Expression of C/EBPα results in a loss of STAT5A^1*6-induced self-renewal and long-term expansion. (A) Human CB CD34⁺ cells were transduced with both STAT5A^1*6-IRES2-EGFP and C/EBPα-ER-IRES2-NGF-R, and cells in gate R3 were sorted on the MoFlo. Cells were plated in the presence or absence of 4-OHT in methylcellulose assays in 96-well plates in limiting dilutions of 1, 3, 9, 27, 81, and 243 cells per well. Two weeks later, CFC counts were scored. (B) Cells were transduced with both MiGR1/NGF-R empty vectors or STAT5A^1*6-IRES2-EGFP and C/EBPα-ER-IRES2-NGF-R as indicated. Cells were sorted and plated in bulk methylcellulose cultures, and the ratio between BFU-E and CFU-GM type colonies is shown in the absence or presence of 4-OHT. (C) Experiment performed as described in panel B, but sorted cells were plated onto MS5 stroma in 96-well plates in limiting dilutions of 5 to 1000 cells per well. After 5 weeks, methylcellulose was added to the wells, and 2 weeks later wells were scored as positive or negative to determine LTC-IC frequencies. Data from a representative experiment are shown. (D) Representative microscopy images of several MS5 cocultures at weeks 1 and 5.