BMI1 promotes long-term in vitro expansion of human CD34⁺ progenitor cells. (A) Cord blood CD34⁺ cells were transduced with either control (open symbols) or BMI1 (closed symbols) vectors and grown in stroma-free liquid cultures using a mix of cytokines as described in “Methods.” Cells were counted weekly and 3 independent experiments are shown. Week 0 represents the number of transduced cells that were plated. (B) Cumulative expansion of the same cultures is shown. (C) The May-Grünwald Giemsa stained picture shows a cytospin of 16-week cultured cells where monocytes, macrophages, granulocytes and some blast-like cells can be observed, indicated by the arrows. (D) A representative experiment where the percentage of CD34⁺ cells was maintained at approximately 4% over a period of 20 weeks in BMI1 overexpressing cells is shown. (E) After 16 weeks, cultures were analyzed for progenitor content in CFC assays in methylcellulose by plating 10 000 cells from the culture without further resorting. Progenitors were only detected in BMI1-expressing cells. A CFU-GEMM colony from a representative CFC experiment is shown in the inset. (F) Transduced and GFP⁺ sorted CB CD34⁺CD38⁻ cells were propagated in the same cytokine-driven liquid culture conditions as in panel A for 20 weeks. (G) After transduction unsorted CD34⁺ cells were grown in cocultures on MS5 stromal cells. The cultures were weekly demidepopulated and analyzed for GFP expression by FACS. (H) Experiment as in panel G, but now transduced CD34⁺CD38⁻ cells were plated on MS5 stroma. (I) Nonadherent cells from week 5 cocultures were used to perform CFC assays (top panel). After 2 weeks, CFCs were harvested and replated into new methylcellulose assays (bottom panel). Only the BMI1-expressing cells contained replating capacity in secondary CFC assays, and a colony is shown in the inset. A representative experiment of 4 performed experiments is shown.