Hematopoietic Stem And Progenitor Cells Egress Research Articles

Abstract 1320: Hypoxic extracellular vesicles induce a pro-metastatic phenotype in neuroblastoma: from the delivery of miR-210-3p to the preconditioning of a metastatic niche

Abstract Neuroblastoma (NB) is a pediatric tumor of neural crest origin accounting for 7% of cancers and 15% of cancer deaths. Solid malignant tumors commonly contain hypoxic areas that stimulate release of extracellular vesicles (EVs), lipid bound vesicles secreted by cells into the extracellular space. In our study we demonstrated that EVs from hypoxic NB cells promote pro-metastatic features in vitro and in an in vivo zebrafish model. We characterized the microRNA cargo of EVs from NB cells and compared their expression in hypoxia vs. normoxia, finding that miR-210-3p was the most upregulated. By using both in vivo and in vitro models we demonstrated that EVs released by hypoxic NB cells induced changes that favor a malignant phenotype in normoxic NB cells by transferring miR-210-3p. We showed that EVs isolated from cells transfected with miR-210-3p-mimic and cultured in normoxic condition, increased the migration ability and invasiveness of NB cells like EVs from hypoxic NB cells. Cells treated with EVs isolated from cells transfected with miR-210-3p-inhibitor and cultured in hypoxic condition were unable to stimulate migration and invasion as efficiently as EVs derived from control transfected cells, despite cells were cultured in hypoxic conditions. We then focused on the effects of EVs in metastatic niches formation in vivo. Intravascular injection within the zebrafish embryo allows a non-invasive visualization of EVs dispersion, uptake, and interactions with host cells. We first demonstrated that EVs released under hypoxic conditions promote angiogenesis and are more easily internalized by endothelial cells than those purified from normoxic cells. Furthermore, we proved via microscopy imaging and cell sorting that the group injected with hypoxic EVs had higher numbers of macrophages. We then focused on the region of the embryo called caudal hematopoietic tissue (CHT) as a potential metastatic site. After EVs injection, we evaluated through qPCR the expression of mmp9, required for HSPC (hematopoietic stem and progenitor cells) egress from the CHT, and cxcl8 that enhances HSPC colonization in the CHT. Results highlighted that the expression of these genes increased after hypoxic EVs injection. We showed that hypoxic EVs induce i. angiogenesis in vivo, required for tumor and metastasis growth, and ii. increased number of macrophages, a major contributor to the tumor microenvironment which produce growth factors and chemokines promoting tumor angiogenesis. Our data demonstrate that hypoxic EVs can modify the behavior of recipient cells in the CHT, as a potential metastatic site, to prepare a ‘fertile soil’ for cancer cells colonization. In conclusion, our findings suggest that EVs released by hypoxic NB cells induce changes that favor a malignant phenotype in normoxic NB cells by transferring miR-210-3p both in vitro and in vivo. Citation Format: Anna Fietta, Pina Fusco, Giuseppe Germano, Elisa Cimetta. Hypoxic extracellular vesicles induce a pro-metastatic phenotype in neuroblastoma: from the delivery of miR-210-3p to the preconditioning of a metastatic niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1320.

Replenishment of Damaged Bone Marrow Niches By Circulating Hematopoietic Stem Cells

Hematopoietic Stem and Progenitor Cells (HSPC) support life-long production of blood cells through multipotent differentiation and self-renewal. Adult HSPC reside predominantly in the bone marrow (BM) but low levels of HSPC can be found in the bloodstream (Wright et al, Science 2001). The spontaneous egress of HSPC from the BM into the circulation follow circadian cycles which are in anti-phase with the chemokine Cxcl12 produced by the niche microenvironment (Mendez-Ferrer et al, Nature 2008). This phenomenon is regulated exogenously by the molecular clock, which orchestrates the circadian release of noradrenaline by the sympathetic nervous system. Medullary HSPC are crucial for haematopoiesis under normal and stress conditions; in contrast, the physiological function of circulating HSPC remains largely unknown. Here we aimed at exploring the role of circulating HSPC.To study the role of circulating HSC under stress conditions we used a mouse model in which genotoxic damage was specifically applied to the lower limbs (femur and tibiae) without affecting other parts of the body. Affected bones were collected at different times after irradiation and the number of LSK (LinNEG Sca1+ c-Kit+) and myeloid progenitors (LinNEG Sca1NEG c-Kit+) were determined by flow cytometry. We found that LSK numbers were restored in the irradiated marrows from two weeks after irradiation, indicating that endogenous circulating HSPC can home into and re-populate genotoxic-damaged niches. To further confirm this observation we performed parabiotic assays in which we surgically conjoined unperturbed or sub-lethally irradiated mice together with non-irradiated wild type (WT) GFP reporter mice. Parabionts were surgically separated after 3 weeks of shared circulation and irradiated mice were analysed for partner-derived HSC repopulating activity. Our results indicate that circulating HSPC efficiently travel through the circulation of the parabiotic partner, home to the BM and repopulate damaged niches.We next examined the contribution of circulating HSC in the absence of exogenous stress. This, however, represented a challenge as no models have been reported to display basal deficiency of circulating HSPC in blood. While analysing mice with genetic deficiency in CXCR2, a chemokine receptor needed for the egress of neutrophils from the BM, we noticed that mice haploinsuficient for CXCR2 (CXCR2HET) displayed a 6-fold reduction in the number of circulating HSPC compared with WT mice. Additional experiments demonstrated that this chemokine receptor was expressed at low levels on HSPC as determined by flow cytometry and quantitative PCR, and was functional as determined by in vitro migration assays. CXCR2 was also expressed in wild-type HSPC with long-term reconstituting capacity (LT-HSC), as BM cells that migrated to the CXCR2 ligand CXCL1 had LT reconstituting capacity following BM transplantation. Remarkably, CXCR2HET resulted in undetectable levels of the receptor on HSPC and impaired in vitro and in vivo migration, which suggested that HSC with LT-repopulating activity need two copies of CXCR2 for homeostatic egress from the BM. These observations also provided a model to study the functional contribution of blood HSC during homeostasis. We therefore conjoined WT or CXCR2HET with S cid mice by parabiosis. Scid mice display an intrinsic HSPC defect that becomes evident in the presence of normal, competing HSC (Qing et al, Blood 2012). After 1 month of shared circulation, the parabionts were surgically separated and partner-derived LT repopulating activity was measured in the S cid partners. While exogenously injected marrow cells from WT or CXCR2HET mice were able to home into and repopulate the BM of the Scid mice, only WT partners were capable to efficiently outcompete Scid HSPC.Our results thus indicate that an important function of endogenously circulating HSPC is to repopulate niches that either become vacant after a stress or are populated by intrinsically defective HSPC, and that this process relies on CXCR2-mediated homeostatic egress. These studies pave the way to understanding the biology of circulating HSC and to extend their application in haematological deficiencies, leukaemia or bone marrow regeneration. DisclosuresNo relevant conflicts of interest to declare.

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Bone marrow failure (BMF) in Fanconi anemia (FA) patients results from dysfunctional hematopoietic stem and progenitor cells (HSPCs). To identify determinants of BMF, we performed single-cell transcriptome profiling of primary HSPCs from FA patients. In addition to overexpression of p53 and TGF-β pathway genes, we identified high levels of MYC expression. We correspondingly observed coexistence of distinct HSPC subpopulations expressing high levels of TP53 or MYC in FA bone marrow (BM). Inhibiting MYC expression with the BET bromodomain inhibitor (+)-JQ1 reduced the clonogenic potential of FA patient HSPCs but rescued physiological and genotoxic stress in HSPCs from FA mice, showing that MYC promotes proliferation while increasing DNA damage. MYC-high HSPCs showed significant downregulation of cell adhesion genes, consistent with enhanced egress of FA HSPCs from bone marrow to peripheral blood. We speculate that MYC overexpression impairs HSPC function in FA patients and contributes to exhaustion in FA bone marrow.

Long-Term Hydroxyurea Use Is Associated with Lower Levels of Hematopoietic Stem and Progenitor Cells in Patients with Sickle Cell Disease

Background Sickle cell disease (SCD) is curable by transplantation and potentially by gene therapy, and is generally treated by a combination of blood transfusion and Hydroxyurea (HU). Characterizing the hematopoietic system in SCD patients is important because the long-term effect of HU treatment are not known, and because of lower than expected efficacy of transduction and transplantation in Hematopoietic Stem and Progenitor Cells (HSPCs) in recent gene therapy trials. Previous studies have shown that the number of bone marrow (BM) CD34+ cells is elevated in SCD patients and that HU treatment is associated with decreased level of CD34+ cells in the peripheral blood (PB) and BM relative to steady state patients. However, hematopoiesis in SCD patients naive or treated with HU or transfusion remains poorly understood. Here, we report on the characterization of the HSPC compartment in patients with SCD by prospective isolation of 49f+ long-term Hematopoietic Stem Cells (49f+LT-HSCs), Multipotent Progenitors (MPPs), Common Myeloid Progenitors (CMPs), Megakaryocyte-Erythroid Progenitors (MEPs), and Granulocyte-Monocyte Progenitors (GMPs). Methods After obtaining consent, PB and/or BM were collected from 69 patients with HBSS/SB0, aged 12 to 45years, and 25 healthy adult African American controls. Patients were divided into chronic transfusion therapy (n=19), HU (n=31) and naïve (n=19) groups. Frozen mono-nuclear cells were analyzed by flow cytometry on a BD LSRII using CD 49f, 90 45Ra, 123, 235a, 38, 34, 33 and lineage antibodies. Results FACS analysis revealed that the number PB CD34+ cells was 2.5 CD34+/uL of blood in the HU group as compared to 19 CD34+/uL in the exchange and naive groups, and 7.3 CD34+/uL in the control group (q-value <0.05 in all cases). Analysis with additional markers revealed that the decrease in circulating HSPCs in the HU group affected the entire hematopoietic system since the number of 49f+LT-HSCs, MPPs, CMPs, MEPs were all significantly lower in the HU group. The decrease in cell number in the HU group, however, was not homogeneous. The proportion of LT-HSCs was higher in the HU and transfusion groups when compared to the naive and control groups. The HU group also had the lowest proportion of MPPs and GMPs, as well as the highest proportion of MEPs. We then investigated hematopoiesis as a function of the length of HU treatment to elucidate the long-term treatment effect of this cytotoxic agent. Patients > 18 years of age that had been treated on HU for at least three years exhibited a strong statistically significant negative correlation between years on HU and CD34+/uL (R2 = 0.41), LT-HSC/uL (R2 = 0.35), MPP/uL (R2 =0.43), CMP/uL (R2 = 0.37), MEP/uL (R2 = 0.25) and GMP/uL (R2 0.39, p<0.01 in all cases). Importantly there was no correlation between WBC counts, age, HU dose, or serum erythropoietin level versus the numbers of any HSPC/uL. Lastly, we compared the number of HSPCs in paired PB and BM samples 10 controls and 4 SCD patients. This revealed that the numbers of CD34+, HSCs, MPPs, CMPs, GMPs and MEPs in the PB and BM were well correlated (r2 in 0.6-0.8 range) suggesting that in first approximation, results obtained in the PB reflect changes in the BM rather than changes in egress of HSPCs from the BM. Discussion We have observed lower numbers of circulating CD34+,49f+LT- HSCs, MPPs, CMP, GMP and MEPs in individuals with HBSS on HU therapy when compared to naive, chronic transfusion and, to a lesser extent, controls. Furthermore we observed subtle differences in the proportion of various circulating stem and progenitor cells (HSPCs) suggesting that the various treatments affects hematopoiesis in complex ways. The strong negative correlations between the length of HU treatment and the numbers of HSPCs can be explained either by decreased cell mobilization to the periphery, or by a depletion of the HSPC numbers in the BM overtime. Most patients undergoing gene therapy trials are currently taken off HU and placed on transfusion therapy for several months to increase CD34+ cell collection and LT-HSC transduction efficiency. We observed a greater number of circulating 49f+LT-HSC/uL of blood in the transfusion group than in the HU group, but the proportion of 49f+LT-HSC relative to the number of CD34+ were similar in both groups. Functional studies may help determine whether 49f+LT-HSCs from the transfusion group are qualitatively different from of the HU group and more amenable to gene therapy. Figure Disclosures Manwani: Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding. Minniti:Doris Duke Foundation: Research Funding.

Targeting VLA4 integrin and CXCR2 mobilizes serially repopulating hematopoietic stem cells.

Mobilized peripheral blood has become the primary source of hematopoietic stem and progenitor cells (HSPCs) for stem cell transplantation, with a five-day course of granulocyte colony stimulating factor (G-CSF) as the most common regimen used for HSPC mobilization. The CXCR4 inhibitor, plerixafor, is a more rapid mobilizer, yet not potent enough when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects. We sought to improve hematopoietic stem cell transplantation by developing a new mobilization strategy in mice through combined targeting of the chemokine receptor CXCR2 and the very late antigen 4 (VLA4) integrin. Rapid and synergistic mobilization of HSPCs along with an enhanced recruitment of true HSCs was achieved when a CXCR2 agonist was co-administered in conjunction with a VLA4 inhibitor. Mechanistic studies revealed involvement of CXCR2 expressed on BM stroma in addition to stimulation of the receptor on granulocytes in the regulation of HSPC localization and egress. Given the rapid kinetics and potency of HSPC mobilization provided by the VLA4 inhibitor and CXCR2 agonist combination in mice compared to currently approved HSPC mobilization methods, it represents an exciting potential strategy for clinical development in the future.

HIF prolyl hydroxylase inhibitor FG-4497 enhances mouse hematopoietic stem cell mobilization via VEGFR2/KDR

AbstractIn normoxia, hypoxia-inducible transcription factors (HIFs) are rapidly degraded within the cytoplasm as a consequence of their prolyl hydroxylation by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes. We have previously shown that hematopoietic stem and progenitor cells (HSPCs) require HIF-1 for effective mobilization in response to granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonist AMD3100/plerixafor. Conversely, HIF PHD inhibitors that stabilize HIF-1 protein in vivo enhance HSPC mobilization in response to G-CSF or AMD3100 in a cell-intrinsic manner. We now show that extrinsic mechanisms involving vascular endothelial growth factor receptor-2 (VEGFR2), via bone marrow (BM) endothelial cells, are also at play. PTK787/vatalanib, a tyrosine kinase inhibitor selective for VEGFR1 and VEGFR2, and neutralizing anti-VEGFR2 monoclonal antibody DC101 blocked enhancement of HSPC mobilization by FG-4497. VEGFR2 was absent on mesenchymal and hematopoietic cells and was detected only in Sca1+ endothelial cells in the BM. We propose that HIF PHD inhibitor FG-4497 enhances HSPC mobilization by stabilizing HIF-1α in HSPCs as previously demonstrated, as well as by activating VEGFR2 signaling in BM endothelial cells, which facilitates HSPC egress from the BM into the circulation.

Nocturnal Melatonin Renews Bone and Blood Forming Stem Cells Reservoir By Metabolic Reprograming

Bone marrow (BM) residing hematopoietic stem and progenitor cells (HSPC) replenish the blood with mature cells with a finite life span on a daily basis while maintaining the reservoir of undifferentiated stem cells. We recently showed that light/darkness onset induce two different BM HSPC peaks. Morning-induced norepinephrine and TNF secretion metabolically facilitate HSPC differentiation and egress to replenish the circulation with new mature leukocytes. Night augmented BM melatonin renews BM CD150+ hematopoietic stem cell (HSC) reservoir and their long-term repopulation potential (Golan et al, Cell Stem Cell, In Press). How melatonin primes BM HSPC to change their phenotype and function to re-acquire an undifferentiated and primitive state, is poorly understood. The hormone melatonin is an important mediator of bone formation and mineralization, and ultimately regulates the balance of bone remodeling (Cardinali DP et al, J. Pineal Res., 2003). The cross talk between HSPC and their BM stromal microenvironment is tightly regulated and determines HSPC fate. Therefore, we examined whether melatonin plays a role in regulation of murine BM mesenchymal stem and progenitor cells (MSPC, CD45-/Sca-1+/PDGFRα+), known to support HSPC maintenance in their BM niches. Mice treated with melatonin for 5h during the morning had increased levels of BM MSPC endowed with higher colony-forming unit fibroblast (CFU-F) potential in vitro. Interestingly, the metabolic state of these progenitor cells was altered by melatonin demonstrating reduced glucose uptake ability and lower mitochondria content. To test if differences in stromal cells content exist between day and night, we examined BM MSPC and found increased levels at 11PM, the time of melatonin BM peak, with higher Sca-1high surface expression levels, as compared to daylight 11AM. These changes were associated with augmented CFU-F levels by MSPC harvested at 11PM and accompanied by reduced glucose uptake levels and mitochondria content. Our preliminary results suggest that melatonin at night increases BM MSPC levels and reduces their metabolic activity to maintain them in a primitive and undifferentiated state. Moreover, we found that melatonin-elevated HSPC at 11PM also share lower glucose uptake ability with reduced mitochondria content and lower mitochondrial membrane potential (evaluated by TMRE). We hypothesize that melatonin reprograms the metabolic state of both HSPC and their stromal MSPC microenvironment to renew and maintain a primitive state of both populations at night. One of the factors inhibited by melatonin is the bioactive lipid Sphingosine 1-Phosphate (S1P), which in turn inhibits melatonin production. We found that mice with low S1P levels (S1Plow) due to lack of the SPHK1 enzyme have high BM melatonin levels also during the day in contrast to wild type (WT) mice. S1Plow mice had higher levels of primitive stromal progenitor cells including CFU-F and lower levels of differentiating osteoblast precursors compared to WT mice. In addition, these mice had less BM Reactive Oxygen Species (ROS)high committed hematopoietic progenitor cells, but more primitive ROSlow EPCR+ HSC endowed with higher long-term repopulation capacity in both primary and serially transplanted recipients. Next, we examined how light/dark cues affect the homing of transplanted BM HSPC into the BM of irradiated hosts 18h after transplantation. We found that donor HSPC harvested at 11PM have elevated homing ability compared to 11AM harvested cells. Importantly, MSPC also better homed to the BM of irradiated recipients when we transplanted donor BM cells obtained at 11PM compared with 11AM. As a result, accelerated BM repopulation kinetics was documented one week post transplantation in mice transplanted with BM cells harvested at 11 PM.Taken together, our results reveal that in vivo melatonin renews and maintains the BM reservoir and function of primitive MSPC and HSPC by metabolically reprogramming these cells during the night on a daily basis. Since the primed HSPC and MSPC at night showed improved function and BM homing potential, these features might be mimicked by human BM cells in order to harness them for improved clinical transplantation protocols. DisclosuresNo relevant conflicts of interest to declare.

Notch Receptor-Ligand Engagement Maintains Hematopoietic Stem Cell Quiescence and Niche Retention.

Notch is long recognized as a signaling molecule important for stem cell self-renewal and fate determination. Here, we reveal a novel adhesive role of Notch-ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O-fucosylglycans on Notch EGF-like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand-expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand-bearing osteoblastic or stromal cells inhibits wild type but not O-fucosylglycan-deficient HSPC cycling, independent of RBP-JK -mediated canonical Notch signaling. Furthermore, Notch-ligand neutralizing antibodies induce RBP-JK -independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor-ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O-fucosylglycans on Notch.

Loss of Notch Receptor-Ligand Engagement Leads to Increased Hematopoietic Stem and Progenitor Cell Egress and Mobilization

BACKGROUNDNotch has long been recognized as an important molecule that regulates stem cell self-renewal and differentiation. Despite that Notch is required for the embryonic hematopoietic stem cell (HSC) generation, and that Notch is expressed in the adult HSC, the role of Notch pathway in adult HSC remains unclear. Recently it has been shown that Notch activation and the ability of interacting with Notch ligand Jagged1 is a signature of human primitive HSC and supports HSC regenerative potential. Here we study the physiological significance of Notch in the adult HSC population focusing on how Notch-ligand engagement regulates HSC quiescence and niche retention. METHODSTo better understand the role of Notch in adult HSC homeostasis, we examined HSC frequency, quiescence maintenance, niche occupancy, and HSC mobilization in mice with either conditional lack of RBP-JK, which mediates the canonical Notch signaling activity, or in mice with conditional lack of Pofut1 that catalyzes O-fucosylation of Notch EGF-like repeats and the generation of O-fucose glycans important for the binding of Notch ligands. We also tested Notch ligand neutralizing antibodies and Notch1 and Notch2 inhibitory antibodies to examine the effect of blocking Notch receptor-ligand engagement or the block of Notch signaling activation on HSC homeostasis. RESULTSWe report here that Pofut1-deficient hematopoietic stem and progenitor cells (HSPCs) display enhanced cell cycling and proliferation cell autonomously. These changes are accompanied by G-CSF-independent increased HSPC egress from the marrow to the periphery and other hematopoietic organs, and their enhanced sensitivity to mobilizing stimuli of G-CSF plus the CXCR4 antagonist, AMD3100. This phenotype is caused by reduced adhesion of Pofut1-deficient HSPC to Notch ligand-expressing stromal cells and the osteoblastic lineage cells. Adhesion to these cells by wild type but not Pofut1-deficient HSPCs can be blocked by recombinant Notch ligand Dll1 or Dll4. In addition, adhesion to these cells inhibits wild type but not Pofut1-deficient HSPC cycling, independent of RBP-Jk-mediated Notch signaling. Further, Pofut1-deficient HSPCs exhibit normal expression of key adhesion molecules and normal SDF-1-mediated chemotaxis, but show scattered and distal occupancy in the endosteal or osteoblastic niche. In support of roles for Notch-ligand engagement in facilitating HSPC niche retention, we show that mice receiving 4 doses of neutralizing antibodies to the Notch ligand Dll4 or Jagged1, but not Dll1, display ~2-3-fold increased HSC and progenitor egress when compared to mice receiving isotype control antibody, and further display 60% increased HSC mobilization when compared to mice receiving control antibody and treated similarly with G-CSF and AMD3100. Dll4 blockade also increases the sinusoidal endothelial cell population and HSPC cell cycling. In comparison, only a mild HSPC proliferation and egress is found in RBP-JK-deficient mice, or in mice receiving Notch2 inhibitory antibody. However, Notch2 blockade but not Notch1 blockade induces unique features of HSC and myeloid progenitor mobilization responding to G-CSF plus AMD3100. CONCLUSIONSBased on these findings, we conclude that HSPC quiescence and retention in the marrow niche is facilitated by the interaction between Notch-expressing HSPCs and Jagged1- or Dll4-expressing niche cells, and is likely also contributed by Notch signaling activation. In addition, Notch receptor-ligand engagement in this process is strengthened by O-fucose modification of Notch receptors. Finally, the observations from our studies may provide a therapeutic indication. Inadequate mobilization in HSPC transplantation remains a clinical problem. Our findings that targeting Notch receptor-ligand interaction and/or inhibiting Notch2 activation increase HSPC emigration suggests a novel approach for enhancing mobilization of stem and progenitor cells for those patients who respond poorly to current mobilizing regimes. DisclosuresShim:Genentech: Employment, Equity Ownership. Yan:Genentech: Employment, Equity Ownership. Lowe:Genentech: Employment, Equity Ownership. Siebel:Genentech: Employment, Equity Ownership.

Macrophages and Bone Marrow Stem Cell Niches: The Roles of PGE2 and Coagulation

Blood-forming hematopoietic stem cells (HSC) functionally express the chemokine receptor CXCR4, which is required to control their directional motility, cell cycle, adhesion, and bone marrow (BM) repopulation. HSC are mostly BM-retained in a quiescent, non-motile mode via CXCR4-induced adhesion interactions with BM niche cells, which functionally express surface CXCL12. Surface CXCL12-mediated adhesion interactions with BM stromal cells protect quiescent CXCR4+ stem cells from DNA damaging agents while preserving their developmental potential. On the other hand, CXCL12 secretion by BM stromal cells and its release to the blood increase CXCR4 expression and signaling in hematopoietic stem and progenitor cells (HSPC), inducing their egress and clinical mobilization. Finally, CXCR4+ HSC also follow CXCL12 gradients to the BM when they home back from the blood. Intriguingly, dynamic CXCR4/CXCL12 signaling cascades control not only HSC retention in the BM, but also their homing to the BM and their release and mobilization to the blood. In addition to their central role in host defense, myeloid cells participate in organ homeostasis, including HSC localization. Monocyte-derived bone resorbing osteoclasts cleave and release endosteal membrane-bound CXCL12, stem cell factor, and osteopontin, factors needed for HSC adhesion and BM retention, leading to CXCL12/CXCR4-mediated HSPC mobilization. Recently, we identified a rare population of BM αSMA+ macrophages that highly express, which protects HSC from inflammatory insults via COX-2-mediated PGE2 secretion and reactive oxygen species inhibition. In vitro PGE2 upregulates CXCR4 on enriched human cord blood CD34+ and murine HSC via cAMP activation, leading to enhanced CXCL12-induced migration, homing and BM repopulation. However, preliminary results reveal that in vivo PGE2 treatment reduced CXCR4 expression on BM HSC by increasing surface CXCL12 expression by stromal cells. Surface CXCL12 upregulation is due to PGE2-mediated secretion and autocrine signaling of lactate, which is followed by cAMP inhibition in BM stromal cells. Indeed, antagonizing COX-2 or activating cAMP induced HSPC mobilization via BM CXCL12 secretion and increased CXCR4 expression and signaling in HSPC. Coagulation cascades also navigate HSC localization. We revealed that anticoagulant microenvironments in the BM mediate HSC adhesion and retention via inhibition of nitric oxide (NO) production and HSC migration. Physiologic stress induced extensive production of the pro-coagulant factor thrombin, which activates NO generation, CXCL12 secretion and enhanced CXCR4+ stem cell motility and mobilization. We found multinucleated pre-osteoclast cell clusters in femoral metaphysis expressing Tissue Factor (TF) a potent initiator of coagulation leading to thrombin generation. Osteoclast maturation and stress signals also activate TF induced pro-coagulation cascades, mediating HSPC egress and recruitment to the circulation. Daily light and darkness cues regulate many physiological processes, including osteoclast/osteoblast bone remodeling, BM CXCL12 production and secretion and CXCR4+ HSC egress. In addition to the previously identified morning peak of HSC egress to the blood, we identified two peaks of BM HSC proliferation: a morning peak in conjunction with stem cell egress and an evening peak of HSC expansion without egress. Higher BM levels of the HSC-protecting aSMA+ monocyte/macrophages documented in the evening and their associate “low-CXCR4/high CXCL12” BM microenvironment provide the mechanism for these fluctuating patterns. Finally, we found higher PGD2 levels and reduced PGE2 levels in monocyte/macrophages in the morning, whereas at night we documented the opposite patterns. PGD2 induced CXCL12 secretion and HSC egress, while PGE2 induced their retention. Our studies attribute central roles for BM myeloid cells in HSC regulation. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem and progenitor cell mobilization in mice.

Hematopoietic stem cell transplantation (HSCT) can be performed with hematopoietic stem and progenitor cells (HSPC) acquired directly from bone marrow, from umbilical cord blood or placental tissue, or from the peripheral blood after treatment of the donor with agents that enhance egress of HSPC into the circulation, a process known as "mobilization." Mobilized peripheral blood stem cells (PBSC) have become the predominate hematopoietic graft for HSCT, particularly for autologous transplants. Despite the success of PBSC transplant, many patients and donors do not achieve optimal levels of mobilization. Thus, accurate animal models and basic laboratory investigations are needed to further investigate the mechanisms that lead to PBSC mobilization and define improved or new mobilizing agents and/or strategies to enhance PBSC mobilization and transplant. This chapter outlines assays and techniques for exploration of hematopoietic mobilization using mice as a model organism.

Blood Cell Replenishment and Bone Marrow Stem Cell Pool Renewal Are Regulated By Different Circadian Peaks Via Norepinephrine and TNFα/S1P Signaling

Hematopoietic stem and progenitor cells (HSPC) are mostly retained in a quiescent, non-motile mode in the bone marrow (BM), shifting to a cycling, differentiating and migratory state on demand. How HSC replenish the blood with new mature leukocytes on a daily basis while maintaining a constant pool of primitive cells in the BM throughout life is not clear. Recently, we reported that the bioactive lipid Sphingosine 1-Phosphate (S1P) regulates HSPC mobilization via ROS signaling and CXCL12 secretion (Golan et al, Blood 2012). We hypothesize that S1P influences the daily circadian egress of HSPC and their proliferation. We report that S1P levels in the blood are increased following initiation of light at the peak of HSPC egress and are reduced towards the termination of light when circulating HSPC reach a nadir. Interestingly, mice with constitutively low S1P plasma levels due to lack of one of the enzymes that generates S1P (Sphingosine kinase 1), do not exhibit fluctuations of HSPC levels in the blood between day and night. We report that HSPC numbers in the BM are also regulated in a circadian manner. Unexpectedly, we found two different daily peaks: one in the morning, following initiation of light, which is accompanied by increased HSPC egress and the other at night after darkness, which is associated with reduced HSPC egress. In both peaks HSPC begin to cycle and differentiate via up-regulation of reactive oxygen species (ROS) however, the night peak had lower ROS levels. Concomitant with the peak of primitive stem and progenitor cells, we also observed (to a larger extent in the night peak), expansion of a rare activated macrophage/monocyte αSMA/Mac-1 population. This population maintains HSPC in a primitive state via COX2/PGE2 signaling that reduces ROS levels and increases BM stromal CXCL12 surface expression (Ludin et al, Nat. Imm. 2012). We identified two different BM peaks in HSPC levels that are regulated by the nervous system via circadian changes in ROS levels. Augmented ROS levels induce HSPC proliferation, differentiation and motility, which take place in the morning peak; however, they need to be restored to normal levels in order to prevent BM HSPC exhaustion. In the night peak, HSPC proliferate with less differentiation and egress, and activated macrophage/monocyte αSMA/Mac-1 cells are increased to restore ROS levels and activate CXCL12/CXCR4 interactions to maintain a HSPC primitive phenotype. Additionally, S1P also regulates HSPC proliferation, thus mice with low S1P levels share reduced hematopoietic progenitor cells in the BM. Interestingly S1P is required more for the HSPC night peak since in mice with low S1P levels, HSPC peak normally during day time but not at darkness. We suggest that the first peak is initiated via elevation of ROS by norepinephrine that is augmented in the BM following light-driven cues from the brain (Mendez-Ferrer at al, Nature 2008). The morning elevated ROS signal induces a decrease in BM CXCL12 levels and up-regulated MMP-9 activity, leading to HSC proliferation, as well as their detachment from their BM microenvironment, resulting in enhanced egress. Importantly, ROS inhibition by N-acetyl cysteine (NAC) reduced the morning HSPC peak. Since norepinephrine is an inhibitor of TNFα, upon light termination norepinephrine levels decrease and TNFα levels are up-regulated. TNFα induces activation of S1P in the BM, leading to the darkness peak in HSPC levels. S1P was previously shown also to induce PGE2 signaling, essential for HSPC maintenance by the rare activated αSMA/Mac-1 population. Indeed, in mice with low S1P levels, we could not detect a peak in COX2 levels in these BM cells during darkness. We conclude that S1P not only induces HSPC proliferation via augmentation of ROS levels, but also activates PGE2/COX2 signaling in αSMA/Mac-1 population to restore ROS levels and prevent HSPC differentiation and egress during the night peak. We hypothesize that the morning HSPC peak, involves proliferation, differentiation and egress, to allow HSPC to replenish the blood circulation with new cells. In contrast, the second HSPC night peak induces proliferation with reduced differentiation and egress, allowing the renewal of the BM HSPC pool. In summary, we identified two daily circadian peaks in HSPC BM levels that are regulated via light/dark cues and concomitantly allow HSPC replenishment of the blood and immune system, as well as maintenance of the HSPC constant pool in the BM. Disclosures:No relevant conflicts of interest to declare.

Human and Murine β-Defensin-Derived Peptides Induce Rapid Mobilization Of Murine Hematopoietic Stem and Progenitor Cells Via Activation Of CXCR4 Signaling and CXCL12 Release

BackgroundRapid mobilization of hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to the peripheral blood by anti-CXCR4 agents such as AMD3100 is a complex process, which requires CXCL12 secretion, activation of proteolytic enzymes and supporting cellular compartments (Dar et. al, Leukemia 2011). Notably, components of innate immune system were also shown to be involved (Ratajczak et. al, Leukemia 2010). Human β-defensin-3 (hBD3) is an antimicrobial peptide possessing also anti-CXCR4 effects on human T cells in vitro (Feng et. al, JI 2006), suggesting its HSPC mobilizing potential. Here, we describe a novel approach for targeting CXCR4 in vivo by utilizing β-defensin-derived peptides, resulting in rapid HSPC mobilization. ResultsWhile AMD3100 blocked CXCL12-mediated signaling and migration of enriched BM mononuclear cells (MNCs) in vitro, we unexpectedly detected rapid phosphorylation of AKT, p38 and ERK1/2 in BM stromal cells (BMSCs). Interestingly, single administration of AMD3100 to mice resulted in enhancement of CXCR4 phosphorylation within minutes in both BM residing mesenchymal stem/progenitor cells (MSCs) and HSPCs, thus suggesting a CXCR4 agonistic activity. Aiming to test HSPC mobilizing potential of hBD3 and avoiding potential toxicity of systemic administration, we synthesized short linear peptides, comprising the C-terminal parts of hBD3 and the murine ortholog β-defensin-14 (mBD14), as well as a cyclic peptide of hBD3, comprising the same amino acids as the linear one, to serve as a control. All full-length β-defensins and tested peptides (both linear and cyclic) specifically bound CXCR4 (demonstrated by docking approach and anti-CXCR4 antibody competition assay) and efficiently blocked CXCL12-mediated activity of enriched BM MNCs in vitro including cell migration and CXCR4-dependent HIV infection. Intriguingly, full-length β-defensins and derived linear peptides (but not cyclic) revealed a strong stimulatory effect on BMSCs in vitro: triggering phosphorylation of AKT, p38 and ERK1/2 along with enhancing secretion of functional CXCL12. Administration of linear peptides to mice led to a fast activation of CXCR4 signaling in BMSCs and MSCs as well as in HSPCs accompanied by CXCL12 release to the circulation, increased activity of proteolytic enzymes and consequent rapid mobilization of progenitors as well as long-term repopulating stem cells. In addition, linear peptides augmented AMD3100-induced rapid mobilization. Importantly, the control cyclic peptide, which bound CXCR4 but failed to activate BMSCs in vitro, did not induce HSPC mobilization in vivo. Moreover, it inhibited both steady-state egress and AMD3100-induced mobilization of HSPCs. A series of in vivo inhibitory analyses confirmed dependence of hBD3- and mBD14-derived peptide-induced HSPC mobilization on the activation of CXCL12/CXCR4 axis and revealed involvement of uPA and JNK signaling as well as ROS generation. ConclusionsOur study demonstrated for the first time the capability of β-defensin-derived peptides to activate in vivo CXCL12/CXCR4 signaling in both hematopoietic and non-hematopoietic BM cells, leading to rapid HSPC mobilization. We suggest that activation of CXCR4 signaling in non-hematopoietic BM cells is crucial for inducing HSPC mobilization. Accordingly, CXCR4-binding agents capable of triggering CXCR4 signaling in non-hematopoietic BM cells in vitro, would induce rapid HSPC mobilization. The results presented here help to better understand the mechanisms of rapid HSPC mobilization and have the potential of improving existing clinical protocols to increase the yield of HSPC harvest for transplantation. Disclosures:Scadden:Fate Therapeutics: Consultancy, Equity Ownership.

Diabetes impairs hematopoietic stem cell mobilization by altering niche function.

Success with transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) in patients depends on adequate collection of these cells after mobilization from the bone marrow niche by the cytokine granulocyte colony-stimulating factor (G-CSF). However, some patients fail to achieve sufficient HSPC mobilization. Retrospective analysis of bone marrow transplant patient records revealed that diabetes correlated with poor mobilization of CD34+ HSPCs. In mouse models of type 1 and type 2 diabetes (streptozotocin-induced and db/db mice, respectively), we found impaired egress of murine HSPCs from the bone marrow after G-CSF treatment. Furthermore, HSPCs were aberrantly localized in the marrow niche of the diabetic mice, and abnormalities in the number and function of sympathetic nerve termini were associated with this mislocalization. Aberrant responses to β-adrenergic stimulation of the bone marrow included an inability of marrow mesenchymal stem cells expressing the marker nestin to down-modulate the chemokine CXCL12 in response to G-CSF treatment (mesenchymal stem cells are reported to be critical for HSPC mobilization). The HSPC mobilization defect was rescued by direct pharmacological inhibition of the interaction of CXCL12 with its receptor CXCR4 using the drug AMD3100. These data suggest that there are diabetes-induced changes in bone marrow physiology and microanatomy and point to a potential intervention to overcome poor HSPC mobilization in diabetic patients.

The Chemotactic Lipid S1P Regulates Hematopoietic Progenitor Cell Egress and Mobilization Via Its Major Receptor S1P1 and by SDF-1 Inhibition In a p38/Akt/mTOR Dependent Manner

AbstractAbstract 553Egress of hematopoietic stem and progenitor cells (HSPC) from the bone marrow (BM) reservoir to the circulation in steady state conditions is a key requirement for normal hematopoiesis. It is dramatically enhanced by G-CSF-induced mobilization, which is widely used for clinical HSPC transplantation. An interplay between cytokines, chemokines (mainly SDF-1 (CXCL12) and its major receptor CXCR4), adhesion molecules, matrix metalloproteinases and neurotransmitters, tightly regulate HSPC egress and mobilization. Recent observations indicate an essential role for sphingolipids, and particularly sphingosine-1-phosphate (S1P) and its major receptor S1P1 in leukocyte trafficking in vivo. Furthermore, several pharmacological agents that target S1P and S1P1 attenuate development of autoimmune and cardiovascular diseases as well as cancer. Based on these findings, we hypothesized that HSPC motility, both in steady state and in stress-induced conditions, is regulated by S1P/S1P1 signaling. We found that cells expressing S1P1 receptor are mainly located near sinusoids in the murine BM, suggesting involvement of S1P/S1P1 axis in HSPC steady state egress. To identify the role of S1P1 in HSPC homeostatic release, we injected mice with the inhibitor FTY720 and discovered a significant decrease in primitive Sca-1+/c-Kit+/Lineage- (SKL) cell numbers in the peripheral blood along with their accumulation in the BM, 24 hr post a single i.p injection. To examine the S1P/S1P1 axis involvement in stress induced mobilization, we tested S1P levels following G-CSF administration. S1P concentrations were decreased in BM supernatants and increased in the peripheral blood, suggesting the formation of a gradient towards the blood, with a potential HSPC mobilization capacity. Accordingly, a 5-fold decreased transcription level of sphingosine kinase 1 (Sphk1, S1P producing enzyme) and a milder increased transcription level of sphingosine phosphatase 1 (SPP1, S1P degrading enzyme) were observed in the BM of G-CSF treated mice. These changes in both S1P modulating enzymes expression levels were mediated by mTOR signaling, independent of the PI3K pathway. Another effect of G-CSF mobilization was enhancing the percentage of BM HSPC expressing surface S1P1 receptor, which was abolished upon inhibition of mTOR by Rapamycin. These findings imply that the reduction in S1P BM levels enabled increased S1P1 receptor expression and HSPC recruitment to the blood. Co-injections of FTY720 with G-CSF revealed decreased numbers of primitive SKL and immature colony-forming cells in the blood, indicating reduced HSPC mobilization. Accordingly, administration of G-CSF to Sphk1 KO mice, which have low S1P plasma concentrations, led to decreased mobilization of primitive SKL cells and progenitors to the blood. We also investigated the cross talk between S1P/S1P1 and SDF-1/CXCR4 axes. Disruption of the S1P/S1P1 axis during G-CSF administration (by co-injections of FTY720 or by using Sphk1 KO mice) reduced HSPC mobilization however, BM mononuclear cells obtained from these mice exhibited enhanced migration to a gradient of SDF-1 in vitro. These results imply that SDF-1/CXCR4 activation is not sufficient for HSPC mobilization. Previously, we have shown that CXCR4 neutralizing antibodies co-administrated on days 4 and 5 of G-CSF treatment, significantly but not completely inhibited HSPC mobilization (Petit et al., Nat Immunol, 2002). Interestingly, such treatment in Sphk1 KO mice completely inhibited mobilization and increased primitive SKL cells in the BM. These results suggest that S1P/S1P1 axis has an important role in parallel to SDF-1/CXCR4 axis during stress-induced mobilization since inactivation of both pathways resulted in total abrogation of HSPC recruitment to the blood. Finally, we show that S1P can inhibit SDF-1 transcription in murine BM stromal cells via activation of the p38/Akt/mTOR signaling pathway. Since SDF-1 reduction in the BM is essential for HSPC mobilization, S1P-induced inhibition of its transcription allows the progenitor cells to detach and migrate. Taken together, our findings reveal involvement of S1P and its major receptor S1P1 in HSPC egress and stress-induced mobilization. These findings may help broaden our understanding regarding the mechanisms behind HSPC motility and thus improve clinical mobilization protocols and drug development based on targeting the S1P/S1P1 axis. Disclosures:No relevant conflicts of interest to declare.

GSK3β Signaling Regulates the Motility of Hematopoietic Progenitors Via Prune.

AbstractAbstract 1553One of the hallmarks of hematopoietic stem and progenitor cells (HSPC) is their motility. In steady state, HSPC are mostly retained in the bone marrow (BM), allowing ongoing hematopoiesis, concomitantly with slow release to the circulation as part of homeostasis and host defense mechanisms. While stress-induced recruitment and clinical mobilization processes are extensively studied, steady state egress mechanisms are poorly understood. In this study, we demonstrate that inhibition of Glycogen Synthase Kinase 3b (GSK3β) directly or via upstream Insulin-like Growth Factor-1 (IGF-1) signaling limited murine HSPC egress to the circulation. Indeed, inhibition of GSK3β resulted in reduced HSPC migration capacity towards a gradient of the chemokine stromal derived factor-1 (SDF-1, also termed CXCL12) in vitro and was found to reduce HSPC mobilization by IGF-1 receptor antagonist treatment. Interestingly, GSK3β signaling also regulated SDF-1 transcription by BM stromal cells in vitro and in vivo, probably as part of HSPC maintenance, since murine CXCR4 signaling is essential for hematopoietic stem cell quiescence. We revealed that the involvement of GSK3β in directional HSPC motility is mediated by the downstream phosphodiesterase Prune. Prune, which is over-expressed in several human cancers, was recently found to localize in focal adhesion sites, promoting the motility of malignant cells. Herein, we show that Prune is also expressed in normal leukocytes, including HSPC. Accordingly, inhibition of Prune resulted in reduced SDF-1 induced migration of murine HSPC in vitro as well as reduced steady state egress in vivo. Prune activity was also shown to regulate the actin cytoskeleton by contributing to its polymerization. In general, highly regulated actin turnover is essential for spontaneous and directional motility mechanisms. Altogether, we present GSK3β and Prune as novel players in physiological HSPC motility, dictating an active rather than passive nature for steady state egress from the BM reservoir to the blood circulation as part of homeostasis. Disclosures:No relevant conflicts of interest to declare.

Novel Trafficking Routes for Hematopoietic Stem and Progenitor Cells

Bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) are known to enter the blood and to home back to the BM. However, whether these migratory HSPCs also follow extramedullary traffic routes is unknown. Our group has recently shown that mouse thoracic duct (TD) lymph contains clonogenic HSPCs that possess short- and long-term multilineage reconstitution capacity in primary and secondary transplantation assays. Using BM transplantation, homing experiments, and parabiotic mice, we established that TD HSPCs originate in the BM and traffic constitutively to multiple extramedullary tissues, where they reside for several days until entering draining lymphatics to return to the blood. While these migratory properties of HSPCs resemble those of lymphocytes, circulating HSPCs access different target tissues because, unlike lymphocytes, they do not require secondary lymphoid organs to recirculate. The egress of HSPCs from extramedullary tissues into lymph depends on sphingosine-1-phosphate (S1P) receptors, particularly S1P(1). We have shown that under physiological conditions migratory HSPCs contribute to the continuous restoration of specialized hematopoietic cells that reside in peripheral tissues. Upon exposure to toll-like receptor (TLR) agonists, migratory HSPCs proliferate locally within extramedullary tissues and generate innate immune effector cells. Thus, HSPCs can survey peripheral organs to replenish tissue-resident hematopoietic cells and act as a source of mature leukocytes during host defense against pathogens.

Immunosurveillance by Hematopoietic Progenitor Cells Trafficking through Blood, Lymph, and Peripheral Tissues

Constitutive egress of bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) into the blood is a well-established phenomenon, but the ultimate fate and functional relevance of circulating HSPCs is largely unknown. We show that mouse thoracic duct (TD) lymph contains HSPCs that possess short- and long-term multilineage reconstitution capacity. TD-derived HSPCs originate in the BM, enter the blood, and traffic to multiple peripheral organs, where they reside for at least 36 hr before entering draining lymphatics to return to the blood and, eventually, the BM. HSPC egress from extramedullary tissues into lymph depends on sphingosine-1-phosphate receptors. Migratory HSPCs proliferate within extramedullary tissues and give rise to tissue-resident myeloid cells, preferentially dendritic cells. HSPC differentiation is amplified upon exposure to Toll-like receptor agonists. Thus, HSPCs can survey peripheral organs and can foster the local production of tissue-resident innate immune cells under both steady-state conditions and in response to inflammatory signals.

Suprasynergistic Peripheral Blood Stem Cell Mobilization in Normal and Fanconi Anemia Knockout Mice by the Combination of G-CSF Plus the CXCR4 Antagonist AMD3100 and the CXCR2 Agonist GRO β

In mice, the CXCR4 antagonist AMD3100 and the CXCR2 agonist GROβ rapidly mobilizes short and long term repopulating hematopoietic stem and progenitor cells (HSPC). Synergy in mobilization is observed using GRO plus G-CSF or AMD plus G-CSF, and we have recently shown synergy in rapid mobilization using AMD plus GROβ. In general, a common feature of mobilization is that only a relatively small percentage of HSPC egress from marrow. We therefore evaluated whether added benefit in HSPC mobilization could be attained by using all three mobilizers in combination. Although this alters the paradigm of rapid mobilization, it addresses shortcomings of poor mobilization response, requirements for multiple aphereses and the need for large numbers of HSPC in transplant and gene therapy applications. We mobilized BALB/c mice with AMD (5 mg/kg SC, 60 min), GROβ (2.5 mg/kg SC, 15 min), G-CSF (100 ug/kg/day, bid, SC x 4 days) or the G-CSF regimen followed by GROβ, AMD or GRO+AMD administered on day 5 and harvest of peripheral blood 15 (GRO; GRO+AMD) or 60 (AMD) min later. Significant CFU-GM/mL blood were mobilized by G-CSF (4362±996), GRO (2562±396) and AMD3100 (991±121) used alone as expected. Single administration of GRO or AMD to mice mobilized by G-CSF and harvest of blood 15 (GRO) and 60 (AMD) min later, resulted in synergistic mobilization of (12,246±2751) and (12,379±953) CFU-GM, respectively. Rapid mobilization by simultaneous injection of GRO+AMD was similar in magnitude (10,709±1041) at 15 min post administration to mobilization by GRO or AMD in combination with a multiday G-CSF regimen. Administration of the combination of GRO+AMD to mice mobilized by G-CSF resulted in suprasynergistic mobilization of 32,510±3569 CFU-GM/mL after 15 min, representing ~5% of total marrow CFU-GM, with no adverse effects. Fanconi Anemia patients mobilize poorly to G-CSF. FancC −/− mice present a phenotype similar to FancC patients and mobilize poorly to G-CSF, which can be improved by the addition of AMD. We evaluated mobilization by GRO, AMD and G-CSF alone and in combination in +/+ C57Bl and FancC −/− mice using the regimens described above. Mobilization by G-CSF was 45% lower in FancC −/− mice (858±21) compared to +/+ controls (1451±80) and AMD+G-CSF synergistically mobilized CFU-GM more effectively in FancC −/− mice (5078±597) than controls (2981±267). Similarly, CFU-GM mobilization by GRO was lower in FancC −/− mice and GRO+G-CSF synergistically mobilized CFU-GM more effectively in FancC −/− mice. The combination of GRO+AMD mobilized CFU-GM within 15 min that was similar in magnitude to mobilization by AMD+G-CSF in wild type (2077±541 vs 2511±176) as well as FancC −/− mice (4924±577 vs 5078±1597). Mobilization by addition of the rapid acting combination of GRO+AMD to mice mobilized by G-CSF was suprasynergistic reaching 44,669±2974 and 41,068±5630 CFU-GM/mL blood in wild type and −/− mice, respectively. In preliminary studies, transduction of mobilized blood cells with FancC and transplant in FancC −/−mice demonstrated durable engraftment. These studies identify highly effective, rapid GRO+AMD mobilization regimens for standalone application in normal donors and combination regimens for potential application in patients who respond poorly to G-CSF or when large quantities of HSPC are required, for example in gene therapy applications.

Signals from the Sympathetic Nervous System Regulate Hematopoietic Stem Cell Egress from Bone Marrow

Hematopoietic stem and progenitor cells (HSPC), attracted by the chemokine CXCL12, reside in specific niches in the bone marrow (BM). HSPC migration out of the BM is a critical process that underlies modern clinical stem cell transplantation. Here we demonstrate that enforced HSPC egress from BM niches depends critically on the nervous system. UDP-galactose ceramide galactosyltransferase-deficient (Cgt(-/-)) mice exhibit aberrant nerve conduction and display virtually no HSPC egress from BM following granulocyte colony-stimulating factor (G-CSF) or fucoidan administration. Adrenergic tone, osteoblast function, and bone CXCL12 are dysregulated in Cgt(-/-) mice. Pharmacological or genetic ablation of adrenergic neurotransmission indicates that norepinephrine (NE) signaling controls G-CSF-induced osteoblast suppression, bone CXCL12 downregulation, and HSPC mobilization. Further, administration of a beta(2) adrenergic agonist enhances mobilization in both control and NE-deficient mice. Thus, these results indicate that the sympathetic nervous system regulates the attraction of stem cells to their niche.