Stepwise transduction of the D171N mutant followed by BMI1 in human CD34⁺ cells. (A) Human CD34⁺ cells were precultured for 3 to 4 days in expansion medium and transduced with GFP-tagged D171N-mutant. After 3 or 4 days, GFP⁺ cells were sorted and cultured in long-term culture medium for 28 days. Then, CD34⁺ cells were reselected by the CD34 MicroBead Kit again and transduced with DsRed-tagged BMI1. We also transduced the DsRed vector as a control. Finally, 35 days after the D171N transduction, GFP⁺/DsRed⁺ cells were sorted and cultured in methylcellulose or long-term culture medium. (B) CFC replating assay in 3 independent experiments. (C) Representative flow cytometry analyses of the first colonies. (D) Proliferation fold in 3 independent experiments. Day 0 was the day of the second (DsRed vectors) transduction. (E) Flow cytometric analysis for CD34 expression, and Wright-Giemsa–stained cytospins on day 39 as captured with a BX51 microscope and a DP12 camera (Olympus) at ×400 and ×1000 original magnifications.