Efficient retrovirus-mediated transduction of primitive human peripheral blood progenitor cells in stroma-free suspension culture

Retroviral transduction of hematopoietic cells has resulted in unsatisfacto ry gene marking in clinical studies. Since cytokine-stimulated stem cells h ave engrafted poorly in animal models, we investigated phenotypic changes d uring culture of peripheral blood progenitor cells (PBPC). Human CD34(+) HL A-DRlow cells, immunomagnetically separated from PBPC collections, were fou nd to extrude rhodamine-123, which is characteristic for primitive hematopo ietic cells. Cells were grown in suspension cultures supplemented with cyto kines. While interleukin-3-containing factor combinations promoted cell pro liferation they caused loss of rhodamine-123 extrusion and reduced the freq uencies of cobblestone area-forming cells (CAFC), Several other cytokines f ailed to stimulate cell divisions, which are required for retroviral transd uction. A combination including Flt-3 ligand (FL), interleukin-6 and stem c ell factor (SCF) preserved an immature phenotype for 5 to 6 days and stimul ated cell divisions, which was improved upon addition of leukemia inhibitor y factor and interleukin-ll. Furthermore, the CAFC frequency among cells tr eated with these cytokines was increased as compared with widely used cockt ails containing interleukin-3, interleukin-6 and SCF. Rhodamine-123 appeare d to be a particularly sensitive indicator for differentiation of PBPC, For analysis of gene transfer, amphotropic retroviruses conferring an MDR1 cDN A were added repeatedly for 6 days to cytokine-treated PBPC stroma-free cul tures. Proviral cDNA was detected by polymerase chain reaction in 68% of co bblestone areas derived from CD34(+) HLA-DRlow cells that had been exposed to Flt-3 ligand, interleukin-6 and SCF. In summary conditions were identifi ed that facilitate efficient transduction of early PBPC with amphotropic re troviruses while preserving a primitive phenotype for extended periods.