Wnt/β-Catenin Activation by iCRT3 Enhanced the Pluripotency of Bovine Expanded Pluripotent Stem Cells

The Wnt/β-catenin signaling pathway is involved in regulating the pluripotency of mammalian stem cells. Fine-tuning of Wnt/β-catenin modulates the transition of naïve, formative or primed states with distinct lineage bias. However, its specific function in large domestic animals such as bovines remains unclear. Here we systematically investigated the role of Wnt/β-catenin signaling and its key effector TCF1 in bovine expanded pluripotent stem cells (bEPSCs) using a combination of small molecules (CHIR99021, XAV939, IWR-1, iCRT3). The results showed that prolonged Wnt/β-catenin activation with CHIR99021 induced morphological changes and downregulated the expression of core pluripotency genes POU5F1 (OCT4) and SOX2 in bEPSCs, while the existence of Wnt/β-catenin inhibitors XAV939 and IWR-1 upregulated these two genes. Knockdown of TCF1, a major nuclear effector of CTNNB1 (β-catenin), reduced the expression of pluripotency genes (POU5F1, SOX2) and key Wnt/β-catenin components (TCF3, LEF1 and CTNNB1). Combined treatment with CHIR99021 and the canonical β-catenin/TCF inhibitor iCRT3 resulted in the overactivation of Wnt/β-catenin signaling, and promoted the expression of core pluripotency genes, revealing extensive rewiring of the Wnt/β-catenin pathway in bovines. Consistent with these findings, global transcriptomics revealed that CHIR99021 combined with iCRT3 enhanced the expression of key pluripotency-related genes and further activated Wnt/β-catenin signaling target genes while simultaneously suppressing mitogenic pathways such as PI3K-Akt and MAPK signaling. Transcriptome profiling also demonstrated that this combination drives bEPSCs toward a hybrid naïve/formative pluripotency state. Together, these results demonstrate that Wnt/β-catenin signaling homeostasis is critical for bovine pluripotency regulation, which provides a foundation for refining livestock stem cell culture conditions and understanding the evolution of pluripotency networks.