Expression of c-kit mRNA and protein during the differentiation of human hematopoietic progenitor cells

To investigate how c-kit and c-kit ligand play a role in the function of hematopoietic stem cells, we determined the expression of c-kit in sorted human hematopoietic stem cells, CD34⁺CD33^- cells and CD34⁺CD33⁺ cells. CD34⁺ cells constituted approximately 1% of the population of gated bone marrow cells and contained colony-forming cells. Two-color analysis by a fluorescence-activated cell sorter (FACS) revealed that about one-third to one-half of the total CD34⁺ cell population were positive for the CD33 antigen. To analyze the relative accumulation of c-kit mRNA in sorted cells, we used the reverse transcription-polymerase chain reaction (RT-PCR) method, followed by Southern blot analysis. There was a linear relationship between the amount of input RNA and products amplified in the range of 10³ to 10⁵ cells. Using this procedure, we carried out an analysis of c-kit mRNA expression in CD34⁺CD33^-, CD34⁺CD33⁺, CD34^-CD33⁺, and CD34^-CD33^- cells. Enhanced expression for c-kit mRNA was observed solely in CD34⁺CD33^- cells. In contrast, flow cytometry shows that c-kit protein was expressed most abundantly in CD34⁺CD33⁺ cells. Colony-forming cells were generated on a human stromal cell layer for 5 weeks initiated with CD34⁺CD33^- cells but not with CD34⁺CD33⁺ cells. During co-culture with stromal cells, CD34⁺CD33^- cells differentiated into CD34⁺CD33⁺ cells. From these findings, it is concluded that CD34⁺CD33⁺ cells are direct progenies of CD34⁺CD33^- cells. In this differentiation pathway, the expression of c-kit mRNA decreased and the c-kit protein increased.