Inhibition of Chk1 by Go6976 promotes differentiation and apoptosis in mouse embryonic stem cells: implications for developmental toxicology

Purpose: Go6976 is a staurosporine-derived kinase inhibitor that interferes with checkpoint signaling pathways. Although Go6976 has been widely used as a pharmacological inhibitor, its effects on embryonic stem cell maintenance and survival remain unclear. This study investigated the effects of Go6976 on pluripotency, differentiation, and apoptosis in mouse embryonic stem cells (mESCs).Methods: mESCs were treated with Go6976 under leukemia inhibitory factor (LIF)-supplemented conditions. Pluripotency was assessed by alkaline phosphatase (AP) staining and analysis of pluripotency marker expression. Differentiation was evaluated by measuring lineage-specific gene expression. Apoptosis was analyzed by cell cycle profiling and Annexin V staining. To clarify whether the effects of Go6976 were associated with protein kinase C inhibition, its effects were compared with those of the protein kinase C inhibitor GF109203X. In vitro kinase assays were also performed to evaluate checkpoint kinase 1 (Chk1) activity. All experiments were performed independently at least 3 times, and statistical significance was determined using one-way analysis of variance, as appropriate.Results: Go6976 significantly reduced AP-positive colonies despite the presence of LIF and decreased the expression of pluripotency markers, including Oct4 and Sox2. In contrast, Go6976 increased the expression of differentiation markers, including Brachyury and FGF5.Conclusion: Pharmacological inhibition of Chk1 by Go6976 promoted differentiation and apoptosis in mESCs. These findings support a role for Chk1-associated signaling in stem cell homeostasis and suggest its potential relevance to developmental toxicology.