Ex vivo expansion of CFU and sustained repopulation activity by loss of p190-B. FL LSK cells were incubated in serum-free medium with stem cell factor, IL-3, IL-6, IL-11, and megakaryocyte growth and development factor for 7 days. (A) Total cell number at day 7 of culture enumerated by hemocytometer (mean ± SEM; n = 6 independent experiments). (B) Analysis of cell division of cultured FL LSK cells. FL LSK cells were labeled with fluorescent dye CFSE and fluorescence intensity of CFSE⁺ LSK cells was analyzed at 0, 24, 36, and 48 hours of culture. Data shown are the representative flow profiles of fluorescent intensity of CFSE (2 independent experiments). (C) Cell-cycle analysis of cultured FL LSK cells. After 6 days of culture, cells were labeled with BrdU in vitro for 30 minutes and stained for LSK. Data represent the percentage of BrdU⁺ cells in LK or LSK cell population (mean ± SD; n = 3; the difference is not statistically significant [ns]). (D) Apoptosis analysis of cultured FL LSK cells at day 6 of culture. The cells were stained with annexin V and 7-AAD. Histogram represents the percentage of annexin V⁺ cells in LSK cell population (mean ± SD; n = 3; the difference is not statistically significant [ns]). (E) CFU plating efficiency of FL LSK cells before and after in vitro culture. Data in 2 left panels represent the number of colonies/input cells on day 0 and day 7 during the culture. Data represent mean ± SEM (n = 3 independent experiments). Data on right panel represent CFU expansion of cultured FL LSK cells expressed as fold increase (mean ± SD; n = 3; 1 representative experiment from 3 independent experiments). (F) Competitive repopulation assay of cultured FL LSK cells. Cultured cells plus freshly isolated competitor BM cells were injected into lethally irradiated mice. Histogram represents the percentage of donor (CD45.2⁺) cells in the PB at the indicated time points after transplantation (mean ± SD; n = 5). *P < .05.