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

Abstract

Summary 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 immuno‐reactivity 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.