title: 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.
abstract: 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.
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Sellers
authors.forename: John F
authors.initials: JF
authors.lastname: Tisdale
authors.forename: Brian A
authors.initials: BA
authors.lastname: Agricola
authors.forename: Robert E
authors.initials: RE
authors.lastname: Donahue
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis; D002478:Cells, Cultured; D005353:Fibronectins / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D039041:Integrin alpha4beta1 / Q000502:physiology; D008253:Macaca mulatta; D010446:Peptide Fragments / Q000502:physiology*; D014161:Transduction, Genetic
chemical_list: D018952:Antigens, CD34; D005353:Fibronectins; D039041:Integrin alpha4beta1; D010446:Peptide Fragments