Transduction of CD34(+) and CD34(-)/lin(-) hemopoietic progenitors by lentivirus vectors

Background While hemopoietic stem cells have been thought to reside predomi nantly in the CD34(+) population, recent data suggests that repopulating ce lls may, in fact, also reside in the lin(-)/CD34(-) population. Transductio n of both these populations by murine retroviral vectors is limited by quie scence of hemopoietic stem cells. Methods We therefore sought to transduce these populations using a VSV-G ps eudotyped, HIV-based, human lentiviral vector, encoding eGFP. CD34(+) cells and lin(-)/CD34(-) cells were selected from the same BM samples by immunom agnetic beads (StemSep) to deplete lineage-positive cells and by CD34 selec tion columns (Miltenyi) to separate CD34(+) and CD34(-) populations. We tra nsduced target cells, with or without prestimulation with cytokines, using conventional suspension culture, or fibronectin plates, or flow-through tra nsduction. Transduction efficiency was analyzed by flow cytometry and clono genic assay. Results We found that transduction on fibronectin plates was more efficient than flow-through transduction, or suspension cultures, for both cell popu lations. Mean transduction rates on fibronectin plates, analyzed by flow cy tometry, were 14% and 5% for CD34(+) cells and lin(-)/CD34(-) cells respect ively, without prestimulation, and 31% and 20% with prestimulation. By cont rast, a murine retroviral vector transduces CD34(+) cells with lower effici ency (mean 16.1% with prestimulation) and does not induce any significant t ransduction of the CD34(-)/lin(-) population (mean <2%). When lentiviral tr ansduction was assayed in short- and long-term clonogenic assays there was minimal transduction of CD34 cells without prestimulation, increasing to 20 % with prestimulation. Discussion Lentiviral eGFP vectors can transduce hematopoietic progenitors effectively and efficiency is improved by cytokine prestimulation and the use of fibro nectin. Moreover, the human viral vectors can transduce a candidate stem-ce ll population that is resistant to murine retroviral transduction.