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Background: CHIR99021, a GSK-3 specific inhibitor, promotes mouse and rat ESC self-renewal through inhibition of glycogen synthase kinase 3 (Gsk3) and subsequent negatively modulating Tcf3 via canonical Wnt/(a)-catenin signaling.However, given the GSK3 is a multifunctional enzyme with a diverse number of actions in intracellular signaling systems, the mechanism of GSK3 inhibition promoted ESC self-renewal remains elusive.