Generation Of The Reconstructed Mouse And Human Liver Based On A Mouse Model Of Liver Injury

The mice harboring humanized liver (mouse-human chimeric liver) provide us with powerful tools for the following studies:(1) human liver development and regeneration;(2) the pathogenesis of human liver diseases (e.g., hepatitis, cirrhosis and liver cancer);(3) generation of susceptible animal models for hepatitis virus (HBV and HCV);(4) pharmacokinetics;(5) providing the theoretical basis and the practice exploration for the reconstructed human liver in big animals (such as miniature pigs). The above-mentioned information encourages us to generate the reconstructed mouse and human liver based on a mouse model of liver injury.Part one Generation of mouse-mouse chimeric liver animal model by transplanting mouse induced pluripotent stem cells (miPSCs) into mouse blastocystsAIM:On the one hand, the mouse/mouse chimeric liver animal model will help us observe the function of the stem cells to repair damaged liver in vivo; on the other hand, this model will also provide the theoretical and practical basis for the establishment of human/mouse chimeric liver animal model.Methods:Mouse-mouse chimeric liver animals were achieved by transplanting the genetically modified mouse induced pluripotent stem cells (LG-miPSCs) into Fah(-/-) mouse blastocysts, and subsequently, a variety of methods, including the detection of the reporter genes (ZsGreen and Fluc), PCR-based genotyping for Fah and Fluc genes, HE staining, immunofluorescence (IFC) assay as well as Western blot, were used to test miPSCs contribution in the recipients.Results:Of38live-born recipients,2had survived to adulthood after NTBC withdrawal from drinking water. The double-reporter gene of zsGreen and Fluc was not detected by the stereology fluorescence microscope and the small animals in vivo imaging system. PCR-based genotyping for Fah and Fluc genes demonstrated the presence of wild-type Fah gene and Fluc gene in the genome of potential mouse-mouse chimeric liver. The structure of the chimeric liver is basically consistent with the liver of normal mice, while the liver of Fah (-/-) mouse with NTBC or NTBC withdrawal was disorganized by HE staining. Data derived from immunofluorescence and Western blot showed the expression of Fah gene in the chimeric mouse liver. The liver chimeric rate of two surviving mice was84.69%and88.43%, respectively.Conclusions:mouse-mouse chimeric liver animal model with high chimeric rate of mouse hepatocytes derived from LG-miPSCs is successfully achieved, which lays a solid foundation for generating human-mouse chimeric liver animal model.Part two Generation of human-mouse chimeric liver animal model by transplanting human bone marrow mesenchymal stem cells (hMSCs) into mouse blastocystsAIM:To establish the human-mouse chimeric liver animal model with an in vivo environment more closely resembling to the situations in human, which provides invaluable and effective approaches for the following issues:(1) human liver development and regeneration;(2) the pathogenesis of human liver diseases;(3) production of susceptible animal models for hepatitis virus;(4) the evaluation of drug metabolism; etc.Methods:Human-mouse chimeric liver animal were achieved by transplanting the genetically modified human bone marrow mesenchymal stem cells (RLG-hMSCs) into blastocysts of Fah (-/-) mice, and subsequently, a variety of methods, including the detection of the reporter genes (RFP、ZsGreen and Fluc), PCR-based genotyping for hAlu, Fah and Fluc genes, HE staining, immunohistochemical (IHC) assay as well as Western blot, were used to test RLG-hMSCs contribution in the recipients.Results:By using lentivirus-mediated foreign gene delivery, we successfully prepared the RLG-hMSCs carrying three reporter genes (RFP、ZsGreen and Fluc) under control of different promoters. The remaining experiment is in progress.