The presence of the carboxy-terminal fragment of fibronectin allows maintenance of non-human primate long-term hematopoietic repopulating cells during extended ex vivo culture and transduction

Ex vivo expansion of primitive hematopoietic cells remains of interest for gene therapy and transplantation. Previous studies reported loss of repopulating activity following culture of cells for more than 4-7 days in the presence of cytokines or stromal cells. In the current study, we investigated whether prolonged culture and transduction in the presence of the carboxy-terminal portion of fibronectin (FN) could maintain or expand retrovirally transduced repopulating hematopoietic stem cells (HSCs).The impact of culture and transduction on rhesus macaque CD34+ peripheral blood stem cells (PBSCs) was assessed in the presence of FN and stimulatory cytokines. A competitive repopulation design using up to three retroviral vectors allowed direct comparison of repopulating activity between cells transduced and cultured for 4 days vs 10 days.In the first animal, all cells were cultured and transduced for 10 days, with one vector used on days 0-4 and a second on days 4-10. There was stable long-term marking from both vectors, indicating that cells cycling both early and late could engraft. In three animals, we compared cells that were cryopreserved following a 4-day transduction to cells that were continued in culture for an additional 6 days. Total marking derived from the 10-day expanded cells was significantly higher than marking from the 4-day cultured cells.These results suggest that culture on FN support allows prolonged ex vivo maintenance and even expansion of transduced repopulating stem cells.