In Vivo and In Vitro Stem Cell Function of c-kit- and Sca-1–Positive Murine Hematopoietic Cells

c-kitis 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 × 103of Linc+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-kitis 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.