C/EBPα induces myeloid differentiation of primary MEPs. (A) Lineage-negative (Lin^neg) cell population was stained with the following cocktail of directly conjugated monoclonal antibodies: PE anti-cKit, FITC anti-CD34, APC anti–Sca-1, PE-Cy7 anti-FcRII/III (2.4G2), and PE-Cy5 anti–IL-7Rα. These cells were then sorted by high-speed sorter (MoFlo; Cytomation) for the IL-7Rα^–/c-Kit⁺/Sca-1^–/CD34^–/FcRII/III^lo cell population identified as MEPs. Normal MEPs were transduced with retrovirus containing pMSCV-C/EBPα-GFP or pMSCV-GFP. GFP⁺ MEPs were isolated and cultured for 3 days in cIMDM containing mSCF (100 ng/mL), hEpo (10 U/mL), and hTpo (100 ng/mL). Lineage differentiation was analyzed by flow cytometry using Gr-1 and TER119 antibodies and differential cell counts of Wright-Giemsa–stained cytocentrifuge preparations. Arrow indicates segmented neutrophils. (B) GFP⁺ MEPs (5 × 10⁴/plate) were also cultured in 1.1% methyl-cellulose containing mSCF (100 ng/mL), hEpo (5 U/mL) to detect erythroid progenitors as described in “Materials and methods.” The mean number of erythroid colonies ± SE of triplicate plates is shown.