Dosed Deficiency of Iron Restricts Terminal Maturation and Enucleation of Cultured Human Erythroblasts

Abstract 1041

In addition to reduced hemoglobin production, iron deficiency causes anemia though poorly understood defects in erythropoiesis that reduce the number of erythroid cells released from the marrow space. To study the negative effects of iron deficiency upon erythropoiesis, CD34+ cells from three healthy human donors were cultured in serum-free media supplemented with transferrin-bound iron. Dosed titrations of holo-transferrin (1.2, 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 mg/ml) were balanced by apo-transferrin supplementation to maintain total transferrin levels of 1.2 mg/ml in all cultures. Analyses were performed over the 21-day culture period that included assessment of proliferation, differentiation, enucleation and surface phosphatidylserine expression. Diminution of holo-transferrin in the culture medium caused a decrease in total cell counts and reduced cell divisions detected by CFSE analysis. During the first two weeks of culture, the progenitor cells differentiated into a glycophorin A positive population at all holo-transferrin doses. However, several iron-dependent changes were detected in the final stages of differentiation during culture days 14–21. Hemoglobin accumulation was reduced by 72% at the lowest holo-transferrin dose. Flow cytometry demonstrated a decrease in the loss of surface transferrin receptor in iron-deficient cultures. Thiazole orange analysis also demonstrated significantly lower levels of enucleation from 25.9±8.2% (1.2 mg/ml) to 4.4±4.2% (0.1mg/ml). Microscopic examination of the enucleated cells demonstrated a hypochromatic phenotype at lower holo-transferrin concentrations. Phosphatidylserine on the surface membrane (annexin V staining) increased with reduction of holo-transferrin from 6.3±2.6% (1.2 mg/ml) to 78.3±9.4% (0.1 mg/ml, p=0.003). The effects of iron on surface phosphatidylserine expression were limited to holo-transferrin concentrations of <0.6 mg/ml. Isocitrate supplemented at a concentration of 5.0 mM increased the cell counts in all concentrations of holo-transferrin, and partially rescued the iron deficient phenotype. These data suggest that iron deficiency ex vivo causes decreased proliferation, decreased loss of surface transferrin receptor, and decreased enucleation of normoblasts. There was also a significant increase in phosphatidylserine detected on the surface of the iron deficient normoblasts that provides a mechanism whereby macrophages can identify, phagocytose, and efficiently recycle iron from those cells to other normoblasts in the local marrow environment via erythroblastic islands.