Study On The Differentiation Of Human Parthenogenetic Embryonic Stem Cells Into Hepatocyte-like Cells

Background and Purpose:At present,the most effective treatment of acute liver failure is limited to liver transplantation,and liver transplantation due to lack of tissue and organ source,high cost of treatment,risk and other defects to limit its wide range,thus ALF is still a major medical problem to be solved internationally.The use of functional hepatocytes differentiated from stem cells to treat ALF has become a new research direction.Human parthenogenetic embryonic stem cells not only have strong differentiation potential and high self-renewal ability as human embryonic stem cells,but also can avoid ethical controversy and immune rejection and so on,and have excellent application prospect.Hepatocytes derived from hPESCs have become a new method to solve the problem of tissue shortage in clinical liver transplantation.In this study,we aimed to explore the feasibility of hPESCs inducing artificial differentiation into functional hepatocytes in vitro.Methods:1、Mouse embryonic fibroblast(MEF)was obtained by primary culture of 0.05%trypsin-EDTA digestion.2、Three different culture systems of hPESCs were cultured in vitro:hPESCs/MEF(with MEF as feeder layer)culture system,hPESCs/hFF(hFF as feeder layer)culture system,hPESCs/3T3(3T3 as feeder layer)culture system.The growth status of hPESCs in different systems was observed.Morphological observation and alkaline phosphatase(AKP)method were used to determine whether hPESCs could grow steadily and maintain undifferentiated in three culture systems.3、The hPESCs were differentiated into hepatocytes by the combination of activin A,fibroblast growth factor 4,bone morphogenetic protein 2,hepatocyte growth factor,oncostatin,dexamethasone and B27.On differentiation of the various stages in the process of cell morphology change records and through reverse transcription real-time fluorescent quantitative PCR technology(RT-qPCR)detection restrictive endoderm(DE)specific gene,Sox 17 and Gsc,the relative expression of liver cell specificity relative expression of gene AFP and propagated,In the late induced by immunocytochemistry(ICC)immunocytochemistry,detection of liver cell marker protein expression of CK18 and Hepa,at the same time by indocyanine green(ICG)dyeing,glycogen PAS staining and cell culture supernatant on sense of fetal protein alpha-fetoprotein(AFP),albumin(albumin,propagated)with urea content detection to judge whether the cells of liver cell function after induction.Results:1、The MEF was successfully obtained by primary culture.The biological characteristics of MEF were verified by morphological observation and hPESCs growth in hPESCs/MEF culture system.2、HPESCs grew well in hPESCs/MEF,hPESCs/hFF and hPESCs/3T3 cultures,The spontaneous differentiation rate of hPESCs was less than 5%and the growth rate was stable.3、The hPESCs were stable and well grown in the three culture systems,but the hPESCs/hFF were used as human feeder layer,and the other two culture systems were excluded from the problem of animal origin pollution,with other animal-derived culture system does not have the advantage.4、The results of RT-qPCR showed that the expression of Sox 17 and Gsc increased significantly when the culture system containing bFGF and feeder layer was removed,and the relative expression level of Sox 17 and Gsc was the highest in the second day after induction.At the end of induction,the specific expression of AFP and hepatocyte specific gene ALB was the highest.The results of immunohistochemistry showed that CK18 and Hepa expressed in the cytoplasm.ICG staining results showed that the induced cells had the function of metabolizing dyes.The results of glycogen PAS staining showed that the glycogen was in the cytoplasm after induction;The results of AFP,ALB and urea content in the supernatant of cell culture showed that the contents of AFP,ALB and urea in the supernatant were significantly higher than those in the control group.Conclusions:1、MEF was successfully obtained by primary culture with 0.05%trypsin-EDTA solution digestion.2、hPESCs/MEF system,hPESCs/hFF system and hPESCs/3T3 system can effectively support the proliferation of hPESCs and maintain its undifferentiated condition,but the hPESCs/hFF system is a human culture system,which avoids the pollution of animal,And thus more advantages.3、The culture system of bFGF and feeder layer can inhibit the spontaneous differentiation of hPESCs,and Activin A can promote the differentiation of hPESCs to DE.4、In combination with the inducible factors Activin A,FGF-4,BMP-2,HGF,OSM,Dex,B27.Through the phased induction of differentiation,hPESCs can be induced to differentiate into hepatocyte-like cells that are very similar to normal liver cells in cell morphology,hepatocyte-specific genes,marker proteins and liver function.