Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture

TLDR

Bipotent mesendoderm, conserved from C. elegans to zebrafish, is a major precursor of definitive endoderm, yet its differentiation pathways remain poorly characterized. The study established GFP‑tagged goosecoid (Gsc) embryonic stem cell lines to trace mesendoderm differentiation using Gsc expression as a marker. Mesendoderm was induced from ES cells by culturing under defined activin or nodal conditions while monitoring Gsc‑GFP expression.

Abstract

Bipotent mesendoderm that can give rise to both endoderm and mesoderm is an established entity from C. elegans to zebrafish. Although previous studies in mouse embryo indicated the presence of bi-potent mesendoderm cells in the organizer region, characterization of mesendoderm and its differentiation processes are still unclear. As bi-potent mesendoderm is implicated as the major precursor of definitive endoderm, its identification is also essential for exploring the differentiation of definitive endoderm. In this study, we have established embryonic stem (ES) cell lines that carry GFP gene in the goosecoid (Gsc) gene locus and have investigated the differentiation course of mesendodermal cells using Gsc expression as a marker. Our results show that mesendoderm is represented as a Gsc-GFP+E-cadherin(ECD)+PDGFRα(αR)+population and is selectively induced from ES cells under defined conditions containing either activin or nodal. Subsequently, it diverges to Gsc+ECD+αR- and Gsc+ECD-αR+ intermediates that eventually differentiate into definitive endoderm and mesodermal lineages,respectively. The presence of mesendodermal cells in nascent Gsc+ECD+αR+ population was also confirmed by single cell analysis. Finally, we show that the defined culture condition and surface markers developed in this study are applicable for obtaining pure mesendodermal cells and their immediate progenies from genetically unmanipulated ES cells.