Effects of hypoxia on megakaryocyte progenitors obtained from the umbilical cord blood of term and preterm neonates

Background: 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. Objective: 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). Methods: 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. Results: 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 O ₂ concentrations. Conclusions: 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.