Changes in c‐Kit expression and effects of SCF during differentiation of human erythroid progenitor cells

Abstract

We analysed c-Kit expression during erythroid differentiation using immunocytochemical staining and flow cytometric analysis. Burst-forming units-erythroid (BFU-E)-derived cell aggregates were identified in methylcellulose cultures containing human umbilical cord blood CD34+ cells and were stained by the indirect immunoalkaline phosphatase method. To investigate the changes in levels of cell-surface c-Kit expression, we subjected progenitor cells in liquid culture to flow cytometric analysis. In addition, the effects of stem cell factor (SCF) on cell-surface c-Kit expression were analysed in these two culture systems and the effects of SCF on erythroid colony formation were studied in a methylcellulose culture. c-Kit was expressed on the cell surface from BFU-E to erythroid precursors recognized morphologically as basophilic erythroblasts. Flow cytometric analysis showed that c-Kit expression increased until 6 d in liquid culture, and that decreased expression of c-Kit was associated with the increased expression of glycophorin A. Moreover, SCF increased the size of erythroid colonies when added at days 0, 4 and 8 in methylcellulose cultures. These results indicate that the c-Kit/SCF system still plays in proliferation of erythroid progenitor cells at the colony-forming units-erythroid stage. Finally, expression of c-Kit in erythroid progenitor cells cultured without SCF showed a diffuse pattern on the cell surface, whereas we observed positive c-Kit immunoreactivity in the region of the Golgi apparatus of these cells cultured with SCF. Flow cytometric analysis also showed that the levels of cell-surface c-Kit expression decreased in the presence of SCF. These results suggest that SCF induced down-modulation of cell-surface c-Kit expression, despite continuous synthesis of c-Kit protein.