A primitive subset of CD34⁺ CML cells survives complete Bcr-Abl inhibition in growth factor–free medium for 12 days. (A) CD34⁺ CML cells (n = 3) were cultured in SFM ± 150nM and viable cell counts performed on days 4, 8, and 12, and (B) percentages of cells residing within each cell division determined by CFSE staining. After 12 days, cells were washed with PBS, added to CFC and LTC-IC assays, cultured in SFM plus high growth factor cocktail for 7 days. At day 7, (C) Ki-67/7AAD and (D) CFSE were measured by flow cytometry and (E) viable cell counts performed. (Ci) Cells at day 12 without drug treatment; (ii) cells at day 12 following dasatinb treatment; (iii) cells from (i) cultured for additional 7 days in SFM plus high growth cocktail; and (iv) cells from (ii) cultured for additional 7 days in SFM plus high growth factor cocktail. (Di) Cells from panel Ci were cultured for additional 7 days in SFM plus high growth factor cocktail, and (ii) cells from panel Cii were cultured for additional 7 days in SFM plus high growth factor cocktail. (F) The relative increase in CD34⁺CD133⁺CFSE^high was calculated (n = 3, P = .001) and the numbers of (G) CFC (n = 7, P = .048) and (H) LTC-IC (n = 3, P = .025) per 1000 cells at day 12 were calculated. The changes in (I) CFC, (J) CFC replated (n = 2), and (K) LTC-IC numbers compared with baseline cells (1.0) were also calculated. (L) The presence of Bcr-Abl (denoted by the white arrows) was measured by D-FISH in the remaining CML cells following the 12 day time course. A representative profile from (i) non-CML CD34⁺ cells at baseline, (ii) untreated CD34⁺ CML cells and (iii) 150nM dasatinib-treated CD34⁺ cells is demonstrated.