Study Of Directing Hepatoblast-like Cells From Mouse Embryonic Stem Cells And Hepatic Differentiation Mechanism

Viral hepatitis is a kind of hepatic disease that occurs much more frequently in China.It may cause severe hepatitis which has a lethal rate of as much as 50%-70%.Presently the major treatment for hepatic failure patients was the whole liver transplantation.However, the development of such cure is hampered by a shortage of donor liver.Nevertheless, hepatocyte transplantation can also be used as cure for end-stage liver diseases,thus becoming a promising substitute for liver transplantation.Hepatocyte transplantation was first proposed by Bumgardner in 1988.In 1993,Mito performed hepatocyte autotransplantation in clinic,which was the first time used on hepatic disease patients in the world.After that,the technique of hepatocyte transplantation developed swiftly and became the one of focus researches in biomedical area.As the same with liver transplantation,the major problem to be solved was the source of hepatocytes to transplantation.The clinical research demonstrated that best hepatocytes source is from self liver,but which is difficult and deficient in hepatic failure patients.So this problem can be solved partially if we can successfully differentiate embryonic stem cells to functional liver cells.Embryonic stem(ES) cells are pluripotent cells that have the ability to self-renew, differentiation,and proliferation,which means that they can differentiate into all tissue cells,including the ones in clinic wanted,and ES cells can self-renew to sustain cell number and pluripopent,which make sure cells-transplantation therapy supply.Because of this,directional differentiated ES cells are used in tissue reconstruction and cells therapy, and show great promise as the source to fill the needs of hepatocytes.With the help of ES cells as hepatocytes supply and further maturing technique,hepatocytes transplantation will be the best treatment for severe liver diseases in 21^st century.The pioneering work was done by Chinzei lab in 2002,when they obtained hepatocyte-like cells through embryonic body(EB) formation from ES cells,and transplanted the cells into liver injured mouse model.4 weeks later,differentiated hepatocytes were found and integrated into the host.In the same year,similar results were also obtained by Yamada and others.The following years were met with great development in ES cells differentiation into hepatocytes.To get high differentiation efficiency, researchers divided the differentiation process as multiple steps,each step added with induction reagent such as growth factors,thus mimicking liver development in vivo.This also provides as a good method for in vitro study of the molecular mechanisms during liver development.This work has used the mouse ES cell line E14.1,which was cultured feeder-free.To get E14.1-1,E14.1 was stably transfected with mRPF driven by Albumin promoter and EGFP driven by CK19 promoter;to get E14.1-2,E14.1 was stably transfected with EGFP under the control of Albumin promoter and hCD25-tdTOMATO under the control of CK19 promoter.The two stable lines were both induced for differentiation in serum-containing and serum-free medium,and the two methods were compared as to the induction efficiency of endoderm and hepatic progenitor cells.The result showed that the induction efficiency of endoderm and hepatic progenitor cells in serum-containing medium is very low,with 14%and 3%respectively.Then we developed a two-step method in serum-free medium to induce liver progenitor cells.E14.1-1 and E14.1-2 cells were cultured in suspension in order to form EB,and 48 hours later,they were treated with activin A and allowed for another 4 days' differentiation before analyzing the surface marker of definitive endoderm(DE) cells,namely CXCR4, ECD,c-Kit.The FACS analysis showed that both CXCR4⁺/ECD⁺ and CXCR4⁺/c-Kit⁺ cells reached the peak of 75%after 4 days' induction.Endoderm genes Foxa2,Sox17,Hex, Shh,Gata4,Gata6 were expressed,and mesoderm genes Flk1,Mixl1 were also expressed while neural ectoderm gene pax6 was not expressed.Meanwhile,Foxa2,Sox17 and Hex were expressed in CXCR4⁺/c-Kit⁺ cells,which showed no expression of visceral endoderm (VE) cell marker Sox7.Altogether,CXCR4⁺/c-Kit⁺ cells were DE cells differentiated by this way.The DE cells were isolated and differentiated into hepatoblast under the compared conditions of suspension culture and adherent culture.As to suspension culture,endoderm cells were cultured with medium containing bFGF and BMP4 in suspension culture dishes which allowed them to proliferate as spherical colonies.48 hours later,hepatic differentiation-related genes Prox1,Afp,Hnf4,and Ttr were upregulated.Then the colonies were cultured with medium containing HGF and EGF in collagen typeâ… coated dishes to allow for attachment.As to adherent culture,isolated cells were cultured in collagen typeâ… coated dishes with the same medium as those of suspension culture.At day 12,Alb and CK19 double positive cells(as displayed by fluorescent proteins) were detected in spherical colonies,and FACS analysis showed that they counted for 22%,while no double positive cells were detected in cells of adherent culture.In addition,cells differentiated further in spherical colonies than that of adherent culture,according to the result of RT-PCR and fluorescent.The above in vitro liver progenitor cells differentiation from ES cells provided as a good model for study of roles played by Gata-4 and Gata-6 during liver development.We established ES cell lines in which Gata-4 and/or Gata-6 were knocked down through RNAi. According to the result from Q-PCR,in the process of endoderm differentiation,Gata-4 and Gata-6 showed no effect on the expression of such DE genes as Tm4sf2 and Cxcr4, while they influenced the expression of VE gene Emp2 and Amn.The expression of hepatic progenitor genes Afp,Hnf4,Ttr were all downregulated when Gata-4 and Gata-6 were both knocked down,but no distinguishable difference can be observed if only one of them was knocked down,which meant that they were important for hepatic progenitor cells differentiation and had some functional redundancy.This work provides a convenient method for study in fetal liver development as well as for handling with cell therapy.In the meantime,we explored the mechanism of fetal liver development,and thus it provided new contents and data for efficient ES cell differentiation towards hepatocytes.