TPO-, SCF-, and TPO/SCF-displaying HIV-1–derived vectors promote high-level gene transfer into CB CD34⁺ cells. (A) CD34⁺ CB cells were incubated for 72 hours with VSV-G/TPO-displaying (G/TPOHA), VSV-G/SCF-displaying (G/SCFSUx), or VSV-G/TPO/SCF-displaying (G/TPOHA/SCFSUx) vectors at an MOI of 20. Cells were analyzed for the presence of the CD34⁺ surface marker and GFP expression by FACS analysis. The percentage of GFP⁺CD34⁺ cells is indicated into the upper right quadrant. The data presented are representative of 6 independent experiments. (B) CD34⁺ CB cells were incubated for 24 hours versus 3 days with TPO-, SCF-, or TPO/SCF-displaying vectors at an MOI of 20. Control incubations were performed with VSV-G–pseudotyped vectors in the absence or presence of cytokines (rTPO = 10 ng/mL; rSCF = 50 ng/mL). Cells transduced for 24 hours were washed and maintained for another 48 hours in serum-free medium in the absence of cytokines. Cells were analyzed for GFP expression 72 hours after the start of transduction by FACS analysis. Data are shown as means ± SD, n = 3. (C) CD34⁺ CB cells were incubated with TPO-, SCF-, and TPO/SCF-displaying LVs at MOIs of 20 and 4. Control incubations were performed with VSV-G–pseudotyped vector in the absence (–) or presence of recombinant cytokines (rTPO = 10 ng/mL; rSCF = 50 ng/mL). After a 72-hour incubation, the total number of transduced CD34⁺ CB cells was calculated (number of cells at start of infection [5 × 10⁴] × cell expansion × % cell transduction × % cell survival; data are shown as means ± SD, n = 4).