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.
OBJECTIVE: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: These results suggest that culture on FN support allows prolonged ex vivo maintenance and even expansion of transduced repopulating stem cells.[1]References
- 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. Sellers, S.E., Tisdale, J.F., Agricola, B.A., Donahue, R.E., Dunbar, C.E. Exp. Hematol. (2004) [Pubmed]
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