ISOLATION IN A SINGLE STEP OF A HIGHLY ENRICHED MURINE HEMATOPOIETIC STEM-CELL POPULATION WITH COMPETITIVE LONG-TERM REPOPULATING ABILITY

  • 15 August 1989
    • journal article
    • research article
    • Vol. 74  (3) , 930-939
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 .times. 105 "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 (> 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 .times. 105 compromised female marrow cells, and .apprx.100 were sufficient to achieve the same results 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.