ABSTRACT
Recently it was shown that embryonic stem (ES) cells could differentiate into hepatocytes both in vitro and in vivo, however, prospective hepatic progenitor cells have not yet been isolated and characterized from ES cells. Here we presented a novel 4-step procedure for the differentiation of mouse ES cells into hepatic progenitor cells and then hepatocytes. The differentiated hepatocytes were identified by morphological, biochemical, and functional analyses. The hepatic progenitor cells were isolated from the cultures after the withdrawal of sodium butyrate, which was characterized by scant cytoplasm, ovoid nuclei, the ability of rapid proliferation, expression of a series of hepatic progenitor cell markers, and the potential of differentiation into hepatocytes and bile duct-like cells under the proper conditions that favor hepatocyte and bile epithelial differentiation. The differentiation of hepatocytes from hepatic progenitor cells was characterized by a number of hepatic cell markers including albumin secretion, upregulated transcription of glucose-6-phosphatase and tyrosine aminotransferase, and functional phenotypes such as glycogen storage. The results from our experiments demonstrated that ES cells could differentiate into a novel bipotential hepatic progenitor cell and mature into hepatocytes with typical morphological, phenotypic and functional characteristics, which provides an useful model for the studies of key events during early liver development and a potential source of transplantable cells for cell-replacement therapies.
Subject(s)
Butyrates/pharmacology , Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Hepatocytes/cytology , Albumins/biosynthesis , Animals , Bile Ducts/cytology , Biomarkers/metabolism , Cell Cycle/physiology , Cell Differentiation/drug effects , Cells, Cultured , Dipeptidyl Peptidase 4/biosynthesis , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Fluorescent Antibody Technique , Glucose-6-Phosphatase/biosynthesis , Hepatocyte Nuclear Factor 3-beta/biosynthesis , Hepatocytes/drug effects , Hepatocytes/metabolism , Keratin-18/biosynthesis , Keratin-19/biosynthesis , Liver Glycogen/biosynthesis , Mice , Reverse Transcriptase Polymerase Chain Reaction , alpha 1-Antitrypsin/biosynthesis , alpha-Fetoproteins/biosynthesis , gamma-Glutamyltransferase/biosynthesisABSTRACT
Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.
