title: Advantages of gradient vs. 5-fluorouracil enrichment of stem cells for retroviral-mediated gene transfer.
abstract: Retrovirally mediated gene transfer into murine totipotent hematopoietic stem cells (THSC) may be more efficient when the donor stem cells are enriched. We have used a rapid, nontoxic density gradient separation of mouse marrow to enrich stem cells. By characterizing the cell types in various fractions of the gradient, we found the majority of the THSC, spleen colony forming stem cells (CFU-S), erythroid burst forming cells (BFU-E) and dividing cells were in the same fraction. The gradient enrichment technique was then compared with one requiring 5-fluorouracil (5-FU) treatment of donor mice prior to marrow harvest. Cells enriched by both methods were tested for their ability to mediate retroviral gene transfer into normal mice. Gradient enrichment provided only one third as many nucleated cells as 5-FU treatment from the same number of donors. During the subsequent 4-day in vitro exposure to the retrovirus and growth factors, however, the number of gradient enriched cells increased 1.6-fold while the number of 5-FU treated cells decreased 3-fold. In lethally irradiated recipients, there was no difference between gradient and 5-FU enriched donor cells in the proportion of cells that generated CFU-S nor in the percentage of CFU-S that were infected. Secondary hosts did show differences. Gradient-enriched cells maintained more survivors for up to 6 months posttransplantation and more of the survivors were positive for the retrovirus. It is clear that the gradient method provides a rapid means to enrich CFU-S and THSC without exposure to the toxic effects of 5-FU.
authors.affiliation: Jackson Laboratory, Bar Harbor, ME 04609.
authors.forename: J E
authors.initials: JE
authors.lastname: Barker
authors.forename: J H
authors.initials: JH
authors.lastname: Wolfe
authors.forename: L B
authors.initials: LB
authors.lastname: Rowe
authors.forename: E H
authors.initials: EH
authors.lastname: Birkenmeier
mesh_terms: D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000032:analysis / Q000737:chemistry; D015672:Erythroid Precursor Cells / Q000166:cytology; D005472:Fluorouracil / Q000494:pharmacology*; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D013154:Spleen / Q000166:cytology; D014162:Transfection
chemical_list: D004247:DNA; D005966:Glucuronidase; D005472:Fluorouracil