![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).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/118/3/10.1182_blood-2010-09-305383/4/zh89991174680002.jpeg?Expires=1721415735&Signature=FtaRBBrRSZkHh6995gHFNp0P4YQVDCUgck4ENr-PZg-JhjhE-2WQio4JRpr9OnasseEEm668sA1Ojf5RMXaMtRs-QpGdLTV-eWkVBEzE8NykIn5GoZf71naFeVtjHHnFBz0n5CDuZ3rQ2dr5NW5KCQOKj1r3ISHTkcrujQTBXR0PK9v0eCjKnEsi04VnkLuCWeKOZDdNAdiGjYYTs4Hv72JLf4VjN0j5UkAmmD6UTHiWXm4CzbjvO-l6jODJtsslikwe7U-p08fPZmZtKLsDoM4G9JcottTqVRdGxa39eDkqoPP7TlFjSJDheU3drjyWHFiD3jBJlOEUJf6qjAwoSw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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).
