The Study Of Notch Signaling Pathways Inducing Human Embryo Stem Cells Directional Differentiation To Hepatic Duct Cells

Objective:Notch signaling pathway is an evolutionarily highly conserved mechanism for cell-cell communication, which keeps cell proliferation, differentiation and apoptosis, and plays an important role in embryogenesis and many types of cell differentiation fate determination. Our purpose was to explore the role of the Notch signaling pathway in the hepatic cell differentiation.Methods:After human embryo stem cell (hESC) Hl cells were inoculated into the board packaged by Matrigel, the cells were induced in the liquid of cytokine activation A (Activin A) differentiation for 5 days. Then, differentiation endoderm(DE) cells obtained by activin A induction were cultured for 5 days in cultivation system 1 (20ng/mlBMP4, 100ng/mlFGF1) and cultivation system2 (20ng/mlBMP4, 100ng/mlFGF1, Notch signal inhibitors NAPT) respectively. Immunocytochemistry staining and RT-PCR were used to identify the nature of the differentiated cells, hepatic duct cells’ differentiation, the related receptors and gene expression of notch signaling pathway.Results:The hESC mass containing about 50-100 cells was the ideal differentiated cell quantity and it had stronger ability of proliferation and differentiation and Noggin could effectively inhibit hES cells differentiation into DE cells. DE cells deriving from hES cells expressed relevant receptors (notch1. notch2) and downstream genes: hes1, hes5 of notch signaling pathway and the notch signaling molecule lower expressed or did not express after BMP-4 and FGF-1 effected for 3 days. After the Notch signal pathway inhibitor was joined into hepatic duct cells differentiation system, it was found that parts of DE cells had apoptosis or death. Notch signal downstream intracellular protein(NICD) of the remaining cells expression obviously decreased, and most protein distributed in the cytoplasm, not transferred into the nucleus. It was obviously that NICD was not in active. On the contrary, NICD fluorescence intensity of the control group(FGF-1, BMP-4) was significantly higher compared with the experimental group and NICD mainly distributed in nuclear, and the result showed that NICD was in active. At the same time, after they effected by notch inhibitors for 5 days, hepatic cell production rates were significantly lower, even undetectable in the experimental group compared with the control group.Conclusions:1. hES cells could be directionally differentiated into DE cells of high rates by inducing in high concentrations of activin A or in low concentration of serum and noggin could inhibit hES cells differentiation into DE cells.2.The differentiation density of hES cells was closely related with DE formation efficiency.3.FGF-1 and BMP-4 could effectively encourage DE cells differentiation into biliary epithelial cells.4.Notch signaling pathway decided the cytogenesis of DE cells differentiation into biliary epithelial cells.