The Optimized Study On The Method To Differentiate Human Embryonic Stem Cells Into Functional Hepatocyte

Stem cell technology is regarded as the most leading edge of scientific researchin the field of regenerative medicine. Especially, human embryonic stem cells (EScells), which was establish in1998, came to wide attention of researches in all of theword, and also hold a good application prospect in clinical therapeutic research ofdisease.The process of ES cells differentiating into functional hepatocyte consists of fourstages, including endoderm induction, hepatic specification, hepatoblast expansionand hepatic maturation. The induction of each stage depends on kinds of celldifferentiation factors. In this study, B27solution was added in the beginning of thedifferentiation, and HCMTMSingleQuots medium was used in the stage of hepaticmaturation, instead of the application of OSM and Dex. The morphology as well asthe expression of hepatic specific proteins or genes were detected in each stage. Thefunction of ES cell derived hepatocyte was evaluated, such as synthesis of glycogen,metabolism of indocyanine green and absorption of low density lipoprotein, in orderto evaluate the application of our method in clinical therapy.The experimental results showed that human ES cells could be differentiated intofunctional hepatocyte successfully with the optimized method. At the end of the finalstage, the differentiated cells hold typical morphology of hepatocyte. At the stage ofendoderm induction, the endoderm specific proteins FOXA2and SOX17wereexpressed in the cells. During the stage of hepatic specification, the differentiated cellshold some characteristics of hepatic progenitor cells, and expressed AFP and HNF4α.At the final stage of hepatic maturation, hepatic specific proteins ALB and HNF4αwere expressed in over90%of the differentiated cells. From the detection of the cellfunction, ES derived hepatocyte was found to hold similar function to real humanhepatocyte. The final cells derived from ES cells hold the function to synthetiseglycogen, metabolize indocyanine green and absorb low density lipoprotein. Moreimportantly, though the method showed no difference in the regulation of pluripotentgenes (POU5F1, SOX2and NANOG) compared with the classic experimental method, the optimized method could improve the expression of some hepatic specific genes(SOX2and NANOG), so as to refine the differentiation of human ES cells tohepatocyte.Taken together, based on the classic differentiating method, this study focused onthe differentiation of human ES cells to hepatocyte. The efficiency of celldifferentiation could be further improved with the optimization of cell factors used inthe differentiating process, in order to establish an effective method to induce ES celldifferentiated into functional hepatocyte. Therefore, this study established a newmodel for the development research of liver and the drug screening of hepatic diseasein vitro, and also provided some theory and technology for the application of humanES cells in clinical therapy of hepatic disease.