Cell-cycle kinetics and VSV-G pseudotyped retrovirus-mediated gene transfer in blood-derived CD34(+) cells

As hematopoietic stem and progenitor cells have a low mitotic index, we have quantitated the impact of cytokine combinations on cell cycling of CD34(+) cells and, using VSV-G pseudotyped retroviral vectors, correlated our findings with ex vivo gene transfer. We tested nine different combinations of cytokines for induction of human peripheral blood CD34(+) cells into cell cycle over 72 hours. Using the 5-bromodeoxyuridine-Hoechst 33258 (BrdU-Hoechst) assay, we measured the cell-cycle kinetics. The combinations of cytokines tested that were most efficient in inducing the CD34(+) cells into cycle were stem cell factor (SCF) plus one of the following interleukin-1 (IL-1), IL-3, granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF). The maximum numbers of cells in S+G(2)M phase were observed after 48 hours of culture. At least 35 +/- 5% of the CD34(+) cells remained quiescent in the first G(0)/G(1) phase, however, no matter which cytokine combination was used. Cell-cycle analysis of the CD34(+)CD38(-) subset by 7-amino actinomycin D staining did not detect cycling cells during 72 hours of culture with any of the cytokines tested. To investigate whether the cells could be infected by the VSV-C pseudotyped virus containing the neomycin phosphotransferase gene (neo), we exposed CD34(+) cells to the virus for 7-8 hours after 0, 36, and 48 hours of cytokine stimulation. Total CD34(+) cells and the CD34(+)CD38(-) subset were analyzed by polymerase chain reaction (PCR) for reverse-transcribed viral DNA of the neomycin resistance gene (RT-neoDNA). Immediately after exposure to the virus, RT-neoDNA was detectable in CD34(+) cells that had been cultured with or without cytokines for 36 to 48 hours. Forty-eight hours postinfection, however, RT-neoDNA could be detected only with cytokine combinations that induced mitosis of the CD34(+) cells, consistent with the requirement for mitotic activity for retroviral integration. Similar experiments performed with the CD34(+)CD38(-) subset showed that RT-neoDNA could not be detected at any time point. Thus, postinfection RT-neoDNA could be immediately detected in noncycling CD34(+) cells but not in CD34(+)CD38(-) cells. These results suggest that during short-term liquid culture, there may be blocks for reverse transcription of retroviral RNA in CD34(+)CD38(-) cells in addition to the lack of mitotic activity.