Hepatic Differentiation Of Murine Embryonic Stem Cells In Vitro

Hepatic differentiation from embryonic stem cells provides an useful in vitro model for studying hepatogenesis and liver metabolism, which is also important for the investigations of the mechanisms of liver development, the pharmokinetic and cytotoxicity screening test for drug discovery applications, and the elucidation of the molecular mechanisms and signaling pathways that regulate mutual induction between the endoderm and the mesoderm during in vivo hepatogenesis. Furthermore, hepatic differentiation from ES cells provides an efficient cell source for cell replacement therapy, such as hepatocyte transplantation, liver tissue engineering and bioartificial liver. Firstly we developed a novel method for generating embryoid bodies from mouse ES cells cultured on the STO feeder layer, and induced the hepatocyte differentiation from ES cells based on the diverse combination of growth factors required for the early liver development. Afterwards, we established a hepatic differentiation system from mouse ES cells with the induction of histone deacetylase (HDAC) inhibitors and isolated a novel hepatic stem/progenitor cell, which provides an appropriate model for the study of the cellular and molecular regulation of liver development.Presently, the most widely used method for generating various lineage cells from ES cells has been using the formation of embryoid bodies, which recapitulate many aspects of the lineage-specific differentiation programs, and the temporal and spatial gene expression patterns of early embryogenesis, therefore, the EB system provides an in vitro model for the study of embryo development and stem cells differentiation. In the present study, We described a novel method for the generation of embryoid bodies from murine ES-D3 cells based on the STO feeder layer. Compared with those traditional methods, the protocol we described is easier to perform and obtain large amounts of EBs spontaneously, of which above 70% could develop into typical cystic embryoid bodies. The results of in vitro differentiation ability evaluation revealed that the EBs could differentiate into neural-, cardiac- and hepatic-lineage cells. Based on the previous reports on embryonic liver development, we applied acidic- and basic-FGF, HGF, and BMP4 to induce hepatic differentiation of EBs in vitro, and found BMP4, secreted from septum transversum mesenchyme, acted cooperatively with FGFs from precardiac mesoderm to significantly enhance the hepatocyte differentiation, while aFGF and bFGF effected equally in the early hepatic...