OBJECTIVE: To establish an optimal method to isolate and culture human embryonic stem (hES) cells from discarded blastocysts and to differentiate these cells into hepatocyte-like cells.
METHODS: Discarded human blastocysts of days 5–6 were cultured on mouse embryonic fibroblast cell feeder layers. Cells from the inner cell mass were isolated and subsequently cultured in vitro. To induce the formation of embryoid bodies (EB), dissociated ES cells were cultured in hanging drops for 5 days. The resulting EB were plated onto 100 mm plastic gelatin-coated dishes and allowed to attach for the outgrowth culture in the presence of hepatocyte growth factor for an additional 15 days.
RESULTS: The hES cells had typical morphological characteristics: round or elliptic nest-like colonies with distinct cell borders and a smooth surface, significant swelling growth, compact cell aggregation, and relatively big nucleus compared to a small cytoplasm. The hES cells were identified by positive alkaline phosphatase staining. All had normal karyotypes and expressed octamer-binding transcription factor and cell surface markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, and tumor rejection antigens TRA-1-60 and TRA-1-81. The cells could be differentiated to form teratomas in vivo. The hES cells could be induced to differentiate into hepatocyte-like cells and 70–80% of the cells expressed liver-associated proteins.
CONCLUSION: Three hES cell lines have been successfully isolated and cultured from discarded human blastocysts. The hES cells can be efficiently differentiated into hepatocyte-like cells using the EB system. These cells possess the potential for treatment of liver diseases.