The Molecular Mechanism Of Mouse Embryonic Fibroblasts Transdifferentiation To Induced Hepatic Stem Cells By Tetracycline-controlled Hnf1? And Foxa3 Expression Vectors

The transdifferentiation of differentiated mature somatic cells to other types of somatic cells or adult stem cells without the intermediate pluripotency state is called lineage reprogramming.The expression of two liver organogenesis transcription factors,Hnf1? and Foxa3,reprogramming MEFs to induced hepatic stem cells(iHepSCs)is an important achievement in this field.However,the molecular mechanisms involved in its reprogramming process are poorly discovered.The cell reprogramming process is in common with the embryonic development that both include the removal of the original cell epigenetics markers and the appearance of new cell-specific epigenetics modifications.In this notion,DNA methylation and demethylation that result in the change of DNA methylation pattern is considered to be a key mechanism.Recent researches have shown that the Tet family(Tet1,Tet2,Tet3)can catalyze the conversion of 5-methylcytosine(5-mC)to 5-hydroxymethylcytosine(5-hmC),thus achieving initiative demethylation.And more studies proved that Tet1 plays a very important role in somatic cell reprogramming.Our research focuses on the role of Tet family members in reprogramming MEFs into iHepSCs.We constructed doxycycline(Dox)-inducible Hnf1? and Foxa3 transcription factor lentiviral expression vectors.After infection with mouse embryonic fibroblasts,Dox was used to control the expression of the two factors and the reprogramming process was observed.We successfully obtained transdifferentiated cell clones by using these two-factor expression vectors that both express the reporter genes,EGFP and mCherry,and found that the induced cells were morphological similar to iHepSCs,at the transcriptome level,of cholangiocytes markers(CK19),hepatobiliary common markers(CK18),and some liver stem / precursor cell markers(Dlk1,Sox9,EpCAM).These results demonstrate the initially establishment of a tetracycline-controlled MEFs transdifferentiation system for iHepSCs.In addition,we also found that the reprogrammed cell,if cultured in iHepSCs medium withdraw Dox,cell proliferation will be significantly reduced,suggesting the expression of transcription factors is crucial for its stemness maintenance.Thus,this system have laid the groundwork for further studies.We explored the role and mechanism of Tet1 in lineage reprogramming of MEFs to iHepSCs.We found that the expression of Tet1 was activated in different induction systems for reprogramming of MEFs to iHepSCs,which suggested the activation of Tet1 was necessary in the formation of iHepSCs.Our study and the tetracycline-controlled iHepSCs-induced system have led to breakthroughs in the induction of iHepSCs and,on the other hand,explored the epigenetic mechanisms of the iHepSCs induction process,which will help to explore more effective and safe way of lineage reprogramming.