Avoidance of stimulation improves engraftment of cultured and retrovirallytransduced hematopoietic cells in primates

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investig ate this finding, which has direct implications for clinical transplantatio n and gene therapy applications. Transfer of rhesus CD34(+) cells to cultur e in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but dec reased cycling. Using retroviral marking with two different gene transfer v ectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) bef ore reinfusion. In vivo competitive repopulation studies showed that the le vel of marking originating from the cells continued in culture for 2 days w ith SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days an d reinfused without the 2-day culture under nonstimulatory conditions. We o bserved stable in vivo overall gene marking levels of up to 29%. This appro ach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.