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

Abstract

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.

Keywords: TCF1; Wnt/β-catenin signaling pathway; bEPSCs; iCRT3; pluripotency genes.

Figure 1

Combinatorial Modulation of Wnt/β-catenin Signaling Influences the pluripotency of bEPSCs. (A) Phase-contrast images showing the morphology of bEPSCs. Cells were cultured in either the standard medium (Control, containing CHIR99021 + XAV939) or in medium where the inhibitor XAV939 was omitted (CHIR99021). Scale bars, 100 μm. (B) Alkaline phosphatase (AP) staining of cells from the same two groups as in (A). AP-positive colonies appear in purple/red. Scale bars, 100 μm. (C) Temporal evolution of colony morphology in the CHIR99021 condition. Representative images are shown at passages 1, 3, 6, and 9. Scale bars, 100 μm. (D) Immunofluorescence staining of core pluripotency transcription factors. Cells from the four indicated treatment groups (see panel (G) for group labels) were stained for NANOG, POU5F1, and SOX2 (green). Nuclei were counterstained with DAPI (blue). Scale bars, 200 μm. (E) Immunofluorescence analysis of β-catenin subcellular localization. Cells from the four treatment groups were stained for β-catenin (green) and DAPI (blue). Scale bars, 100 μm. (F) Quantification of nuclear fluorescence intensity. The mean fluorescence intensity for NANOG, POU5F1, SOX2, and β-catenin was measured from images as in (D,E) and is shown for the four treatment groups. Data are normalized to the Control group (set as 1). Each treatment contained three replicates. (G) qRT-PCR analysis of gene expression. mRNA levels of pluripotency genes (NANOG, POU5F1, SOX2), early lineage markers (CDX2, GATA4), and Wnt/β-catenin pathway genes (TCF1, TCF3, TCF4, β-catenin, SP5) were assessed in bEPSCs cultured under the four indicated conditions. Gene expression was normalized to the Control group (CHIR99021 + XAV939). Each treatment contained three replicates. Each black dot represents an independent experimental replicate. The p values in (F,G) were calculated by t test (** p < 0.01, **** p < 0.0001; ns, not significant).

Figure 2

TCF1 Knockdown Compromises Pluripotency and Alters Wnt/β-catenin Signaling in bEPSCs. (A) Phase-contrast images of bEPSC colonies. Colonies were transfected with either a non-targeting scramble siRNA (Control) or a TCF1-specific siRNA (si-TCF1-2). Images show colony morphology 48–72 h post-transfection. Scale bars, 100 µm. (B) Knockdown efficiency at the mRNA level. TCF1 mRNA expression was assessed by qRT-PCR in cells transfected as in (A). Expression is shown relative to the scramble control (set as 1). Each treatment contained three replicates. Each black dot represents an independent experimental replicate. (C) Knockdown efficiency at the protein level. Representative Western blot (top) and quantification (bottom) of TCF1 protein levels in the two groups. GAPDH served as a loading control. Band intensity was quantified using ImageJ and normalized to the Control. Each treatment contained three replicates. (D) Effect on core pluripotency factors. qRT-PCR analysis of NANOG, POU5F1, and SOX2 mRNA levels following TCF1 knockdown. Expression was normalized to the scramble control. Each treatment contained three replicates. Each black dot represents an independent experimental replicate. (E) Effect on early lineage markers. qRT-PCR analysis of trophectoderm (CDX2), ectoderm/neural (GFAP), primitive ectoderm (FGF5), endoderm (GATA4), and endoderm/forebrain (PAX6) marker genes. Expression was normalized to the scramble control. Each treatment contained three replicates. Each black dot represents an independent experimental replicate. (F) Effect on Wnt/β-catenin signaling components and targets. qRT-PCR analysis of key pathway genes: transcription factors (TCF3, TCF4, LEF1), the central mediator CTNNB1, and canonical targets (AXIN2, SP5). Expression was normalized to the scramble control. Each treatment contained three replicates. Each black dot represents an independent experimental replicate. The p values in (B) were calculated by t test. The p values in (C–F) were calculated by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant).

Figure 3

Co-treatment with CHIR99021 and iCRT3 Enhances Pluripotency Marker Expression. (A) Morphology and alkaline phosphatase (AP) activity of bEPSCs under long-term culture. Phase-contrast (left) and AP-stained (right) images of cells cultured for multiple passages in the four indicated conditions: (i) standard (CHIR99021 + XAV939), (ii) CHIR99021 + iCRT3, (iii) CHIR99021 alone, and (iv) iCRT3 alone. AP-positive colonies appear in purple/red. Scale bars, 100 μm. (B) Immunofluorescence analysis of core pluripotency transcription factors. Cells from the four treatment groups (A) were stained for NANOG, SOX2, and POU5F1 (green). Nuclei were counterstained with DAPI (blue). Representative merged images are shown. Scale bars, 200 μm. (C) Quantification of nuclear fluorescence intensity for pluripotency factors. The mean fluorescence intensity of NANOG, SOX2, and POU5F1 was measured from images as in (B) and is shown for the four treatment groups. Data are normalized to the standard group (CHIR99021 + XAV939, set as 1). Each treatment contained three replicates. Each black dot represents an independent experimental replicate. (D) qRT-PCR analysis of core pluripotency gene expression. mRNA levels of NANOG, SOX2, and POU5F1 4 were assessed in the four treatment groups. Expression was normalized to the standard group (CHIR99021 + XAV939). Each treatment contained three replicates. Each black dot represents an independent experimental replicate.The p values in (C,D) were calculated by one-way ANOVA.

Figure 4

CHIR99021 and iCRT3 Combination Correlates with Synergistic Upregulation of Wnt/β-catenin Signaling Activity. (A) Immunofluorescence images showing the localization and expression levels of key Wnt/β-catenin signaling components (TCF1, TCF3, TCF4, and CTNNB1) in bEPSCs under indicated culture conditions. Cells were treated with: (i) standard medium (CHIR99021 + XAV939, Control), (ii) CHIR99021 combined with iCRT3, (iii) CHIR99021 alone, or (iv) iCRT3 alone. Nuclei were counterstained with DAPI (blue). Scale bars, 200 μm. (B) Quantitative analysis of nuclear fluorescence intensity for TCF1, TCF3, TCF4, and CTNNB1. Intensities were measured from images in (A), normalized to the Control group (set as 1). Each treatment contained three replicates. (C) iCRT3 enhances the nuclear translocation of β-catenin. Quantitative analysis of the nuclear-to-cytoplasmic (N/C) ratio of β-catenin from immunofluorescence staining. Bovine stem cells were treated as indicated. The N/C ratio was calculated for each individual cell (n > 50 cells per group from 3 independent experiments). (D) qRT-PCR analysis of Wnt/β-catenin pathway gene expression. mRNA levels of TCF1, TCF3, TCF4, CTNNB1, and SP5 were assessed under the four treatment conditions. Expression was normalized to the Control group (CHIR99021 + XAV939). Each treatment contained three replicates. The p values in 4B-D were calculated by one-way ANOVA.

Figure 5

Transcriptomic Analysis of bEPSCs under Different Wnt/β-catenin Signaling Modulations. (A) Principal component analysis (PCA) of RNA-seq data. The plot shows transcriptomic profiles of bEPSCs cultured under four conditions: (i) standard (CHIR99021 + XAV939), (ii) CHIR99021 alone, (iii) CHIR99021 + ICRT3, and (iv) ICRT3 alone. Each point represents one independent biological replicate. Ellipses (optional) indicate 95% confidence intervals for each group. PC1 and PC2 explain the indicated percentage of total variance. (B) Spearman correlation matrix of global gene expression. The matrix quantifies pairwise transcriptomic similarity between all samples. Color intensity and numbers in each cell represent the Spearman correlation coefficient (r), with darker red indicating higher similarity. (C) Heatmap of pluripotency state marker expression. Rows represent a curated set of marker genes for naïve, primed, and formative pluripotency states. Columns represent individual samples grouped by condition. Expression levels are shown as Z-scores (red, high expression relative to the mean; blue, low). (D) Heatmap of lineage-associated marker expression. Rows represent marker genes for core pluripotency, epiblast, mesoderm, and hypoblast lineages. Columns are as in (C). Visualization uses Z-scores (red, high; blue, low). (E) Volcano plot of differentially expressed genes (DEGs). The plot compares the CHIR99021 + ICRT3 group against the standard control (CHIR99021 + XAV939). Each point represents a gene. The x-axis shows log₂ fold change (positive values indicate upregulation in CHIR99021 + ICRT3). The y-axis shows -log₁₀ (p value). Genes with |log₂FC| > 1 and adjusted p value < 0.05 are highlighted in red (upregulated) and blue (downregulated). The total numbers of significant up- and down-regulated genes are indicated. (F) Gene Ontology (GO) enrichment analysis for biological processes. The bar chart shows the top significantly enriched GO terms (adjusted p value < 0.05) among the DEGs identified in (E) for the CHIR99021 + ICRT3 comparison. (G,H) KEGG pathway enrichment analysis. (G) Bar chart of the top enriched KEGG pathways among the upregulated DEGs from the CHIR99021 + ICRT3 comparison. (H) Bar chart of the top enriched KEGG pathways among the downregulated DEGs from the same comparison. For both (G,H), enrichment significance is shown as −log₁₀ (p value) or adjusted p value.