The leukemia-initiating cell frequency and expansion potential are greatly increased in a 2-oncogene compared with a 1-oncogene model of acute myeloid leukemia. (A) Experimental design of limiting dilution assay for LIC and assessment of LIC expansion potential during 6 days of in vitro culture. (B) LIC assay from bone marrow cells expressing 1 (MN1+CTL) or 2 (MN1+ND13) oncogenes. Cells were cultured ex vivo for infection and selection for 21 days. Results of 2 independent experiments were analyzed together after observation of mice for 16 weeks (n = 4-7 per cell dose, 95% CI). LIC frequency and 95% CI were calculated by Poisson statistics. (C) Survival curves for mice that received a transplant of a cell dose at limiting dilution either transduced with MN1+CTL (n = 4) or MN1+ND13 (n = 4). (D) Consecutive CRU assays (5 cell doses) before and after a 6-day culture period from MN1+CTL- compared with MN1+ND13-expressing cells. Cells were cultured ex vivo for infection and selection for 13 days (corresponds to day 0 of expansion experiment), and 105 cells were plated on day 0. LIC frequency was calculated by Poisson statistics. *P < .01 for day-6 LIC frequency MN1+CTL versus MN1+ND13. AB indicates antibiotics for selection of transgene-expressing cells; CTL, control; and ND13, NUP98HOXD13 fusion gene
