Construction Of HCV Susceptible Cell Model And Mice Model Withing Human ESCs-derived Hepatic Lineage Cells

Acute and chronic infection caused by hepatitis C virus (HCV) and gradually developinto liver cirrhosis, liver fibrosis, and even hepatocellular carcinoma, is nowrecognized as a harm to human health of the world’s major infection diseases. Sincethe virus was identified in1989, the pathogenesis of HCV, life cycle, the antiviraldrugs and vaccine research progress had been significantly hampered by the lack ofnature infected cell models in vitro and small animal models in vivo, because humansand chimpanzees are the only species permissive to HCV infection and liver is theorgan infected by this pathogen.As human primary hepatocytes were host cells for HCV, chimeric mice with humanhepatocytes repopulated liver would be an ideal model to explore the pathogenesisand life cycle of HCV. The prerequisite for the chimeric mice model construction areinfectious HCVcc and the mice with inconvertible liver damage. The key point is theselection of cells with high susceptibility to HCV and highly proliferation,differentiation and implanted ability.A number of immunodeficiency and targeted liver injury mice engrafted with humanhepatocytes have been developed as excellent models for analysis of HCV infectionand pathogenesis. The chimeric mice models commonly used include: NOD/SCIDmice, urokinase-type plasminogen activator (uPA) SCID mice, ALB-uPA-SCID mice,fumaryl acetoacytate hydrolasedeficiency-SCID (FRG) mice and AFC8-hu HSC/Hepmice, etc. Most cells for transplantation are adult human primary hepatocytes. Buthuman adult primary hepatocytes were difficult to obtain because of the limitation ofthe liver source. Otherwise, poor functions as well as limited proliferation potential invitro of primary human hepatocytes dramatically restricted its application.Attentions are focused on finding more reliable sources instead of human primaryhepatocytes. Human embryonic stem cells (hESCs), because of in vitro cultureinfinite proliferation, self-renewal and multi-directional differentiation properties, canbe differentiation to almost all cell types of the body, thus become the ideal seeds ofconstructing in vitro cell model and in vivo humanized mouse liver model for HCV. Studies carried out for hepatic differentiation from mouse and human ESCs orinduced pluripotent stem cells (iPSCs) are proved to be successfully at an efficiencyas high as80%, which means ESCs or iPSCs can serve as unlimited cell sources forconstruction HCV chimeric animal models.The separation of HCV JFH-1is the milestone of HCV research, which led to theestablishment of specific cell culture system in vitro. The more clarified about the lifecycle of HCV, the closer interaction between virus proteins and host factors appears tobe. The glycoprotein envelopes E1and E2are the major determinants ofreceptor-mediated cellular entry of HCV. Several cell surface molecules are criticalfor HCV entry: the low-density lipoprotein receptor (LDL-R), glycosaminoglycan(GAG), the tetraspanin CD81, the scavenger receptor B type I (SR-BI), claudin-1andoccludin (OCLN) and so on. The maintenance of the polarity of hepatocytes mainlybased on the function and structure of tight junction (TJ) proteins, which werecomposed of claudin-1&occludin and et al. Expression of human occludin andclaudin-1, in combination with CD81and SR-BI, permits HCV entry into otherwiseimpervious mouse fibroblasts, although viral RNA replication is not observed inmurine cells. Depletion of either claudin-1or occludin with siRNA prevents infectionof susceptible human hepatoma cell lines, indicating that both proteins are requiredfor infection.To study the life cycle and pathogenesis of HCV, construction of a HCV-susceptiblehumanized mouse model become extremely important. In order to achieve this goal,first, we expect to optimize inducing system by the combination of small molecules,cytokines and extracellular matrix (ECM), and obtain fully functional humanhepatocyte-like cells. Based on these full-functional hepatocyte-like cells, we candetect the HCV susceptibility of induced cells at different differentiation stages, findwhich hepatic lineage differentiation stage had the higher susceptibility to HCV. Thenapplication of small molecules or combination of cytokines, improve the cells’susceptibility to HCV. At last, using infected hESCs-derived hepatic cells, in situtransplant back into the FR mice liver, withdraw NTBC and obtain HCV-infectedhuman chimeric liver mice. The specific studies and results are as follows:1. hESCs differentiation to hepatic lineage and explore the potential rules of HCVinfection.According to the lineage differentiation characteristics of liver development in vivo, applications stepwise strategy, in combinations of different small moleculecompounds or cytokines in different stages of differentiation respectively, screeningand establishment of differentiation program, which could induce human ESCs tomature, functional hepatocytes. The hepatocyte-like cells obtained from thisoptimized stepwise strategy were PAS staining positive and had the function ofalbumin secretion, urea production. ICG uptake and excretion was also observed inthese cells. The homogeneity of the induced cells were better, up to91.4%of cellswere positive for albumin and the cells had the function of drug metabolism in vitro.Stepwise differentiation strategy establishment and functional hepatocytes lay thefoundation for subsequent study of HCV susceptibility in vitro.The cells induced by stepwise strategy could stay at a particular stage ofdifferentiation, followed by infecting the cells in different stages by HCVcc. Theresults showed that cells in the early stages of differentiation (ES, DE) could not beinfected by HCVcc. When the cells differentiated to the hepatic stem cells (HS),HCVcc could infect the induced cells. Hepatoblast (HB) phase cells had the higherHCV RNA copies; otherwise, mature hepatocyte-like cells decreased susceptibility toHCV. The expression of occludin (OCLN), which is necessary for HCV entry,unchanged from the hESCs to mature hepatocyte-like cells, but the location of OCLNchanged from the nucleus to cytoplasm, then transferred to cell membrane. So theexpression amount of OCLN does not affect HCV entry and the HCV susceptibility ofhost cells.2. The effect of VEGF on differentiated cells polarity and its HCV susceptibility.In this study, exogenous VEGF were added to induction system, on the one hand, toobserve its effect on hESCs differentiation into hepatocytes, on the other hand toverify its impact on the formation of cell polarity, which promote HCV susceptibilityof hESCs-induced cells. The results of Q-RT PCR and immunofluorescent stainingshowed that after adding VEGF, help maintain the cells at an earlier stage, can alsohelp improve the susceptibility of HCV. VEGF treatment promoted the expression ofOCLN and reduced phosphorylation level of OCLN, thereby disrupt OCLNlocalization, reduce TJ integrity, promote HCV entry and ultimately affect the cell’ssusceptibility to HCV.3. Construction of HCV-infected humanized mouse model with functionalhepatocytes lineage restricted from hESCs. In order to get HCV susceptible humanized mouse model, a new build strategyadopted. Firstly, HB stage cells were infected with HCVcc in vitro followed bytransplantation back to the mice liver via in situ injection. Through regular withdrawNTBC, the transplanted hepatoblasts can effectively integrate to Fah-/-Rag2-/-miceliver. Experimental results demonstrated that the humanized mice using this strategycould successfully rescue the lives of mice at a survival rate of75%.Immunohistochemical staining confirmed that the source of the cells in chimeric miceliver were from human being (FAH, anti-human hepatocyte antibody, ALB, etc. are allpositive). Non-structural protein NS5A and core protein of HCV were also detected inthese cells, but viremia was not detected in these humanized mice model.4. The study of siRNA interference to IFN-β and improvement of the differentiatedcells’ HCV susceptibility in vitro&in vivo.When hepatocytes were infected by HCV, innate immune of hepatocytes occurred andtype I interferon (IFN α/β) produced, which have antiviral effect and clear the virus.Therefore, we used the IFN-β-siRNA, explore whether the decrease of IFN-β couldlead to reduce the clearance of HCV in infected hepatic cells, and transplant such cellscarrying the virus into the FR mouse liver, after integration detect the viremia in thesemice. The results showed that siRNA interference in vitro, persistently HCV RNAcopies in the infected cells could be maintained at105magnitude for up to eight days,indicated that siRNA interference with IFN-β, with a reduction of the innate immuneeffect on HCV clearing. In vivo interference experiments, we still not observed theoccurrence of viremia, the result suggested that the IFN-β-siRNA had an effect onvirus clearance.In conclusion, this study established an efficient differentiation system, which couldinduced human embryonic stem cells into mature hepatocytes, differentiated cellscould functional integration into FR mice liver and constructed humanized liver micemodel successfully. The study also revealed the potential rules of HCV infection inliver cell lineage. It mean that at the earlier stage of hepatocyte differentiation, hepaticstem cells could be infected by HCV, and hepatoblast had the higher susceptibility toHCV. Our research also preliminary clarified the effect mechanisms of the TightJunction between cells and cell polarity on HCV susceptibility. Then we put forward anew strategy to construct HCV susceptible humanized liver mice model, and themodel based on this strategy would provide an efficient platform for the research of HCV life cycle, pathogenesis research and anti HCV replication drug screening andevaluation.