H/F-LVs allow close to 100% transduction of prestimulated hCD34⁺cells. (A) hCD34⁺ cells were prestimulated with a cytokine cocktail (TPO, SCF, Flt3-L [Flt3-L]) and transduced with H/F-LVs or VSV-G-LVs at indicated MOIs in the presence or absence of retronectin. Six days after transduction, the cells were analyzed for the percentage of GFP⁺CD34⁺ cells by FACS (means ± standard deviation [SD]; n = 6). (B) hCD34⁺ cells were prestimulated for 18 hours as follows: SCF or TPO alone, SCF + TPO, or SCF + TPO + Flt3-L. Subsequently, they were transduced with H/F-LVs or VSV-G-LVs at indicated MOIs in the presence of retronectin. Six days after transduction, the cells were evaluated for the percentage of hCD34⁺GFP⁺ cells by FACS (mean ± SD; n = 5). (C-D) Comparison between the transduction level of CD34⁺ cells and the myeloid colonies derived from these cells (GFP⁺ CFCs) for CD34⁺ cells prestimulated with (C) SCF + TPO or (D) SCF + TPO + Flt3-L (mean ± SD; n = 3). (E-F) An MOI of 10 for H/F-LVs and an MOI of 50 for VSV-G-LVs was used. Subsequently, they were transduced with H/F-LVs or VSV-G-LVs at increasing MOIs in the presence of retronectin. Six days after transduction, the cells were analyzed for the percentage of hCD34⁺GFP⁺ cells by FACS (means ± SD; n = 3). Statistical analysis for comparison of VSV-G-LV vs H/F-LVs was performed by using the paired Student t test. *P < .05; **P < .01; ***P < .001.