High-ratio differentiation of embryonic stem cells into hepatocytes in vitro.

Background/purpose: To date, in differentiating system of embryonic stem (ES) cells into hepatocytes, hepatic differentiation ratio was still not shown. Here, after investigating hepatic differentiation from ES cells, we determined the differentiation ratios of hepatocytes and studied how to improve the ratios in ES cell differentiating system.

Methods: Embryonic bodies (EBs) formed from ES cells for 5 days were plated onto culture dishes and some growth factors were added into medium for hepatic differentiation. Expressions of hepatic genes and proteins were analysed using reverse transcriptase-polymerase chain reaction, immunocytochemistry (ICC) and radioimmunoassay. The relative counts of hepatocyte-like cells among all EBs cells were analysed by flow cytometry by which hepatic differentiation ratios were determined. Then, we observed the spatial distribution of ICC-positive cells in EB cells cluster and isolated the cells of positive areas in other EBs clusters without ICC examined. At last, isolated cells were re-cultured with previous condition and hepatic differentiation ratios were also determined.

Results: The hepatic genes and proteins were, respectively, expressed in cytoplasm. Hepatic differentiation ratio was first determined at day 11 to be 12.1% and the level reached maximum to be 33.4% at day 21. In isolated cells culture system, hepatic genes and proteins expressed stronger than that expressed in EBs cluster and hepatic differentiation ratio was got to 72.6% at day 21.

Conclusions: Isolating hepatocyte-like cells from EBs cell cluster and re-culturing them could produce hepatocytes with high differentiation ratio. This culture system may produce a new source of cell types for hepatocytes replacement therapies in hepatic failure.