Promoting effects of activated Nodal signal pathway on definitive endoderm induction from mouse embryonic stem cells

AIM: To study the process of promoting mouse embryonic stem cells (ESC) to specify to definitive endoderm by up-regulating of Nodal signal pathway in order to find the best cultivated systems of differentiation of mouse ESC to definitive endoderm cells. METHODS: The cells were divided into different groups based on the culture medium: ESC group (serum-free medium + LIF), natural differentiation group (serum-free medium) and activin A group (serum-free medium +50μg/L activin A). The cells and the sterilized coverslips with cells were collected at 1, 3, 5 and 7 d of the cultivation. The proportion of CXCR4⁺ cells was detected by flow cytometry. The expression of CXCR4 was determined by immunocytochemical method, and the protein expression of OCT4 and CXCR4 was detected by Western blot. RESULTS: The proportion of CXCR4⁺ cells showed no dramatic change in ESC group along with the extending of cultivation day, while there were gradually increased in natural differentiation group and activin A group and the highest level was observed at 5 d. Among the 3 groups, the proportion of CXCR4⁺ cells at 5 d was the highest in activin A group. The brown or tan staining in the cells observed under microscope was considered as positive CXCR4 by immunocytochemistry. The protein levels of OCT4 and CXCR4 in ESC group along with the extending of cultivation days was observed. The expression levels of OCT4 were gradually decreased in the cells in natural differentiation group and activin A group, while those of CXCR4 were gradually increased with the highest level at 5 d. It was highest in the cells in activin A group. CONCLUSION: The proportion of definitive endoderm was the highest at 5 d of the induction during in vitro mouse ESC differentiation. Up-regulation of Nodal signal pathway by adding activin A at the early stage of mouse ESC differentiation promotes mouse ESC to specify to definitive endoderm with CXCR4 molecular marker.