Efficient T-cell differentiation in vitro of SCID-X1 BM CD34⁺HSPCs after transduction with BaEV-LVs encoding γC receptor. SCID-X1 BM CD34⁺ HSPCs were transduced (BaEV) or not (mock) with BaEV-LVs encoding IL-2RγC at an MOI of 10 for 6 hours after 16 hours of prestimulation with cytokines in either RetroNectin or Dll4 plus RetroNectin-coated wells (see supplemental Figure 6A). The X-SCID mutation is a breakpoint mutation in intron 3 of the IL-2RG gene, which is a γC leaky mutation. Flow cytometry analyses indicating the percentage (A) and absolute cell number (B) of CD34⁺CD7⁺ and CD34^–CD7⁺ T-cell progenitors after 4 days of culture in the presence (+ Dll4) or absence (–Dll4) of Dll4. After 4 days of culture, the cells were further cultured with OP9-hDL1 stromal cells to pursue the T-cell differentiation process. Analyses of VCN per cell in the genome by duplex quantitative PCR in sorted CD7⁺ cells at week 5 (C) and IL-2RγC mRNA expression by semi-quantitative RT-PCR in hCD45⁺ cells at week 7 (D) of differentiation (compared with CB CD34⁺ T-cell differentiation cultures in parallel). During coculture with OP9-hDL1 stromal cells, mock and BaEV-LV transduced cells were analyzed once per week by flow cytometry. Analyses of absolute cell numbers of CD4⁺CD8⁺ (E) and CD3⁺ cells (F) at the indicated weeks after OP9-hDL1 cocultures. (G) TCR repertoire analysis by multiplex PCR followed by GeneScan analysis in CD7⁺ cells sorted at week 5 after OP9-hDL1 cocultures. All the populations were gated on live 7-amino-actinomycin-D^– cells.