Summary Hematopoietic 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 using human and non-human primate HSCs. dmPGE2 significantly increased total human hematopoietic colony formation in vitro and enhanced engraftment of unfractionated and CD34+ hCB following xenotransplantation. In non-human primate autologous transplantation, dmPGE2-treated CD34+ MPBSCs showed stable multilineage engraftment over one year post-infusion. Together, our analyses indicated that dmPGE2 mediates conserved responses in HSCs from human and non-human primates, and provided sufficient preclinical information to support proceeding to an FDA-approved phase 1 clinical trial.

Apoptosis is critical to the resolution of inflammation, as it promotes the removal of neutrophils (PMN) by the reticuloendothelial system. In contrast, PMN persistence characterizes the early stages of chronic inflammation. Adult PMN with delayed senescence retain some functionality, although this has not been described for neonatal PMN. We hypothesized that neonatal PMN with prolonged survival retain cytotoxic and inflammatory function. To test one aspect of inflammatory function, we determined surface CD11b expression on 0-h and 24-h PMN after chemotactic formyl-methionine-leucine-phenylalanine (fMLP) stimulation. Although fMLP induced a greater percentage upregulation of CD11b on 0-h adult PMN, this was similar between nonapoptotic cord blood and adult PMN at 24 h. Furthermore, percentage up-regulation of CD11b was more robust for 24-h than for 0-h cord blood PMN. In contrast, there was no difference in responsiveness between 0-h and 24-h adult PMN. In studies of cytotoxic potential, we determined the expression of reactive oxygen intermediates (ROI) in phorbol 12-myristate 13-acetatestimulated cord blood and adult PMN at 0 h and in 24-h nonapoptotic PMN, using the dihydrorhodamine 123 assay.Stimulated cord blood PMN generated more ROI than did adult PMN at both 0 h and 24 h; in addition, ROI levels in 24-h cord blood PMN were similar to those of 0-h adult PMN. We conclude that PMN with prolonged survival retain specific cytotoxic and inflammatory functions, and these are enhanced in cord blood PMN. We speculate that neonatal PMN with prolonged survival have the functional capacity to contribute to the pathogenesis of inflammatory disorders. Abbreviations CLD, chronic lung disease DHR, dihydrorhodamine 123 fMLP, formyl-methionine-leucine-phenylalanine PE, phycoerythrin PMA, phorbol 12-myristate 13-acetate PMN, neutrophil(s) ROI, reactive oxygen intermediates 7-AAD, 7-aminoactinomycin PMN serve as the primary line of host defense during the early stages of sepsis and inflammation (1). After activation by systemic or local factors, primed endothelial cells interact with PMN through adhesion molecules that include selectins and integrins (2). These adhesive interactions initiate a process that allows the extravasation of PMN into inflamed tissue with the goal of neutralizing the inciting stimulus. In the case of inflammation mediated by bacterial or fungal microorganisms, phagocytosis and subsequent killing of these invaders occurs through mechanisms that partly involve ROI and proteases (3,4).The concept that apoptosis of inflammatory PMN and their timely removal by resident macrophages are critical to the resolution of inflammation has been well established (5,6). During the resolution phase, PMN typically undergo the phenotypic changes associated with apoptosis, which promotes their removal by resident macrophages (7). PMN can also undergo necrosis and lysis, a process that induces cytotoxicity in surrounding tissues (8), although apoptosis is the more common and physiologically preferable route. All cells a...

The Fas-Fas ligand (FasL) pathway of apoptosis is abnormally activated in diseases associated with impaired immune tolerance or chronic inflammation. Pregnancy-related hypertension is a spectrum of disease that commonly causes significant morbidity in women and in their newborn infants, is associated with generalized inflammation, and may be causally related to impaired maternal-fetal tolerance. Our recent observation of enhanced trophoblast expression of FasL in one form of pregnancy-related hypertension led us to hypothesize that this group of disorders might be associated with abnormal activation of the Fas-FasL pathway. To test this hypothesis, we prospectively quantified soluble and leukocyte-associated Fas receptor and FasL in the maternal and umbilical cord blood (CB) sera of 20 gestations complicated by preeclampsia and of 18 normal control gestations, using ELISA and flow cytometric analyses. We determined higher soluble FasL levels in paired maternal and CB sera of hypertensive gestations compared with control gestations (p Ͻ 0.01); in contrast, soluble Fas levels were similar between groups. Surface expression of FasL was lower on maternal (p Ͻ 0.01) and CB (p Ͻ 0.05) neutrophils from affected gestations, whereas surface Fas expression was lower on maternal (p Ͻ 0.02), but not CB, neutrophils and lymphocytes. We conclude that expression of Fas and FasL in sera and on leukocytes is altered in gestations complicated by preeclampsia, and speculate that activation of the Fas-FasL pathway mediates associated pathologic processes in affected women and in their neonates. Activation of the Fas-Fas Ligand pathway involves cellular interactions between the Fas receptor and FasL, and can result in apoptosis or in an inflammatory response (1). The Fas receptor (CD95, Apo-1, FasR), a type I integral membrane protein belonging to the tumor necrosis factor receptor family, is expressed on numerous cell types including hematopoietic cells, and its expression increases during inflammation (1). In contrast, expression of FasL (CD95L), a type II transmembrane molecule belonging to the tumor necrosis factor receptor superfamily, is limited to certain leukocytes and tissues with immune privilege (2). Fas-FasL interactions leading to clonal deletion of antigen-presenting cells have been implicated in the establishment of graft tolerance (3), and provide one explanation for the immune privilege status of certain tissues, including the placenta, cornea, and testis (2). In contrast, abnormal function of the Fas-FasL pathway is involved in the pathogenesis of a variety of disorders, including cytopenias, autoimmune diseases, graft-versus-host disease, and graft rejection (1,(3)(4)(5)(6).Mice with the gld mutation are deficient in FasL function and have smaller litters with decreased viability (7). Studies in these mice suggest that the Fas-FasL pathway contributes to preservation of the fetal allograft by preventing the trafficking of activated leukocytes between the mother and fetus during normal gestation, observations also ...

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The thrombocytopenia observed in neonates following an acute hypoxic event is not likely due to a direct deleterious effect of hypoxia on Mk progenitors.

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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<0.001) was found following dmPGE2 treatment of hCB cells. We have recently shown that PGE2 signals through cAMP to regulate wnt mediated control of HSC formation and function both in vitro and in vivo (Goessling et al., Cell 2009); to determine whether hCB cells increase cAMP activity in response to dmPGE2 treatment, fresh whole human cord blood units were exposed to increasing concentrations of dmPGE2 for 5, 15 and 30 mins, and intracellular cAMP levels were measured by chemiluminescence. dmPGE2 caused a dose-dependent increase in cAMP concentration, comparable to that of the known cAMP activator forskolin. Further, qPCR analysis of dmPGE2 treated CD34+ hCB cells demonstrated up regulation of camp-responsive elements, as well as genes indicative of wnt pathway activation (AXIN1), HSC induction (RUNX1) and homing (CXCR4). dmPGE2 was previously shown to enhance the multilineage serial transplantable long-term repopulating ability of murine HSCs (North et al., Nature 2007). To mimic clinical transplantation protocols, which typically utilize unmanipulated (non-lineage depleted, non-CD34 enriched) samples, fresh whole cord blood samples were employed for the initial xenotransplantation studies. Ex vivo dmPGE2 treatment significantly enhanced the number of engrafted recipients compared to matched controls (27/46 vs 13/42; *p=0.01) in comparative transplantation assays; use of purified CD34+ hCB cells for transplantation confirmed the effect of dmPGE2 on HSCs. These data suggest that treatment of hCB products with dmPGE2 will be both safe and effective in achieving expansion of HSCs for transplantation in the clinical setting; an FDA- approved phase 1 clinical trial evaluating dmPGE2 (FT1050, Fate Therapeutics) for this purpose is currently accruing patients at Dana-Farber/Partners Cancer Center. 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.