195 EFFECTS OF GSK3 INHIBITOR ON THE PLURIPOTENCY MAINTENANCE OF BUFFALO EMBRYONIC STEM-CELL-LIKE CELLS

Abstract

In this study, to explore the effects and mechanism of Wnt/β-catenin signalling pathway on the maintenance of pluripotency of buffalo embryonic stem-cell-like cells (buffalo ESC-like cells), the GSK3 inhibitors BIO and CHIR99021 were added throughout the experiment – i.e. from buffalo inner cell mass (ICM) culture to ESC-like line generation. The buffalo ICM were respectively cultured in the medium containing 0.5 μg mL^–1 BIO and 5 mmol L^–1 CHIR99021. The percentage of ICMs attachment and primary colony formation were observed, and found that there was no significant difference in the ICMs attachment rate among of the BIO, CHIR99021, and the control groups (91.18% and 92.98% v. 94.59%; P > 0.05). Treating ICMs with CHIR99021 resulted in more primary colony formation rate compared with the control group (77.71% v. 55.41%; P < 0.05). The proliferation rate of primary colonies of buffalo ESC-like cells was detected by bromodeoxyuridine immunofluorescence techniques. The results show that the proliferation rate of primary colonies in the group of buffalo ESC-like cells treated with CHIR99021 was significantly higher than that of the control group on Day 1, Day 3, Day 4, and Day 5 (P < 0.05), and it was also evidently higher than that of control group only on Day 1 (P < 0.05) in the group of BIO, but there was no significant difference in other days (P > 0.05). The mRNA expression level of proliferation marker PCNA of ESC-like cells was significantly up-regulated in both CHIR99021 and BIO treatment groups (P < 0.05), however, treating buffalo ESC-like cells with CHIR99021 significantly up-regulated the expression of pluripotent gene Oct4 and Sox2 (P < 0.05), but had no effect on pluripotent gene Nanog expression (P > 0.05). Oct4 expression was significantly increased (P < 0.05), and the expression of Sox2 and Nanog were significantly decreased (P < 0.05) in the group of BIO treatment. Furthermore, the relative protein level of β-catenin (the downstream effector of Wnt/β-catenin signalling pathway) and the mRNA expression level of c-Myc (the downstream target gene of Wnt/β-catenin signalling pathway) were significantly increased when buffalo ESC-like cells respectively treated with CHIR99021 and BIO (P < 0.05). In conclusion, treating buffalo ESC-like cells with GSK3 inhibitors CHIR99021 can promote proliferation of buffalo ESC-like cells, maintain their undifferentiated state, and up-regulate the expression levels of β-Catenin and c-Myc in buffalo ESC-like cells. These results indicate that Wnt/β-catenin signalling pathway plays an important role in regulation of self-renewal of buffalo ESC-like cells.

This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2012GXNSFFA060004).