Coexpression of BCR-ABL and BMI1 in human CD34⁺ cells promotes long-term stem cell self-renewal and myeloid and lymphoid expansion in vitro. (A) BMI1/BCR-ABL coexpression results in the immortalization of human CB CD34⁺ cells on MS5 stroma under myeloid conditions. The difference in proliferative capacity between the groups is shown. (B) A detailed representation of the experiment in panel A is shown. After 5 weeks, both suspension and adherent human CD45⁺ cells were harvested from the cultures, and the cells were seeded onto new stroma to evaluate their replating capacity. Arrows indicate time points when replating was performed. (C) An example of week 18 phase-dark CAFCs that could be harvested and replated to initiate new expanding cocultures. (D) The number of hematopoietic progenitors was evaluated in CFC assays in methylcellulose. A representative experiment is shown, where transduced CB CD34⁺ cells were analyzed after 9 weeks of expansion on MS5 BM stroma. (E) Cytospin preparations of suspension cells from cocultures described in (A) at week 16, indicating the presence of blast-like, as well as erythroid and myeloid precursor, cells. (F) Stem cell frequencies were determined in LTC-IC assays using freshly transduced CB CD34⁺ cells. (G) Long-term expansion of the transduced cells under lymphoid culture conditions. (H) Representative cytospin at week 8 from double-transduced cultures. (I) Clonogenic assays in methylcellulose were performed to evaluate the capacity to form progenitor colonies in the presence of IL-7. Microscopic images of the colonies are shown as the insets in the graph. (J) Immediately after transduction, the cells were plated in stroma-free liquid cultures in the presence of IL-3 and SCF. Long-term expanding BMI1/BCR-ABL cultures could be maintained for more than 23 weeks.