Hepatic Stellate Cells Induced By TGF-B1 Regulate Hepatic Progenitor Cell Differentiation Into Cholangiocytes

ObjectiveTo investigate the effects of mouse hepatic stellate cells(mHSCs)activated by transcription factor ?1 signaling(TGF-?1)on mouse hepatic progenitor cells(mHPCs)differentiation.MethodsPart 1:Lentivirus-concentrated solutions of TGF-?1,GFP,TGF-?R1 sh3RNA and RFP were successfully transfected into mHSCs.Morphological alterations of HSCs were examined by the expression of a-SMA through immunofluorescence staining.mRNA experssion of TGF-?1??-SMA?TGF-?R1?Smad2?Smad3?Jaggedl?VEGF?HGF?Wnt3a were detected by qRT-PCR.Protein expressions of TGF-?1?a-SMA?TGF-PRl?Smad2/3?p-Smad2/3?Jaggedl were tested by Western blotting.Part 2:Primary mHPCs were isolated from fetal livers based on the cell surface antigen delta-like proteinl(Dlkl)by a fluorescence-activated cell sorter(FACS)and alpha-fetoprotein(AFP)?albumin(ALB)?cytokeratin19(CK19)expression were observed by immunofluorescence method.After transwell co-culture with mHSCs for 6 d,these cells were divided into mHPCs+mHSCs-TGF-?l group,mHPCs+mHSCs-RFP,mHPCs+mHSCs-TGF-?Rlsih3 group and mHPCs group.AFP?ALB?CK19 expression were observed by immunofluorescence staining;mRNA expression of differentiation markers AFP?ALB?CK19?SOX9?Hes1 were measured by qRT-PCR;Cell functions of glycogen synthesis and storage after differentiation were tested by PAS staining.Part 3:Lentivirus-concentrated solutions of pHBLV-CMVIE-GFP was successfully transfected into mHPCs-E14.5 cell line and transfection efficiency was assessed by flow cytometry and immunofluorescence.Hepatic stem cells Characteristics of mHPCs-E14.5 cell line was detected by immunofluorescence assay.Acute liver injury mouse model was established through intraperitoneal injection of CC14 in combination with 2/3 partial hepatectomy,followed by 200ul DMEM medium transplantation(Sham operation group),mHPCs-E14.5 transplantation,co-transplantation of mHPCs-E14.5 and mHSCs-TGF-?l,co-transplantation of mHPCs-E14.5 and mHSCs-RUP and co-transplantation of mHPCs-E14.5+mHSCs-TGF-?Rl sih3 into C57 mouse spleens for cell transplantation assay.At the seventh day of cell transplantation,engrafment of transplanted cells into mouse spleens was detected by H&E staining and confocal immunofluorescence assay.Experession of AFP?ALB?CK19?SOX9??-SMA.Jaggedl was measured by immunohistochemistry and qRT-PCR to identify differentiation of transplanted cells.ALT and AST were tested by automatic biochemical analyzer to evaluate the liver functions recovery.ResultsPart 1:mRNA expressions of TGF-?1??-SMA?TGF-?Rl?Smad2?Smad3?Jaggedl?VEGF?HGF?Wnt3a and protein expressions of TGF-?l?a-SMA?TGF-?-R1?Smad2/3?p-Smad2/3?Jaggedl were significantly higher in TGF-?i over-expression group compared to its control group,but these expression were much lower in TGF-?Rl-silenced mHSCs compared to the control group.There was significantly statistical difference between three groups(P<0.01).in the overexpression group,a-SMA showed obvious positive expression in the cytoplasm,but expression was only weakly positive in the control group and the gene-silencing group,suggesting that TGF-?1 activated mHSCs into myofibroblast through Jagged-1/Notch and more cell factors were secreted after mHSCs activation.Part 2:In immunofluorescence assay,it was clear that newly sorted DLKl+cells expressed AFP and low levels of ALB,but not CK19,further indicating that most of the DLK1+ cells were mHPCs.At the 6 d of co-culture with mHSCs,master biliary cell marker CK19 was highly expressed in the cytoplasm of mHPCs in mHPCs+mHSCs-TGF-?l group,but mature hepatocyte marker ALB expression was significantly increased in mHPCs co-cultured with TGF-?R1-knockdown mHSCs.QRT-PCR analysis showed that after co-culturing with TGF-?l-overexpressing mHSCs for 6 d,expression of duct-specific CK19 was significantly increased,as well as SOX9 and Hesl in mHPCs.However,ALB experession was most obvious in mHPCs after interrupting TGF-?1 signaling via gene silencing in mHSCs compared to control mHPCs.Hepatic stem cell AFP was highly expressed in mHPCs+mHSCs-RFP group and mHPCs group.There was significantly statistical difference between three groups(P<0.05).In the cells cultured with TGF-?R1-knockdown mHSCs,90%of mHPCs could synthesize and store glycogen,but only 20-30%of TGF-?l-affected mHPCs were positive for PAS staining.In two other control groups,more than half the cells had these functions.There was significantly statistical difference between these groups(P<0.05).The above results showed that mHSCs induced by TGF-?1 promoted differentiation of mHPCs into cholancyotes through Jaggedl/Notch signaling.Part 3:After transfection of empty-lentivirus GFP,stable monoclonal colonies were formed in mHPCs-E14.5 cell line.Transfection efficiency was>95%,suggesting that the cells were suitable for further animal cell transplantation studies.In our immunofluorescence study,it was obvious that mHPC-E14.5 cell lines highly expressed hepatic stem cell marker AFP and low levels of ALB and CK19,which was the same as primary mHPCs.At the seventh day of cell transplantation,H&E staining and confocal immunofluorescence study showed that transplanted cells engrafted successfully into recipient mouse livers without any abnormal tumor cells.Immunohistochemical staining and qRT-PCR results showed that experssion of a-SMA,Jaggedl and master cholangiocyte markers were all highly expressed in mHPCs-E14.5+mHSCs-TGF-?1 co-transplantation group,but the mature hepatocyte marker expression was the most highest in mHPCs-E14.5+ mHSCs-TGF-?Rlsih3 co-transplantation group.Hepatic stem cell AFP was highly expressed in mHPCs-E14.5+mHSCs co-transplantation group and mHPCs-E14.5 transplantation group.There was significantly statistical difference between three groups(P<0.05).AST and ALT levels were decreased remarkably after cell transplantation,moreover,the decrease was more obvious in the experimental co-transplantation group.Overall,transplanted cells engrafted and differentiated into hepatocytes and cholangiocytes in the splenic parechyma successfully in vivo and accelerated recovery from CC14/partial hepatectomy induced acute liver injury.Conclusion1.TGF-?1 signaling activates mHSCs into myofibroblasts through Jaggedl/Notch pathway and mHSCs secrete more cell factors after activation to fulfill its biological functions.2.mHSCs promote mHPCs differentiation into cholangiocytes through TGF-?-Jaggedl/Notch signaling axis in vitro.3.mHPCs are successfully engrafted into mouse spleens after co-transplantation with mHSCs.Importantly,hepatic stellate cells induced by TGF-?1 promote the differentiation of fetal hepatic progenitor cells into cholangiocytes and accelerate recovery from acute liver injury in vivo.