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Endoderm precursors expressing  FoxA2  and  Sox17  develop from the epiblast through the gastrulation process. In this study, we developed an experimental system to model the endoderm‐generating gastrulation process using epiblast stem cells (EpiSCs). To this end, we established an EpiSC line i22, in which enhanced green fluorescent protein is coexpressed with  Foxa2 . Culturing i22 EpiSCs as aggregates for a few days was sufficient to initiate  Foxa2  expression, and further culturing of the aggregates in Matrigel promoted the sequential activation of transcription factor genes involved in endoderm precursor development, e.g.,  Eomes ,  Gsc , and  Sox17 . In aggregation culture of i22 cells for 3 days, all cells expressed POU5F1, SOX2, and E‐cadherin, a signature of the epiblast, whereas expression of GATA4 and SOX17 was also activated moderately in dispersed cells, suggesting priming of these cells to endodermal development. Embedding the aggregates in Matrigel for further 3 days elicited migration of the cells into the lumen of laminin‐rich matrices covering the aggregates, in which FOXA2 and SOX17 were expressed at a high level with the concomitant loss of E‐cadherin, indicating the migratory phase of endodermal precursors. Prolonged culturing of the aggregates generated three segregating cell populations found in post‐gastrulation stage embryos: (1) definitive endoderm co‐expressing high SOX17, GATA4, and E‐cadherin, (2) mesodermal cells expressing a low level of GATA4 and lacking E‐cadherin, and (3) primed epiblast cells expressing POU5F1, SOX2 without E‐cadherin. Thus, aggregation of EpiSCs followed by embedding of aggregates in the laminin‐rich matrix models the gastrulation‐dependent endoderm precursor development.

Modeling early stages of endoderm development in epiblast stem cell aggregates with supply of extracellular matrices