scholarly journals Hematopoietic recovery after transplantation is primarily derived from the stochastic contribution of hematopoietic stem cells

2021 ◽  
Author(s):  
Stefan Radtke ◽  
Mark Enstrom ◽  
Dnyanada P. Pande ◽  
Margaret L. Cui ◽  
Ravishankar Madhu ◽  
...  
Keyword(s):  
Clonal Diversity ◽  
Enhanced Sampling ◽  
Primate Model ◽  
Hematopoietic Stem ◽  
Neutrophil Recovery ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution ◽  
Initial Recovery ◽  
Two Phases

Reconstitution after hematopoietic stem cell (HSC) transplantation is assumed to occur in two distinct phases: initial recovery mediated by short-term progenitors and long-term repopulation by multipotent HSCs which do not contribute to hematopoietic reconstitution during the first 6-9 months. We have previously reported the transplantation and exclusive engraftment of the HSC-enriched CD34+CD45RA-CD90+ phenotype in a nonhuman primate model. Here, we closely followed the clonal diversity and kinetics in these animals. Enhanced sampling and high density clonal tracking within the first 3 month revealed that multipotent HSCs actively contributed to the early phases of neutrophil recovery and became the dominant source for blood cells as early as 50 days after transplant. Longitudinal changes in clonal diversity supported a stochastic engraftment of HSCs with the majority of HSCs clones vanishing early during neutrophil recovery and a smaller fraction of HSC clones expanding into bigger pools to support long-term hematopoiesis. In contrast to the bi-phasic model, we propose that hematopoietic recovery after myeloablation and transplantation is primarily derived from HSCs in a stochastic manner rather than in two phases by independent cell populations.

scholarly journals High-Density Clonal Analysis Reveals Highly Active Contribution of Multipotent Hematopoietic Stem Cells during Early Phases of Hematopoietic Recovery after Transplantation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3258-3258
Author(s):  
Stefan Radtke ◽  
Mark Enstrom ◽  
Dnyanada Pande ◽  
Margaret Cui ◽  
Hans-Peter Kiem
Keyword(s):  
Gene Therapy ◽  
Clonal Diversity ◽  
High Density ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution

Abstract Recovery after conditioning and transplantation of hematopoietic stem and progenitor cells (HSPC) is thought to be biphasic, with short-term engrafting progenitors driving the recovery for 6-9 months and multipotent hematopoietic stem cells (HSCs) providing long-term repopulation. Recent clonal tracking data from autologous human gene therapy trials seems to support this model (Biasco et al. 2016, Cell Stem Cell; Six et al. 2020, Blood). These recent reports investigating the contribution of HSCs in patients are based on the longitudinal tracking of thousands of gene-marked cells using retroviral integration site analysis (ISA). While this technology is very reliable to follow gene therapy patients and monitor the potential outgrowth of dominant or malignant clones, low sensitivity and high error rates require significant data exclusion and sophisticated statistical tests to ensure data reliability (Adair et al. 2020, Molecular Therapy MCD). Lack of sensitivity can be overcome by increasing the frequency (high density) of sampling. However, limited material from patients remains a bottleneck for improved data quality and, consequently, correct interpretation of such complex datasets. To overcome the limitations of ISA and determine the onset of HSC contribution we performed high-density sampling for ISA in nonhuman primates (NHPs) transplanted with gene-modified HSCs. In the first month of hematopoietic recovery weekly blood samples were taken to enhance data density and increase the reliablity to detect clones with low abundance. Animals were followed up to 5 years to confirm that identified HSC clones persist long-term. Finally, clonal tracking data from the NHPs was used to inform a simulation of hematopoietic reconstitution, determine the temporal involvement of HSCs, and refine the phases of hematopoietic recovery after myeloablation and HSC transplantation. In contrast to the current biphasic model, contribution of multipotent HSCs clones was detected in the very first blood samples taken 2 to 3 weeks post-transplant during neutrophil recovery. HSC clones found in these early time points persisted long-term throughout the entire follow-up and were detected in bone marrow CD34 + cells 4 years later. Most surprisingly, multipotent HSCs became the dominant source for mature blood cells in the peripheral blood as early as 50 days post-transplant. To understand the observed kinetics of HSC contribution and change in clonal diversity in our dataset, we simulated the clonal outgrowth and differentiation of multipotent clones. Simulations predicted that hematopoietic recovery is primarily HSC driven and HSC contribution follows a stochastic pattern. Finally, to confirm that HSCs proliferation and differentiation is a stochastic process, in vitro experiments in colony-forming cell (CFC) assays were carried out. As predicted, the decision of individual HSCs to either grow into a larger pool or differentiate and get lost followed the same kinetics as observed in vivo. Here, we show evidence that long-term persisting multipotent HSCs actively contribute during early hematopoietic reconstitution after myeloablation and HSC transplantation. Enhanced sampling showed that multipotent HSCs produce neutrophils during recovery and become the predominant source of mature blood cells as early as 50 days post-transplant. Most importantly, observed changes in the clonal diversity during early recovery suggest a stochastic engraftment of HSCs rather than a bi-phasic reconstitution initially driven by short-term progenitors. These findings should have important implications for the design of ex vivo and in vivo HSC gene therapy and genome editing approaches. Figure 1 Figure 1. Disclosures Radtke: 47 Inc.: Consultancy; Ensoma Inc.: Consultancy. Kiem: Homology Medicines: Consultancy; VOR Biopharma: Consultancy; Ensoma Inc.: Consultancy, Current holder of individual stocks in a privately-held company.

scholarly journals CXCR4-SDF-1 Signaling in Nestin+ Mesenchymal Stem Cell Is Required for HSC Maintenance during Homeostasis and Regeneration after Irradiation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3883-3883 ◽  
Author(s):  
Pratibha Singh ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Stromal Cell ◽  
Cxcr4 Expression ◽  
Wild Type ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution

Hematopoietic stem cells (HSC) reside in a complex microenvironment (niche) within the bone marrow (BM), where multiple populations of microenvironmental stromal cells regulate and finely tune their proliferation, differentiation and trafficking. Recent studies have shown that mesenchymal stem cells (MSC) are an essential component of the HSC niche. Intrinsic HSC CXCR4-SDF-1 signaling has been implicated in self-renewal and quiescence; however, the role of microenvironment CXCR4-SDF-1 signaling in supporting HSC function remains unclear. We previously demonstrated that microenvironmental stromal cell-derived CXCR4 is important for HSC recovery, as transplantation of wild-type HSC into CXCR4 deficient recipients showed reduced HSC engraftment. In this study, we now show that CXCR4-SDF-1 signaling in nestin+ MSC regulates HSC maintenance under normal homeostatic conditions and promotes hematopoietic regeneration after irradiation. Multivariate flow cytometry analysis of marrow stroma cells revealed that mouse BM MSCs identified as CD45-Ter119-CD31-Nestin+PDGFR+CD51+ express the CXCR4 receptor, which was confirmed by RT-PCR analysis. To investigate the role of MSC CXCR4 signaling in niche maintenance and support of HSC function, we utilized genetic mouse models, in which CXCR4 could be deleted in specific stromal cell types. Selective deletion of CXCR4 from nestin+ MSC in adult tamoxifen inducible nestin-cre CXCR4flox/flox mice resulted in reduced total MSC in BM (Control vs. Deleted: 647±128 vs. 209±51/femur, respectively, n=5, p<0.05), which was associated with a significant reduction in Lineage-Sca-1+c-Kit+ (LSK) cells (Control vs. Deleted: 18,033±439 vs. 4523±358/femur, respectively n=5, p<0.05). Selective CXCR4 deletion in nestin+ MSC also resulted in enhanced LSK cell egress to the peripheral circulation (Control vs. Deleted: 1022±106 vs. 2690±757/ml blood, respectively n=5, p<0.05), with no detectable difference in HSC cell cycle or apoptosis. However, the repopulation ability of HSC obtained from mice where CXCR4 was deleted in nestin+ MSC was reduced by >2 fold. In contrast, deletion of CXCR4 from osteoblasts using osteocalcin cre CXCR4flox/flox mice had no effect on HSC numbers in BM and blood.To investigate the role of nestin+ MSC CXCR4 signaling in BM niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or nestin+ MSC CXCR4 deleted recipients after lethal irradiation (950 rad) and analyzed HSC homing, niche recovery and hematopoietic reconstitution. Deletion of CXCR4 from nestin expressing MSC resulted in significantly reduced LSK cell homing at 16 hrs post transplantation (Control vs. Deleted: 8643±1371 vs. 3004±1044/ mouse, respectively, n=5, p<0.05). Robust apoptosis and senescence after total body irradiation was observed in nestin expressing MSCs lacking CXCR4 expression. At 15 days post-transplantation, chimeric mice with nestin+ MSC lacking CXCR4 expression displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. In conclusion, our study suggests that CXCR4-SDF-1 signaling in nestin+ MSC is critical for the maintenance and retention of HSC in BM during homeostasis and promotes niche regeneration and hematopoietic recovery after transplantation. Furthermore, our data suggest the modulating CXCR4 signaling in the hematopoietic niche could be beneficial as a means to enhance HSC recovery following clinical hematopoietic transplantation or radiation/chemotherapy injury. Disclosures No relevant conflicts of interest to declare.

Tritium contamination of hematopoietic stem cells alters long-term hematopoietic reconstitution

2011 ◽  
Vol 87 (6) ◽  
pp. 556-570 ◽  
Author(s):  
Fabio Di Giacomo ◽  
Christine Granotier ◽  
Vilma Barroca ◽  
David Laurent ◽  
François D. Boussin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution

scholarly journals HMGA2 promotes long-term engraftment and myeloerythroid differentiation of human hematopoietic stem and progenitor cells

2019 ◽  
Vol 3 (4) ◽  
pp. 681-691 ◽  
Author(s):  
Praveen Kumar ◽  
Dominik Beck ◽  
Roman Galeev ◽  
Julie A. I. Thoms ◽  
Mehrnaz Safaee Talkhoncheh ◽  
...  
Keyword(s):  
Cell Subset ◽  
High Mobility ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Growth And Survival ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells

Abstract Identification of determinants of fate choices in hematopoietic stem cells (HSCs) is essential to improve the clinical use of HSCs and to enhance our understanding of the biology of normal and malignant hematopoiesis. Here, we show that high-mobility group AT hook 2 (HMGA2), a nonhistone chromosomal-binding protein, is highly and preferentially expressed in HSCs and in the most immature progenitor cell subset of fetal, neonatal, and adult human hematopoiesis. Knockdown of HMGA2 by short hairpin RNA impaired the long-term hematopoietic reconstitution of cord blood (CB)–derived CB CD34+ cells. Conversely, overexpression of HMGA2 in CB CD34+ cells led to overall enhanced reconstitution in serial transplantation assays accompanied by a skewing toward the myeloerythroid lineages. RNA-sequencing analysis showed that enforced HMGA2 expression in CD34+ cells induced gene-expression signatures associated with differentiation toward megakaryocyte-erythroid and myeloid lineages, as well as signatures associated with growth and survival, which at the protein level were coupled with strong activation of AKT. Taken together, our findings demonstrate a key role of HMGA2 in regulation of both proliferation and differentiation of human HSPCs.

Systemic Administration of Pleiotrophin Induces Hematopoietic Stem Cell Regeneration In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1498-1498
Author(s):  
Heather A Himburg ◽  
Pamela Daher ◽  
Sarah Kristen Meadows ◽  
J. Lauren Russell ◽  
Phuong Doan ◽  
...  
Keyword(s):  
Stem Cell ◽  
Growth Factor ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Fold Increase ◽  
Cell Regeneration ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution

Abstract Abstract 1498 Poster Board I-521 Significant progress has been made toward delineating the intrinsic and extrinsic signaling pathways that regulate hematopoietic stem cell (HSC) self-renewal. However, much less is known regarding the process of HSC regeneration or the extrinsic signals that regulate hematopoietic reconstitution following stress or injury. Elucidation of the microenvironmental signals which promote HSC regeneration in vivo would have important implications for the treatment of patients undergoing radiation therapy, chemotherapy and stem cell transplantation. We recently reported that pleiotrophin, a soluble heparin-binding growth factor, induced a 10-fold expansion of murine long-term repopulating HSCs in short term culture (Himburg et al. Blood (ASH Annual Meeting Abstracts), Nov 2008; 112: 78). Based on this observation, we hypothesized that PTN might also be a regenerative growth factor for HSCs. Here we tested the effect of systemic administration of PTN to non-irradiated and irradiated C57Bl6 mice to determine if PTN could promote HSC regeneration in vivo. C57Bl6 mice were irradiated with 700 cGy total body irradiation (TBI) followed by intraperitoneal administration of 2 μg PTN or saline x 7 days, followed by analysis of BM stem and progenitor cell content. Saline-treated mice demonstrated significant reductions in total BM cells, BM c-kit+sca-1+lin- (KSL) cells, colony forming cells (CFCs) and long term culture-initiating cells (LTC-ICs) compared to non-irradiated control mice. In contrast, PTN-treated mice demonstrated a 2.3-fold increase in total BM cells (p=0.03), a 5.6-fold increase in BM KSL stem/progenitor cells (p=0.04), a 2.9-fold increase in BM CFCs (p=0.004) and an 11-fold increase in LTC-ICs (p=0.03) compared to saline-treated mice. Moreover, competitive repopulating transplantation assays demonstrated that BM from PTN-treated, irradiated mice contained 5-fold increased competitive repopulating units (CRUs) compared to saline-treated, irradiated mice (p=0.04). Taken together, these data demonstrate that the administration of PTN induces BM HSC and progenitor cell regeneration in vivo following injury. Comparable increases in total BM cells, BM KSL cells and BM CFCs were also observed in PTN-treated mice compared to saline-treated controls following 300 cGy TBI, demonstrating that PTN is a potent growth factor for hematopoietic stem/progenitor cells in vivo at less than ablative doses of TBI. In order to determine whether PTN acted directly on BM HSCs to induce their proliferation and expansion in vivo, we exposed mice to BrDU in their drinking water x 7 days and compared the response to saline treatment versus PTN treatment. PTN-treated mice demonstrated a significant increase in BrDU+ BM KSL cells compared to saline-treated controls (p=0.04) and cell cycle analysis confirmed a significant increase in BM KSL cells in S phase in the PTN-treatment group compared to saline-treated controls (p=0.04). These data indicate that PTN serves as a soluble growth factor for BM HSCs and induces their proliferation and expansion in vivo while preserving their repopulating capacity. These results suggest that PTN has therapeutic potential as a novel growth factor to accelerate hematopoietic reconstitution in patients undergoing myelosuppressive radiotherapy or chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Rejuvenation of Aged Vascular Niches to Enhance Hematopoietic Function

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 781-781
Author(s):  
Michael Gustave Poulos ◽  
Michael Gutkin ◽  
Christopher Y. Park ◽  
Jason M. Butler
Keyword(s):  
Culture System ◽  
Molecular Mechanisms ◽  
Mapk Signaling ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Hematopoietic Recovery ◽  
Vascular Niche ◽  
Age Related ◽  
In Vivo Animal Model

Abstract The molecular mechanisms regulating the aging of hematopoietic stem cells (HSCs) are poorly understood. To date, most studies describing age-related alterations have focused on HSC-intrinsic alterations, showing that the absolute number of immunophenotypically defined HSCs increases with age but that aged HSCs exhibit decreased long-term reconstitution potential, self-renewing capacity, altered transciptomes, cell-cycling and responses to cellular stress and DNA damage. Furthermore, old HSCs exhibit a significant myeloid bias at the expense of lymphopoiesis, which is thought to predispose the aging hematopoietic to the development of myeloid neoplasms. While these studies show that cell-intrinsic changes contribute to the aging of the hematopoietic system, most have not adequately addressed the effects of the aging microenvironment. A large body of evidence has demonstrated functional interactions between the HSC and its niche, suggesting that local and systemic factors may regulate HSC function; however, the role of the bone marrow (BM) microenvironment in regulating HSC aging has not been fully elucidated. Understanding the relationship between the BM microenvironment and the HSC during aging may aid in efforts to prevent or reverse the age-related functional decline observed in the hematopoietic system. We have shown that Akt-activated endothelial cells (ECs) within the hematopoietic microenvironment, are indispensable for supporting HSC self-renewal during both steady-state and regenerative hematopoiesis and that EC-specific Mapk signaling drives the differentiation of HSCs into lineage-committed progeny. Here, we demonstrate that young BMECs maintain high levels of Akt signaling, whereas aged ECs exhibit preferential signaling through Mapk. Utilizing a novel HSC/EC co-culture system, we demonstrate that aged BMECs co-cultured with hematopoietic stem and progenitor cells (HSPCs) isolated from young mice inhibit the expansion of repopulating HSCs and are unable to expand aged HSPCs that give rise to long-term, multilineage engraftment. Of note, when we co-cultured aged HSPCs with young BMECs we found that we were able to maintain their functional capacity when assessed by competitive repopulation assays. These data suggest that BMECs play an important role in regulating HSC function. Based on these observations, we set out to test if endothelial Mapk inhibits the vascular niche from supporting functional hematopoiesis. We generated a mouse model in which Mapk was selectively overexpressed in ECs (Mapk VCC) and demonstrated that these mice exhibit a defect in phenotypic and functional HSCs, resembling phenotypes similar to aged HSCs. In particular, transplanted HSCs from Mapk VCC mice lead to diminished engraftment ability with an in increase in myeloid contribution at the expense of B and T cells. To directly test if the functional defects in the HSCs were due to the Mapk-activated vascular niche, we isolated BMECs from these mice and found that Mapk-activated ECs have a decreased ability to support the ex vivo expansion of functional HSCs, with less HSCs in quiescence and more differentiation into granulocytic myeloid cells. Transcriptome and proteomic analyses revealed that aged and Mapk-activated BMECs have similar defects in their pro-HSC angiocrine repertoire, suggesting a possible mechanism for their diminished capacity to instruct and maintain a balanced and healthy hematopoietic system. Furthermore, we utilized an endothelial-based cellular therapy approach to rejuvenate the BM microenvironment and demonstrated that transplantation of young BMECs can enhance hematopoietic recovery and restore HSC function following myeloablative injury in aged mice. Taken together, our in vivo animal model and EC/HSC co-culture system will allow us to screen for angiocrine factors that support the functional attributes of the HSC. Additionally, we have unlocked a potential therapeutic application for the transplantation of ECs following myeloablative treatment. Transplantation of BMECs may create a more permissive microenvironment that promotes an increase in the number of engrafted HSPCs following BM transplantation and accelerates the rate of hematopoietic recovery following radiation or chemotherapeutic regimens decreasing the morbidity/mortality associated with life threatening pancytopenias in the elderly. Disclosures No relevant conflicts of interest to declare.

scholarly journals Long-term persistence and function of hematopoietic stem cell-derived chimeric antigen receptor T cells in a nonhuman primate model of HIV/AIDS

2017 ◽  
Vol 13 (12) ◽  
pp. e1006753 ◽  
Author(s):  
Anjie Zhen ◽  
Christopher W. Peterson ◽  
Mayra A. Carrillo ◽  
Sowmya Somashekar Reddy ◽  
Cindy S. Youn ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Chimeric Antigen Receptor ◽  
Nonhuman Primate ◽  
Nonhuman Primate Model ◽  
Primate Model ◽  
Hematopoietic Stem ◽  
And Function

scholarly journals Multiple allogeneic progenitors in combination function as a unit to support early transient hematopoiesis in transplantation

2016 ◽  
Vol 213 (9) ◽  
pp. 1865-1880 ◽  
Author(s):  
Takashi Ishida ◽  
Satoshi Takahashi ◽  
Chen-Yi Lai ◽  
Masanori Nojima ◽  
Ryo Yamamoto ◽  
...  
Keyword(s):  
Hematopoietic Cell Transplantation ◽  
Initial Time ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Single Donor ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Combination Function ◽  
Donor Source

Cord blood (CB) is a valuable donor source in hematopoietic cell transplantation. However, the initial time to engraftment in CB transplantation (CBT) is often delayed because of low graft cell numbers. This limits the use of CB. To overcome this cell dose barrier, we modeled an insufficient dose CBT setting in lethally irradiated mice and then added hematopoietic stem/progenitor cells (HSCs/HPCs; HSPCs) derived from four mouse allogeneic strains. The mixture of HSPCs rescued recipients and significantly accelerated hematopoietic recovery. Including T cells from one strain favored single-donor chimerism through graft versus graft reactions, with early hematopoietic recovery unaffected. Furthermore, using clinically relevant procedures, we successfully isolated a mixture of CD34+ cells from multiple frozen CB units at one time regardless of HLA-type disparities. These CD34+ cells in combination proved transplantable into immunodeficient mice. This work provides proof of concept that when circumstances require support of hematopoiesis, combined multiple units of allogeneic HSPCs are capable of early hematopoietic reconstitution while allowing single-donor hematopoiesis by a principal graft.

scholarly journals Novel Strategy for Successful Long-Term Hematopoietic Recovery after Transplanting a Limited Number of Hematopoietic Stem/Progenitor Cells

2014 ◽  
Vol 20 (9) ◽  
pp. 1282-1289 ◽  
Author(s):  
Hakmo Lee ◽  
Ho Seon Park ◽  
Ok Kyung Choi ◽  
Ju Eun Oh ◽  
Sung Soo Chung ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Novel Strategy

scholarly journals Suppression of Luteinizing Hormone Enhances HSC Recovery after Hematopoietic Injuries

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 370-370
Author(s):  
Enrico Velardi ◽  
Jennifer Jia-ying Tsai ◽  
Kimon V. Argyropoulos ◽  
Shieh Jae-Hung ◽  
Sophie Lieberman ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Sex Steroids ◽  
Radiation Injury ◽  
Pool Size ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Lh Receptor ◽  
Pharmacological Inhibition ◽  
Hematopoietic Recovery

Abstract A tightly regulated network of intrinsic and extrinsic signaling pathways exists to preserve HSC pool size and function. This is particularly relevant during hematopoietic injuries when dormant HSCs transiently start to proliferate to replenish blood cells; as unbalanced HSC proliferation can lead to stem cell exhaustion, long-term myelosuppression and death. Although there has been growing interest in how circulating sex hormones influence HSC function (Nakada et al., 2014; Sanchez-Aguilera et al., 2014), this pathway remains poorly understood. Here we describe a heretofore unknown role for the upstream hormone regulator, luteinizing hormone (LH), in regulating HSC biology. We found that both human and mouse HSCs highly expressed the LH receptor, and its expression was decreased or nearly absent in downstream progenitors (Figure 1a). LH significantly promoted HSC colony forming potential in cobblestone area-forming cell and colony-forming cell assays that, together with expression of the receptor, suggested that LH increased HSC expansion in vitro by acting directly on the most primitive HSCs. To investigate whether LH levels could impact on HSC pool size during hematopoietic stress in vivo, we challenged mice using models that force HSCs out of their quiescent status, Poly I:C and sub-lethal dose of total body irradiation (SL-TBI, 550cGy). We found that ablation of LH production using a luteinizing hormone-releasing hormone-antagonist (LHRH-Ant) retained significantly more HSCs in G0 in both models (Figure 1b,c). Previous reports have shown that induction of HSC quiescence after high-dose irradiation correlates with increased hematopoietic recovery and enhanced mouse survival (Chen et al., 2008; Himburg et al., 2014; Johnson et al., 2010). Given its effectiveness in promoting HSC quiescence and the fact that LHRH-Ant are widely available and clinically approved, we hypothesized that LHRH-Ant could represent a rational non-cellular medical countermeasure for mitigating radiation injury and promoting hematopoietic regeneration when administered after hematopoietic insult. To test this hypothesis, we used a lethal TBI (L-TBI) dose of 840cGy that mediated lethality in more than 90% of B6 male mice. We found that pharmacological inhibition of LH using LHRH-Ant 24h after L-TBI spared the most primitive long term HSCs (Figure 1d) thus promoting hematopoietic recovery and mouse survival (Figure 1e). Consistent with our original hypothesis we also found a significantly higher proportion of Ki-67− quiescent HSCs in the LHRH-Ant-treated group with fewer proliferative HSCs compared to controls. Given the wide-ranging hormonal changes induced by LHRH suppression and the previously reported effects mediated by sex steroid ablation on hematopoietic stem/progenitor cell (HSPC) compartment (Khong et al., 2015), we next evaluated whether the LHRH-Ant effects on mouse survival after L-TBI were independent from the suppression of the downstream sex steroids. Administration of LHRH-Ant improved survival rates in surgically castrated mice following radiation injury, while surgical castration alone did not, indicating that the regenerative effects were independent from downstream sex steroids. To confirm whether the protective effects of LHRH-Ant treatment depended on suppression of LH, we administered the LH receptor agonist human chorionic gonadotropin (hCG) to LHRH-Ant treated mice that had been given L-TBI one-day prior. Consistent with our hypothesis, administration of hCG abrogated the beneficial effects of LHRH-Ant on survival after radiation injury. Taken together our studies showed that HSCs are a physiological target of LH, which promotes their proliferation. Furthermore, pharmacological inhibition of LH signaling using a single dose of an LHRH-Ant represents a rational and feasible approach to preserve the HSC pool after high dose radiation, thereby mitigating acute hematopoietic radiation syndrome. Disclosures Van Den Brink: Seres: Research Funding; Novartis: Consultancy; Regeneron: Consultancy; Flagship Ventures: Consultancy; Boehringer Ingelheim: Consultancy; Merck: Consultancy.

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