BaEV-LVs are highly superior over RDTR-LVs and VSV-G-LVs for the transduction of unstimulated hCD34⁺ cells. Freshly isolated CB hCD34⁺ cells were directly transduced by the indicated LV pseudotypes at an MOI of 10. For VSVG-LVs, an MOI of 100 was used. Analysis for the percentage GFP⁺ hCD34⁺-cells was performed 3 days after transduction by FACS. A representative dot blot is shown for each pseudotype in A and the average transduction levels are indicated in B (means ± SD, n = 6). (B) Three days after transduction, the hCD34⁺ cells were seeded in myeloid differentiation medium. The percentage of GFP⁺ CFCs is presented (means ± SD, n = 4; P < .001 when comparing BaEV-LVs with VSV-G- and RDTR-LVs). (C) Cell cycle progression was monitored 3 days after transduction of the hCD34⁺ cells with the different pseudotypes by simultaneously visualizing RNA (Pyronine-Y) and DNA (7-AAD) content of the cells. As controls, unstimulated and stimulated (SCF+TPO+Flt3-L) hCD34⁺ cells without exposure to vector (no vector) were included. The percentages of cells in the G0/G1a, G1b, and S/G2/M phase of the cell cycle are indicated in the dot blots. (D) Freshly isolated CB hCD34⁺ cells were transduced with BaEVTR-LVs and BaEVRless-LVs at an MOI = 10 for 24 hours. The cells were then injected into the liver of newborn NSG mice. On reconstitution for 12 weeks, the different hematopoietic tissues (BM, spleen, and thymus) of these engrafted mice were analyzed for human cell engraftment by anti-hCD45 staining (Table 1). Transduction levels of the different cell lineages in the BM of NSG reconstituted mice are presented (upper histograms). GFP⁺ immature early progenitor cells (CD34⁺CD19⁻CD10⁻), pre/pro-B cells (CD34⁺CD19⁺CD10⁻), pro-B cells (CD34⁻CD19⁺CD10⁻), immature and mature B cells (CD34⁻CD19⁺CD10⁺), and myeloid progenitors (CD13⁺) are shown. Transduction levels of different cell lineages in the spleen of NSG reconstituted mice (lower histogram). The percentage of GFP⁺ pre/pro-B cells (CD34⁺CD19⁺CD10⁻), pro-B cells (CD34⁻CD19⁺CD10⁻), immature and mature B cells (CD34⁻CD19⁺CD10⁺), monocytes, and granulocytes (CD14⁺) are shown. Data for 4 representative mice from Table 1 for each vector type are shown. All transductions were performed in the presence of RetroNectin.