Transgene expression in differentiated hematopoietic lineages after transduction of human CD34⁺ cells with HIV-derived vectors.

Human CD34⁺ cells from umbilical cord blood (10⁵ cells) were transduced with 10⁶ TU of HIV vectors (corresponding to an MOI of 10) containing either the EF1α or the PGK promoters and differentiated into various hematopoietic lineages as described in “Materials and methods.” After differentiation, HIV-EF1α– (first and second column) or HIV-PGK–transduced cells (third column) were analyzed by flow cytometry for both GFP (x-axis) and lineage-specific marker expression (glycophorin, CD14, CD42b, CD15, and CD1a, respectively, for erythroids, monocytes, megakaryocytes, granulocytes, and DCs). Isotype control antibodies were used in the first column. For each promoter, cell populations expressing high levels of lineage-specific markers were gated (upper rectangle in 2-dimensional plots), and monoparametric frequency histograms of GFP expression for these cells were generated (located above the 2-dimensional plots). The percentage of GFP⁺ cells (upper number) and median of fluorescence intensity of GFP (lower number) was determined. For the experiment generating erythroids, monocytes, megakaryocytes, and granulocytes, the fraction of GFP⁺ CD34⁺ cells before differentiation was 22% for EF1 and 16% for PGK. For the experiment generating DCs, the fraction of GFP⁺ precursors before differentiation was 32% for EF1 and 29% for PGK. These data are representative of 4 independent experiments.