ALBERT

Description: Whether hematopoietic stem cells (HSCs) home selectively to bone marrow (BM) early after transplantation remains an issue of debate. Better understanding of homing mechanisms may benefit BM transplantation protocols in cases of limited graft cell number or nonmyeloablative conditioning regimens. Using flow cytometry and serial transplantation to stringently identify HSCs, trafficking patterns of long-term engrafting cells were mapped between BM and spleen early after transplantation. Low-density BM cells were tracked in irradiated or nonirradiated mice 1, 3, 6, and 20 hours after transplantation, at which time recipient BM and spleen were analyzed for recovery of primitive donor cells by phenotype and adhesion molecule expression. In addition, phenotypically defined HSC-enriched or HSC-depleted grafts were tracked 20 hours after transplantation in recipient BM and spleen and analyzed for recovery and long-term repopulating potential in mice undergoing serial transplantation. Regardless of irradiation status, recovery of donor Sca-1+ lin- cells was higher at most time points in recipient BM than in spleen, while recovery of total Sca-1+ cells was variable. A significantly higher percentage of BM-homed donor Sca-1+ cells expressed CD43, CD49e, and CD49d 20 hours after transplantation than spleen-homed cells, which contained significantly more non-HSC phenotypes. Furthermore, BM-homed cells were significantly enriched for cells capable of secondary multilineage hematopoiesis in mice undergoing serial transplantation compared with spleen-homed cells. These results support the notion of specific homing of HSCs to BM by 20 hours after transplantation and provide a basis for the enhanced engraftment potential afforded some Sca-1+ lin- cells subfractionated on the basis of adhesion molecule expression.

Description: Although it is clear that chemokines, cytokines and adhesion molecules play an essential role in regulating hematopoietic stem and progenitor cell (HSC/P) self-renewal, lineage commitment, apoptosis and mobilization, the intracellular signals that regulate these processes are poorly defined. Here, we demonstrate that the deficiency of hematopoietic specific Src family kinases (SFKs) in Lin- HSC/Ps results in reduced chemotaxis and adhesion via CXCR4 and β1 integrins, respectively (n=3, p

Description: Background: We previously reported a pattern of lymphocyte expansion in transplant recipients prior to neutrophil engraftment following cyclophosphamide-fludarabine (Cy/Flu) nonmyeloablative allotrans-plantation (NMAT) in humans. T-cells and NK with approximately 85-90% donor chimerism were the predominant leukocyte population detectable in peripheral circulation in patients with acute myeloid leukemia (AML)/myelodysplasia (MDS) between days +6 and +8. This observation appears unique and reproducible and the mechanism is unknown. In an exploratory study, we analyzed TH1, TH2 and homeostatic cytokines present on day +7 in the serum of engrafting patients by Multiplex. Methods: Sixteen patients with a variety of hematological malignancies who underwent Cy/Flu NMAT according to a phase II protocol (NCT00975975) at Indiana University between 2009 and 2011 were included. Frozen serum samples collected from patients on day +7 were thawed and cytokine levels measured using a commercially available multiplex platform (MPXHCYTO-60K, Millipore Corp, Billerica, MA, USA). Samples were run in triplicate and compared against sample matrix alone; sample matrix alone was spiked with known levels of the specific cytokines and standardized quality controls revealed low and high ranges for each cytokine. Clinical parameters included age, diagnosis, donor source, stem cell dose, daily temperature, serum albumin and days to engraftment. Data were analyzed using SAS. Results: Median age was 60 years. Hematological diseases included acute myeloid leukemia/myelodysplasia (n=8), concomitant myelodysplasia and multiple myeloma (n=1), chronic lymphocytic leukemia (n=3), non-Hodgkin’s lymphoma (n=1), cutaneous T-cell lymphoma (n=1), acute lymphoblastic leukemia (n=1) and primary myelofibrosis (n=1). Patients received peripheral blood mononuclear cell grafts from matched related (n=8) or unrelated (n=8) donors. Median CD34+ dose was 4.7x108 cells/kg (range 2.0-8.1) and median CD3+ dose was 1.7x108 cells/kg (range 0.8 to 4.3). Median time to engraftment was 13.5 days (range 10 to 18). Table-1 summarizes clinical features and levels of various cytokine at day +7. Ten patients had a fever (〉38.2oC) by day +7. Median baseline (day -7) albumin was 3.5 mg/dl (range 3.1 to 4.1). We observed a median decrease in albumin by 1.0 mg/dl (range 0.1 to 1.6; P38.2C Alb 500IFNγTNFαIL4IL6FLT3LIL2IL7IL10IL15sIL2RαIL8TH1TH2Growth FactorsOther*Cell dose ( X108/kg)by Day +7pg/dL1MUD3.03.4YesYes13N/AN/AN/AN/AN/AN/AN/AN/AN/AN/AN/A2MRD2.42.2NoYes14060271263005923375323MUD4.70.8NoNo15070053200329225204MUD6.11.7YesYes1212203411290070261230285MRD5.91.2NoNo171415011599100863351533736MRD4.41.5YesNo150202210624004613732751547MRD5.21.4YesYes11070023300613481218MRD8.12.3YesYes11N/AN/AN/AN/AN/AN/AN/AN/AN/AN/AN/A9MRD3.52.6YesYes17N/AN/AN/AN/AN/AN/AN/AN/AN/AN/AN/A10MUD4.70.9YesYes1551923174152328106416093144314211MRD5.01.9NoNo165220688933027296452412MUD2.04.3YesYes133099059506183317511253913MUD5.02.0NoYes184234072129340902111112123614MUD4.21.6YesYes11106106485526230380280773570715MUD6.01.0YesYes10725399514120174895826539916MRD4.71.7NoNo110110151036001563172316 MUD: matched unrelated donor, MRD: matched related donor, Alb: Albumin, ANC: absolute neutrophil count, N/A: not available *other activation/inflammatory cytokines Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2443 Poster Board II-420 Transplanted hematopoietic progenitor cells (HPC) and stem cells (HSC) provide short- and long-term hematopoietic support, respectively, in myeloablated recipients after transplantation. Despite the reliance on these cells for successful clinical engraftment and reconstitution of transplant recipients, little is known regarding their proliferation kinetics in vivo during the period of engraftment, or how this relates to the vast literature describing steady state hematopoiesis. We have previously established methodology that can track donor HSC and HPC in mice after transplantation using retention and loss of CFSE fluorescence to identify CFSEbright and CFSEdim cells, respectively. Cells identified as CFSEbright on d5 post-transplantation were shown to be exclusively enriched for donor long-term repopulating potential, thus comprising all the HSC within the donor cell population. In the current study, we used this methodology to examine the long-term repopulating potential and progenitor activity of CFSEbright, CFSEmid, and CFSEdim cells isolated from primary recipients on days 3, 5, 7, and 10 after transplantation of low density bone marrow (LDBM) cells. We aimed to determine when HSC activity moved from CFSEbright cells into the CFSEmid, as a means of estimating the time point at which donor HSC undergo self-renewal divisions in recipient BM. Likewise, using clonogenic assays, HPC content of the three CFSE fractions was followed to determine the kinetics and nature of proliferation of donor progenitor cells. As expected, the percentage and absolute number of CFSEbright and CFSEmid cells decreased by day 10 to approximately 1-10% of day 3 values, while CFSEdim cells increased ∼200-fold to comprise 〉95% of donor cells by day 10 (n=14-16 mice/time point). Interestingly, when the HPC content of the various CFSE populations was examined, all HPC activity at day 3 post-transplant resided in the CFSEmid cells, suggesting that HPC divide rapidly upon transplantation and leave the CFSEbright pool within 1-2 days. Progenitor activity began to appear in the CFSEdim population by d5 post-transplant, increasing 5- to 20-fold in absolute number by day 10, roughly paralleling the increase in absolute number of CFSEdim cells during this same time frame. While the frequency of total HPC in CFSEmid and CFSEdim populations was similar to that of steady state LDBM, 5- to 15-fold more of these progenitors were identified as CFU-GEMM and HPP-CFC compared to steady state BM, suggesting that engrafting cells expand their primitive HPC content at a faster rate than steady state BM. In contrast to the rapid proliferation kinetics of engrafting HPC, results of competitive transplantation studies of the various CFSE fractions suggest that long-term multi-lineage engraftment potential moves from the CFSE-bright to the CFSE-mid population around day 7-8 post-transplantation. Using the number of transplanted CFSE graft cells and their 6mo chimerism values to determine an enrichment factor for HSC potential, we estimate that d5 CFSEbright cells are 28-fold more enriched for HSC activity than steady state LDBM, while d7 CFSEmid cells are 8-fold more enriched. These data suggest that within the first 7 days post-transplant, 1 in 3.5 cell divisions of CFSEbright cells are self-renewal in nature. In contrast, using the same formula, CFSEdim cells were found to possess ∼1% of the HSC activity of steady state LDBM when analyzed up to 1 month post-transplantation, suggesting that CFSEdim cells are functionally weakened at these early time points post-transplant, and thus unable to provide significant chimerism in secondary recipients. Taken together, these data suggest that donor HSC undergo self-renewal divisions at approximately 1 week post-transplant and at a much higher rate than during steady state hematopoiesis. In addition, transplanted HPC were found to proliferate between 1-2 days post-transplant, and appear to give rise to a pool of progenitors 5 to 15-fold more enriched for primitive HPC than that present in steady state LDBM. These results add to our understanding of HSC/HPC engraftment and the kinetics of self-renewal and differentiation divisions in vivo, and may have clinical implications in designing methodologies to optimize hematopoietic engraftment and reconstitution. Disclosures: No relevant conflicts of interest to declare.

Description: Hematopoietic stem cell (HSC) numbers and function are reported to be dysregulated/compromised in old mice (e.g. ≥20 months of age: Cho, et. al., Blood 2008; Dykstra, et. al., J. Exp. Med. 2011; Sun, et. al., Cell Stem Cell 2014, amongst other papers). HSC numbers apparently increase with age, with decreased lymphoid production and compromised engrafting capability. However, what we know about aged HSCs is based on evaluation of these cells after they have first been collected and processed in ambient air. This is an hyperoxic environment, much increased in oxygen compared to the in vivo bone marrow (BM) hypoxic (~ 1-5 %) environment that the HSCs reside in. Moreover, there is little information on hematopoietic progenitor cells (HPCs) in terms of numbers and functional capacity in aged animals. Because of the need to better understand HSCs and HPCs in aging, where hematopoietic cell function appears to be compromised, and in context of blood disorders associated with aging, it is important to assess HSCs and HPCs removed from the body for analysis in situations as close as possible to that in which these cells find themselves in the body. It has recently become apparent that collection of BM cells from relatively young mice (e.g. 6-12 weeks of age), even for short periods (≤20 minutes) in ambient air, exposes these cells to the phenomenon of extra physiologic oxygen shock/stress (EPHOSS). EPHOSS triggers the opening of the mitochondrial permeability transition pore, resulting in enhanced production of mitochondrial reactive oxygen species that results in rapid cell differentiation, with decreased numbers of phenotypically- and functionally-defined long term (LT) repopulating, self-renewing HSCs and concomitant increases in rapidly cycling HPCs (Mantel, et. al. Cell 161:1553, 2015). By rigorous attention to detail in collection and processing mouse BM cells from young mice under constant hypoxic conditions of 3 % oxygen, it became clear that, in fact, there were on average 3-5 fold greater numbers of phenotyped- and functional LT HSCs. But, there were many fewer HPCs, and these HPCs were in a slow or non-cycling state when compared to cells collected and processed in ambient air, or when collected in 3% oxygen and then exposed to ambient air. We thus hypothesized that BM HSCs and HPCs from aged mice collected/processed in ambient air may not reflect their true numbers and functional characteristics in vivo. This led us to re-evalaute hematopoiesis in aged mice, compared to that in young mice, but in which BM cells were collected and processed for numbers and functions of HSCs and HPCs in a more physiological oxygen tension of 3 %, as reported (Mantel, et. al. Cell, 2015). We evaluated BM from CB6, Balb/c, and C57Bl/6 mice at 20-25 months vs. 6-16 weeks of age, collected/processed in ambient air or 3% oxygen. Collection in air demonstrated that older mice had 2.6-2.8 and 1.5-1.9 fold more LT- and short term (ST)-HSCs, with 1.8-2.2 fold fewer phenotypically-defined common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMPs) and common lymphoid progenitors (CLPs), and 2-3 fold fewer functional myeloid HPCs (CFU-GM, BFU-E, CFU-GEMM) than younger mice. Moreover, HPCs of older mice, as assessed by colony assay, were in a slow cycling state. In contrast, BM of young and old mice collected/processed in hypoxia, demonstrated similar numbers of LT-HSC, ST-HSCs, CMPs, GMPs, and CLPs. Moreover, while CFU-GM, BFU-E, and CFU-GEMM in young mice were decreased after hypoxic collection/processing and were in slow cycle, those of older mice were greatly increased in numbers, and were in rapid cycle. Engrafting and other cell and intracellular studies are ongoing, but it is clear that hematopoietic cell studies previously reported in aged mice will have to be re-evaluated for better mechanistic understanding of their actual numbers and cell and intracellular characteristics as reflected in an in vivo hypoxic environment. Disclosures Broxmeyer: CordUse: Other: SAB Member .

Description: Introduction: High dose total body irradiation (TBI) causes severe damage to the hematopoietic system, resulting in ablation of both red and white blood cells and a high probability of infection, hemorrhage, and death. This is known as the hematopoietic syndrome of the acute radiation syndrome (HS-ARS). TBI is used therapeutically, and incidental exposure through nuclear accidents or radical terrorism is a current threat for which preparation is critical. Safe, effective, and pragmatic agents against HS-ARS are needed. We have identified prostaglandin E2 (PGE2) as a promising medical countermeasure to promote hematopoietic recovery and increase survival from lethal irradiation. Here, using the PGE2 analog 16,16-dimethyl PGE2 (dmPGE2), pre-irradiation dosing strategies were assessed in survival studies with an established mouse model of HS-ARS, and time course analyses were performed to elucidate the early cellular events associated with PGE2 radioprotection. Methods: C57BL/6 mice (n = 20/group) were exposed to LD70/30 (872 cGy) or LD90/30 (904 cGy) and treated with one of the following four dmPGE2 dosing strategies: a single dose of 35 μg at time -30 min, -1 hr, or -3 hr, or a double-dose of 20 μg each at time -45 min and -15 min pre-irradiation. Thirty-day survival was evaluated. The double-dose regimen was further utilized to assess cellular effects over time following LD70/30. Peripheral blood (PB) and bone marrow (BM) cells were harvested on days 1, 2, 4, 7, and 10 post-irradiation (n = 3/group/day). BM was analyzed by flow cytometry with immunostaining for surface markers (Sca-1, c-Kit, CD150, CD48 and blood cell lineage markers), cell cycle (Hoechst/PyroninY), and apoptosis (AnnexinV/7-AAD). PB differentials were assessed by a veterinary hematology analyzer. Results: All dmPGE2 regimens conferred 100% survival to mice receiving LD70/30, and all regimens provided a distinct survival advantage to mice receiving LD90/30 with the double-dose strategy conferring 100% survival at LD90/30. The double-dose was then chosen to assess cellular effects of dmPGE2 throughout the critical 10-day time period following LD70/30. Within the BM, marker-defined HSC numbers were significantly preserved by day 1 post-irradiation compared to vehicle controls, with a drop to vehicle levels observed on day 2 only. Cell cycling frequency among primitive hematopoietic cells was also conserved on day 1, decreased on day 2, then restored to non-irradiated control levels by days 7-10, while that of the unprotected mice remained significantly lower. Differentiated BM cells (lineage marker-positive) were substantially less apoptotic by day 2 and, in significant contrast to the untreated mice, returned to control levels of basal apoptosis by days 7-10. Overall BM cell populations were found to be largely replenished within one week as compared to the vehicle-treated mice, which remained significantly depressed. Further, 73-88% of detectable BM cells from the untreated mice were non-viable by day 4 post-irradiation based on sub-diploid DNA content and did not recover through day 10; dmPGE2-treated mice maintained significantly greater BM cell viability at each time point, returning to only 17-26% sub-diploid by day 7 (non-irradiated controls demonstrated ~5% sub-diploid DNA content). A corresponding effect on PB resulted in significant rescue from neutropenia, anemia, and thrombocytopenia by or before day 10. Conclusions: A single treatment with PGE2 prior to lethal irradiation can facilitate virtually complete survival, though a double treatment regimen may be most effective; further work will test whether this may be primarily due to the combined higher dose or to the altered timing. This PGE2-mediated survival involves early protection of both primitive and differentiated hematopoietic BM cells, affecting cell number, cell cycling, and apoptosis. Early hematopoietic BM recovery subsequently rescues PB components, facilitating ultimate survival. With further study, PGE2 may serve to provide an effective radioprotectant with the potential to safeguard patients undergoing TBI therapy, as well as military personnel and first responders, from the risk of radiation-associated mortality. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2638 The highly proliferative nature of hematopoietic stem (HSC) and progenitor (HPC) cells, particularly during stress induced hematopoiesis, makes them highly sensitive to radiation, and in extreme circumstances results in the Hematopoietic Syndrome of the Acute Radiation Syndrome (HS-ARS). In addition to the therapeutic use of high dose total body irradiation (TBI), the proliferation of nuclear weapons, increasing use of nuclear power, and worldwide radical terrorism has resulted in a rising need and increased research emphasis on developing countermeasures to a radiological mass casualty event. HS-ARS is characterized by life-threatening neutropenia, thrombocytopenia and lymphocytopenia, and possible death due to infection and/or bleeding. While HSC and HPC are susceptible to radiation exposure, surviving populations of these cells can recover hematopoiesis if given critical time to repair DNA damage, self-renew, expand and differentiate. We previously reported (Hoggatt et al, Blood 2009) that PGE2 increases HSC self-renewal and expression of the anti-apoptotic protein Survivin, resulting in reduced apoptosis and increased HSC number. Since PGE2 production is increased following radiation exposure, and tumors over-producing PGE2 are radioresistant, we hypothesized that PGE2 production may be an endogenous mechanism for recovery from radiation damage, and that enhancement of PGE2 signaling could improve post-irradiation hematopoiesis and survival. Mid-lethally irradiated mice were treated with a single dose of the long-acting PGE2 analog, 16,16 dimethyl PGE2 (dmPGE2) or vehicle 6 hrs post-TBI and morbidity and mortality monitored for 30 days (n=20 mice/group). Treatment with dmPGE2 resulted in 95% survival (P=0.001) compared to only 50% survival in control mice. The number of marrow CFU-GM, BFU-E and CFU-GEMM were significantly higher in surviving mice from the dmPGE2 treated group compared to control mice (2.0±0.1 fold increase in CFC). While PGE2 is beneficial for HSC self-renewal and anti-apoptosis and our data clearly indicate that dmPGE2 treatment enhances hematopoietic recovery and survival post-TBI, we and others have previously shown that PGE2 is inhibitory to myelopoiesis. Therefore, we hypothesized that while exposure to PGE2 early after TBI is beneficial and can increase the number of surviving HSC, sustained exposure to PGE2 is inhibitory to HPC expansion, and may limit hematopoietic recovery. To test this hypothesis, we treated lethally irradiated mice with meloxicam, a cyclooxygenase inhibitor that blocks PGE2 production, for 4 consecutive days, starting either 6 hrs post-irradiation or delayed for 48 hours. While only 5% of control mice survived 30 days post-TBI, 35% of mice treated with meloxicam 6 hrs post-irradiation and 50% of mice receiving delayed meloxicam treatment survived. A faster and more robust recovery of white blood cells (WBC), neutrophils (PMN) and platelets (PLT) was observed at 15 and 30 days post-TBI with delayed meloxicam administration compared to control [15 days: (WBC 4.12 vs 1.15) (PMN 1.25 vs 0.27) (PLT 285 vs 85) x103/ul; 30 days: (WBC 11.3 vs 3.6) (PMN 6.8 vs 1.3) (PLT 819 vs 249) x103/ul], while administration 6 hrs post-irradiation resulted in more modest increases. In addition, analysis of marrow 30 days post-TBI demonstrated a significant enhancement in CFC in both non-delayed and delayed treatment groups compared to control (1.4 and 3.1 fold increase, respectively). These data suggest that inhibition of PGE2 synthesis post-TBI is beneficial for hematopoietic recovery and survival, but that allowing the positive effects of PGE2 on HSC to occur within the first 48 hours of TBI before inhibiting biosynthesis, results in a more efficacious treatment; a model supported by our results demonstrating enhanced recovery and survival with a single treatment of dmPGE2 shortly following TBI. Faced with the complexities of a mass casualty event and difficulty of individual dosimetry and triage, interventions that can mitigate or reduce the severity of exposure, but that are benign to those individuals with limited or no exposure are required. Our results define 2 different treatment modalities which are both highly effective and safe to administer, and can be readily available. In addition, the hematopoietic recovery demonstrated in these studies suggests a potential therapeutic benefit of cyclooxygenase inhibitors in TBI settings. Disclosures: No relevant conflicts of interest to declare.

Description: Aging is an inevitable process associated with eventual deterioration of normal physiological functions. Aged hematopoiesis is associated with increased numbers of hematopoietic stem cells (HSC), but with decreased HSC functional activity (e.g. decreased engrafting capability in lethally irradiated mice and a shift in the myeloid:lymphoid bias of the engrafting HSC of the old mice, such that there are more myeloid but fewer lymphoid cells generated from HSC of the old mice). Production of HSC and progenitor (HPC) cells ex vivo is more efficient when cells are cultured in a hypoxic environment of ~ 5% oxygen tension than when cells are grown at ambient air (~21% oxygen). The bone marrow (BM) microenvironment niche that nurtures the survival and production of HSC and HPC and hematopoiesis during adult life is a hypoxic environment (~1-5% oxygen tension) compared to that of ambient air. However, almost all results of studies of young and aged mouse hematopoiesis have been based on numbers and activity of HSC and HPC that have been collected and processed in ambient air. Our recent work evaluating hematopoiesis in BM cells of young adult mice and with human cord blood cells found, through a phenomenon we designated Extra Physiological Oxygen Shock/Stress (EPHOSS), that there is a large loss of HSC with an increase of HPC within minutes of the collection of these cells in ambient air (Mantel et al., Cell, 2015). This led us to reason that perhaps what we know about aging hematopoiesis might not be entirely accurate and that a re-evaluation of aged HSC, HPC, and hematopoiesis was in order. We hypothesized that hematopoiesis in aged (~20-27 months of age) mice may not be as dysregulated as reported but that collection and processing of BM from the aged mice is more sensitive than similar cells from young (~6-16 weeks) mice to EPHOSS-induced events generated by the collection of the cells in ambient air. We evaluated BM from three different mouse strains (CB6, BALB/c, and C57Bl/6) at 20-25 months vs. 6-16 weeks of age, collected/processed in ambient air or hypoxia (3% oxygen). BM from old mice collected/processed under hypoxic conditions exhibited phenotypically increased long-term HSC and common lymphoid progenitor (CLP) numbers and decreased common myeloid progenitor (CMP) and granulocyte-macrophage progenitor (GMP) numbers when compared to old BM collected/processed under ambient air conditions. BM collected from old C57Bl/6 mice under hypoxia had increased engrafting capability more closely matching that of young BM. This was associated with a 3.14-fold increase in the number of competitive repopulating units (representative of functional HSC) in old BM collected under hypoxic conditions compared to old BM collected in ambient air as determined through limiting dilution analysis. The myeloid:lymphoid ratio of old BM collected under hypoxia matched that of young BM collected under air. This was associated with decreased cycling of CFU-GM, BFU-E, and CFU-GEMM in old BM collected/processed in hypoxia. Enhanced numbers/function of old BM HSCs collected in hypoxia is associated with changes in expression of CXCR4 (and HSC homing capability), CCR5, stress protein levels (e.g. HSP40 etc) and ROS (both total and mitochondrial). All of these noted changes demonstrated that the old BM collected/processed under hypoxic conditions more closely resemble functionally young BM. Thus, age-related differences between the HSC/HPC populations are not as drastic as previously reported and reflect the increased sensitivity of hematopoiesis from aged mice to an artificial ambient air collection procedure. Disclosures No relevant conflicts of interest to declare.

Description: High-dose radiation treatment for hematological malignancies such as leukemia, lymphoma or multiple myeloma induces damage to the bone marrow microenvironment that limits hematopoietic regeneration. We previously demonstrated that single administration of dimethyl prostaglandin E2 (dmPGE2) at 24 hrs post lethal irradiation (796 cGys; LD70/30) in mice increases survival and accelerates hematopoietic recovery. Since bone marrow niches regulate hematopoiesis, we investigated whether the effects of PGE2 administration on hematopoietic recovery and survival were dependent on PGE2 signaling in bone marrow stromal cells. Total body irradiation (TBI) severely disrupts bone marrow niche components including MSC, osteoblasts and endothelial cells. Multivariate flow cytometry revealed that exposure of mice to 650 cGy TBI reduced the total number of bone marrow MSC defined as CD45- Ter119- CD31- PDGFR+ CD51+ MSC by 8 fold and reduced functional fibroblast colony formation (CFU-F) by 8.5 fold. Osteoblast (OB) and endothelial cell (EC) counts were reduced by 6.5 fold and 6 fold respectively. Treatment of mice with dmPGE2 at 24 hours post-irradiation substantially rescued MSC and EC, which were 3.5 and 2.2-fold higher compared to un-treated irradiated mice. Histological and flow cytometric analysis indicated that total OB, OB precursors and mesenchymal progenitor cells (MPC) in bone marrow were also enhanced by PGE2 administration. Interestingly, dmPGE2 failed to rescue MSC and OB from aged (24-27 mo/old) mice compared to young (4-6 mo/old) mice. Since PGE2 signals through four receptors (EP1-4), each with unique signaling pathways, we hypothesized that the hematopoietic niche regeneration was due to activation of PGE2 signaling via one or more EP receptors. Treatment of mice with the EP4 receptor agonist L-902,688 following irradiation enhanced the recovery of bone marrow MSC by 3.1 fold and EC by 2.0 fold compared to untreated irradiated control mice. EP1, EP2 and EP3 receptor agonists failed to enhance hematopoietic niche regeneration in irradiated mice. To further investigate the role of EP4 signaling in bone marrow niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or conditional inducible EP4-/- recipients after lethal irradiation, and analyzed stromal cells recovery and hematopoietic reconstitution. At 15 days post-transplantation, chimeric mice with EP4-/- stroma displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. PGE2 or EP4 agonist administration increased the expression of the endogenous anti-apoptotic protein Survivin, and enhanced survival of MSC and EC. In conclusion, our study suggests that PGE2 signaling through the EP4 receptor increases hemotopoietic recovery after TBI by enhancing the survival and expansion of MSC and endothelial cells. Furthermore, our data suggest the modulation/regulation PGE2 signaling in hematopoietic niche components could be beneficial to enhance HSC recovery following clinical hematopoietic transplantation. Disclosures No relevant conflicts of interest to declare.