EXPRESSION OF C-KIT MESSENGER-RNA 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 popu lation of gated bone marrow cells and contained colony-forming cells. Two-color analysis by a fluorescence-activated cell sorter (FACS) reve aled that about one-third to one-half of the total CD34(+) cell popula tion were positive for the CD33 antigen. To analyze the relative accum ulation of c-kit mRNA in sorted cells, we used the reverse transcripti on-polymerase chain reaction (RT-PCR) method, followed by Southern blo t analysis. There was a linear relationship between the amount of inpu t RNA and products amplified in the range of 10(3) to 10(5) cells. Usi ng 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 (+)CD35(-) 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 initia ted with CD34(+)CD33(-) cells but not with CD34(+)CD33(+) cells. Durin g co-culture with stromal cells, CD34(+)CD33(-) cells differentiated i nto CD34(+)CD33(+) cells. From these findings, it is concluded that CD 34(+)CD33(+) cells are direct progenies of CD34(+)CD33(-) cells. In th is differentiation pathway, the expression of c-kit mRNA decreased and the c-kit protein increased.