Hes1- and BCR-ABL-transduced KSLs, CMPs, or GMPs were immortalized independently of IL-3. (A) Colony-forming assay of KSLs, CMPs, and GMPs transduced with BCR-ABL alone or Hes1 and BCR-ABL, cultured in methylcellulose with or without cytokine cocktail containing SCF, TPO, IL-3, and IL-6. Hes1⁺BCR-ABL⁺ cells could be serially replated more than 4 times both with or without cytokines. In contrast, whereas KSLs, but not CMPs or GMPs, transduced with BCR-ABL alone, formed colonies in the presence of cytokines, neither KSLs, nor CMPs, nor GMPs formed colonies without cytokine supplementation. Bars represent the number of colonies obtained per 10³ cells after each round of plating in methylcellulose. A representative result from 3 independent and reproducible experiments is shown. Error bars represent the SD from duplicate cultures. (B) Sustained growth of Hes1⁺BCR-ABL⁺ cells in liquid culture without cytokine supplementation. The numbers of cells were determined every 7 days by trypan blue staining, and 10⁵ cells per well were seeded into a 6-well plate. Liquid culture was reproducibly continued for more than 6 months. (C) Typical colonies derived from KSLs, CMPs, and GMPs transduced with BCR-ABL alone (left panels) or BCR-ABL and Hes1 (right panels) in the presence of SCF, TPO, IL-3, and IL-6. Images were obtained with an IX70 microscope and a DP70 camera (Olympus); an objective lens, UPlanFl (Olympus); original magnification ×100. (D) Giemsa staining of Hes1⁺BCR-ABL⁺ KSLs, CMPs, and GMPs. Images were obtained with a BX51 microscope and a DP12 camera (Olympus); an objective lens, UPlanFl (Olympus); original magnification ×1000. (E) Flow-cytometric analysis of Hes1⁺BCR-ABL⁺ KSLs cultured in methylcellulose supplemented with SCF, TPO, IL-3, and IL-6. The dot plots represent Gr-1, CD11b, c-Kit, Sca-1, CD3, CD4, CD8a, B220, CD19, CD34, Ter119, and CD14 labeled with a corresponding PE-conjugated monoclonal antibody versus expression of GFP/BCR-ABL. Hes1⁺BCR-ABL⁺ CMPs and GMPs showed a similar expression pattern (supplemental Figure 2C-D). The analyzed cells were GFP and NGFR sorted at 48 to 60 hours from the initiation of BCR-ABL- or Hes1+BCR-ABL transduction and cultured for the following lengths of time before the analysis: (A) 0 days, (B) 4 weeks, (C) 1 week, (D) 1 week, and (E) 1 week.