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

¹ Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, 51 Blossom St, Boston, MA 02114, U.S.A., Rutgers, The State University of New Jersey, Department of Chemical and Biochemical Engineering, Rutgers University, The State University of New Jersey, 98 Brett Road, Piscataway, NJ 08854, U.S.A
² Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital, Harvard Medical School, 51 Blossom St, Boston, MA 02114, U.S.A.

^* To whom correspondence should be addressed. E-mail: ireis{at}sbi.org.

	Abstract

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 cells (mES) using fibronectin-coated collagen gels. This technique results in a homogenous 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, while follistatin increased the Foxa2 positive endoderm fraction to 78%. Our work suggests that activin delays the induction of endoderm through it transient precursors, the epiblast and mesendoderm. Long term differentiation, displays a two-fold reduction in hepatic gene expression and three-fold reduction in hepatic protein expression of activin-treated cells compared to follistatin-treated cells. Moreover, subcutaneous transplantation of activin-treated cells in a syngeneic mouse generated a heterogeneous teratoma-like mass, suggesting these were a more primitive population. In contrast, follistatin-treated cells resulted in an encapsulated epithelial-like mass, suggesting 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.