Potential therapeutic applications of embryonic stem cell (ESC)-derived 
hepatocytes are limited by their relatively low output in differentiating ESC 
cultures, as well as by the danger of contamination with tumorigenic 
undifferentiated ESCs. To address these problems, we developed transgenic murine 
ESC clones possessing bicistronic expression vector that contains the 
alpha-fetoprotein gene promoter driving a cassette for the enhanced green "live" 
fluorescent reporter protein (eGFP) and a puromycin resistance gene. Under 
established culture conditions these clones allowed for both monitoring of 
differentiation and for puromycin selection of hepatocyte-committed cells in a 
suspension mass culture of transgenic ESC aggregates ("embryoid bodies" [EBs]). 
When plated on fibronectin, the selected eGFP-positive cells formed colonies, in 
which intensely proliferating hepatocyte precursor-like cells gave rise to 
morphologically differentiated cells expressing alpha-1-antitrypsin, 
alpha-fetoprotein, and albumin. A number of cells synthesized glycogen and in 
some of the cells cytokeratin 18 microfilaments were detected. Major hepatocyte 
marker genes were expressed in the culture, along with the gene and protein 
expression of stem/progenitor markers, suggesting the features of both 
hepatocyte precursors and more advanced differentiated cells. When cultured in 
suspension, the EB-derived puromycin-selected cells formed spheroids capable of 
outgrowing on an adhesive substrate, resembling the behavior of fetal mouse 
hepatic progenitor cells. The established system based on the highly efficient 
selection/purification procedure could be suitable for scalable generation of 
ESC-derived hepatocyte- and hepatocyte precursor-like cells and offers a 
potential in vitro source of cells for transplantation therapy of liver 
diseases, tissue engineering, and drug and toxicology screening.