of the dorsal aorta (VDA). Critically, the cellular mechanism(s) allowing HSPC egress and migration to secondary niches are incompletely understood. Matrix metalloproteinases (MMPs) are inflammation-responsive proteins that regulate extracellular matrix (ECM) remodeling, cellular interactions, and signaling. Here, inhibition of vascular-associated Mmp2 function caused accumulation of fibronectin-rich ECM, retention of runx1/cmyb+ HSPCs in the VDA, and delayed caudal hematopoietic tissue (CHT) colonization; these defects were absent in fibronectin mutants, indicating that Mmp2 facilitates endothelial-to-hematopoietic transition via ECM remodeling. In contrast, Mmp9 was dispensable for HSPC budding, being instead required for proper colonization of secondary niches. Significantly, these migration defects were mimicked by overexpression and blocked by knockdown of C-X-C motif chemokine-12 (cxcl12), suggesting that Mmp9 controls CHT homeostasis through chemokine regulation. Our findings indicate Mmp2 and Mmp9 play distinct but complementary roles in developmental HSPC production and migration.

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.

Erythropoietin induces production of hepatocyte growth factor from bone marrow mesenchymal stem cells in vitro

BackgroundHepatocyte Growth Factor (HGF) plays a pivotal role in hematopoiesis, motility, growth and mobilization of hematopoietic stem/progenitor cells (HSPCs). HGF mainly is produced by bone marrow mesenchymal stem cells (BM-MSCs). MSCs express erythropoietin (EPO) receptor. In this study, we aimed to assess the effect of EPO on HGF secretion in BM-MSCs. MethodsThe BM-MSCs treated with EPO (4 IU/ml) for 6, 24 and 48 h. HGF gene expression and protein level were assessed using quantitative real time PCR (qRT-PCR) and Enzyme-linked immunosorbant Assay. In order to show the effect of secreted HGF on migration of HSPCs, hematopoietic stem cells (HSCs) were isolated from cord blood and evaluated using transwell migration assay. ResultsWe observed a significant increase in level of HGF in cell supernatant after 48 h compared to control group (P < 0.05). Also, qRT-PCR results demonstrated a significant elevation in HGF expression level after 24 and 48 h treatment with EPO compared to control group (P < 0.05). Finally, migration assay results showed a significant increase in migration of HSCs in treated group after 48 h. ConclusionOur data indicated that EPO may play an important role in stem cell mobilization through up regulating HGF in MSCs and inducing migration of HSCs.

The Involvment of Hematopoietic-Specific PLC -β2 in Homing and Engraftment of Hematopoietic Stem/Progenitor Cells.

Migration and bone marrow (BM) homing of hematopoietic stem progenitor cells (HSPCs) is regulated by several signaling pathways, and here we provide evidence for the involvement in this process of hematopoietic-specific phospholipase C-β2 (PLC-β2). This enzyme is involved in release of intracellular calcium and activation of protein kinase C (PKC). Recently we reported that PLC-β2 promotes mobilization of HSPCs from BM into peripheral blood (PB), and this effect is mediated by the involvement of PLC-β2 in the release of proteolytic enzymes from granulocytes and its role in disintegration of membrane lipid rafts. Here we report that, besides the role of PLC-β2 in the release of HSPCs from BM niches, PLC-β2 regulates the migration of HSPCs in response to chemotactic gradients of BM homing factors, including SDF-1, S1P, C1P, and 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 in PLC-β2-KO mice and a high baseline level of heme oxygenase 1 (HO-1), an enzyme that negatively regulates cell migration.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-016-9689-x) contains supplementary material, which is available to authorized users.

Downregulation of Heme Oxygenase 1 (HO-1) Activity in Hematopoietic Cells Enhances Their Engraftment After Transplantation.

Heme oxygenase 1 (HO-1) is an inducible stress-response enzyme that not only catalyzes the degradation of heme (e.g., released from erythrocytes) but also has an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury. HO-1 has a well-documented anti-inflammatory potential, and HO-1 has been reported to have a negative effect on adhesion and migration of neutrophils in acute inflammation in a model of peritonitis. This finding is supported by our recent observation that hematopoietic stem progenitor cells (HSPCs) from HO-1 KO mice are easy mobilizers, since they respond better to peripheral blood chemotactic gradients than wild-type littermates. Based on these findings, we hypothesized that transient inhibition of HO-1 by nontoxic small-molecule inhibitors would enhance migration of HSPCs in response to bone marrow chemoattractants and thereby facilitate their homing. To directly address this issue, we generated several human hematopoietic cell lines in which HO-1 was upregulated or downregulated. We also exposed murine and human BM-derived cells to small-molecule activators and inhibitors of HO-1. Our results indicate that HO-1 is an inhibitor of hematopoietic cell migration in response to crucial BM homing chemoattractants such as stromal-derived factor 1 (SDF-1) and sphingosine-1-phosphate (S1P). Most importantly, our in vitro and in vivo animal experiments demonstrate for the first time that transiently inhibiting HO-1 activity in HSPCs by small-molecule inhibitors improves HSPC engraftment. We propose that this simple and inexpensive strategy could be employed in the clinical setting to improve engraftment of HSPCs, particularly in those situations in which the number of HSPCs available for transplant is limited (e.g., when transplanting umbilical cord blood).

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.

CD34 Is a Ligand for Vascular Selectins on Human Hematopoietic Stem/Progenitor Cells

The cell surface glycoprotein CD34 is widely recognized as a marker of hematopoietic stem/progenitor cells (HSPCs), but its physiological role on HSPCs remains elusive. Although CD34 is well recognized for mediating homing of L-selectin+ naïve T cells to high endothelial venules of secondary lymphoid organs, the natural ligand for HSPC specific-CD34 is unknown. Studies in both humans and mice indicate that vascular selectins (E-selectin and P-selectin) are constitutively expressed on marrow endothelial cells and intravital studies revealed that HSPC migration to marrow occurs at specialized microvascular beds expressing E-selectin. Given that the identity of these glycoprotein E-selectin ligands (E-selL) on human HSPCs is limited, we used mass spectrometry (MS)-based proteomics to fully characterize all these potential ligands expressed on primary human CD34+ HSPCs and on the CD34+ KG1a cell line (human acute myelogenous leukemia that serves as a model for human HSPCs). We obtained a very rich resource for further investigation and chose to highlight CD34 as an E-selL candidate. The physiological binding of CD34 to E-selectin was addressed at both the molecular level and cellular level using novel binding assays. At the molecular level we developed a novel-binding assay to capture endogenous candidate glycoprotein ligands from whole cell lysates prepared from human HSPCs onto a surface plasmon resonance (SPR) chip and characterized its binding kinetics to recombinant E-sel. At the cellular level we used the blot rolling assay to monitor the ability of cells stably transfected with E-selectin (CHO-E) to roll on CD34 immunoprecipitated from HSPCs to demonstrate that CD34 binds to E-selectin with affinity comparable to well-described E-selectin ligands. Further biochemical analysis demonstrated that E-selectin binding was restricted to a specific glycoform that expresses sialyl Lewis X (sLex). This interaction was dependent on calcium, sialylation and O-glycans as removal of these abolished binding to E-selectin. CD34 binding to the other vascular selectin, P-selectin was dependent on O-glycan and tyrosine sulfation making CD34 the first selectin ligand since P-selectin glycoprotein ligand-1 (PSGL-1) reported to bind all three selectins. Among the stem cell specific processes such as self-renewal and differentiation that CD34 has been implicated to play a significant role in, these data are the first to define a role for human CD34 in mediating the direct migration of HSPCs to the bone marrow and suggests that a natural ligand for CD34 on human HSPCs are vascular selectins. DisclosuresNo relevant conflicts of interest to declare.

Transient Ex Vivo Inhibition of Heme Oxygenase 1 (HO-1) in Hematopoietic Stem/Progenitor Cells (HSPCs) By Small-Molecule Inhibitors Enhances Their Migratory Responsiveness to Bone Marrow (BM)-Secreted Chemoattractants a Novel and Simple Strategy to Improve Homing of HSPCs

Background. Heme oxygenase 1 (HO-1) is an inducible stress-response enzyme that not only catalyzes the degradation of heme (e.g., released from erythrocytes) but also has an important function in various physiological and pathophysiological states associated with cellular stress, such as ischemic/reperfusion injury. HO-1 has a well-documented anti-inflammatory potential and inhibits complement cascade (ComC)-mediated inflammatory responses. Moreover, HO-1 has been reported to have a negative effect on adhesion and migration of neutrophils in acute inflammation in a model of peritonitis. Radiation chimeras created after transplantation with HSPCs having a mutation in one of the alleles of HO-1 engrafted much faster; however, a persistent decrease in HO-1 activity in these animals resulted in their enhanced sensitivity to stress and susceptibility to irradiation (Blood 2008, 112, 4494-4502). Moreover, we recently demonstrated that HO-1-deficient HSPCs show enhanced in vitro migration up an SDF-1 gradient (Stem Cell Rev & Rep. 2015, 11, 110-118).Hypothesis. Based on these findings, we hypothesized that transient inhibition of HO-1 by non-toxic, small-molecule inhibitors would enhance in vivo migration of HSPCs to bone marrow (BM)-derived chemoattractants and thus would facilitate their homing and accelerate hematopoietic recoveryMaterials and Methods. To address this issue, we first generated several human hematopoietic cell lines in which HO-1 was upregulated or downregulated. We also exposed murine and human BM-derived cells to small-molecule inhibitors or activators of HO-1 and performed dose and timing toxicity studies. Next, murine BM mononuclear cells (MNCs) and human umbilical cord blood (UCB) MNCs were exposed to the small-molecule HO-1 inhibitor Sn(IV) protoporphyrin IX dichloride (SnPP) and tested for their chemotactic response in Transwell migration assays to all currently known HSPC chemoattractants, including stromal-derived factor 1 (SDF-1), sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and the extracellular nucleotides ATP and UTP. For in vivo assays, lethally irradiated mice were transplanted with BM MNCs exposed or not exposed to SnPP, and in recipient animals we evaluated i) the number of day-12 colony-forming units in spleen (CFU-S) and colony-forming units for granulocyte/macrophage (CFU-GM) progenitors in BM and ii) the kinetics of peripheral blood (PB) count recovery by measuring the number of leucocytes, lymphocytes, and platelets. We also performed competitive repopulation studies with a limited number of transplanted BM MNCs using the CD45.1 and CD45.2 congenic mouse models.Results and Conclusions. We demonstrate here that HO-1 is a negative regulator of HSPC migration, and thus, by transiently inhibiting its activity in HSPCs with the non-toxic small-molecule inhibitor (SnPP), it is possible to accelerate homing and subsequent engraftment of HSPCs. We propose that this simple and inexpensive strategy could be employed in the clinical setting to improve seeding efficiency of transplanted HSPCs and their engraftment, particularly in those situations in which the number of HSPCs available for transplant is limited (e.g., from UCB or grafts harvested from poor mobilizers). DisclosuresNo relevant conflicts of interest to declare.

TopBP1 Governs Hematopoietic Stem/Progenitor Cells Survival in Zebrafish Definitive Hematopoiesis.

In vertebrate definitive hematopoiesis, nascent hematopoietic stem/progenitor cells (HSPCs) migrate to and reside in proliferative hematopoietic microenvironment for transitory expansion. In this process, well-established DNA damage response pathways are vital to resolve the replication stress, which is deleterious for genome stability and cell survival. However, the detailed mechanism on the response and repair of the replication stress-induced DNA damage during hematopoietic progenitor expansion remains elusive. Here we report that a novel zebrafish mutantcas003 with nonsense mutation in topbp1 gene encoding topoisomerase II β binding protein 1 (TopBP1) exhibits severe definitive hematopoiesis failure. Homozygous topbp1cas003 mutants manifest reduced number of HSPCs during definitive hematopoietic cell expansion, without affecting the formation and migration of HSPCs. Moreover, HSPCs in the caudal hematopoietic tissue (an equivalent of the fetal liver in mammals) in topbp1cas003 mutant embryos are more sensitive to hydroxyurea (HU) treatment. Mechanistically, subcellular mislocalization of TopBP1cas003 protein results in ATR/Chk1 activation failure and DNA damage accumulation in HSPCs, and eventually induces the p53-dependent apoptosis of HSPCs. Collectively, this study demonstrates a novel and vital role of TopBP1 in the maintenance of HSPCs genome integrity and survival during hematopoietic progenitor expansion.