Hematopoietic Stem And Progenitor Cells Proliferation Research Articles

Acute exercise mobilizes hematopoietic stem and progenitor cells and alters the mesenchymal stromal cell secretome.

Transplantation of hematopoietic stem and progenitor cells (HSPC), collected from peripheral blood, is the primary treatment for many hematological malignancies; however, variable collection efficacy with current protocols merits further examination into factors responsible for HSPC mobilization. HSPCs primarily reside within the bone marrow and are regulated by mesenchymal stromal cells (MSC). Exercise potently and transiently mobilizes HSPCs from the bone marrow into peripheral circulation. Thus the purpose of the present study was to evaluate potential factors in the bone marrow responsible for HSPC mobilization, investigate potential sites of HSPC homing, and assess changes in bone marrow cell populations following exercise. An acute exercise bout increased circulating HSPCs at 15 min (88%, P < 0.001) that returned to baseline at 60 min. Gene expression for HSPC homing factors (CXCL12, vascular endothelial growth factor-a, and angiopoietin-1) were increased at 15 min in skeletal muscle and HSPC content was increased in the spleen 48 h postexercise (45%, P < 0.01). Acute exercise did not alter HSPCs or MSCs quantity in the bone marrow; however, proliferation of HSPCs (40%, P < 0.001), multipotent progenitors (40%, P < 0.001), short-term hematopoietic stem cells (61%, P < 0.001), long-term hematopoietic stem cells (55%, P = 0.002), and MSCs (20%, P = 0.01) increased postexercise. Acute exercise increased the content of the mobilization agent granulocyte-colony stimulating factor, as well as stem cell factor, interleukin-3, and thrombopoeitin in conditioned media collected from bone marrow stromal cells 15 min postexercise. These findings suggest that the MSC secretome is responsible for HSPC mobilization and proliferation; concurrently, HSPCs are homing to extramedullary sites following exercise.

Hoxa cluster genes determine the proliferative activity of adult mouse hematopoietic stem and progenitor cells

AbstractDetermination of defined roles for endogenous homeobox (Hox) genes in adult hematopoietic stem and progenitor cell (HSPC) activity has been hampered by a combination of embryonic defects and functional redundancy. Here we show that conditional homozygous deletion of the Hoxa cluster (Hoxa−/−) results in a marked reduction of adult HSPC activity, both in vitro and in vivo. Specifically, proliferation of Hoxa−/− HSPCs is reduced compared with wild-type (WT) cells in vitro and they are less competitive in vivo. Notably, the loss of Hoxa genes had little impact on HSPC differentiation. Comparative RNA sequencing analyses of Hoxa−/− and WT hematopoietic stem cells (CD150+/CD48−/Lineage−/c-kit+/Sca-1+) identified a large number of differentially expressed genes, three of which (Nr4a3, Col1a1, and Hnf4a) showed >10-fold reduced levels. Engineered overexpression of Hoxa9 in Hoxa−/− HSPCs resulted in partial phenotypic rescue in vivo with associated recovery in expression of a large proportion of deregulated genes. Together, these results provide definitive evidence linking Hoxa gene expression to proliferation of adult HSPCs.

Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture.

Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

ER71/ETV2 Is Essential for Hematopoietic Regeneration after Injury

Hematopoietic stem and progenitor cell (HSPC) expansion and regeneration is requisite for effective hematopoietic transplantation and recovery from anticancer chemotherapy drug induced bone marrow injury. In chemotherapy-mediated hematopoietic injury model, we found that key developmental genes that are critical for HSPC generation were upregulated in HSPCs when treated with 5-FU. ETS transcription factor Er71/ETV2 is essential for the development of endothelial and blood cells during mammalian embryogenesis. Here, we generated different tissue specific Er71 conditional knockout (CKO) mice as well as transgenic mice that coexpress Er71, GATA2 and Scl (EGS) in a doxycycline inducible manner to investigate the Er71 role in adult hematopoiesis. While Vav-Cre; Er71 CKO and Tie2-Cre; Er71 CKO mice did not display any hematopoietic defects in homeostatic conditions, Vav-Cre; Er71 CKO bone marrow was significantly compromised in hematopoietic reconstitution potential compared to controls. In addition, Vav-Cre; Er71 CKO and Tie2-Cre; Er71 CKO but not VECadherin-Cre; Er71 CKO mice showed reduced HSPC proliferation and deficient HSPC and myeloid cell recovery after 5-FU injury. On the other hand, temporal Er71, GATA2 and Scl (EGS) coexpression promoted HSPC proliferation, hematopoietic reconstitution potential and regeneration after 5-FU injury. These results demonstrate that hematopoietic Er71 is critical for HSPC expansion and regeneration. Future studies warrant Er71 application for in vitro hematopoietic stem cell expansion and bone marrow regeneration in response to hematopoietic injury. DisclosuresNo relevant conflicts of interest to declare.

Zebrafish embryonic stromal trunk (ZEST) cells support hematopoietic stem and progenitor cell (HSPC) proliferation, survival, and differentiation

Forward genetic screens in zebrafish have been used to identify genes essential for the generation of primitive blood and the emergence of hematopoietic stem cells (HSCs), but have not elucidated the genes essential for hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation because of the lack of methodologies to functionally assess these processes. We previously described techniques used to test the developmental potential of HSPCs by culturing them on zebrafish kidney stromal (ZKS) cells, derived from the main site of hematopoiesis in the adult teleost. Here we describe an additional primary stromal cell line we refer to as zebrafish embryonic stromal trunk (ZEST) cells, derived from tissue surrounding the embryonic dorsal aorta, the site of HSC emergence in developing fish. ZEST cells encouraged HSPC differentiation toward the myeloid, lymphoid, and erythroid pathways when assessed by morphologic and quantitative reverse transcription polymerase chain reaction analyses. Additionally, ZEST cells significantly expanded the number of cultured HSPCs in vitro, indicating that these stromal cells are supportive of both HSPC proliferation and multilineage differentiation. Examination of ZEST cells indicates that they express numerous cytokines and Notch ligands and possess endothelial characteristics. Further characterization of ZEST cells should prove to be invaluable in understanding the complex signaling cascades instigated by the embryonic hematopoietic niche required to expand and differentiate HSPCs. Elucidating these processes and identifying possibilities for the modulation of these molecular pathways should allow the in vitro expansion of HSPCs for a multitude of therapeutic uses.

Abstract 941: G1T28-1, a novel CDK4/6 inhibitor, protects murine hematopoietic stem and progenitor cells from cytotoxic chemotherapy

Abstract G1T28-1 is a clinical stage, small molecule inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6). Hematopoietic stem and progenitor cells (HSPC) require CDK4/6 for proliferation, allowing the transient arrest of HSPC in the G1 phase of the cell cycle by CDK4/6 inhibition. The CDK4/6 transient arrest may reduce the sensitivity of HSPC to DNA damaging chemotherapies by limiting G1 to S-phase progression in the setting of unrepaired DNA damage. A reduction in chemotherapy-induced HSPC death would in turn reduce chemotherapy-induced myelosuppression (CIM), the major dose-limiting toxicity of most cytotoxic anti-cancer agents. G1T28-1 is highly potent, exhibits exquisite selectivity, and has favorable pharmacology allowing the induction of a predictable and well-defined transient arrest of HSPC with greater uniformity and distinct profile from the arrest induced by less potent and selective CDK4/6 inhibitors. To characterize HSPC inhibition induced by G1T28-1 treatment, we studied the compound's pharmacodynamic properties in mice. Murine HSPC proliferation was measured in vivo using EdU incorporation. This work showed that G1T28-1 induces a significant, rapid and reversible G1 arrest in all early hematopoietic lineages in a dose-dependent manner. Above a determined threshold dose, higher doses do not augment the percentage of specific HSPC fractions in G1, but instead extend the duration of the block of G1 to S-phase traversal of these populations. The degree of G1 arrest differed for specific HSPC fractions, multipotent progenitors, and early lymphoid progenitors being more sensitive than populations of early myeloid and erythroid progenitors. To determine the ability of G1T28-1 to ameliorate CIM, we assessed the effect of concomitant G1T28-1 administration in a well-characterized murine model of 5FU-induced myelosuppression. A single dose of G1T28-1 given 30-minutes prior to 5FU provided multilineage protection resulting in a more rapid recovery of complete blood counts (CBCs) compared to animals who received 5FU + vehicle. This effect persisted through multiple cycles of 5FU. After four cycles of 5FU, even at count recovery, the CBC's in animals treated with vehicle + 5FU were significantly worse compared to cycle one, whereas this chronic myelosuppressive effect was completely ameliorated in animals that received G1T28-1 prior to each 5FU dose. In conclusion, we have demonstrated that G1T 28-1 is a highly potent CDK4/6 inhibitor that causes robust and transient inhibition in a broad range of HSPC. Arrest at the time of 5FU administration significantly lessens CIM; benefitting all hematopoietic lineages and attenuating the acute myelosuppression of each cycle of chemotherapy as well as reducing a chronic reduction in peripheral blood counts caused by repeated, serial chemotherapy dosing. Citation Format: Jessica A. Sorrentino, Shenghui He, John E. Bisi, Patrick J. Roberts, Jay C. Strum, Norman E. Sharpless. G1T28-1, a novel CDK4/6 inhibitor, protects murine hematopoietic stem and progenitor cells from cytotoxic chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 941. doi:10.1158/1538-7445.AM2015-941

Targeting splenic hematopoietic stem cells in cardiovascular disease

Inflammation plays a crucial role in cardiovascular disease. For example, inflammatory monocytes, after being recruited to atherosclerotic plaques, secrete proteinases that erode the fibrous cap. This may lead to plaque rupture and subsequent myocardial infarction (MI). Patients with acute myocardial infarction have elevated levels of blood leukocytes [1]. Peripheral blood leukocyte count after MI correlates with in-hospital mortality, underscoring role of inflammatory cells in post-MI myocardial remodeling. The spleen provides steady flow of leukocytes to the infarct and atherosclerotic plaques. Since monocyte turnover at the sites of inflammation is very high [2], hematopoietic stem and progenitor cells (HSPC) differentiate into myeloid cells to keep up with the increased demand. MI drives splenic HSPC into the cell cycle [3] and induces their lineage differentiation. Subsequent imaging studies [4, 5] suggested splenocyte proliferation after acute myocardial infarction in patients. Although splenic myelopoiesis exaggerates inflammation in cardiovascular disease, the components of splenic hematopoietic niche are not well understood. Since macrophages play a major role in bone marrow HSC maintenance, we investigated if macrophages regulate hematopoiesis in the spleen [6]. Toward this end, we developed lipidoid nanoparticles containing siRNA against CD115, the receptor for MCS-F, which is involved in macrophage differentiation, proliferation and survival. CD115 knockdown significantly reduced splenic macrophage numbers in mice injected with the Toll like receptor ligand lipopolysaccharide. Concomitantly, splenic HSC, HSPC and granulocyte macrophage progenitor numbers were diminished after knocking down CD115. Levels of VCAM-1, an HSC retention factor, were reduced in the spleen; however, HSC proliferation and apoptosis were unaltered, indicating impaired retention of splenic HSC after CD115 knockdown. Reduced splenic retention was in line with elevated HSPC levels in the blood after siCD115 treatment. Additionally, we found similar diminished splenic HSC numbers after depletion of splenic macrophages in CD169-iDTR mice with diphtheria toxin, bolstering the hypothesis that macrophages maintain splenic HSC. CD115 knockdown curtailed myeloid cell supply to the infarct in mice on day 4 after coronary ligation. Furthermore, the treatment also abated inflammation in atherosclerotic lesions in ApoE−/− mice fed with high fat diet. This reduction of inflammation was likely a consequence of mitigated extramedullary myelopoiesis due to impaired HSC maintenance after CD115 knockdown. In agreement with reduced inflammation, the treatment reduced plaque size and necrotic core area, and thickened fibrous cap. These are features of stable atherosclerotic plaques. Since VCAM-1 was the only HSC retention factor that was downregulated in splenocytes after CD115 knockdown, we investigated which hematopoietic cells express VCAM-1. Out of all hematopoietic cells tested, only macrophages expressed VCMA-1 at high levels. An adoptive transfer experiment revealed that splenic HSPC reside in close proximity with VCAM-1+ macrophages. In fact, more than 90% of HSPC reside within 10 μm distance from this macrophage subset. These data indicate that VCAM-1+ macrophages maintain splenic HSC. To investigate this further, we formulated siRNA against VCAM-1 in lipidoid nanoparticles which enrich in macrophages but not endothelial cells. VCAM-1 knockdown in macrophages significantly reduced myelopoiesis and HSC numbers in the spleen. Of note, the treatment did not change HSC numbers in the bone marrow, indicating involvement of other cell types and redundant mechanisms of retention for bone marrow HSC maintenance. Further, macrophage-specific VCAM-1 knockdown reduced inflammation in atherosclerosis and improved characteristics of stable atherosclerotic plaques. In summary, the study suggests that VCAM-1+ macrophages are important components of splenic HSC niche. In absence of VCAM-1 or splenic macrophages, HSC depart from the spleen, resulting in decreased myelopoiesis after MI and in mice with atherosclerosis. We previously showed that MI-induced splenic HSPC proliferation is stem cell factor-dependent [3]. Endothelial cells are major source of stem cell factor, which maintain HSC in the bone marrow [7]. However, it is not known if SCF secreted by splenic endothelial cells after MI triggers myelopoiesis. The bone marrow HSC niche is a complex microenvironment and orchestrated by different cells such as nestin+ mesenchymal stem cell, endothelial cells and regulatory T cells. It remains to be investigated which splenic cells besides macrophages maintain HSC.

MicroRNAs and Metabolites in Serum Change after Chemotherapy: Impact on Hematopoietic Stem and Progenitor Cells.

Hematopoietic regeneration after high dose chemotherapy necessitates activation of the stem cell pool. There is evidence that serum taken after chemotherapy comprises factors stimulating proliferation and self-renewal of CD34+ hematopoietic stem and progenitor cells (HSPCs) – however, the nature of these feedback signals is yet unclear. Here, we addressed the question if specific microRNAs (miRNAs) or metabolites are affected after high dose chemotherapy. Serum taken from the same patients before and after chemotherapy was supplemented for in vitro cultivation of HSPCs. Serum taken after chemotherapy significantly enhanced HSPC proliferation, better maintained a CD34+ immunophenotype, and stimulated colony forming units. Microarray analysis revealed that 23 miRNAs changed in serum after chemotherapy – particularly, miRNA-320c and miRNA-1275 were down-regulated whereas miRNA-3663-3p was up-regulated. miRNA-320c was exemplarily inhibited by an antagomiR, which seemed to increase proliferation. Metabolomic profiling demonstrated that 44 metabolites were less abundant, whereas three (including 2-hydroxybutyrate and taurocholenate sulphate) increased in serum upon chemotherapy. Nine of these metabolites were subsequently tested for effects on HSPCs in vitro, but none of them exerted a clear concentration dependent effect on proliferation, immunophenotype and colony forming unit formation. Taken together, serum profiles of miRNAs and metabolites changed after chemotherapy. Rather than individually, these factors may act in concert to recruit HSPCs into action for hematopoietic regeneration.

Conversion of Danger Signals into Cytokine Signals By Hematopoietic Stem and Progenitor Cells for Regulation of Stress-Induced Hematopoiesis

Mammals respond to infection by rapid production of innate immune cells. Hematopoietic stem and progenitor cells (HSPCs) have the ability to respond to pathogens directly through toll-like receptors. However, how the pathogen sensing ability of HSPCs may contribute to immune cell output remains unknown. Using a novel single-cell proteomic platform and mouse genetic models, we have shown that in response to toll-like receptor stimulation, short-term HSCs and multipotent progenitor cells produce copious amounts of diverse cytokines through NF-κB signaling. Interestingly, the cytokine production ability of HSPCs trumps mature immune cells in both magnitude and breadth. Among cytokines produced by HSPCs, IL-6 is a particularly important regulator of myeloid differentiation and HSPC proliferation. This is especially important in mediating rapid early myeloid cell recovery during neutropenia after chemotherapy or HSC transplant. This study has uncovered an important property of HSPCs that enables them to convert danger signals into versatile cytokine signals for the regulation of stress-induced hematopoiesis. To translate these findings into human hematopoietic system, we have recently developed a human cytokine chip and our preliminary analysis suggests that purified human bone marrow HSCs also have the capacity to secrete cytokines. DisclosuresNo relevant conflicts of interest to declare.

Gtpase Immunity-Associated Protein Family Member 4 Contributes to the Enhanced Expansion of HSPCs in RUNX1 Deficient Mice

RUNX1 is the transcription factor described as the master regulator of hematopoiesis. Due to its central role during blood development, numerous RUNX1 mutations have been reported in hematologic abnormalities. Mice null for Runx1 die during embryogenesis, lacking definitive HSCs. Conditional Runx1Δ/Δ mice are viable, but exhibit a variety of blood abnormalities. The most salient defect in these Runx1Δ/Δ mice is expansion of the hematopoietic stem and progenitor cell (HSPC) population, measured as an increase in number of lineage negative, Sca1 positive, cKit positive (LSK) cells. A shortened form of RUNX1 (RUNX1SF) lacking the C-terminal and part of the N-terminal domain (41-214) acts as a dominant negative regulator of RUNX1 and hence also models RUNX1 loss-of-function.A differential gene expression analysis of HSPCs derived from Runx1Δ/Δ compared to wild type mice uncovered GTPase immunity-associated protein family member 4 (GIMAP4) as one of the genes most highly upregulated. Previous studies have focused almost exclusively on the role of GIMAP4 as a pro-apoptotic protein during T-cell development. This study illuminates a novel non-apoptotic role of GIMAP4 in a formerly unstudied HSPC context. Runx1Δ/Δ mice were crossed with Gimap4-/- mice to generate a double knockout (dKO) mouse line. These dKO mice exhibited attenuated HSPC proliferation in comparison to Runx1Δ/Δ mice, suggesting that GIMAP4 functions in this HSPC expansion phenotype. BMT experiments using lethally irradiated C57 mice and RUNX1SF transduced wild type versus Gimap4-/-bone marrow confirmed this result.GIMAP4 also worked independently and coordinately with RUNX1 to influence individual progenitor populations. Common lymphoid progenitors (CLP) were affected only by GIMAP4. Gimap4-/- mice exhibited an expansion of the CLP population, consistent with its pro-apoptotic role in lymphoid populations. Conversely, both RUNX1 and GIMAP4 coordinately exerted an effect on myeloid progenitor populations. Runx1Δ/Δ mice harbored expanded granulocyte-macrophage progenitor (GMP) and common myeloid progenitor (CMP) populations. This expansion was not observed when GIMAP4 was also ablated. This suggests a pro-proliferative role of GIMAP4 specifically in myeloid populations. These opposing roles of GIMAP4 in lymphoid versus myeloid cells suggest a more contextual, cell-specific role of this GTPase protein. Ultimately, this study provides insight into how RUNX1 and GIMAP4 may coordinate to maintain HSPC homeostasis. DisclosuresNo relevant conflicts of interest to declare.

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.

Related-to-receptor tyrosine kinase receptor regulates hematopoietic stem and progenitor sensitivity to myelosuppressive injury in mice

Maintaining a careful balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) is necessary for lifelong blood formation. Previously, we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a noncanonical Wnt ligand receptor termed 'related-to-receptor tyrosine kinase' (Ryk). Expression of Ryk on HSPCs invivo is associated with a lower rate of proliferation, and, following treatment with fluorouracil (5-FU), the percentage of Ryk(+/high) HSPCs increased and the percentage of Ryk(-/low) HSPCs decreased. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. We found that Ryk expression on HSPCs is associated with lower rates of apoptosis following 5-FU and radiation. Transient inhibition of Ryk signaling invivo resulted in increased hematopoietic-stem-cell proliferation and decreased hematopoietic-stem-cell function in bone marrow transplant assays. Furthermore, inhibition of Ryk signaling sensitized HSPCs to 5-FU treatment in association with increased levels of reactive oxygen species. Together, these results demonstrated an association between Ryk expression and survival of HSPCs following suppressive injury.

PRL2/PTP4A2 phosphatase is important for hematopoietic stem cell self-renewal.

Hematopoietic stem cell (HSC) self-renewal is tightly controlled by cytokines and other signals in the microenvironment. While stem cell factor (SCF) is an early acting cytokine that activates the receptor tyrosine kinase KIT and promotes HSC maintenance, how SCF/KIT signaling is regulated in HSCs is poorly understood. The protein tyrosine phosphatase 4A (PTP4A) family (aka PRL [phosphatase of regenerating liver] phosphatases), consisting of PTP4A1/PRL1, PTP4A2/PRL2, and PTP4A3/PRL3, represents an intriguing group of phosphatases implicated in cell proliferation and tumorigenesis. However, the role of PTP4A in hematopoiesis remains elusive. To define the role of PTP4A in hematopoiesis, we analyzed HSC behavior in Ptp4a2 (Prl2) deficient mice. We found that Ptp4a2 deficiency impairs HSC self-renewal as revealed by serial bone marrow transplantation assays. Moreover, we observed that Ptp4a2 null hematopoietic stem and progenitor cells (HSPCs) are more quiescent and show reduced activation of the AKT and ERK signaling. Importantly, we discovered that the ability of PTP4A2 to enhance HSPC proliferation and activation of AKT and ERK signaling depends on its phosphatase activity. Furthermore, we found that PTP4A2 is important for SCF-mediated HSPC proliferation and loss of Ptp4a2 decreased the ability of oncogenic KIT/D814V mutant in promoting hematopoietic progenitor cell proliferation. Thus, PTP4A2 plays critical roles in regulating HSC self-renewal and mediating SCF/KIT signaling.

Conversion of Danger Signals into Cytokine Signals by Hematopoietic Stem and Progenitor Cells for Regulation of Stress-Induced Hematopoiesis

During an infection, the body increases the output of mature immune cells in order to fight off the pathogen. Despite convincing evidence that hematopoietic stem and progenitor cells (HSPCs) can sense pathogens directly, how this contributes to hematopoietic cell output remains unknown. Here, we have combined mouse models with a single-cell proteomics platform to show that, in response to Toll-like receptor stimulation, short-term HSCs and multipotent progenitor cells produce copious amounts of diverse cytokines through nuclear factor κB (NF-κB) signaling. Interestingly, the cytokine production ability of HSPCs trumps mature immune cells in both magnitude and breadth. Among cytokines produced by HSPCs, IL-6 is a particularly important regulator of myeloid differentiation and HSPC proliferation in a paracrine manner and in mediating rapid myeloid cell recovery during neutropenia. This study has uncovered an important property of HSPCs that enables them to convert danger signals into versatile cytokine signals for the regulation of stress hematopoiesis.

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.

Hematopoietic Stem Cells and Their BM Stromal Microenvironment Share a Dynamic Inverse Metabolic State During Quiescence and Proliferation Via ROS Transfer Between The Two Populations

Hematopoietic stem and progenitor cells (HSPC) are mostly retained in the bone marrow (BM) in a ROSlow quiescent mode via adhesion interactions with stromal cells, while mature blood cells are generated on demand via ROShigh stem cell proliferation and differentiation. The metabolic state of BM stromal cells and their cross-talk with quiescent and cycling HSPC is poorly understood. The bioactive lipid Sphingosine 1-Phosphate (S1P) is an important inducer of ROS in HSPC, increasing their motility (Golan et al, Blood 2012). We report that by increasing S1P levels in the murine BM, we also increased HSPC levels via augmented ROS production and cell cycling. Unexpectedly, we discovered an opposite effect of S1P on BM stromal cells, which contained less mesenchymal stem and progenitor cells (MSPCs) and also lower ROS levels. To further test the opposite metabolic relationship between BM HSPC and stromal cells, we utilized 3 repeated (once every 24h) G-CSF stimulations, which induce HSPC proliferation without mobilization. As expected, we found increased HSPC levels in the BM accompanied by augmented ROS levels. Importantly, we found again the opposite effect between HSPC and BM stromal cells, i.e. reduced MSPC levels that were less cycling and contained lower levels of ROS. These results suggest an inverse metabolic state between BM HSPC and their stromal microenvironment. While HSPC utilize oxidative phosphorylation and ROS generation during proliferation and differentiation, their stromal microenvironment share a low metabolic state, most probably due to anaerobic glycolysis. Mimicking alarm situations by clinical G-CSF treatment blocks bone remodeling to allow HSPC development and mature blood replenishment. As expected, mice with reduced S1P levels due to lack of one of its biosynthetic enzymes (Sphingosine kinase 1), exhibit lower levels of BM HSPCs accompanied by reduced ROS levels. More importantly, these mice share an augmented population of undifferentiated, primitive ROSlow, quiescent non-motile HSCs identified as EPCR+/LSK cells, which results in higher levels of long-term HSC repopulation in transplanted wild type mice (with normal S1P levels). Concomitantly, S1P low mice have BM MSPCs with increased ROS levels and higher colony-forming unit fibroblasts (CFU-F) potential in vitro. We conclude that when HSPC are maintained quiescent due to a low energy metabolic state of anaerobic glycolysis, the BM stromal microenvironment utilizes a high energetic metabolism by oxidative phosphorylation and ROS generation. Our data suggests that the dynamic, inverse metabolic state between HSPC and the stromal microenvironment is induced by energy transfer between the two populations. HSPC can eliminate excess ROS post 5-FU treatment, by transferring ROS to BM stromal cells via connexin43 (Cx43) gap-junctions (Taniguchi et al, PNAS 2012). In agreement, we found that mice treated with a broad inhibitor of connexin gap-junctions (CBX), induced BM HSPC proliferation via ROS elevation, concomitant with down regulation of ROS in MSPCs. Interestingly, in mice with low S1P levels, CBX inhibition of connexins did not elevate HSPC ROS levels or their numbers, implying that cell contact blockage is essential, but not sufficient to induce proliferation. To induce HSPC proliferation and differentiation, an activator of ROS, such as S1P, is also needed to initiate cell cycle entry and blockage of Cx43, further inducing CXCL12 secretion to the circulation and HSPC migration. Our results imply that once ROS reaches a certain level in HSPC, it is scavenged by the stromal microenvironment to prevent exhaustion of the hematopoietic stem cell pool and in parallel to initiate new bone development and remodeling. Altogether, we have discovered a dynamic, opposite metabolic state between BM HSPCs and their supporting stromal microenvironment during quiescence, proliferation and differentiation of the two populations, which are both ROS regulated on a one population at a time basis. Thus, either blood cell production or bone development takes place at the expense of the other. This metabolic seesaw is regulated via ROS transfer from HSCs to their supportive microenvironment via Cx43 gap-junctions, which also regulates surface CXCL12 expression that is essential for stem cell quiescence. Disclosures:No relevant conflicts of interest to declare.

Cathecholamines Differently Regulate Human AML and Normal Hematopoietic Progenitor Cell Motility Via miR126 and RGS16

Abstract Introduction Motility, proliferation, bone marrow (BM) retention and egress to the circulation of hematopoietic stem and progenitor cell (HSPCs) are key elements in normal hematopoiesis and also in the pathogenesis of acute myeloid leukemia (AML). HSPCs are tightly regulated in the BM niche by various molecules. Among them, CXCL12 and its major receptor CXCR4, expressed by both HSPCs and BM niche components, govern HSPC retention in the BM. Administration of CXCR4 antagonists lead to mobilization of both normal HSPCs and leukemia-inducing stem cells (LICs) from the BM to the circulation. We have previously found that normal human HSPCs functionally express b2-adrenergic receptors (b2-AR) that are up-regulated by myeloid cytokines such as G-CSF. The catecholaminergic neurotransmitters epinephrine and norepinephrine (NE) activate both Wnt and GSK3β signaling pathways via b2-AR, leading to enhanced HSPC proliferation, motility and BM repopulation (Spiegel et al, Nat Immunol 2007; Lapid et al, JCI 2013). These findings indicate HSPC regulation by dynamic interactions of the sympathetic nervous and hematopoietic systems. However, the role of catecholamines in regulation of AML remains elusive. Results We found that several human AML cell lines from different FAB subtypes express b2-AR. NE, a b2-AR activating ligand, increased b2-AR expression and the CXCL12-induced migration of AML primary patients’ cells and cell lines. In addition, NE treatment significantly enhanced CXCL12induced actin polymerization, which drives most of the cellular movements. Looking for a downstream effector of b2-AR, we focused on RGS16, a G-protein signaling regulator, which negatively regulates CXCL12/CXCR4 axis (Berthebaud et al., Blood 2005). Concurrently with the increase in cell migration, NE decreased RGS16 expression (both in protein and mRNA level) in monocytic AML cells. However, no effect on either cell migration or RGS16 expression was observed in non-monocytic AML cells. These data provided evidence for the involvement of catecholamines in the regulation of AML cell migration and showed a correlation between AML FAB subtypes, cell motility and RGS16 expression. One of the regulators of RGS16 levels is miR126, which is highly expressed in AML and normal HSPCs and plays an important role in mobilization and proliferation of normal HSPCs. Indeed, in search for the mechanisms underlying the above observed differences, we found that the enhancing effect of NE on CXCL12-induced migration of monocytic AML cells was accompanied by up-regulation of miR126 expression concurrently with down-regulation of RGS16 expression, whereas in non-monocytic AML cells we observed the opposite effects, suggesting that NE differently regulates AML cells belonging to different FAB subtypes. Upon studying human normal HSPCs, we found that in steady state, normal cells express low levels of β2-AR and NE did not affect either RGS16 expression or CXCL12-induced migration of both mononuclear and CD34+ cells derived from human cord blood and BM. Conclusions Our results demonstrate that while normal and AML cells share common mechanisms that govern their motility, there are unrevealed yet mechanisms, apparently cell-type dependent, which uniquely lead to opposite effects in normal HSPCs, monocytic and non-monocytic AML cells. Altogether, these findings suggest that targeting of miR126 and RGS16 pathways by specific agonists and antagonists may serve as a new approach for selective eradication of LICs. Disclosures: No relevant conflicts of interest to declare.

Maintenance Of Bone Marrow Residency and Self-Renewal Of Leukemic Stem Cells By Cdc42 Gtpase

The leukemic stem cell (LSC) depends on specific interactions with extracellular matrix, soluble factors, and cellular components of the microenvironment, or niche. These interactions promote LSC self-renewal and survival, thus contributing to chemoresistance and treatment failure. Understanding the signaling pathways that promote LSC maintenance in response to niche interactions may reveal novel targets for therapy. Recent studies indicate a critical role for the small Rho GTPase, Cdc42, in the maintenance of normal hematopoietic stem and progenitor cells (HSPCs). Cdc42 coordinates actin cytoskeleton organization, adhesion, migration, self-renewal, cell polarity, proliferation, and survival of normal HSPCs in response to niche signaling through multiple cell surface receptors, including CXCL12/CXCR4, SCF/KIT, and fibronectin/integrin. Cdc42 activity is increased in both murine and human models of MLL-AF9 (MA9) acute myeloid leukemia (AML). Cdc42 expression is also increased in human patient AML samples across cytogenetic subtypes, compared to normal hematopoietic cell subsets, in analysis of curated datasets in the HemaExplorer database. In earlier work, we have shown that Cdc42 inhibition leads to peripheral mobilization of leukemia cells out of the marrow niche (Blood 114, 13). In the present study, we investigate whether Cdc42 inhibition also disrupts intrinsic LSC self-renewal. To interrogate Cdc42 in LSC self-renewal, MA9 cell lines were established following transduction of bone marrow HSPCs harvested from tamoxifen-inducible Cdc42 knockout mice, with Cre null donors as controls. Upon tamoxifen (TAM) treatment, Cdc42KO-MA9 cells had decreased CFU and small, diffuse colony morphology. Mice transplanted with untreated MA9 cells were divided to receive injections of TAM vs control. The Cdc42KO-MA9 cohort remains alive at over 180 days post-transplant, whereas vehicle control mice died of AML with latency similar to Cre null MA9 cell recipients (p<0.005). In vivo deletion of Cdc42 from MA9 leukemia in secondary recipients prolonged disease latency (p<0.005). AML cells recovered from vehicle control mice showed decreased growth in culture, reduced CFU content, and increased apoptosis following treatment with TAM to delete Cdc42. Cdc42KO-MA9 leukemia cells also had higher side scatter and Gr-1 expression, and decreased c-Kit, suggesting differentiation. These data indicate that Cdc42 is required for murine LSC maintenance. We used Tet-inducible shRNA targeting of Cdc42 in human cell lines expressing MA9 and mutant NRas (MA9/NRas). Cdc42 knockdown reduced MA9/NRas colony-forming ability, blocked actin polymerization and migration in response to CXCL12, and induced apoptosis. MA9/NRas cells co-expressing inducible Cdc42 shRNA and luciferase were transplanted into NSGS mice on doxycycline chow to induce knockdown. Bioluminescence imaging showed delayed AML progression in the knockdown group compared to non-targeting shRNA and regular chow controls. Thus, Cdc42 deficiency in human MA9 LSC reproduces the phenotype seen in the mouse genetic model. We used a novel small-molecule Cdc42-activity specific inhibitor, CASIN, to test pharmacologic inhibition of Cdc42 in AML. Consistent with knockdown data, in vitro CASIN treatment blocked MA9 cell colony-forming ability, actin polymerization, and migration. CASIN treatment led to specific induction of apoptosis in MA9 cells, while normal human umbilical cord blood CD34+ cells showed no significant toxicity in the dose range tested. Together, these studies show that Cdc42 signaling is critical to intrinsic LSC self-renewal and engagement of the niche, and Cdc42 inhibition represents a rational therapeutic principle to target LSC maintenance. Disclosures:No relevant conflicts of interest to declare.

Hmga2 Is a RUNX1 Target Gene In Hematopoietic Stem and Progenitor Cells

RUNX1, also known as AML1, is a DNA binding transcription factor that is expressed in hematopoietic stem and progenitor cells (HSPCs). As demonstrated by several mouse models, RUNX1 is necessary for definitive hematopoiesis and proper homeostasis of HSPCs. Furthermore, mutations of RUNX1have been implicated in patients with a variety of blood-related malignancies and disorders. These findings have established RUNX1 as a master regulator of hematopoiesis. As a transcription factor, RUNX1 exerts its function in hematopoiesis by binding to regulatory regions in order to guide the expression of its direct target genes. Most confirmed RUNX1 target genes are mainly expressed in differentiated blood cells. Direct targets of RUNX1 in HSPCs, however, have largely remained unexplored. Identifying direct target genes of RUNX1 offers an insightful view of how this master regulator influences HSPC function. To elucidate RUNX1 target genes in HSPCs, we have analyzed gene expression signatures from wildtype and RUNX1-deficient HSPCs (Lineage-/cKit+/Sca1+) in a previous report (Matsuura et al., Blood, 2012). With the goal of continuing the characterization of RUNX1 target genes, in this current study, we performed genome-occupancy analysis with chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) using RUNX1 antibodies and a murine HSPC cell line. Bioinformatics analysis of the ChIP-seq data revealed 6370 significant RUNX1 binding peaks (<1% FDR). The majority of these peaks were located in areas outside of promoter regions. The top de novo generated sequence motif from these peaks corresponds with the known RUNX binding consensus motif TG(T/C)GGT, suggesting that our ChIP-seq dataset is highly reliable. The combination of differential gene expression and RUNX1 genome occupancy data has revealed a list of candidate RUNX1-regulated target genes. We hypothesize that RUNX1 directly modulates the expression of these genes in normal hematopoiesis.One of the genes identified is Hmga2. We observed three RUNX1 binding peaks in the upstream, intron, and downstream regions relative to the Hmga2 gene locus. Furthermore, we confirmed strong up-regulation of Hmga2 in RUNX1-deficient HSPCs using reverse transcription coupled with quantitative polymerase chain reaction. HMGA2, also known as High Mobility Group AT-hook 2, is a non-histone chromatin protein. Its expression is highest during embryonic development and in undifferentiated cells. Over-expression of HMGA2 in transgenic mice or in bone marrow transplantation models have been reported to cause expansion of HSPCs. These reports indicate that HMGA2 is a significant mediator of HSPC proliferation. Interestingly, a major characteristic of mice without RUNX1 in their hematopoietic cells is the expansion of HSPCs, suggesting that HMGA2 may contribute to this phenotype. To further validate Hmga2 as a RUNX1 target gene, we cloned the Hmga2 promoter sequence and DNA fragments corresponding to the three RUNX1 binding peaks into luciferase reporter constructs and performed transfection studies using K562 and 293T cells. Interestingly, while these studies demonstrated strong responses to RUNX1 in promoter-luciferase assays, the effect of RUNX1 on Hmga2 promoter activity in these two cell lines was opposite. In addition, eliminating two RUNX binding motifs in the Hmga2 promoter did not affect RUNX1-mediated promoter-luciferase activity, indicating that there are additional regulatory mechanisms that may be important for RUNX1's effect on the Hmga2 promoter. To examine the function of the three regions containing RUNX1 binding peaks in the Hmga2 gene locus, we also used luciferase reporter constructs including these regions in transfection studies. Increase of transcriptional activity was detected in the presence of the two regions that were upstream and downstream of the Hmga2 gene, suggesting that RUNX1 can act as a positive regulator through these regions. In contrast, the RUNX1 binding fragment in the intron region of Hmga2 reduced promoter-luciferase activity. This outcome indicates that RUNX1 acts as a suppressor through the Hmga2 intron element. In summary, these results establish Hmga2 as a novel RUNX1 target gene in HSPCs and mark the first study of the transcriptional regulation of the Hmga2 gene by RUNX1 through differential control regions. Disclosures:No relevant conflicts of interest to declare.

The Ryk Receptor Regulates Hematopoietic Stem and Progenitor Cell Proliferation and Their Sensitivity To Myeloablative Agents

The molecular mechanisms that control the balance between quiescence and proliferation of hematopoietic stem and progenitor cells (HSPCs) are critical for maintaining life-long hematopoiesis. In a recent study (Povinelli, et al. Stem Cells, In Press, 2013) we demonstrated that the Wnt5a ligand inhibits HSPC proliferation through a functional interaction with a non-canonical Wnt ligand receptor termed Related to Receptor Tyrosine Kinase (Ryk). Expression of Ryk on HSPCs in vivo was associated with a decreased rate of proliferation. Following treatment with fluorouracil (5-FU), the percentage of Ryk+ HSPCs increased at the expense of Ryk-/low HSPCs. Based on these data, we hypothesized that one function of the Ryk receptor is to protect HSPCs from the effects of myeloablative agents. To test this hypothesis, we injected 6-8 week old C57BL/6 mice with 150 mg/kg of 5-FU and analyzed bone marrow 48 hours later for the presence of apoptotic HSPCs, defined as lineage negative (Lin-), Sca-1+, CD48- cells positive for active caspase-3. There was a 2.5-fold decrease in the percentage of apoptotic Ryk+ HSPCs (12.9 ± 1.7%) compared to Ryk-/low HSPCs (32.4 ± 5.3%, p < 0.001, n = 3). To test whether this effect was limited to 5-FU, we performed a similar study in which we irradiated C57BL/6 mice with 3 cGy of total body irradiation (TBI) and analyzed bone marrow 72 hours later for apoptotic HSPCs (for this experiment, defined by a Lin-, c-kit+, Sca-1+, CD150+, CD48- immunophenotype or LSK, SLAM). Comparable to the effects of 5-FU, there was a significant 3.0-fold reduction in the percentage of apoptotic Ryk+ HSPCs (3.1 ± 0.2%) compared to Ryk-/low HSPCs (9.2 ± 1.5%, p < 0. 001, n = 3) in mice receiving 3 cGy TBI. These results demonstrated an association between Ryk expression and survival of HSPCs following myeloablative injury. To determine whether in vivo targeting of the Ryk receptor would increase the sensitivity of HSPCs to myeloablative injury, we utilized a neutralizing rabbit anti-Ryk antibody (α-Ryk). We injected C57BL/6 mice with 5 mg/kg α-Ryk or rabbit IgG isotype for 2 consecutive days. Twenty-four hours after the second dose, we determined the frequency and cell cycle status of LSK SLAM cells. Treatment with α-Ryk significantly increased the percentage of LSK SLAM cells in the S/G2/M phases compared to control (α-Ryk: 17.8 ± 2.2%; isotype IgG: 11.6 ± 2.7%, p < 0.05, n = 3). This was associated with a decrease in the percentage of LSK, SLAM cells in G1 following treatment with α-Ryk (α-Ryk: 40.5 ± 3.2%, isotype IgG: 51.3 ± 2.2; p < 0.01, n = 3). The percentage of G0 LSK SLAM cells was unchanged (α-Ryk: 37.9 ± 2.6, isotype IgG: 35.7 ± 3.1% n = 3) indicating that inhibiting Ryk promoted the exit of LSK SLAM cells from G1. Treatment with α-Ryk also increased the percentage of whole bone marrow cells expressing the LSK SLAM phenotype by 1.4-fold compared to controls (p < 0.05, n = 3). To determine if α-Ryk treatment altered HSPC function, we transplanted whole bone marrow cells from C57BL/6 mice treated with two days of α-Ryk or isotype IgG at a 1:1 ratio with whole bone marrow from untreated Ubc-GFP transgenic mice into lethally irradiated B6.SJL mice. Four weeks after transplant, we analyzed peripheral blood cells for the percentage of CD45.2+ GFP- cells. There was no difference in engraftment by transplanted bone marrow cells from mice treated with α-Ryk or isotype IgG (α-Ryk: 61.6 ± 6.1% n = 4, isotype IgG: 52.8 ± 13.6%, n = 5), indicating that the neutralizing antibody does not inhibit short-term HSPC function on its own. We then tested whether blocking Ryk function resulted in greater sensitivity of HSPCs to 5-FU. We treated B6.SJL mice with 5 mg/kg α-Ryk or isotype IgG for 2 consecutive days, followed by 150 mg/kg of 5-FU. Forty-eight hours after 5-FU treatment, we transplanted 2x106 C57BL/6 whole bone marrow cells into treated B6.SJL mice without additional conditioning. Four weeks after transplant, we determined the percentage of donor-derived CD45.2+ peripheral blood cells. Treatment of recipient mice with α-Ryk prior to 5-FU treatment resulted in increased engraftment of donor bone marrow by 3.6-fold compared to isotype (p < 0.05, n = 5), suggesting that inhibition of Ryk resulted in increased elimination of host HSPCs by 5-FU. Collectively, these data suggest a model in which inhibition of the Ryk receptor results in increased proliferation of HSPCs, rendering them more sensitive to the effects of myeloablative agents such as chemotherapy or TBI. Disclosures:No relevant conflicts of interest to declare.