Coexpression of BCR-ABL and BMI1 results in long-term expansion and self-renewal of HSCs, but not CMPs, GMPs, or MEPs. (A) Sorting strategies of the transduced cells on the basis of combinatorial expression of cell-surface markers. Four different populations of cells were sorted from each group, as indicated. (B) CFC assay from the MiGR1/MiNR1 group revealed high purity of the sorted populations. (C) HSCs, CMPs, GMPs, and MEPs from all the groups were grown on MS5 stroma to evaluate their proliferative capacity. Maximal expansion was observed in cultures initiated with HSCs, and moreover, only the BMI1/BCR-ABL HSCs could be maintained over the long term. (D) Cells were plated weekly in methylcellulose, and colonies were scored on the basis of their morphology; a representative example at week 4 is shown. The HSC fraction yielded the highest progenitor frequencies, and only this fraction contained self-renewal properties, as determined by secondary replating assays. (E) Sorting scheme for Lin⁻ HSCs that were used in transduction experiments. (F) Transduced Lin⁻ HSCs were cultured on MS5 BM stromal cells under myeloid or lymphoid conditions. Arrows indicate time of replating.