Stimulation of mouse bone marrow cells with Kit ligand, FLT3 ligand, and thrombopoietin leads to efficient retrovirus-mediated gene transfer to stem cells, whereas interleukin 3 and interleukin 11 reduce transduction of short- and long-term repopulating cells

The effects of cytokine stimulation during retroviral transduction on in vi vo reconstitution of mouse hematopoietic stem cells was tested in a murine competitive repopulation assay with alpha-thalassemia as a marker to distin guish donor and recipient red blood cells (RBCs) and the enhanced green flu orescent protein (EGFP) as a marker for gene transfer. After transplantatio n, EGFP was detected in up to 90% of circulating RBCs, platelets, and leuko cytes, and in primitive progenitors in bone marrow (BM), spleen, and thymus of individual transplanted mice for observation periods of more than 6 mon ths. Large quantitative differences in reconstitution were observed after t ransplantation with graded numbers (1000-30,000) of EGFP(+) cells precondit ioned with various combinations of Kit ligand (KL), FLT-3 ligand (FL), thro mbopoietin (TPO), interleukin 3 (IL-3), and IL-11. Relative to nonmanipulat ed BM cells, repopulation of EGFP(+) cells was maintained by KL/FL/TPO stim ulation, but, similar to 30-fold reduced after stimulation with KL/FL/TPO/I L-11, KL/FL/TPO/IL-3, or KL/FL/IL-3/IL-11. These differences were not cause d by changes in the ability of immature hematopoietic cells to home to the BM, which was only moderately reduced. In conclusion, these quantitative tr ansplantation studies of mice demonstrate the importance of optimal ex vivo cytokine stimulation for gene transfer to stem cells with retention of the ir in vivo hematopoietic potential, and also emphasize that overall in vitr o transduction frequency does not predict gene transfer to repopulating ste m cells.