title: Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates.
abstract: Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: M
authors.initials: M
authors.lastname: Takatoku
authors.forename: S
authors.initials: S
authors.lastname: Sellers
authors.forename: B A
authors.initials: BA
authors.lastname: Agricola
authors.forename: M E
authors.initials: ME
authors.lastname: Metzger
authors.forename: I
authors.initials: I
authors.lastname: Kato
authors.forename: R E
authors.initials: RE
authors.lastname: Donahue
authors.forename: C E
authors.initials: CE
authors.lastname: Dunbar
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D001483:Base Sequence; D002453:Cell Cycle / Q000187:drug effects; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D005353:Fibronectins / Q000494:pharmacology; D015316:Genetic Therapy; D005822:Genetic Vectors; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D006801:Humans; D008253:Macaca mulatta; D010446:Peptide Fragments / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics; D019089:Stem Cell Factor / Q000494:pharmacology; D014161:Transduction, Genetic
chemical_list: D018952:Antigens, CD34; D016207:Cytokines; D017931:DNA Primers; D005353:Fibronectins; D010446:Peptide Fragments; D019089:Stem Cell Factor