In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells.
c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker-negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c-kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c-kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.
D000818:Animals; D000950:Antigens, Ly / Q000032:analysis*; D001854:Bone Marrow Cells; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D007375:Interleukin-1 / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008810:Mice, Inbred C57BL; D011505:Protein-Tyrosine Kinases / Q000032:analysis; D011518:Proto-Oncogene Proteins / Q000032:analysis*; D019009:Proto-Oncogene Proteins c-kit; D011519:Proto-Oncogenes; D011994:Recombinant Proteins / Q000494:pharmacology; D019089:Stem Cell Factor
D000950:Antigens, Ly; D016298:Hematopoietic Cell Growth Factors; D007375:Interleukin-1; D007377:Interleukin-3; D015850:Interleukin-6; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D016179:Granulocyte Colony-Stimulating Factor; D016173:Macrophage Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D011505:Protein-Tyrosine Kinases; D019009:Proto-Oncogene Proteins c-kit