Objective: To investigate in vitro methods of inducing mouse embryonic stem cell(s) (ESC) into hepatocytes.
Methods: E14 mouse ESC were cultivated in suspension and plated to form aggregates, the embryoid bodies. They were allowed to outgrow on the plated culture with the stepwise addition of growth factors-- acidic fibroblast growth factor (aFGF), hepatocyte growth factor (HGF) and oncostatin M (OSM) into the culture medium. Morphology was investigated by phase contrast microscopy. Gene expressions of endodermal and liver specific mRNA were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Indocyanine green (ICG) uptake assay and periodic acid-Schiff reaction (PAS) were performed to assess the differentiation and function of the cells.
Results: Morphology analysis revealed a difference between ESC-derived hepatic cells and original ESC in that the former showed distinct round or polygonal shapes with clear boundaries, some arranged tightly in cords, while the latter grew in clones without clear boundaries between cells. Those ESC-derived hepatic cells expressed endodermal and liver specific genes mRNA--TTR, AAT, AFP, ALB, G6P and TAT. ICG uptake assay and PAS reaction were positive for those ESC-derived hepatic cells. The ICG positive cells were about 85.1% in number.
Conclusion: ESC-derived hepatic cells possess characteristics of hepatocytes, which would promise the eventual clinical use of ESC in treating damaged liver tissues.