Rapid Mobilization Reveals a Highly Engraftable Hematopoietic Stem Cell.

Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.

Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock.

Hematopoietic stem cells (HSCs) reside in hypoxic niches within bone marrow and cord blood. Yet, essentially all HSC studies have been performed with cells isolated and processed in non-physiologic ambient air. By collecting and manipulating bone marrow and cord blood in native conditions of hypoxia, we demonstrate that brief exposure to ambient oxygen decreases recovery of long-term repopulating HSCs and increases progenitor cells, a phenomenon we term extraphysiologic oxygen shock/stress (EPHOSS). Thus, true numbers of HSCs in the bone marrow and cord blood are routinely underestimated. We linked ROS production and induction of the mitochondrial permeability transition pore (MPTP) via cyclophilin D and p53 as mechanisms of EPHOSS. The MPTP inhibitor cyclosporin A protects mouse bone marrow and human cord blood HSCs from EPHOSS during collection in air, resulting in increased recovery of transplantable HSCs. Mitigating EPHOSS during cell collection and processing by pharmacological means may be clinically advantageous for transplantation.

CXCR4 expression in the bone marrow microenvironment is required for hematopoietic stem and progenitor cell maintenance and early hematopoietic regeneration after myeloablation.

The bone marrow (BM) microenvironment/niche plays a key role in regulating hematopoietic stem and progenitor cell (HSPC) activities; however, mechanisms regulating niche cell function are not well understood. In this study, we show that niche intrinsic expression of the CXCR4 chemokine receptor critically regulates HSPC maintenance during steady state, and promotes early hematopoietic regeneration after myeloablative irradiation. At steady state, chimeric mice with wild-type (WT) HSPC and marrow stroma that lack CXCR4 show decreased HSPC quiescence, and their repopulation capacity was markedly reduced. Mesenchymal stromal cells (MSC) were significantly reduced in the BM of CXCR4 deficient mice, which was accompanied by decreased levels of the HSPC supporting factors stromal cell-derived factor-1 (SDF-1) and stem cell factor (SCF). CXCR4 also plays a crucial role in survival and restoration of BM stromal cells after myeloablative irradiation, where the loss of BM stromal cells was more severe in CXCR4-deficient mice compared to WT mice. In addition, transplantation of WT donor HSPC into CXCR4-deficient recipient mice demonstrated reduced HSPC homing and early hematopoietic reconstitution. We found that CXCR4 signaling attenuates irradiation-induced BM stromal cell loss by upregulating the expression of the antiapoptotic protein Survivin via the PI3K pathway. Our study suggests that SDF-1-CXCR4 signaling in the stromal microenvironment cells plays a crucial role in maintenance of HSPCs during homeostasis, and promotes niche regeneration and early hematopoietic reconstitution after transplantation. Modulation of CXCR4 signaling in the HSPC microenvironment could be a means to enhance hematopoietic recovery after clinical hematopoietic cell transplantation.

Survivin Is Required for Mouse and Human Bone Marrow Mesenchymal Stromal Cell Function.

Although mesenchymal stromal cells (MSCs) have significant potential in cell-based therapies, little is known about the factors that regulate their functions. While exploring regulatory molecules potentially involved in MSC activities, we found that the endogenous multifunctional factor Survivin is essential for MSC survival, expansion, lineage commitment, and migration. Pharmacological or genetic blockade of Survivin expression in mouse and human bone marrow MSC enhances caspase 3 and 7 expression and reduces proliferation resulting in fewer MSC and clonogenic colony-forming unit-fibroblasts (CFU-F), whereas ectopic Survivin overexpression in MSC results in their expansion. Survivin is also required for the MSC proliferative responses to basic fibroblast growth factor and platelet derived growth factor. In a wound healing model, Survivin inhibition results in suppression of MSC migration to the wound site. In addition, loss of Survivin in MSCs compromises their hematopoiesis-supporting capacity. These results demonstrate that Survivin is a key regulator of mouse and human MSC function, and suggest that targeted modulation of Survivin in MSCs may have clinical utility to enhance MSC recovery and activity following insult or stress. Stem Cells 2018;36:123-129.

Differential stem- and progenitor-cell trafficking by prostaglandin E2.

To maintain lifelong production of blood cells, haematopoietic stem cells (HSCs) are tightly regulated by inherent programs and extrinsic regulatory signals received from their microenvironmental niche. Long-term repopulating HSCs reside in several, perhaps overlapping, niches that produce regulatory molecules and signals necessary for homeostasis and for increased output after stress or injury. Despite considerable advances in the specific cellular or molecular mechanisms governing HSC-niche interactions, little is known about the regulatory function in the intact mammalian haematopoietic niche. Recently, we and others described a positive regulatory role for prostaglandin E2 (PGE2) on HSC function ex vivo. Here we show that inhibition of endogenous PGE2 by non-steroidal anti-inflammatory drug (NSAID) treatment in mice results in modest HSC egress from the bone marrow. Surprisingly, this was independent of the SDF-1-CXCR4 axis implicated in stem-cell migration. Stem and progenitor cells were found to have differing mechanisms of egress, with HSC transit to the periphery dependent on niche attenuation and reduction in the retentive molecule osteopontin. Haematopoietic grafts mobilized with NSAIDs had superior repopulating ability and long-term engraftment. Treatment of non-human primates and healthy human volunteers confirmed NSAID-mediated egress in other species. PGE2 receptor knockout mice demonstrated that progenitor expansion and stem/progenitor egress resulted from reduced E-prostanoid 4 (EP4) receptor signalling. These results not only uncover unique regulatory roles for EP4 signalling in HSC retention in the niche, but also define a rapidly translatable strategy to enhance transplantation therapeutically.

Delta-1 provides pleasant stem cell environs.

In this issue of Blood, Csaszar et al show that in ex vivo cultures of CD341 cells, simultaneous reduction of interleukin-6 receptor (IL-6R) expression on myeloid progenitor cells by Delta-1 treatment combined with removal of soluble IL-6R using a dynamically fed culture system, reduces mature myeloid cell production, leading to enhanced expansion of primitive hematopoietic stem cells.

Pharmacologic increase in HIF1α enhances hematopoietic stem and progenitor homing and engraftment.

Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for a number of immunologic disorders. For effective transplant, HSCs must traffic from the peripheral blood to supportive bone marrow niches. We previously showed that HSC trafficking can be enhanced by ex vivo treatment of hematopoietic grafts with 16-16 dimethyl prostaglandin E2 (dmPGE2). While exploring regulatory molecules involved in dmPGE2 enhancement, we found that transiently increasing the transcription factor hypoxia-inducible factor 1-α (HIF1α) is required for dmPGE2-enhanced CXCR4 upregulation and enhanced migration and homing of stem and progenitor cells and that pharmacologic manipulation of HIF1α is also capable of enhancing homing and engraftment. We also now identify the specific hypoxia response element required for CXCR4 upregulation. These data define a precise mechanism through which ex vivo pulse treatment with dmPGE2 enhances the function of hematopoietic stem and progenitor cells; these data also define a role for hypoxia and HIF1α in enhancement of hematopoietic transplantation.

CD166 regulates human and murine hematopoietic stem cells and the hematopoietic niche.

We previously showed that immature CD166(+) osteoblasts (OB) promote hematopoietic stem cell (HSC) function. Here, we demonstrate that CD166 is a functional HSC marker that identifies both murine and human long-term repopulating cells. Both murine LSKCD48(-)CD166(+)CD150(+) and LSKCD48(-)CD166(+)CD150(+)CD9(+) cells, as well as human Lin(-)CD34(+)CD38(-)CD49f(+)CD166(+) cells sustained significantly higher levels of chimerism in primary and secondary recipients than CD166(-) cells. CD166(-/-) knockout (KO) LSK cells engrafted poorly in wild-type (WT) recipients and KO bone marrow cells failed to radioprotect lethally irradiated WT recipients. CD166(-/-) hosts supported short-term, but not long-term WT HSC engraftment, confirming that loss of CD166 is detrimental to the competence of the hematopoietic niche. CD166(-/-) mice were significantly more sensitive to hematopoietic stress. Marrow-homed transplanted WT hematopoietic cells lodged closer to the recipient endosteum than CD166(-/-) cells, suggesting that HSC-OB homophilic CD166 interactions are critical for HSC engraftment. STAT3 has 3 binding sites on the CD166 promoter and STAT3 inhibition reduced CD166 expression, suggesting that both CD166 and STAT3 may be functionally coupled and involved in HSC competence. These studies illustrate the significance of CD166 in the identification and engraftment of HSC and in HSC-niche interactions, and suggest that CD166 expression can be modulated to enhance HSC function.

Prostaglandin E2 enhances long-term repopulation but does not permanently alter inherent stem cell competitiveness.

Hematopoietic stem cell (HSC) transplantation is a lifesaving therapy for malignant and nonmalignant hematologic diseases and metabolic disorders. Although successful, hematopoietic transplantation can be hindered by inadequate stem cell number or poor engrafting efficiency. To overcome these deficits, we and others have previously reported the HSC-enhancing ability of a short-term exposure of prostaglandin E2 (PGE2); this strategy has now progressed to phase 1 clinical trials in double cord blood transplantation. To further analyze the short- and long-term effects of HSC exposure to PGE2, we followed the repopulation kinetics of PGE2-treated hematopoietic grafts through 5 serial transplantations and compared inherent long-term competitiveness in a HSC head-to-head secondary transplantation model. Treatment with PGE2 did not result in a long-term increase in HSC competitiveness, lineage bias, or enhanced proliferative potential, demonstrating that pulse exposure to PGE2 results in transient increases in HSC homing and engraftment potential.

Prostaglandin-modulated umbilical cord blood hematopoietic stem cell transplantation.

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) for use in allogeneic transplantation. Key advantages of UCB are rapid availability and less stringent requirements for HLA matching. However, UCB contains an inherently limited HSC count, which is associated with delayed time to engraftment, high graft failure rates, and early mortality. 16,16-Dimethyl prostaglandin E2 (dmPGE2) was previously identified to be a critical regulator of HSC homeostasis, and we hypothesized that brief ex vivo modulation with dmPGE2 could improve patient outcomes by increasing the "effective dose" of HSCs. Molecular profiling approaches were used to determine the optimal ex vivo modulation conditions (temperature, time, concentration, and media) for use in the clinical setting. A phase 1 trial was performed to evaluate the safety and therapeutic potential of ex vivo modulation of a single UCB unit using dmPGE2 before reduced-intensity, double UCB transplantation. Results from this study demonstrated clear safety with durable, multilineage engraftment of dmPGE2-treated UCB units. We observed encouraging trends in efficacy, with accelerated neutrophil recovery (17.5 vs 21 days, P = .045), coupled with preferential, long-term engraftment of the dmPGE2-treated UCB unit in 10 of 12 treated participants.

Inhibition of DPP4/CD26 and dmPGE2 treatment enhances engraftment of mouse bone marrow hematopoietic stem cells.

Enhancing the engraftment of hematopoietic stem cells (HSC) is especially important when times to engraftment are prolonged due either to limiting numbers of HSC in the donor graft or to intrinsic slower engrafting time of the tissue sources of HSC. Both inhibition of dipeptidylpeptidase (DPP) 4/CD26 and treatment of cells with 16,16 dimethyl prostaglandin E2 (dmPGE2) have been shown to enhance hematopoietic stem cell engraftment in murine transplantation models and have been evaluated in clinical settings for their influence on engraftment of cord blood cells, a tissue source of HSC known to manifest an extended time to engraftment of donor cells compared to that of bone marrow (BM) and mobilized peripheral blood for hematopoietic cell transplantation (HCT). Herein, we present new experimental data, using a CD45(+) head-to-head congenic model of donor mouse BM cells for engraftment of lethally irradiated mice, demonstrating that similar levels of enhanced engraftment are detected by pulsing donor BM cells with diprotin A, a DPP4 inhibitor, or with dmPGE2 prior to infusion, or by pretreating recipient mice with sitagliptin, also a DPP4 inhibitor, by oral gavage. Moreover, the combined effects of pretreating the donor BM cells with dmPGE2 in context of pretreating the recipient mice with sitagliptin after the administration of a lethal dose of radiation resulted in significantly enhanced competitively repopulating HCT compared to either treatment alone. This information is highly relevant to the goal of enhancing engraftment in human clinical HCT.

Blockade of prostaglandin E2 signaling through EP1 and EP3 receptors attenuates Flt3L-dependent dendritic cell development from hematopoietic progenitor cells.

Dendritic cell (DC) homeostasis, like all mature blood cells, is maintained via hierarchal generation from hematopoietic precursors; however, little is known about the regulatory mechanisms governing DC generation. Here, we show that prostaglandin E(2) (PGE(2)) is required for optimal Flt3 ligand-mediated DC development and regulates expression of the Flt3 receptor on DC-committed progenitor cells. Inhibition of PGE(2) biosynthesis reduces Flt3-mediated activation of STAT3 and expression of the antiapoptotic protein survivin, resulting in increased apoptosis of DC-committed progenitor cells. Reduced DC development caused by diminished PGE(2) signaling is reversed by overexpression of Flt3 or survivin in DC progenitors and conversely is mimicked by STAT3 inhibition. PGE(2) regulation of DC generation is specifically mediated through the EP1 and EP3 G protein PGE(2) receptors. These studies define a novel DC progenitor regulatory pathway in which PGE(2) signaling through EP1/EP3 receptors regulates Flt3 expression and downstream STAT3 activation and survivin expression, required for optimal DC progenitor survival and DC development in vivo.

Concise review: Sowing the seeds of a fruitful harvest: hematopoietic stem cell mobilization.

Hematopoietic stem cell transplantation is the only curative option for a number of malignant and nonmalignant diseases. As the use of hematopoietic transplant has expanded, so too has the source of stem and progenitor cells. The predominate source of stem and progenitors today, particularly in settings of autologous transplantation, is mobilized peripheral blood. This review will highlight the historical advances which led to the widespread use of peripheral blood stem cells for transplantation, with a look toward future enhancements to mobilization strategies.

Pharmacokinetics and Biodistribution of 16,16 dimethyl Prostaglandin E2 in Non-Irradiated and Irradiated Mice and Non-Irradiated Non-Human Primates.

Exposure to high-dose ionizing radiation can lead to life-threatening injuries and mortality. Bone marrow is the most sensitive organ to radiation damage, resulting in the hematopoietic acute radiation syndrome (H-ARS) with the potential sequelae of infection, hemorrhage, anemia, and death if untreated. The development of medical countermeasures (MCMs) to protect or mitigate radiation injury is a medical necessity. In our well-established murine model of H-ARS we have demonstrated that the prostaglandin E2 (PGE2) analog 16,16 dimethyl-PGE2 (dmPGE2) has survival efficacy as both a radioprotectant and radiomitigator. The purpose of this study was to investigate the pharmacokinetics (PK) and biodistribution of dmPGE2 when used as a radioprotector in irradiated and non-irradiated inbred C57BL/6J mice, PK in irradiated and non-irradiated Jackson Diversity Outbred (JDO) mice, and the PK profile of dmPGE2 in non-irradiated non-human primates (NHPs). The C57BL/6J and JDO mice each received a single subcutaneous (SC) dose of 35 ug of dmPGE2 and were randomized to either receive radiation 30 min later or remain non-irradiated. Plasma and tissue PK profiles were established. The NHP were dosed with 0.1 mg/kg by SC administration and the PK profile in plasma was established. The concentration time profiles were analyzed by standard non-compartmental analysis and the metrics of AUC0-Inf, AUC60-480 (AUC from 60-480 min), Cmax, and t1/2 were evaluated. AUC60-480 represents the postirradiation time frame and was used to assess radiation effect. Overall, AUC0-Inf, Cmax, and t1/2 were numerically similar between strains (C57BL/6J and JDO) when combined, regardless of exposure status (AUC0-Inf: 112.50 ng·h/ml and 114.48 ng·h/ml, Cmax: 44.53 ng/ml and 63.96 ng/ml; t1/2: 1.8 h and 1.1 h, respectively). PK metrics were numerically lower in irradiated C57BL/6J mice than in non-irradiated mice [irradiation ratio: irradiated values/non-irradiated values = 0.71 for AUC60-480 (i.e., 29% lower), and 0.6 for t1/2]. In JDO mice, the radiation ratio was 0.53 for AUC60-480 (i.e., 47% lower), and 1.7 h for t1/2. The AUC0-Inf, Cmax, and t1/2 of the NHPs were 29.20 ng·h/ml, 7.68 ng/ml, and 3.26 h, respectively. Despite the numerical differences seen between irradiated and non-irradiated groups in PK parameters, the effect of radiation on PK can be considered minimal based on current data. The biodistribution in C57BL/6J mice showed that dmPGE2 per gram of tissue was highest in the lungs, regardless of exposure status. The radiation ratio for the different tissue AUC60-480 in C57BL/6J mice ranged between 0.5-1.1 (50% lower to 10% higher). Spleen, liver and bone marrow showed close to twice lower exposures after irradiation, whereas heart had a 10% higher exposure. Based on the clearance values from mice and NHP, the estimated allometric scaling coefficient was 0.81 (95% CI: 0.75, 0.86). While slightly higher than the current literature estimates of 0.75, this scaling coefficient can be considered a reasonable estimate and can be used to scale dmPGE2 dosing from animals to humans for future trials.

Recovery from hematopoietic injury by modulating prostaglandin E(2) signaling post-irradiation.

While high dose total body irradiation (TBI) is used therapeutically, the proliferation of nuclear weapons, increasing use of nuclear power, and worldwide radical terrorism underscore the need to develop countermeasures to a radiological mass casualty event. The hematopoietic syndrome of the acute radiation syndrome (HS-ARS) results from severe compromise to the hematopoietic system, including lymphocytopenia, neutropenia, thrombocytopenia, and possible death from infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow hematopoietic stem cells (HSC) and progenitor cells (HPC) may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E(2) (PGE(2)) has been shown to have pleiotropic effects on hematopoiesis, acting to inhibit apoptosis and promote self-renewal of HSC, while inhibiting HPC proliferation. We assessed the radio-mitigating potential of modulating PGE(2) signaling in a mouse model of HS-ARS. Treatment with the PGE(2) analog 16,16 dimethyl PGE(2) (dmPGE(2)) 6h post-irradiation or inhibition of PGE(2) synthesis via delayed administration of the non-steroidal anti-inflammatory drug (NSAID) Meloxicam resulted in increased survival of lethally irradiated mice. Both early dmPGE(2) and delayed Meloxicam treatment were associated with increased HPC activity 35days following irradiation, demonstrating enhanced recovery of hematopoiesis. Our results define two different treatment modalities that are highly effective and safe to administer, and can be readily available.

The Factory of Blood Production: Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) support the lifelong production of hundreds of billions of blood cells per day. This unique, incredible ability of HSCs also creates an incredible therapeutic potential for patients. To advance this potential, effective methods to study HSCs are continually evolving. This chapter summarizes the variety of protocols and techniques covered in this book used to evaluate HSCs - modification, characterization, interaction with their niche, and in vivo function.

Hematopoietic Stem Cell Identification Postirradiation.

Radiation exposure is particularly damaging to cells of the hematopoietic system, inducing pancytopenia and bone marrow failure. The study of these processes, as well as the development of treatments to prevent hematopoietic damage or enhance recovery after radiation exposure, often require analysis of bone marrow cells early after irradiation. While flow cytometry methods are well characterized for identification and analysis of bone marrow populations in the nonirradiated setting, multiple complications arise when dealing with irradiated tissues. Among these complications is a radiation-induced loss of c-Kit, a central marker for conventional gating of primitive hematopoietic populations in mice. These include hematopoietic stem cells (HSCs), which are central to blood reconstitution and life-long bone marrow function, and are important targets of analysis in these studies. This chapter outlines techniques for HSC identification and analysis from mouse bone marrow postirradiation.

Establishing a Murine Model of the Hematopoietic Acute Radiation Syndrome.

The hematopoietic system is one of the most sensitive tissues to ionizing radiation, and radiation doses from 2 to 10 gray can result in death from bleeding and infection if left untreated. Reviewing the range of radiation doses reported in the literature that result in similar lethality highlights the need for a more consistent model that would allow a better comparison of the hematopoietic acute radiation syndrome (H-ARS) studies carried out in different laboratories. Developing a murine model of H-ARS to provide a platform suited for efficacy testing of medical countermeasures (MCM) against radiation should include a review of the Food and Drug Administration requirements outlined in the Animal Rule. The various aspects of a murine H-ARS model found to affect consistent performance will be described in this chapter including strain, sex, radiation type and dose, mouse restraint, and husbandry.

The magnitude of CXCR4 signaling regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.

CXCR4 antagonists sensitize FLT3/ITD+ AML cells to FLT3 inhibitors; however, CXCR4 signaling can induce apoptosis in AML cells, raising the question of whether CXCR4 signaling exerts divergent effects on FLT3/ITD+ cells. The present study investigated the paradoxical function of CXCR4 in resistance to FLT3 inhibitors. The FLT3 inhibitor quizartinib significantly decreased the number of FLT3/ITD+ Ba/F3 cells, whereas 1 ng/ml CXCL12 showed a significant protective effect against quizartinib. In contrast, CXCL12 over 100 ng/ml significantly decreased FLT3/ITD+ cell viability with concomitant downregulation of Runx1. Moreover, the survival of FLT3/ITD+ Ba/F3 or MOLM13 cells with low surface CXCR4 expression incubated with quizartinib was significantly enhanced by 100 ng/ml CXCL12; however, this protective effect of CXCL12 against quizartinib was barely detected in cells with high surface CXCR4 expression. Although silencing Runx1 downregulated CXCR4 expression, RUNX1 expression levels were significantly higher in CXCR4LOW FLT3/ITD+ Ba/F3 cells incubated with 100 ng/ml CXCL12 than in CXCR4HIGH cells, coincident with an increase in FLT3 phosphorylation. Silencing RUNX1 partially abrogated resistance to quizartinib in CXCR4LOW cells incubated with CXCL12, whereas ectopic RUNX1 significantly restored resistance in CXCR4HIGH cells. These results indicate that CXCR4 signaling of different magnitudes paradoxically regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.

Further Characterization of Multi-Organ DEARE and Protection by 16,16 Dimethyl Prostaglandin E2 in a Mouse Model of the Hematopoietic Acute Radiation Syndrome.

Survivors of acute radiation exposure suffer from the delayed effects of acute radiation exposure (DEARE), a chronic condition affecting multiple organs, including lung, kidney, heart, gastrointestinal tract, eyes, and brain, and often causing cancer. While effective medical countermeasures (MCM) for the hematopoietic-acute radiation syndrome (H-ARS) have been identified and approved by the FDA, development of MCM for DEARE has not yet been successful. We previously documented residual bone marrow damage (RBMD) and progressive renal and cardiovascular DEARE in murine survivors of H-ARS, and significant survival efficacy of 16,16-dimethyl prostaglandin E2 (dmPGE2) given as a radioprotectant or radiomitigator for H-ARS. We now describe additional DEARE (physiological and neural function, progressive fur graying, ocular inflammation, and malignancy) developing after sub-threshold doses in our H-ARS model, and detailed analysis of the effects of dmPGE2 administered before (PGE-pre) or after (PGE-post) lethal total-body irradiation (TBI) on these DEARE. Administration of PGE-pre normalized the twofold reduction of white blood cells (WBC) and lymphocytes seen in vehicle-treated survivors (Veh), and increased the number of bone marrow (BM) cells, splenocytes, thymocytes, and phenotypically defined hematopoietic progenitor cells (HPC) and hematopoietic stem cells (HSC) to levels equivalent to those in non-irradiated age-matched controls. PGE-pre significantly protected HPC colony formation ex vivo by >twofold, long term-HSC in vivo engraftment potential up to ninefold, and significantly blunted TBI-induced myeloid skewing. Secondary transplantation documented continued production of LT-HSC with normal lineage differentiation. PGE-pre reduced development of DEARE cardiovascular pathologies and renal damage; prevented coronary artery rarefication, blunted progressive loss of coronary artery endothelia, reduced inflammation and coronary early senescence, and blunted radiation-induced increase in blood urea nitrogen (BUN). Ocular monocytes were significantly lower in PGE-pre mice, as was TBI-induced fur graying. Increased body weight and decreased frailty in male mice, and reduced incidence of thymic lymphoma were documented in PGE-pre mice. In assays measuring behavioral and cognitive functions, PGE-pre reduced anxiety in females, significantly blunted shock flinch response, and increased exploratory behavior in males. No effect of TBI was observed on memory in any group. PGE-post, despite significantly increasing 30-day survival in H-ARS and WBC and hematopoietic recovery, was not effective in reducing TBI-induced RBMD or any other DEARE. In summary, dmPGE2 administered as an H-ARS MCM before lethal TBI significantly increased 30-day survival and ameliorated RBMD and multi-organ and cognitive/behavioral DEARE to at least 12 months after TBI, whereas given after TBI, dmPGE2 enhances survival from H-ARS but has little impact on RBMD or other DEARE.