Generation of glycogen- and albumin-producing hepatocyte-like cells from embryonic stem cells

We present a novel twostep protocol for the differentiation of embryonic stem (ES) cells into the hepatic lineage. Differentiated hepatocytelike cells express genes and proteins characteristic for endodermal and hepatic cells and acquire a functional hepatic phenotype as demonstrated by albumin secretion and glycogen storage. During differentiation, alpha-fetoprotein, albumin, transthyretin, alpha-1-antitrypsin, cytochrome P450 subunits 2b9 and 2b13 and tyrosine aminotransferase transcripts are upregulated. Quantitative RTPCR data revealed a fetal hepatic phenotype corresponding to day 1314 of liver development. Terminally differentiated hepatocytelike cells show a binucleated, cuboidal morphology labeled by albumin, alpha-1-antitrypsin, liver amylase, dipeptidyl peptidase IV, cmet and cytokeratin 18. ES-derived intermediate cell types transiently and partially coexpress nestin with albumin and alpha-fetoprotein, respectively, but not cytokeratin 19. This finding suggests an ESderived potential hepatic progenitor cell type, which is partially nestin, albumin and alpha-fetoproteinpositive, but cytokeratin 19-negative.