STAT5b and to a lesser extent STAT1 mediate massive leukemia stem cell expansion. (A-B) Cytokine stimulation assay of wild-type or Stat5b^−/− murine bone marrow cells stably transduced with MN1+HOXA9. Cells were cultured in the presence of the indicated cytokines (6 ng/mL IL-3, 10 ng/mL IL-6, 20 ng/mL SCF, or 10 ng/mL GM-CSF), and viable cells were counted and replated every 3 days (mean ± SD, n = 3). (C-D) Cytokine stimulation assay of wild-type or Stat1^−/− murine bone marrow cells stably transduced with MN1+HOXA9. Cells were cultured in the presence of the indicated cytokines (6 ng/mL IL-3, 10 n/mL IL-6, 20 ng/mL SCF, or 10 ng/mL GM-CSF), and viable cells were counted and replated every 3 days (mean ± SD, n = 3). (E) Consecutive limiting dilution assays for LIC (4 cell doses) before and after a 6-day culture period from wild-type compared with Stat5b^−/− bone marrow cells expressing MN1+HOXA9. Cells were cultured ex vivo for infection and selection for 16 days (corresponds to day 0 of expansion experiment), and 10⁵ cells were plated on day 0. LIC frequency was calculated by Poisson statistics. (F) Consecutive limiting dilution assays for LIC (4 cell doses) before and after a 6-day culture period from wild-type compared with Stat1^−/− bone marrow cells expressing MN1+HOXA9. Cells were cultured ex vivo for infection and selection for 16 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+HOXA9 in null versus MN1+HOXA9 in wild-type cells.