Induction Of Human Gallbladder Epithelial Stem Cells Into Functional Hepatocyte-like Cells

The liver is an important digestive organ of the human body,which is responsible for bile production,glucose and lipid metabolism,toxin clearance,blood purification and other important life activities.There are more than 100 kinds of liver diseases,including viral hepatitis,alcoholic liver disease,fatty liver,hereditary diseases,and liver cirrhosis and so on.These diseases can cause varying degrees of liver damage,and some serious diseases can lead to liver failure and even life-threatening.At present,the most effective treatment for irreversible liver failure is liver transplantation.However,its wide clinical application is greatly limited due to the shortage of donor liver resources,immune rejection and high cost of operation.Clinical data suggests that hepatocyte transplantation can relieve the symptoms of liver failure to some extent.Cell therapy by obtaining functional mature hepatocytes or hepatocyte-like cells is expected to become a new method for the treatment of liver diseases.It has been reported that there are stem cells in the gallbladder epithelium that can express the markers of liver progenitor cells,and these cells have the potential of multi-directional differentiation.The purpose of this study is to explore how to change the fate of cells by small molecular compounds in vitro and induce human gallbladder epithelial cells（hGBECs）to differentiate into functional hepatocyte-like cells.In order to achieve the above purposes,the research is carried out from the following aspects:1)Three-dimensional culture of hGBECs:through isolation of gallbladder tissue cells,collection of cell suspension,removal of red blood cells,differential centrifugation to obtain hGBECs,optimize the culture scheme to expand the scale of cultured cells.2)Stable amplification of hGBECs:to evaluate the growth status of hGBECs through long-term passage and cryopreservation and resuscitation.3)To explore the stem cell gene expression of hGBECs:RNA from different generations of cells was collected to reverse transcribed into c DNA,and the expression of dry markers,hepatic stem cell markers and hepatic progenitor cell markers at gene level was detected by RT-PCR,and the protein expression of related markers was identified by immunofluorescence staining.4)To explore the chemical induction method of hGBECs differentiation into hepatocyte-like like cells in vitro:Based on the development studies of the liver and bile duct system,we selected small molecules that can promote or inhibit the signaling pathway of regulating tissue development stage,and screened out the combination of small molecules that can promote the expression of genes related to liver function.5)To detect whether the differentiated cells have hepatocyte-related functions:the functional status of hGBECs after induced differentiation was analyzed by glycogen staining,BODIPY staining,ELISA to detect ALB secretion and medicine metabolism.6)To test whether hepatocyte-like cells are functional in vivo:the differentiated cells were transplanted into mouse.Through the study,we found that:1)the three-dimensional culture system maintained the long-term expansion of hGBECs in organ-like form in vitro,and the cryopreserved and resuscitated cells could form organs with normal structure and passed on to more than 10generations.2)different generations of hGBECs organs stably expressed bile duct/hepatic stem cell markers CD133,CK19,LGR5,Ep CAM and hepatic progenitor cell markers SOX9 and HNF4αat m RNA level.Immunofluorescence staining results further confirmed the expression of hepatic stem cells and hepatic progenitor cell markers in hGBECs.3)after three rounds of screening and repeated experiments,it was determined that inhibition of Notch signal pathway and TGF-βsignal pathway could induce hGBECs to differentiate into liver-like cells,and triiodothyronine and thyroxine promoted cell maturation.4)compared with undifferentiated cells,the expression of hepatocyte function-related genes（ALB,FAH,CYP1A2,CYP3A4,etc.）was significantly up-regulated in hepatoid cells.Fluorescence staining showed the localization of ALB,AAT,CYP3A4 and FAH in hepatoid cells.5)after the functional identification of hepatoid cells,it was found that there was a large amount of glycogen accumulation in hepatoid cells,and BODIPY staining showed the distribution of lipid droplets.The results of albumin ELISA showed that the ability of hepatoid cells to secrete albumin was improved.The metabolism tests of omeprazole and testosterone showed that the activity of cytochrome P450 family 1A2enzymes in hepatoid cells was strong and 3A4 activity was weak.6)Hepatocyte-like cells rescued CCl₄-treated liver-injured mouse.In this study,the hGBECs culture system was established.We proved that hGBECs were effectively promoted to differentiate into hepatocyte-like cells by inhibiting Notch signal pathway and TGF-βsignal pathway,which enriched the source of hepatocyte-like cells in vitro,and laid a research foundation for the study of liver-related diseases,hepatocyte drug toxicity and metabolism,and hepatocyte replacement therapy.