Wnt3a Activates the WNT-YAP/TAZ Pathway to Sustain <i>CDX2</i> Expression in Bovine Trophoblast Stem Cells

Abstract

Trophoblast stem cells (TSCs), the precursors of placental cells, are effective for studying placental formation <i>in vitro</i>. Using a dual inhibition (2i) medium and mixed L-Wnt3a/mouse embryonic fibroblast feeder cells, we previously established the bovine trophoblast cell line BTS-1. In this study, we used bovine fetal fibroblasts and added Wnt3a to the 2i medium to establish another bovine TSC line (BTSW). BTSW cells expressed pluripotency markers, including <i>NANOG</i>, <i>SOX2</i>, <i>OCT4</i>, <i>TRA-1-60</i>, <i>TRA-1-81</i>, <i>SSEA4</i>, <i>CDH1</i>, and <i>KRT18</i>, and TSC markers <i>CDX2</i>, <i>TEAD4</i>, and <i>ESRRB</i>. Methylation sequencing of the promoter regions of <i>NANOG</i>, <i>OCT4</i>, and <i>CDX2</i> revealed no significant differences between BTS-1 and BTSW cells. Removal of Wnt3a from the culture medium resulted in downregulation (<i>p</i> < 0.05) of <i>NANOG</i>, <i>OCT4</i>, <i>CDX2</i>, and TSC marker genes, and upregulation of TSC differentiation markers, including <i>MASH2</i>, <i>GCM1</i>, and <i>PAG</i>. Western blotting indicated activation of the WNT-YAP/TAZ signaling pathway in BTS-1 and BTSW cells, consequently activating <i>TEAD4</i> transcription. However, this pathway was not activated in BCFF cells, an established bovine embryonic stem-like cell line that expresses <i>OCT4</i>, <i>SOX2</i>, and <i>NANOG</i>, but not <i>CDX2</i>. Thus, Wnt3a may play a critical role in bovine TSC maintenance by activating and regulating <i>CDX2</i> expression through the WNT-YAP/TAZ signaling pathway.