Normal marrow CD34⁻CD244^±CD117⁺ populations are GM and erythroid precursors. (A, top) FACS plots with analysis gates showing lineage markers and CD34 expression, CD150/CD48 expression and forward scatter (FSC), and CD244 and CD117 expression in normal BM. (Bottom) Mean and SEM of each population as a percentage of MNCs. (B) Photomicrographs of FACS-sorted Lin⁻CD34⁻244⁺117⁺ (promyelocyte-like), Lin⁻CD34⁻244⁺117⁻ (mature granulocytic series and monocytes), and Lin⁻CD34⁻CD244⁻117⁺ (erythroblast-like) stained with May-Giemsa-Grunwald. The box with dotted lines shows two amalgamated microscope fields from the same cytospin slide. Images are representative of three separate experiments using BM from three separate donors. Bars, 20 µm. (C and D) FACS-sorted Lin⁻CD34⁺ (n = 3 donors) and Lin⁻CD34⁻ (CD244⁺117⁺ and CD244⁺117⁻, n = 6; CD244⁻117⁺, n = 4) populations were tested for CD11b, CD15, and CD14 (C) and CD71 (D) expression. (E) Normalized gene expression (to GAPDH) of SPI1/PU.1, MPO, CSF3R, IL3RA, ELANE, and ITGAM (expressed in GM lineage cells); EPOR and SPTB (expressed in erythroid cells); and PF4 in Lin⁻CD34⁻CD244⁺117⁺, Lin⁻CD34⁻CD244⁺117⁻, and Lin⁻CD34⁻CD244⁻117⁺ cells (n = 3 donors). *, P < 0.05; **, P < 0.01; two-sided Student’s t test. (F) Liquid culture output (human CD45⁺ cells and a percentage of total nucleated cells [TNC]) of 200 Lin⁻CD34⁺ and Lin⁻CD34⁻ populations assessed by FACS studied on D7, 14, 21, and 28 (n = 3–4). CD56⁺, NK cells; CD19⁺, B cells; CD14⁺, monocytes; CD11b⁺15⁺, granulocytes; and CD11b⁻15⁺, promyelocytes.(G) Colonies produced when 300 cells from Lin⁻CD34⁺ and Lin⁻CD34⁻ populations were plated in methylcellulose under myeloid colony formation conditions. Lineage affiliation was assessed by microscopy and confirmed in colony cytospins. E, erythroid; M, macrophage. Colonies were counted at D7 and D14 after plating (n = 5 donors). Error bars represent SEM. (H) Photomicrographs of representative D7 and D14 CFU-GM, D14 CFU-E colonies, and cytospins from the indicated populations (from five separate experiments using BM from five donors). Bars: (CFU-GM D7) 200 µm; (CFU-GM D14) 2,000 µm; (CFU-M/GM D14) 10 µm; (CFU-E D14) 100 µm; (CFU-E D14, right) 10 µm.