Background: While hemopoietic stem cells have been thought to reside predominantly in the CD34(+) population, recent data suggests that repopulating cells may, in fact, also reside in the lin(-)/CD34(-) population. Transduction of both these populations by murine retroviral vectors is limited by quiescence of hemopoietic stem cells.
Methods: We therefore sought to transduce these populations using a VSV-G pseudotyped, HIV-based, human lentiviral vector, encoding eGFP. CD34(+) cells and lin(-)/CD34(-) cells were selected from the same BM samples by immunomagnetic beads (StemSep) to deplete lineage-positive cells and by CD34 selection columns (Miltenyi) to separate CD34(+) and CD34(-) populations. We transduced target cells, with or without prestimulation with cytokines, using conventional suspension culture, or fibronectin plates, or flow-through transduction. Transduction efficiency was analyzed by flow cytometry and clonogenic assay.
Results: We found that transduction on fibronectin plates was more efficient than flow-through transduction, or suspension cultures, for both cell populations. Mean transduction rates on fibronectin plates, analyzed by flow cytometry, were 14% and 5% for CD34(+) cells and lin(-)/CD34(-) cells respectively, without prestimulation, and 31% and 20% with prestimulation. By contrast, a murine retroviral vector transduces CD34(+) cells with lower efficiency (mean 16.1% with prestimulation) and does not induce any significant transduction of the CD34(-)/lin(-) population (mean < 2%). When lentiviral transduction 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 fibronectin. Moreover, the human viral vectors can transduce a candidate stem-cell population that is resistant to murine retroviral transduction.