To Develop Strategies To Treat Liver Diseases With HESC-derived Hepatic-like Cells

ObjectiveTake the advantages of humanized mice model to study the strategies of hepatic cells derived from hESCs in treating liver disease in order to provide basic scientific evidence for clinical application and development.Contents and methods1.Differentiate CP-hESCs and WT-hESCs into Hepato-Blasts and Hepatic Like Cells a.We optimized the three-step protocol of differentiation to obtain early stage Hepato-Blasts(HBs)and mature Hepatic Like Cells(HLCs)using WT-hESCs and CP-hESCs.b.The differentiation efficiency was determined by qRT-PCR,immunostaining and the Flow Cytometry Analysis.c.The PAS staining,ICG uptake assay and Rifampicin induced the expression of CYP450 were used to test the function of differentiated HLCs.2.The study of differentiated HBs in rescuing acute liver injury and homing abilitya.WT-HBs and CP-HBs were transplanted through the spleen after the NSG acute liver injury model was induced by intraperitoneally injecting sublethal dosage of DMN.The survival curve was recorded and the ability of HBs transplantation in rescuing the liver injury was analyzed.b.Using ELISA to detect the concentration of secreted human Albumin(hALB)in peripheral blood of NSG mice at different days post WT-HBs or CP-HBs transplantation.Frozen section and immunostaining were executed to detect the homing of transplanted WT-HBs and CP-HBs in harvested NSG liver.The homing rate was calculated as well.3.The study of immune-tolerance of CP-HBs in Humanized Mice after intramuscular implantationa.NSG mice were reconstituted with human robust immune system using CD34+hematopoietic stem cells and fetal thymus from the same donor.b.WT-HBs and CP-HBs were intra-muscular implanted in Humanized Mice.Immunostaining of HBs marker was used to detect the survived cells.Immunostaining of the T cell infiltration was conducted to analyze the immune reaction.4.The study of homing ability and immune-tolerance of intra-splenically transplanted CP-HBs in Hu-mice.a.Humanized mice liver injury model was established as NSG mice.b.WT-HBs and CP-HBs were intra-splenically transplanted in Humanized Mice.Immunostaining of indicated markers was executed to detect the homing HBs and immune reaction.5.The long-term observation of transplanted CP-HBs in Humanized Micea.By prolonging the observation time after transplantation,the survival ability of CPHBs in Humanized Mice was analyzed and its homing rate was compared with those transplanted with WT-HBs.6.The study of new strategy for embryonic stem cells in treating HBVa.Using Crispr/Cas9 to generate HBV receptor(NTCP)knock out hESCs cell line and its pluripotency was determined.b.The differentiation ability of NTCP-/-hESCs was determined.Results and conclusions1.CP-hESCs can be efficiently differentiated into Hepato-blasts(HBs)and mature Hepatic Like Cells(HLCs)in vitro.There is no difference in differentiation efficiency between CP-hESCs and WT-hESCs and the efficiency is more than 90%.In addition,They both have similar functions.2.Both CP-HBs and WT-HBs are able to reverse the death caused by acute liver injury in NSG mice.Both show similar homing rates in the liver and are able to proliferate and mature into functional hepatocytes in vivo.Both can secret human albumin as well.It proves that CP-HBs could be an important cell source for hepatocytes transplantation therapy.3.CP-HBs can obtain immune tolerance after intra-muscularly transplanted in Humanized Mice,which was reconstituted with robust human immune system.It proves that CP-HBs could to some extent alleviate the immune rejection problem in clinical hepatocytes transplantation therapy.4.CP-HBs proliferate to mature hepatocytes and rescue liver injury in Hu-mice,while WT-HBs are immune rejected.It further reflects the application prospect of CP-HBs in clinical treatment of liver injury.5.CP-HBs can survive long-term up to 5 weeks in the liver of humanized mice,which stands for an important prerequisite for the therapeutic effect of hepatocytes transplantation in clinical applications.For the record,none of them developed into tumors.6.A Hepatitis B Virus receptor(NTCP)knockout embryonic stem cell line is established.Its pluripotent phenotype is unaffected and its ability to differentiate into hepatic cells is initially verified,which lay the foundation for the future test whether it is resistant to HBV infection in vitro and whether HBV reinfection can be avoided after transplantation in vivo.