Figure 4
Figure 4. Self-renewal of Ap2a2-transduced HSCs in vivo. (A) Analyses of secondary transplantation recipients. Donor CD45.1 reconstitution in numbered primary donor (pd) mice and transplanted secondary recipient (sr) mice. Horizontal bar shows the mean level of reconstitution for each (sr) group. (B) In vivo clonal analyses of self-renewal. Southern blots with GFP-specific probe on BM from 1 (pd) mouse from each Ap2a2-transduced well (1-4 in Figure 3B) and their respective (sr) mice (as numbered and indicated by respective color codes). (C) CRU assay in secondary recipients. From 3 further independent in vitro to in vivo assays with vector-transduced and Ap2a2-transduced CD45.2 donor CD150+48−Lin− HSCs transplanted into CD45.1 recipients: 1 representative primary CD45.1 recipient mouse from each assay was taken at 20 weeks after transplantation (supplemental Figure 1A). From these 3 vector-transduced and 3 Ap2a2-transduced primary recipient mice, BM cells were flow sorted for donor CD45.2 cells. Equivalent limiting donor cell numbers, together with 200 000 CD45.1 competitor BM cells, were transplanted into sublethally irradiated secondary CD45.1 recipients for assessment of donor reconstitution. Analyses were done 16 weeks after secondary transplantation via WEHI bioinformatics ELDA software (http://bioinf.wehi.edu.au/software/elda/). (D) Quantification of CD45.2 donor reconstitution in secondary recipients with respective transduced-HSCs from CRUs in panel C. Significance for recipients transplanted with, respectively, 100 000, 200 000, and 500 000 Ap2a2- compared with vector-transduced cells are P = .02, P = .03, and P = .01 (*). (E) Donor-derived compared with recipient-derived LSK cells in BM from secondary recipients analyzed at 20 weeks after transplantation. Given the minimal CD45.2 donor repopulation in vector-transduced recipient mice (D), the 2 vector-transduced mice together with the competitor CD45.1 subpopulations from the Ap2a2-transduced recipients were assessed together as the competitor CD45.1 population and compared with the Ap2a2-transduced CD45.2 donor populations for respective LSK%. *P = .02.

Self-renewal of Ap2a2-transduced HSCs in vivo. (A) Analyses of secondary transplantation recipients. Donor CD45.1 reconstitution in numbered primary donor (pd) mice and transplanted secondary recipient (sr) mice. Horizontal bar shows the mean level of reconstitution for each (sr) group. (B) In vivo clonal analyses of self-renewal. Southern blots with GFP-specific probe on BM from 1 (pd) mouse from each Ap2a2-transduced well (1-4 in Figure 3B) and their respective (sr) mice (as numbered and indicated by respective color codes). (C) CRU assay in secondary recipients. From 3 further independent in vitro to in vivo assays with vector-transduced and Ap2a2-transduced CD45.2 donor CD150+48−Lin− HSCs transplanted into CD45.1 recipients: 1 representative primary CD45.1 recipient mouse from each assay was taken at 20 weeks after transplantation (supplemental Figure 1A). From these 3 vector-transduced and 3 Ap2a2-transduced primary recipient mice, BM cells were flow sorted for donor CD45.2 cells. Equivalent limiting donor cell numbers, together with 200 000 CD45.1 competitor BM cells, were transplanted into sublethally irradiated secondary CD45.1 recipients for assessment of donor reconstitution. Analyses were done 16 weeks after secondary transplantation via WEHI bioinformatics ELDA software (http://bioinf.wehi.edu.au/software/elda/). (D) Quantification of CD45.2 donor reconstitution in secondary recipients with respective transduced-HSCs from CRUs in panel C. Significance for recipients transplanted with, respectively, 100 000, 200 000, and 500 000 Ap2a2- compared with vector-transduced cells are P = .02, P = .03, and P = .01 (*). (E) Donor-derived compared with recipient-derived LSK cells in BM from secondary recipients analyzed at 20 weeks after transplantation. Given the minimal CD45.2 donor repopulation in vector-transduced recipient mice (D), the 2 vector-transduced mice together with the competitor CD45.1 subpopulations from the Ap2a2-transduced recipients were assessed together as the competitor CD45.1 population and compared with the Ap2a2-transduced CD45.2 donor populations for respective LSK%. *P = .02.

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