Phenotypic isolation, morphology, and potential of 6 distinct hematopoietic progenitor populations. (A) Lin^–IL-7Rα^–Sca-1^– BM cells were analyzed for expression of c-Kit versus IL-3Rα (i) or c-Kit versus antibody isotype control (ii). Cells shown in i correspond to 2% of all nucleated BM cells. Lin^–Sca-^+–IL-7Rα^– cells were sorted into c-Kit⁺IL-3Rα^– (green gate in i) and c-Kit⁺IL-3Rα⁺ (red gate in i) populations. Cells shown in iii and xi correspond to 0.52% and 0.68% of all nucleated BM cells, respectively. Purified c-Kit⁺IL-3Rα^– cells (iii) were restained for expression CD71 versus CD41 (iv), and sorted into CD71⁺CD41^– (v), CD71^–CD41^– (vii), or CD71^–CD41⁺ (ix) cells. Sorted c-Kit⁺IL-3Rα⁺ cells (xi) were stained for expression of FcγR (CD16/CD32) versus CD41 (xii), and further separated into FcγR^hiCD41^– (xiii), FcγR^loCD41^– (xv), or FcγR^loCD41⁺ (xvii) cells. Numbers in i, iv, and xii are relative percentages of the populations in the indicated regions. Numbers shown in v, vii, ix, xiii, xv, and xvii are percentages of each population per total nucleated BM cells. Cytospins from each population were stained with May-Grünwald-Giemsa stain (vi,viii,x,xiv,xvi,xviii). The scale bar (shown in xviii) corresponds to 15 μM and applies to panels vi, viii, x, xiv, xvi, and xviii. Based on their potential, the 6 progenitors were designated as erythrocyte progenitors (EPs; v), IL-3Rα^– common myeloid progenitors (CMP IL-3Rα^–; vii), IL-3Rα ^– megakaryocyte progenitors (MP1; ix), granulocyte-monocyte progenitors (GMPs; xiii), IL-3Rα⁺ common myeloid progenitors (CMP IL-3Rα⁺; xv), and IL-3Rα⁺ megakaryocyte progenitors (MP2; xvii). (B) Purified BM progenitors (as shown in panel A) were plated at 2000 cells/culture into methylcellulose assays promoting the generation of BFU-E (i), CFU-Meg (ii), or CFU-GM (iii) colonies. Colonies were counted after 8 days. BFU-E colonies were typed by benzidine staining for globin. CFU-Meg and CFU-GM colonies were identified by morphology. EPs lack potential for BFU-E, CFU-Meg, or CFU-GM colonies. Data shown are representative for one of 3 experiments. Formation of more mature CFU-Ss day 8 (iv), and more immature CFU-Ss day 12 (v) was analyzed by transfer of HSCs (2500 cells/mouse; number of recipients [n] = 2), CMP IL-3Rα^– (2500 cells/mouse; n = 2), CMP IL-3Rα⁺ (2500 cells/mouse; n = 2), GMP (2500 cells/mouse; n = 2), EP (2500 cells/mouse; n = 2), MP1 (2500 cells/mouse; n = 2), or MP2 (2500 cells/mouse; n = 2). Injection of total BM (2 × 10⁵ cells/mouse; n = 2) or PBS (n = 2) served as positive and negative controls, respectively. After 8 or 12 days macroscopic colonies were counted. CFU-Ss are restricted to HSC and CMP subpopulations (iv-v). Data shown are representative for one of 2 experiments.