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<p>Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that marks pluripotent embryonic stem cells (ESCs). Regulation of <i>Epcam</i> expression yields a spatiotemporal patterning during embryogenesis that is thoroughly mimicked in a 3D model of spontaneous differentiation of embryoid bodies (EBs). Here, we present a role of EpCAM in exit from pluripotency of murine ESCs (mESCs) to establish cardiomyocytes in EBs. Comparative transcriptomic analysis of wildtype and <i>Epcam</i>-knockout mESCs at strategic time points of spontaneous differentiation uncovered molecular deficiencies of <i>Epcam</i>-knockout ESCs in “Wnt signaling” and “Heart development”. Multi-level bioinformatic analyses revealed central lineage-defining transcription factors <i>Eomes</i>, <i>Foxa2</i>, and <i>Gata6</i> as differentially expressed genes (DEGs) that are misregulated in <i>Epcam</i>-knockout mESCs. Gene expression association of <i>Epcam</i> with <i>Eomes</i>, <i>Foxa2</i>, and <i>Gata6</i> was prominent at day three of spontaneous differentiation, representing primitive streak formation in EBs. Interrogation of public single-cell RNA sequencing (scRNAseq) datasets supported a co-expression of <i>Epcam</i> and <i>Eomes</i> at early stages of murine embryogenesis in epiblast, primitive streak, nascent mesoderm, extraembryonic ectoderm and endoderm. Newly generated scRNAseq of wildtype mESCs in spontaneous differentiation delineated the formation of epiblast, primitive streak, endo- and mesoderm cells, and cardiomyocytes. Expression and pseudotime analysis positioned <i>Epcam</i> expression slightly ahead of <i>Eomes</i> at the transition of early to late primitive streak, along with rising Wnt signaling. Accordingly, conditional re-expression of <i>Epcam</i> or <i>Eomes</i> but not of <i>Foxa2</i> or <i>Gata6</i> complemented differentiation defects of <i>Epcam</i>-knockouts and confirmed an involvement of Wnt signaling in the EpCAM-dependent activation of <i>Eomes</i>. Hence, defective exit of pluripotency in <i>Epcam</i>-deficient ESCs is linked to <i>Eomes</i> regulation via Wnt signaling.</p><p></p>

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EpCAM supports exit from pluripotency of embryonic stem cells via Eomes

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月11日

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  • Ningyue Gong,
  • Mahesh Gouda,
  • Ana Marija Balaz,
  • Jiahang Song,
  • Gisela Kranz,
  • Julia Hess,
  • Philipp Baumeister,
  • Kristian Unger,
  • Vera Katalina,
  • Martin Canis,
  • Olivier Gires

摘要

Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that marks pluripotent embryonic stem cells (ESCs). Regulation of Epcam expression yields a spatiotemporal patterning during embryogenesis that is thoroughly mimicked in a 3D model of spontaneous differentiation of embryoid bodies (EBs). Here, we present a role of EpCAM in exit from pluripotency of murine ESCs (mESCs) to establish cardiomyocytes in EBs. Comparative transcriptomic analysis of wildtype and Epcam-knockout mESCs at strategic time points of spontaneous differentiation uncovered molecular deficiencies of Epcam-knockout ESCs in “Wnt signaling” and “Heart development”. Multi-level bioinformatic analyses revealed central lineage-defining transcription factors Eomes, Foxa2, and Gata6 as differentially expressed genes (DEGs) that are misregulated in Epcam-knockout mESCs. Gene expression association of Epcam with Eomes, Foxa2, and Gata6 was prominent at day three of spontaneous differentiation, representing primitive streak formation in EBs. Interrogation of public single-cell RNA sequencing (scRNAseq) datasets supported a co-expression of Epcam and Eomes at early stages of murine embryogenesis in epiblast, primitive streak, nascent mesoderm, extraembryonic ectoderm and endoderm. Newly generated scRNAseq of wildtype mESCs in spontaneous differentiation delineated the formation of epiblast, primitive streak, endo- and mesoderm cells, and cardiomyocytes. Expression and pseudotime analysis positioned Epcam expression slightly ahead of Eomes at the transition of early to late primitive streak, along with rising Wnt signaling. Accordingly, conditional re-expression of Epcam or Eomes but not of Foxa2 or Gata6 complemented differentiation defects of Epcam-knockouts and confirmed an involvement of Wnt signaling in the EpCAM-dependent activation of Eomes. Hence, defective exit of pluripotency in Epcam-deficient ESCs is linked to Eomes regulation via Wnt signaling.