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SummaryHematopoietic stem cells (HSCs) are used in transplantation therapy to reconstitute the hematopoietic system. Human cord blood (hCB) transplantation has emerged as an attractive alternative treatment option when traditional HSC sources are unavailable; however, the absolute number of hCB HSCs transplanted is significantly lower than bone marrow or mobilized peripheral blood stem cells (MPBSCs). We previously demonstrated that dimethyl-prostaglandin E2 (dmPGE2) increased HSCs in vertebrate models. Here, we describe preclinical analyses of the therapeutic potential of dmPGE2 treatment by using human and nonhuman primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34+ hCB after xenotransplantation. In nonhuman primate autologous transplantation, dmPGE2-treated CD34+ MPBSCs showed stable multilineage engraftment over 1 year postinfusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and nonhuman primates and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.

Severe hypoxic insults to the fetus and neonate are associated with the development of thrombocytopenia. The thrombocytopenia in some cases is the result of disseminated intravascular coagulation, but that mechanism fails to account for all, perhaps the majority, of cases.We hypothesized that human fetal megakaryocyte (Mk) progenitors are directly adversely affected by transient anoxia.To test this, we isolated CD34pos cells from the umbilical cord blood of 10 healthy term neonates, and exposed these to 0% or 20% O2 for 24 h, with or without recombinant thrombopoietin (rTpo, 50 ng/mL). After 24 h, a portion of the CD34pos cells were harvested for flow cytometric evaluation of apoptosis. The remaining cells were cultured for an additional 10-12 days, under normoxic conditions, in a collagen-based serum-free system containing rTpo, IL-3, and IL-6. In this way, we sought to determine the effect of transient anoxia on clonogenic capacity of Mk progenitors.Contrary to our hypothesis, anoxia did not increase either apoptosis or cell death of the CD34pos cells. The addition of rTpo was protective, with a significant decrease in apoptosis and cell death (P0.0001), and an increase in the number of Mk colonies cultured (P = 0.04). There was no difference between the normoxic and anoxic groups in proliferative potential of the Mk progenitor cells.The thrombocytopenia observed in neonates following an acute hypoxic event is not likely due to a direct deleterious effect of hypoxia on Mk progenitors.

Abstract 369 Hematopoietic stem cells (HSCs) comprise the base of the entire hematopoietic system and alone possess the ability to both self-renew and differentiate into all mature blood lineages, thereby maintaining immune function, tissue perfusion and hematopoietic homeostasis. HSCs are therapeutically valuable for the treatment of hematological malignances, immunodeficiencies and bone marrow failure. Prostaglandin (PG) E2 has been shown to enhance HSC engraftment in allogeneic murine transplantation models by our lab (North et al., Nature 2007) and others (Hoggatt et al., Blood 2009); PGE2 was additionally found to influence the balance of apoptosis and proliferation in the HSC population via modulation of wnt activity (Goessling et al., Cell 2009), and to modify CXCR4-responsive homing to the hematopoietic niche following transplantation (Hoggatt et al., Blood 2009). In order to translate the therapeutic potential of a stabilized version of PGE2, dmPGE2, we sought to determine the safety and efficacy of ex vivo dmPGE2 exposure in human cord blood (hCB) stem cells. Compared to matched control cord samples, no significant negative impact on hCB cell viability was observed following dmPGE2 treatment (10?M for 1 hour) using either fresh or frozen cord blood units; of note, the CD34+ stem and progenitor compartment seemed particularly able to tolerate the treatment protocol. To determine whether dmPGE2 treatment was not only safe, but potentially valuable for preserving hCB cell viability, apoptosis was measured by FACS analysis for 7AAD and AnnexinV in pooled CD34-enriched (CD34+) hCB samples treated in parallel with 1?M dmPGE2 or the vehicle control (DMSO); at 6 and 9 hours post exposure, cells treated with dmPGE2 showed a significant reduction in apoptosis compared to controls. Cell proliferation assays confirmed results seen in prior murine studies and demonstrated that dmPGE2 not only suppressed apoptosis, but enhanced HSC self-renewal. To determine if dmPGE2 exposure altered the functional characteristics of human cord blood samples, in vitro culture assays were conducted; pooled CD34+ samples were exposed over a time series (12 and 30 mins, 1, 3, 6 and 12 hours) to the vehicle control and dmPGE2 (1?M) then plated at limiting dilutions (2000, 800, 320). A significant 2-fold enhancement in total colony number (p Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties. Zon:FATE Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Stemgent: Consultancy. North:Fate Therapeutics: Consultancy, Patents & Royalties.