Activin Alters the Kinetics of Endoderm Induction in Embryonic Stem Cells Cultured on Collagen Gels

Embryonic stem cell‐derived endoderm is critical for the development of cellular therapies for the treatment of disease such as diabetes, liver cirrhosis, or pulmonary emphysema. Here, we describe a novel approach to induce endoderm from mouse embryonic stem (mES) cells using fibronectin‐coated collagen gels. This technique results in a homogeneous endoderm‐like cell population, demonstrating endoderm‐specific gene and protein expression, which remains committed following in vivo transplantation. In this system, activin, normally an endoderm inducer, caused an 80% decrease in the Foxa2‐positive endoderm fraction, whereas follistatin increased the Foxa2‐positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through its transient precursors, the epiblast and mesendoderm. Long‐term differentiation displays a twofold reduction in hepatic gene expression and threefold reduction in hepatic protein expression of activin‐treated cells compared with follistatin‐treated cells. Moreover, subcutaneous transplantation of activin‐treated cells in a syngeneic mouse generated a heterogeneous teratoma‐like mass, suggesting that these were a more primitive population. In contrast, follistatin‐treated cells resulted in an encapsulated epithelial‐like mass, suggesting that these cells remained committed to the endoderm lineage. In conclusion, we demonstrate a novel technique to induce the direct differentiation of endoderm from mES cells without cell sorting. In addition, our work suggests a new role for activin in induction of the precursors to endoderm and a new endoderm‐enrichment technique using follistatin. Disclosure of potential conflicts of interest is found at the end of this article.