Objective: To construct a directional differentiation model from mouse embryonic stem cells into leydig-like cells in vitro.
Methods: Mouse ES-D3 cells were transfected with plasmid containing steroidogenic factor 1 (SF-1) gene, then treated with RA and 8Br-cAMP, while the cells transfected with empty plasmid were used as the negative controls. The morphology of leydig-like cells differentiated from ES-D3 cells was observed with light microscopy. The expression levels of StAR, P450scc and 3β-HSD were detected by RT-PCR, Western Blot and fluorescence microscopy analysis in leydig-like cells derived from the ES cells.
Results: ES-D3 cells were transfected with plasmid containing SF-1 gene successfully, and SF-1 was expressed 24 h after transfection. The SF-1-transfected ES-D3 cells were induced by RA and 8Br-cAMP to differentiate into leydig-like cells. The differentiated cells showed spindle shape with tentacles, which expressed the specific protein marker for leydig cells 3β-HSD1 and P450scc. Meanwhile, in these leydig-like cells, the expression of StAR increased compared with control group. 3β-HSD1, P450scc and StAR were not detected in negative control group.
Conclusion: When the ES-D3 cells are transfected with SF-1 plasmid and then treated with RA and 8Br-cAMP, the cells are able to differentiate into leydig-like cells, indicating that the model of directional differentiation of ES cells into leydig-like cells has been constructed successfully.