Increased erythroid development in C/EBPα^–/– mice. (A) Cytocentrifuge preparations of C/EBPα^–/– FL cells were stained with Wright-Giemsa, and differential cell counts were performed on 400 cells from 3 individual mice. The data are reported as the mean number of cells ± standard error (SE). (B) FL cells (2.5 × 10⁴) from C/EBPα^+/+, C/EBPα ^+/–, and C/EBPα^–/– embryos at E15.5 were cultured in complete IMDM (cIMDM) containing 1.1% methylcellulose, 25% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine (P/S/G), and cultures were supplemented with murine (m) IL-3 (30 ng/mL), stem cell factor (mSCF, 100 ng/mL), and human erythropoietin (hEpo, 5 U/mL). The mean number of BFU-E colonies ± SE of triplicate plates is shown. (C-D) C/EBPα^–/– and C/EBPα^+/+ FL cells were transduced with MSCV-GFP retroviral vectors to track donor erythroid reconstitution. Fetal liver cells were resuspended at 2 × 10⁵/mL in cIMDM and cultured with 8 μg/mL polybrene, 100 ng/mL mSCF, 100 ng/mL thrombopoietin (hTpo), 100 ng/mL hFlt-3L, and 50 ng/mL IL-6. A total of 3 retrovirus infections were performed, after which the cells were cultured with the same cytokine mixture. GFP-positive (GFP⁺) cells were isolated 2 days after the last infection by FACS sorting (FACSAria [BD Biosciences] or MoFlo [Cytomation, Fort Collins, CO]). BM cells were harvested 4 months after transplantation and then analyzed for the expression of the erythroid markers (TER119 and CD71) and myeloid cell markers (Gr-1 and Mac-1). (D) Horizontal bars indicate median values. (E) GFP⁺ BM cells were purified by FACS and cultured in methylcellulose (7.5 × 10⁴/plate) or soft agar (1 × 10⁵/plate) to determine the number of erythroid or myeloid progenitors. For myeloid progenitors, cells were plated in cIMDM containing 10% FCS, P/S/G, and 0.35% sea plaque agarose in the presence or absence of 50 ng/mL hG-CSF, 100 ng/mL M-CSF, 100 ng/mL mSCF, 20 ng/mL mGM-CSF, 30 ng/mL mIL-3. The mean number of BFU-E and CFU-c colonies ± SE of triplicate plates is shown.