Expansion of primitive hematopoietic cells by ITD-Flt3 requires Survivin. (A) Two million bone marrow cells were infected with retrovirus containing wild-type (WT) human Flt3, ITD-Flt3 (N51) in MSCV-IRES-EGFP vector, or vector control for 2 days in the presence of 100 ng/mL rmSCF, rhTPO, and rhG-CSF. After infection, the medium was replaced with the same cytokine cocktail and incubated for an additional 3 days. Total cells were counted and stained to determine the proportion and the absolute number of GFP⁺ lineage Lin^Neg, c-kit⁺, Lin^Neg (KL) or c-kit⁺, Sca-1⁺, Lin^Neg (KSL) cells. Data are expressed as mean ± SEM from 16 independent experiments. *P < .05 compared with wild-type Flt3; †P < .05 compared with vector control. (B,C) Number of GFP⁺ KSL (B) and KL (C) cells transduced with vector control (EGFP⁺), wild-type (WT) Flt3, or ITD-Flt3 (N51) in cultures of littermate control Survivin^flox/flox or Cre-ER Survivin^flox/flox cells after culture for 2 weeks with 4OH-tamoxifen without hematopoietic growth factors. Data are the average of 5 experiments and expressed as mean ± SEM. *P < .05 compared with Survivin^flox/flox. The bottom panel shows the gene deletion of Survivin allele by 4OH-tamoxifen. (D) Representative plots showing the proportion of KSL cells in Survivin^flox/flox and Cre-ER Survivin^flox/flox mouse bone marrow cells overexpressing ITD-Flt3 (N51) and cultured with 1 μM 4OH-tamoxifen for 2 weeks. The box represents c-kit⁺, Sca-1⁺ cells in the Lin^Neg fraction. (E) Annexin-V staining of ITD-Flt3 (N51)–transduced GFP⁺ KL cells, from cultures of Survivin^flox/flox or Cre-ER Survivin^flox/flox marrow cells after culture for 2 weeks with 4OH-tamoxifen without hematopoietic growth factors. The left panel shows representative data from 1 of 2 experiments. The mean ± SEM of annexin V fluorescence intensity (MFI) from 2 experiments is shown in the right panel. *P < .05 compared with Survivin^flox/flox. (F) Sub G1 content of ITD-Flt3 (N51)–transduced KL cells from cultures of Survivin^flox/flox or Cre-ER Survivin^flox/flox cells after culture in the absence of growth factors with or without 4OH-tamoxifen for 14 days. Data are the mean ± SEM from 3 experiments. *P < .05 compared with Survivin^flox/flox. (G) The effect of Survivin deletion and culture with increasing concentration of SU5416 on KSL cell number. Marrow cells from control Survivin^flox/flox or Cre-ER Survivin^flox/flox mice were retrovirally transduced with ITD-Flt3 (N51) and cultured with 1 μM 4OH-tamoxifen and escalating doses of SU5416 in the absence of hematopoietic growth factors for 14 days. Data represent 1 of the 2 experiments with similar results and are expressed as mean ± SEM. *P < .05 compared with Survivin^flox/flox.