Involvement of Wnt pathway in ethanol-induced inhibition of mouse embryonic stem cell differentiation

Ethanol has been reported to have toxicity on embryonic stem cells (ESCs). The present study aims to address the teratogenic effects of ethanol on the growth and cardiac differentiation of ESCs. Mouse embryonic stem D3 cells were employed. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used to determine cytotoxicity. Quantitative real time polymerase chain reaction (qRT-PCR) and Western blotting were used to analyze the expressions of cardiac differentiation-related and Wnt signaling factors. The beating profile of cardiomyocytes was recorded to assess cardiac differentiation. Ethanol induced growth inhibition in both undifferentiated and differentiated ESCs after 5 days of exposure. Ethanol inhibited the loss of pluripotent gene expressions including Nanog, Sox2 and Oct4. The expressions of cardiac markers, Nkx2.5, Mef2c, Tbx5, dHand, αMHC, Cx43 and troponin C1, were suppressed by ethanol treatment. Furthermore, ethanol delayed cardiac differentiation of ESCs till 11 days of differentiation. The expressions of Wnt-related regulators, β-catenin and its target cyclin D1, were downregulated by ethanol. Wnt pathway agonist wnt3a could greatly rescue ethanol-induced inhibition of cardiac differentiation and Wnt-pathway-related protein expressions. These finding suggested that ethanol suppresses mouse ESC differentiation largely by inhibiting Wnt signaling pathway.