BMI1 is required for long-term growth and self-renewal of AML CD34⁺ cells. (A) AML CD34⁺ cells from different FAB subclassification were transduced with scrambled RNAi or BMI1-RNAi lentiviral vectors, and long-term cultures on MS5 bone marrow stromal cells were performed. Expansion was monitored weekly, and cumulative cell counts are shown. (B) Experiment as in panel A, but now transduced AML CD34⁺ cells were cultured on MS5 for a period of 5 to 6 weeks, after which human CD45⁺ cells were harvested and replated onto new MS5 cells, followed by an additional culturing period of 4 to 5 weeks. (C) Representative micrographs of cobblestone area-forming cells present in MS5 cocultures at week 4 initiated with AML CD34⁺ cells transduced with scrambled RNAi or BMI1-RNAi lentiviral vectors. Pictures were taken with a Leica DM-IL microscope (Leica Microsystems) with a 20×/0.30 objective.