| Liver transplantation is the only effective way to the end-stage of liver disease.However, the lack of donor organs and immune rejection has hampered this clinicalmethod. Recently, as a alternative choice of orthotopic liver transplantation,hepatocyte transplantation achieved some therapeutic effect. Then, liver cell basedtreatment also has it’s own limitations,such as short survival of hepatocyte with theculture time increase, cannot be expanded in vitro, and during the chronic liverdisease like cirrhosis, the normal liver cell proliferation was inhibited. Therefore, tofind a new and effective cells is one of the most urgent tasks for liver therapyresearch.Hepatic stem cells are bi-potential stem cells which can differentiated tohepatocytes and cholangiocytes. Induced hepatic stem cells (iHepSC) have thephenotype and characteristic of liver progenitor cells, which was derived frommouse embryonic fibroblasts (MEF)by lineage reprogramming method in ourlaboratory. By the in vitro culture of iHepSC, we find that, it has high proliferationability and normal nuclear karyotype, which can be differentiated to maturehepatocytes and cholangiocytes in vivo and in vitro to replace the damaged cells, anddon’t have the ability to form tumors. Therefore, iHepSC can serve as a new idealcell source for liver cell therapy. However, the stemness mechanism of the inducedhepatic stem cells were poorly known, and this could be a obstacle for the clinicaluse of iHepSC.In order to know the mechanism of stemness maintenance of hepaticstem/progenitor cells, this project is committed to study the gene which expresseddifferently between the hepatic stem/progenitor cells and hepatocytes by single cellRNA analyze method. After the comparison, we received five transcription factorswhich have a significant expressing levels between iHepSC and differentiated cells:Pitx2, Twist1, Stat3β, TSC22d1and GATA6. Based on the results above, we focuson two of them: Pitx2(paired-like homeodomain transcription factor2) and Twist1(The basic Helix-loop-helix (bHLH) transcription factors), for the later research.Byconstructing a lentivirus ShRNA eukaryotic expression vector to specifically knockdown the expression of Pitx2and Twist1in iHepSC, and finally obtained stablesilencing iHepSC cell strains, named iHepSC-Sh-Pitx2and iHepSC-Sh-Twist1.From the comparison of expression of stem cell related gene, self-renew ability and differentiation potential, we systematically analyzed the effect of transcriptionfactor Pitx2and Twist1on stemness maintenance of iHepSC.After knockdown of Pitx2and Twist1, we found that there was no change incell morphology of iHepSC, however, the result of RT-PCR and immunoflourenceindicated that they donnot express Sox9, which is a very important marker of stemcells. Even though they both express the other stem cell marker Lgr5and EpCAM,the expressing level is far below the normal one. And there is no difference of thehepatocyte specific gene Albumin (ALb), cholangiocyte specific gene cytokeratin-19(CK19) and immature hepatocyte marker α-fetoprotein (AFP) in iHepSCs afterknock down of Pitx2and Twist1. This may indicate that the known down oftranscription factors Pitx2and Twist1may have some effect on stemnessmaintenance of iHepSC.The self-renew ability is one of the basic character of hepatic stem cells, so wecompare the proliferation ability between iHepSC and iHepSC-Sh-Pitx2andiHepSc-Sh-Twist1，the results shows that the iHepSC’s growth and clone formingpotential extremely slowed after Pitx2and Twist1knockdown. The result of CCK8indicate that the iHepSC need more proliferation time in24and48hours than thecontrol ones, and this have the statistic significant. The result of Brdu and Ki67havethe same trends like CCK8. All of the results above shown that Pitx2and Twist1canreduce the proliferation capacity of iHepSC.iHepSC can differentiated to mature hepatocytes and cholangiocytes. Wepreliminary check the self differentiation of iHepSC before and after Pitx2andTwist1knockdown discrepantly. After the analyze of premature hepatic markers αAT,TAT and TTR，premature cholangiocytes marker NCAM, CK7and ChromA, maturehepatocytes marker G6P, Cyp7a1and Cyp3a11. The result shows that iHepSC dosenot differentiate to any direction after Pitx2and Twist1knockdown in iHepSC. Thismay indicate that Pitx2and Twist1might be not the main trigger for differentiationof iHepSC without change of culture environment and defined induce medium, theeffect on differentiation of iHepSC need more experiment to prove.During the reprogramming of MEF to iHepSC, there has a process calledmesenchymal-epithelial transition (MET). Therefore, we analyze the MET markersof iHepSC. The result shows that:the epithelial marker Claudin, N-Cadherin,E-Cadherin and β-Catenin of the experimental group was expressed much less thanthe control, and begin to express the mesenchymal marker Snail, Slug and Vimentin, which means that iHepSC began to differentiated to the direction of mesenchymalcells after Pitx2and Twist1was knockdown.This study was present the effect of transcription factor Pitx2and Twist1on theinduced hepatic stem cell maintenance for the first time. After knockdown of Pitx2and Twist1of iHepSC, the self-renew and the expression of hepatic stem cell markerbegan to reduce, and catch some marker of mesenchymal cells. The construction ofiHepSC-Sh-Pitx2and iHepSC-Sh-Twist1can serve as a research model of stemnessmaintenance of hepatic stem cells. Experiment shows that there must be somerelationship between Pitx2and Twist1and liver stem cells. However, the mechanismof transcription factors for stem cell maintenance is still not clear. Our results havegive a new insights and shown a new clue of mechanism for hepatic stem cellsmaintenance and stem cell-based therapy of liver disease. |
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