LV-transduced progenitor T cells allowed accelerated thymopoiesis in vivo in NSG mice compared with LV-transduced CD34⁺cells. (A) CD34⁺ cells were plated on Dll4-coated wells in the presence of a cytokine cocktail (IL-7, TPO, Flt3-L) for 24 hours, and they were subsequently transduced with BaEV- and H/F-LVs encoding GFP at an MOI of 10 or without vector in the presence of Vectofusin, an agent that facilitates transduction.⁷⁸  At 8 days of T-cell differentiation, these transduced T-cell progenitors were injected intrahepatically into NSG recipient mice. In parallel, CD34⁺ cells from the same donor were prestimulated briefly with cytokines that maintain HSC potential (SCF, TPO, Flt3-L) for 24 hours and were transduced with BaEV- and H/F-LVs (MOI, 10) or without vector in the presence of RetroNectin. At 24 hours after transduction, the transduced CD34⁺ cells were engrafted in a second group of NSG recipients. Human thymocyte reconstitution was assessed 6 weeks after injection into NSG mice by FACS. (B) Percentage of GFP⁺ thymocytes (CD4 SP, CD4⁺CD8^–; DP, CD4⁺CD8⁺; DN, CD4^–CD8^–; CD8 SP, CD8⁺CD4^–) for T-cell progenitor engraftment compared with CD34⁺ cell engraftment. (C) Distribution of CD4 SP, DP, DN, and CD8 SP thymic subpopulations for proT cells compared with CD34⁺ cell engraftment. CD4 SP cells were analyzed for CD3 surface expression for proT (left panels) and CD34⁺ (right panels) engrafted cells. ISP CD4, CD4⁺CD3^– cells. (D) FACS analysis of the percentage of CD3⁺ T cells per total hCD45⁺ cells in the thymus. (E) The absolute numbers of mature CD3⁺ T cells per total human thymocytes for proT cells compared with CD34⁺ cell engraftment (mean ± SD; n = 3). (F) Percentage of mature CD3⁺ T cells in the blood per total hCD45⁺ cells for proT cells compared with CD34⁺ cell engraftment (mean ± SD; n = 3). *P < .05.