Enrichment and characterization of murine hematopoietic stem cells that express c-kit molecule

The proto-oncogene c-kit encodes 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-kit molecule was prepared. Flow cytometric analysis showed that the bone marrow cells that expressed the c-kit molecule (approximately 5%) were B220(B)^-, TER119(erythroid)^-, Thy1(negative-low), and WGA⁺ . A small number of Mac-1(macrophage)⁺ 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-123(low) and 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-1(low)Lin^-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-kit molecule is expressed in primitive stem cells and plays an essential role in the early stages of hematopoiesis.