Effects of Hypoxia on Megakaryocyte Progenitors Obtained from the Umbilical Cord Blood of Term and Preterm Neonates

<i>Background:</i> Placental insufficiency is associated with early-onset thrombocytopenia in preterm neonates. Prior studies demonstrated a reduction in circulating megakaryocyte (Mk) progenitors, suggesting decreased platelet production. We hypothesized that decreased Mk production is the result of a direct inhibitory effect of hypoxia on the proliferation of Mk progenitors, or a hypoxia-induced change in the fetal hematopoietic environment. <i>Objective:</i> To test the effects of hypoxia on the clonogenic maturation of Mk progenitors obtained from term and preterm cord blood CD34^pos cells, either cultured alone or in conjunction with CD34^neg light density mononuclear cells (LDMCs). <i>Methods:</i> CD34^pos cells and CD34^neg LDMCs were isolated from the cord blood of term and preterm deliveries, and mobilized peripheral blood CD34^pos cells were obtained from healthy adults. CD34^pos cells were then cultured alone or co-cultured with CD34^neg LDMCs in a semisolid, serum-free media containing rTpo, IL-3, and IL-6. Cultures were exposed to 20%, 5%, or 1% oxygen for 10–12 days. Mk colonies were then quantified following immunohistochemical staining. <i>Results:</i> Pure CD34^pos cells from preterm (n = 5) and term (n = 5) neonates and from adults (n = 4) generated similar numbers of Mk colonies in all three oxygen concentrations. However, the number of Mk colonies in preterm co-cultures was progressively lower with decreasing O₂ concentrations. <i>Conclusions:</i> Hypoxia did not appear to directly inhibit colony formation of Mk progenitors from preterm and term cord blood CD34^pos cells. However, co-culture studies showed a decrease in Mk colony formation with hypoxia, suggesting an indirect inhibitory effect of hypoxia on Mk clonogenic maturation mediated by non-progenitor cells in the hematopoietic microenvironment.