Re-expression of C/EBPα results in a loss of STAT5A^1*6-induced proliferative advantage of human CD34⁺ cells. (A) Human CB CD34⁺ cells were transduced with MiGR1, STAT5A^1*6-IRES2-EGFP, C/EBPα-ER-IRES2-NGF-R, or both STAT5A^1*6 and C/EBPα-ER, as indicated. Cells were plated on MS5 stroma in the presence of 4-OHT, and the proliferation was monitored weekly by FACS analysis for GFP or NGF-R. (B) Experiment performed as described in panel A, but cells were sorted after transduction, and expansion was monitored by determining the cell counts weekly. (C) Combined data of 3 representative experiments in which the relative expansion is indicated as compared to the MiGR1 control that was set to 1 at each time point. Significant differences were determined using a Student t test. (D-E) Experiments as in panel B, but now 4-OHT was added at day 0 (D) or day 3 (E). Cultures were monitored for 14 days. Data are presented as the percentage of untransduced, STAT5A^1*6 (EGFP⁺), C/EBPα-ER (NGF-R⁺), or double-transduced cells within 1 batch of transduced cells that was expanded on MS5 over a 2-week period. (F) Cells were transduced as indicated, plated onto MS5 stroma to allow expansion for 7 days, after which 4-OHT was added for an additional 24 hours. After 4-OHT stimulation, the cell-cycle distribution was determined by FACS. (G) Experiment as in panel F, but now cells were treated with 4-OHT for 48 hours.