Molecular and Cellular Specialization of Iron Regulation In Human Erythroid Progenitor Cells Prior to Hemoglobin Production

Abstract 4255

Based upon the importance of iron for erythropoiesis, it was hypothesized that specific molecular and cellular mechanisms may be manifested during early erythropoiesis in preparation for heme and hemoglobin production. Here iron regulation was studied in immature erythroid progenitor cells prior to the increased production of heme or hemoglobin. CD34+ cells were cultured in SCF, IL-3 and FLT-3 ligand for 7 days, and the erythroid progenitor cells were identified and sorted from three donors according to the differential expression of CD36, CD41, and CD71. Among the sorted populations, the CD71(+)CD36(+)CD41(-)GPA(-) cells grew almost exclusively (>95%) into erythroid colonies in methylcellulose culture. Cytospin examination of those erythroid progenitor cells demonstrated large blasts with deep blue cytoplasm and blebbed membranes consistent with pre-proerythroblasts. Furthermore, heme accumulation and hemoglobin were absent. Western analyses revealed that ferritin, transferrin receptor 1 (TfR1), and transferrin receptor 2 (TfR2) expression were regulated by dosed titrations (0, 10, 20, 40, 60, and 100 %) of iron-saturated transferrin in the culture medium. The intracellular iron-storage protein, ferritin, showed increased expression with higher holo-transferrin saturation. Opposite patterns of iron-dependent regulation for the transferrin receptors was identified. TfR1 protein decreased and TfR2 increased with increased transferrin saturation. While TfR1 was detected at high levels on the plasma membranes, immunofluorescence studies showed TfR2 co-localization with lysosomes (correlation with LAMP1, a lysosomal marker, R = 0.53 ± 0.11, n=22). Decreased holo-transferrin in the culture medium resulted in no significant reduction in the proliferation of these cells until hemoglobin production was induced by erythropoietin. Confocal microscopy demonstrated a distinct peri-centrosomal lysosomal compartment surrounded by mitochondria. Coincidentally, upon sulfide-silver staining, it was observed that iron accumulated within the lysosomal compartment in a transferrin-dependent fashion. No similar iron-containing lysosomal-mitochondrial structure was identified in non-erythroid cells in the same cultures. These results suggest that functional, lineage-specific differentiation occurs at the progenitor stage of hematopoiesis, and that iron import and regulation is highly developed during erythropoiesis prior to hemoglobin production.