High-level hCD34⁺-cell transduction by H/F-LV is maintained in myeloid and lymphoid lineages in primary NSG recipient mice. hCD34⁺ cells were prestimulated with a cytokine cocktail (TPO + SCF + Flt3-L) for 16 hours (A-D) or not (E-H) and transduced with H/F-LVs (MOI, 10) or VSV-G-LVs (MOI, 50) in the presence of retronectin for 36 hours. Subsequently, the cells (2 × 10⁵) were injected into the liver of irradiated newborn NOD/SCID γc^−/− mice (NSG mice). Upon reconstitution for 12 weeks, the BM, spleen, and thymus of these primary engrafted mice were analyzed. (A,E) Total human cell engraftment analyzed by anti-hCD45 staining using FACS; no significant differences in reconstitutions with H/F-LVs vs VSV-G-LVs and vs untransduced were detected (paired Student t test). Gray line indicates mean % hCD45 reconstitution. (B,F) The percentage of transduced human cells (GFP⁺hCD45⁺ cells) was analyzed by FACS in the different hematopoietic tissues (BM, spleen, and thymus). (C,G) Transduction levels of the different hematopoietic cell lineages in the BM of NSG reconstituted mice. The percentage of 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⁺CD20⁺), and myeloid progenitors (CD13⁺) are shown. (D,H) Transduction levels of different cell lineages in the spleen of NSG reconstituted mice. The percentages of GFP⁺ mature B cells (CD34^–CD19⁺CD20⁺), monocytes, and granulocytes (CD14⁺) are shown. M1 to M9 represent individual reconstituted NSG mice for independent transductions with the indicated vectors.