title: Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability.
abstract: A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male "test" cells and 1 to 2 x 10(5) "compromised" female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5-FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.
authors.affiliation: Terry Fox Laboratory, B. C. Cancer Research Centre, Vancouver.
authors.forename: S J
authors.initials: SJ
authors.lastname: Szilvassy
authors.forename: P M
authors.initials: PM
authors.lastname: Lansdorp
authors.forename: R K
authors.initials: RK
authors.lastname: Humphries
authors.forename: A C
authors.initials: AC
authors.lastname: Eaves
authors.forename: C J
authors.initials: CJ
authors.lastname: Eaves
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D005260:Female; D005434:Flow Cytometry; D006183:H-2 Antigens / Q000032:analysis; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000145:classification*; D008027:Light; D008297:Male; D051379:Mice; D010641:Phenotype; D012542:Scattering, Radiation; D018800:Thy-1 Antigens
chemical_list: D000954:Antigens, Surface; D006183:H-2 Antigens; D018800:Thy-1 Antigens