Enrichment and Characterization of Murine Hematopoietic Stem Cells That Express c-kitMolecule

The proto-oncogene c-kitencodes a transmembrane tyrosine kinase receptor for stem cell factor (SCF). The c-kit/SCF signal is expected to have an important role in hematopoiesis. A monoclonal antibody (ACK-2) against the murine c-kitmolecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kitmolecule (approximately 5%) were B220(B)−, TER119(erythroid)−, Thy1negative-low, and WGA+. A small number of Mac-1(macro-phage)+or Gr-1(granulocyte)+cells were c-kit-low positive. Colony-forming unit in culture (CFU-C) and day-8 and day-12 CFU-spleen (CFU-S) existed exclusively in the c-kit-positive fraction. About 20% of the Lin(lineage)−c-kit+cells were rhodamine-123lowand this fraction contained more day-12 CFU-S than day-8 CFU-S. On the basis of these findings, murine hematopoietic stem cells were enriched with normal bone marrow cells. One of two and one of four Thy-1lowLin−WGA+c-kit+cells were CFU-C and CFU-S, respectively. Long-term repopulating ability was investigated using B6/Ly5 congenic mice. Eight and 25 weeks after transplantation of Lin−c-kit+cells, donor-derived cells were found in the bone marrow, spleen, thymus, and peripheral blood. In peripheral blood, T cells, B cells, and granulocyte-macrophages were derived from donor cells. Injection of ACK-2 into the irradiated mice after bone marrow transplantation decreased the numbers of day-8 and day-12 CFU-S in a dose-dependent manner. Day-8 spleen colony formation was completely suppressed by the injection of 100 μg ACK-2, but a small number of day-12 colonies were spared. Our data show that the c-kitmolecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.