Transplantation of the AR Sca1⁺CD19⁺ population results in leukemia. (A) Experimental design. HSCs (CD45.2) were cultured ex vivo for 5 days in the presence of mSCF, mTPO, mFLT3L, mIL6, and mIL11 (with or without γ retroviral [MFG-GFP] transduction) and then transplanted into 10 Gy-irradiated WT mice (CD45.1). (B) A blood chimerism analysis of recipient animals 15 weeks after transplantation on the basis of CD45.1 (recipient) and CD45.2 (donor) alloantigen expression. The T-cell (CD3⁺), B-cell (B220⁺), NK-cell (NK1.1⁺), and monocyte (CD11b⁺) subpopulations were analyzed by immunofluorescence. ■ indicates the WBC count/μL (WBC); and □, the CD45.2⁺ cell count/μL in the blood. Columns represent the mean values and SD for AR mice (n = 11) and A mice (n = 9). (C) Kaplan-Meier survival curves of WT mice transplanted with A-derived Sca1⁺ mock-transduced (▵, n = 9) and GFP-transduced cells (▴, n = 9) or AR-derived Sca1⁺ mock-transduced (□, n = 11) and MFG-GFP vector-transduced cells (■, n = 12). A log-rank test was used to compare the groups (P < .0001). (D) Integration site analysis of pro-B-cell leukemia in the AR-GFP cohort. Gel electrophoresis of PCR-amplified genomic vector integration fragments showed a polyclonal integration pattern for the AR-GFP Sca1⁺ (Sca1⁺) pool before transplantation (lanes S1 and S2) and a characteristic, mono-oligoclonal integration pattern found in 7 transduced AR pro-B-cell tumors (AR*) (lanes 1, 4, 5, 9, 12, 13, 1x); M represents the DNA ladder. (E) AR Sca1⁺CD19⁻ GFP-transduced cells were transplanted into lethally irradiated WT recipients. The Kaplan-Meier curve shows a significantly (*) lower incidence of B-cell leukemia in mice transplanted with the AR Sca1⁺CD19⁻ population (solid line ■, n = 10) than in animals transplanted with the AR Sca1⁺ population (dashed line ■, n = 12, P < .0003 in a log rank test) at 52 weeks posttransplantation. (F) Kaplan-Meier survival curves of WT mice transplanted with AR-derived Sca1⁺CD19⁻ GFP-transduced cells (n = 12), AR-derived Sca1⁺CD19⁻ Rag1-transduced cells (n = 8).