Prospective isolation of human BM populations highly enriched in BFU-E and CFU-E activity. (A) Left: Clonogenic capacity of flow-sorted CMP, MEP, and GMP from purified, BM-derived CD34⁺ cells from TD DBA patients (n = 5) and age-matched controls (CON, n = 4). Right: Abnormal erythroid colonies, which we termed erythroid clusters, are observed exclusively in DBA cultures. (B) EP populations arising downstream of MEP (defined by flow cytometry as Lin⁻CD34⁺CD38⁺CD45RA⁻CD123⁻) were characterized by their expression of CD71, CD41a, CD36, and CD105. (C) CD71⁺CD41a⁻CD105⁻CD36⁻ (n = 6) and CD71⁺CD41a⁻CD105⁺CD36⁺ (n = 5) subpopulations were sorted into complete methylcellulose containing cytokines supportive of erythroid and myeloid development. GMPs were sorted concurrently as a control. Hematopoietic colonies were identified and scored as described in supplemental Materials and methods. (D) Single-cell clonogenic assays of Lin⁻CD34⁺CD38⁺CD45RA⁻CD123⁻CD71⁺CD41a⁻CD105⁻CD36⁻ cells (EEP; n = 5) and Lin⁻CD34⁺CD38⁺CD45RA⁻CD123⁻CD71⁺CD41a⁻CD105⁺CD36⁺ cells (LEP; n = 3). The percentage single-cell clonogenicity is defined as % of wells in which a colony grew following flow sorting of 1 cell per well. (E) May-Grünwald Giemsa staining of flow-sorted EEP and LEP from control adult BM. (F) Proliferative capacity of flow-sorted EEP and LEP in a longitudinal liquid culture (Protocol B; supplemental Materials and methods) assessed by cell counting (n = 4). Cells on day 11 were erythroblasts (ie, CD34⁻CD71⁺GlyA⁺CD14⁻CD11b⁻CD41a⁻). (G) Concurrent cell-cycle analysis on day 11 shows a higher number of erythroblasts derived from EEP in S phase with fewer in G_0/1 compared with erythroblasts derived from LEP (P < .05). (H) Flow-sorted EEP and LEP derived from control adult BM were cultured in the presence of erythropoietin, IL-3, IL-6, and stem cell factor (Protocol A; supplemental Materials and methods). Three days later, flow-cytometric analysis showed that EEP acquired expression of CD105 and CD36, whereas LEP gained higher expression of CD105 and CD36 (blue dots = day 0; green dots = day 3). Plots shown are representative of 2 independent experiments. (I) Messenger RNA expression levels of CD36, GATA-1, and GATA-2 determined by quantitative real-time PCR in purified GMP, MEP, EEP, LEP, and erythroblasts (Lin⁻CD34⁻CD36^hiCD71⁺GlyA⁺ erythroblasts [EB]), derived from 3 control BM samples. Transcript levels are normalized to GAPDH and expressed relative to the corresponding gene expression in MEP. As expected, GMPs do not express CD36 and GATA-1 but express a low level of GATA-2. Data are shown as mean ± SEM. *P < .05. CON, controls; NS, not significant.