BMI1 expression pattern in human CD34⁺ cells and enforced BMI1 expression in human CD34⁺ cells. (A) BMI1 expression levels in CD34⁺ cells of clinical samples. Relative BMI1 expression was measured by triplicated qRT-PCR and calculated as the ratio of BMI1 to GAPDH expression. Data are also expressed as mean ± SD of each patient group. **P < .01. (B) BMI1 expression in transduced CD34⁺ cells was confirmed by qRT-PCR. CD34⁺ cells were repurified from GFP⁺ sorted cells after 5 and 40 days of culture in complete cytokine medium. Bar chart represents the mean ± SD of 3 independent experiments. RNA from pMXs.IG-transduced cells on day 5 served as a control, and the RNA level was defined as 1. *P < .05; **P < .01. (C) pMXs.IRES-DsRed-Express (pMXs.IR) retroviral construct for the expression of BMI1. (D) Representative flow cytometry profile of cells retrovirally transduced with pMXs.IR or BMI1 shows the transduction efficiency. The DsRed⁺ cells shown within the gate were collected. (E) Expression of BMI1 was confirmed by Western blotting using anti-Bmi1 antibody. Anti–β-actin antibody was used as the control. (F) Human CD34⁺ cells transduced with the indicated vector and sorted for DsRed expression were analyzed by CFC replating assay. Ten thousand cells were plated in methylcellulose culture dishes. Data are expressed as mean ± SD of 3 independent experiments. (G) Growth pattern of the transduced cells cultured in complete cytokine medium displayed as proliferation fold originating from 10⁰ just after sorting. The error bars represent the SD from 4 independent experiments. (H) The expression pattern of surface markers as shown by a typical flow cytometry profile, and Wright-Giemsa–stained cytospins of the DsRed⁺ cells on day 42 culture in complete cytokine medium as captured with a BX51 microscope and a DP12 camera (Olympus); original magnification ×1000.