BaEV-LVs allow high-level transduction of hCD34⁺ cells following mild cytokine prestimulation. (A) CB hCD34⁺ cells were prestimulated overnight (18-24 hours) with SCF, TPO, SCF+TPO, or SCF+TPO+Flt3-L and transduced in the presence of RetroNectin by the indicated LV pseudotypes at an MOI of 10. Analysis for the percentage GFP⁺ cells was performed 6 days after transduction (means ± SD, n = 4). hCD34⁺ cells were prestimulated with (B) rSCF+rTPO+rFlt3-L or (C) rSCF+rTPO and transduced by the different LV pseudotypes at different MOIs (1, 5, 10, and 20); for VSV-G-LVs, an additional MOI of 100 was applied. Analysis of percentage GFP⁺ cells was performed 6 days after transduction by FACS (means ± SD, n = 3). (D) CB hCD34⁺ cells were prestimulated with rSCF+rTPO or rSCF+rTPO+rFlt3-L and subsequently transduced with the indicated LV pseudotypes. An MOI of 10 was applied except for VSV-G LVs (MOI = 100). Three days after transduction, the cells were seeded in myeloid differentiation medium for 14 days. The percentage of GFP⁺ CFCs is shown for both stimulation conditions (means ± SD, n = 3). (E) Mobilized hCD34⁺ cells were prestimulated with SCF+TPO+Flt3-L+IL-3 and transduced with the indicated LV pseudotypes at the same vector doses as in D. Myeloid differentiation and transduction analysis was performed as in D. The percentage of GFP⁺hCD34⁺ cells and CFCs are shown (means ± SD, n = 3). All transduction were performed in the presence of RetroNectin, and P < .05 when comparing CFC transduction levels of BaEV-LVs with the other pseudotypes.