Direct Hepatic Differentiation And The Regulation Of Gene Expression

End stage liver disease (ESLD) is an irreversible condition that leads to the eventualfailure of the liver. It is the final stage of many liver diseases, for example, viral hepatitis,autoimmune hepatic disorders, fatty liver disease, drug induced liver injury, andhepatocellular carcinoma (HCC). Current optimal treatment for certain HCCs or ESLD isorthotopic liver transplantation. However limited availability of donor livers andimmunological incompatibilities are two major obstacles to its routine deployment. Thishighlights the important need for alternative therapeutic strategies. Researchers haveproposed that stem cell biology could provide a scalable answer for the treatment of ESLD.Human stem cells are scalable cell populations with capablity of cellular differentiation.This makes them a very attractive in vitro cellular resource and in theory providesunlimited amounts of primary cells. In recent years, efficient hepatic differentiation fromhuman stem cells has been achieved by several research groups including our own.Hepatocyte Nuclear Factor4(HNF4) is a nuclear hormone receptor protein mostlyexpressed in the liver and lowly expressed in gut, kidney, and pancreatic beta cells. As akey member of HNF4family, HNF4a is well studied and shown that it critically regulates alarge number of genes involved in most hepatocyte functions, which gives a great promisefor the maintenance and enhancement of hepatic differentiation and hepatic function. Ourprevious researches on hepatic differentiation from pluripotent stem cells (PSCs) havedemonstrated that HNF4a is lower expression in undifferentiated hepatic status andincreased gradually during the hepatic maturing process.Some modifications of HNF4a have been documented, however the mechanismbehind these regulations remain obscure. The small ubiquitin-related modifier (SUMO),around100amino acids in length and12kDa in molecular weight, have nearly identicalstructural fold of ubiquitin but only share about18%identity of the sequence.SUMOylation is a post-translational modification (PTM) to modify many proteins relatedto various cellular processes, such as differentiation, cell cycle, gene transcription, proteinlocalisation. Our following study showed some promising clues between SUMOylationand the stability of HNF4a. Therefore, my project focuses on the role of HNF4a in hepticdifferentiation and dedifferentiation and aims to clarify the mechanism of SUMOylation onHNF4a in this process. To investigate the role of HNF4a in hepatic differentiation, human embronic stemcells (hESCs) were directly induced to differentiate into hepatocytes. Some ELISA assaysand fluorescence immunocytochemistry were carried out to test some proteins related tohepatic function, such as HNF4a, albumin, cytochrome P4503A4(CYP3A4). We alsoanalysed these proteins in cellular lysates by Western blotting (WB). During the process ofdedifferentiation, we detected the reduced levels of albumin, CYP3A4, TTR, AFP,fibronectin, transthyretin, which are regulated by HNF4a.We also detected that during the hepatic dedifferentiation process, the expression ofHNF4a and SUMO2reduced gradually, highly consistent with the low expressed RNF4. Inaddition, fetal liver in different developmental stages and adult liver tissue was examinedby immunohistochemical staining and showed that in the adult liver tissue SUMO2andRNF4were highly expressed but not the T1, T2liver tissue. Based on our previous workabout SUMOylation on HNF4a, we concluded that HNF4a is regulated by SUMOylationprobably resulting in RNF4mediated ubiquitination followed by its degradation. Furtherexperiments are initiated to extend this but not completed during this project. It will bedesigned to upregulate or downregulate the expression of HNF4a, SUMO2, RNF4toobserve the impact on hepatic differentiation.In conclusion, we created and optimized a direct induced protocol to efficientlyproduce hepatocytes from hESCs. In addition, we demonstrated that HNF4a is critical forhepatic differentiation both driving the formation of hepatocytes and maintaining a maturephenotype. Furthermore, polySUMOylation plays an important role in the PTM of HNF4aby regulating its stability.