[Directional development and differentiation of mouse embryonic stem cells into hepatocytes in vitro].

OBJECTIVE

To investigate the mechanism and regulation of differentiation from embryonic stem (ES) cells to hepatocytes in vitro and to find a new source of cell types for hepatocytes replacement therapies in the treatment of hepatic failure.

METHODS

ES cells of BALB/c mice were cultured and maintained undifferentiated in gelatin-coated dishes in Dubecco's modified Eagle's medium (DMEM) containing 1 000 U/L leukaemia inhibitory factor (LIF). Then, the ES cells were cultured in DMEM without LIF so as to develop into embryonic bodies (EBs). For directional differentiating, EBs were put into 24-well tissue culture dish and differentiate into cells with different forms. On day 7 to day 11 acid fibroblast growth factor (aFGF) with terminal concentration of 100 ng/ml was added, and on day 11 to day 15 hepatocyte growth factor (HGF) terminal concentration of 20 ng/ml was added. EBs in culture without growth factor was used as controls. The development and differentiation status was observed by inversion microscope dynamically. The concentrations of alpha-fetoprotein (AFP) and albumin (ALB) in the supernatants were determined by radioimmunoassay on the 1st to 15th day since the ES cells were inoculated. Immunocytochemistry (ICC) was used to detect the expression of hepatic proteins such as AFP, ALB, cytokeratin 8 (CK8) and cytokeratin 18 (CK18) in the cells on the days 7, 8, 9, 10, 11, 12, 13, 14, and 15. The number of ALB positive cells was counted. The ALB positive rate was calculated as the differentiation ratio of hepatic cells.

RESULTS

ES cells remained undifferentiated and grew to ES clones in the DMEM containing LIF and developed to EBs in the medium without LIF. AFP was detected firstly in the medium on day 8 with a concentration of 3.4 ng/ml and the concentration increased to be 22.8 ng/ml at day 15; ALB was detected firstly in the medium on day 11 with a concentration of 0.2 microg/ml and the concentration increased to be 2.2 microg/ml on day15. ICC showed that AFP, ALB, CK8, and CK18 began to be expressed on day 8, day 10, day 10, and day 11 respectively. The ICC-positive cells have the ultrastructures consistent with those of the normal mouse hepatocytes. The ratio of hepatic differentiation was 30%.

CONCLUSION

ES cells can differentiate directionally into hepatocytes when cultured in gelatin-coated dish and medium with aFGF and HGF. This ES cell differentiating system provides enough functional hepatocytes and may be a good new source of differentiated cell types for hepatocytes replacement therapies in treatment of hepatic failure.