Prostaglandin E2 enhances human cord blood stem cell xenotransplants and shows long-term safety in preclinical nonhuman primate transplant models.

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 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.

Tacrolimus: in vitro effects on myelopoiesis, apoptosis, and CD11b expression.

BACKGROUND: Tacrolimus is a common component of multi-drug immunosuppressive regimens that are used for the prevention of rejection in transplant recipients. Tacrolimus therapy has been associated with anemia after transplantation, and recent clinical evidence in children suggests its association with the development of neutropenia for which an alternative etiology is not apparent. Mechanisms of suspected tacrolimus-related neutropenia have not been previously elucidated. We hypothesized that this variety of neutropenia might be due to a negative effect of tacrolimus on neutrophil production and/or survival. METHODS: We designed in vitro studies to determine the dose-dependent effect of tacrolimus on myeloid cell production and/or apoptosis. CD34+ cells and neutrophils isolated from umbilical cord blood of term gestations were cultured with tacrolimus (0-1,000 ng/ml). To evaluate apoptosis, cells cultured for 24 hours were stained with annexin V-fluorescein isothiocyanate (V-FITC) and 7-amino-actinomycin D (7-AAD) and analyzed by flow cytometry. For clonal analysis, CD34+ cells cultured in cytokine-enhanced semi-solid media were scored for their myeloid/erythroid mix colony forming units (CFU-Mix) and myeloid (CFU-GM) progenitor cell contents. RESULTS: Tacrolimus induced a dose-dependent enhancement of clonogenesis and survival of CD34+ cells at clinically relevant doses. Conversely, tacrolimus had no effect on the survival of mature neutrophils or on the upregulation of CD11b in response to chemotactic stimulation. CONCLUSION: In contrast to our initial hypothesis, we observed that tacrolimus at clinically relevant concentrations enhanced clonogenesis of neutrophil progenitors and promoted their survival. Our in vitro studies suggest that tacrolimus alone is unlikely to be a significant factor in the neutropenia observed during immunosuppressive therapy.

Neonatal neutrophils with prolonged survival exhibit enhanced inflammatory and cytotoxic responsiveness.

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 up-regulation 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-acetate-stimulated 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.

Effects of anoxia on megakaryocyte progenitors derived from cord blood CD34pos cells.

BACKGROUND: 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. OBJECTIVE: We hypothesized that human fetal megakaryocyte (Mk) progenitors are directly adversely affected by transient anoxia. DESIGN AND METHODS: 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. RESULTS: 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 (P < 0.0001), 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. CONCLUSIONS: The thrombocytopenia observed in neonates following an acute hypoxic event is not likely due to a direct deleterious effect of hypoxia on Mk progenitors.

Decreased functional caspase-3 expression in umbilical cord blood neutrophils is linked to delayed apoptosis.

Resolution of inflammatory processes depends on the efficient removal of aging neutrophils by the reticuloendothelial system. Neutrophil apoptosis is key to this process, and its impairment may contribute to the pathogenesis of chronic inflammation. We recently discovered that Fas-mediated apoptosis in umbilical cord blood neutrophils was significantly delayed as compared with those of adults. Because execution of apoptosis relies on caspases, we used reverse transcription PCR, immunoblots, and enzymatic assays to study the integrity of several members of those proteases known to mediate Fas-induced apoptosis in neutrophils. Our results indicate that diminished expression of caspase-3 mRNA and the precursor form of the protein, as well as a lower functional enzymatic activity of caspase-3, correlates with delayed apoptosis in umbilical cord blood neutrophils. Our data suggest that functional expression of caspase-3 in neutrophils may be regulated during ontogeny.