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

## TL;DR

This study shows that a precise balance of Wnt/β-catenin signaling is essential for maintaining pluripotency in bovine stem cells, with a surprising synergy between iCRT3 and CHIR99021.

## Contribution

The study reveals a unique rewiring of the Wnt/β-catenin pathway in bovines and identifies a synergistic drug combination for enhancing pluripotency.

## Key findings

- Prolonged Wnt/β-catenin activation with CHIR99021 downregulates core pluripotency genes in bovine stem cells.
- Combining CHIR99021 with iCRT3 enhances pluripotency gene expression and activates Wnt/β-catenin signaling.
- The drug combination drives bEPSCs toward a hybrid naïve/formative pluripotency state.

## Abstract

Maintaining pluripotent stem cells in bovine is crucial for breeding and biotech applications but remains challenging due to unclear signaling requirements. We investigated the role of the Wnt/β-catenin pathway in bovine stem cells. Surprisingly, we found that these cells need an exact balance of Wnt/β-catenin signaling to maintain pluripotency, rather than merely simple activation or inhibition. Even more surprisingly, the combination of the classic inhibitor iCRT3 and the activator CHIR99021 produced a synergistic effect. Instead of inhibiting the pathway, combining iCRT3 with CHIR99021 appears to enhance its activity and improve stemness. Our study reveals a unique wiring of this critical pathway in bovine, providing new insights for optimizing stem cell culture systems in livestock species.

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.