Study On Differentiation Feature Of The CK19-positive Cell Lineage In The Mouse Liver

Currently,a lot of researches on liver stem-like cells(such as liver progenitor cells)have been done,however,the marker for the real liver stem cells(LSCs)is still unknown as there is no any effective research approach for LSCs.The phenotypic and molecular characteristics of liver stem-like cells reported by different laboratories are significantly different,but they have one common feature,which is that all reported liver stem-like cells could express CK19.Based on this phenomenon,combined with our findings about the liver precursors,I proposed a hypothesis that "hepatic CK19-positive cell lineage could contain authentic liver stem cells".In order to confirm this hypothesis,I employed the CK19 CreERT/loxP knockin mouse tracer system to trace the CK19~+ cells lineage in the mouse liver,and two liver injury models to analysis the differentiation potential of CK19-positive cells.The C57BL/6 mouse strain CK19 CreERT was crossed with Rosa26-GFP mouse,desired CK19CreERT/Rosa26-GFP double positive mice were obtained,and the CK19-positive cell lineage was genetically labeled as well.And also,the labeled GFP-positive cells mainly appeared in the portal area.Then we established two liver injury models,DDC liver injury model and CCl4 injury model,upon the genetically labeled CK19CreERT/Rosa26-GFP mouse to explore the differentiation potential of labeled CK19 positive cell.In the DDC model,bile duct injury can occur and can be followed by atipical ductular proliferation,which can be used for analysis of CK19 positive cells differentiate forward to bile duct epithelial cells.And in the CCl4 model,hepatocellular necrosis can happen and can be followed by the repair of damaged liver parenchyma,which can be used for assess of the differentiation potential of CK19 positive cells forward to hepatocytes.In this study,the DDC liver injury model and CCl4 liver injury model were successfully constructed in CK19CreERT/Rosa26-GFP mouse.In DDC model,we found GFP positive cells presented in the proliferating ductules under fluorescence microscopy.The bile duct was detected by immunofluorescence analysis,and we found GFP positive cells in the bile duct also express CK19(marker of biliary epithelial cells),indicating that there are the cells with cholangiocytic differentiation potential.In CCl4 model,we found GFP positive cells appear in the lobule with fluorescence detection.Then we confirmed the GFP positive cells could express hepatocyte-specific marker ALB by immunofluorescence analysis,indicating that these CK19 positive cells originate from the genetic labeled GFP positivecells and a part of the genetically labeled GFP cells have hepatocytic differentiation potential.Overall,this study indicates that there are liver stem cells in the CK19-positive cell lineage,and our results provides a understanding for the development of a new and effective liver stem cell research system.