Isolation of primary bone marrow–derived erythroblasts and EPO dose-dependent proliferation. (A) Erythroid progenitor cells from wild-type marrow were cultured in serum-free SP34-EX medium in the presence of EPO and SCF. At day 3.5, 90% or more of total cells were represented by CD71^high erythroid progenitors (top panels). Lin^pos depletion increased this representation to 98%, including 71% early-stage Kit^posCD71^high cells (bottom panels). (B) For the Lin^pos-depleted erythroblasts represented in panel A, the capacities of low-dose EPO to promote ³HdT incorporation and increases in cell numbers were defined. EPO dose-dependent formation of BrdU-positive cells also was assessed in short-term labeling experiments (ie, 1.5 and 3 hours). (C) Bone marrow progenitor cells were expanded for 3.5 days in SP34-EX medium. Kit^posCD71^high and Kit^negCD71^high cells were then isolated by MACS and analyzed in cytospin preparations. (D) These 2 discrete erythroblast populations were incubated for 5 hours in the absence of hematopoietic cytokines prior to culture in the presence of BrdU and EPO at 0.1 or 1.0 U/mL. At 2 hours, BrdU incorporation levels were assayed. Cells also were costained with 7-AAD to facilitate estimations of cell-cycle phase distributions. Note the increased representation of Kit^posCD71^high cells in S-phase (80%).