Bissig KD, Wieland SF, Tran P, Isogawa M, Le TT, Chisari FV, et al. Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment. J Clin Invest 2010;120:924-930. (Reprinted with permission.)
A paucity of versatile small animal models of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has been an impediment to both furthering understanding of virus biology and testing antiviral therapies. We recently described a regulatable system for repopulating the liver of immunodeficient mice (specifically mice lacking fumaryl acetoacetate hydrolase [Fah], recombination activating gene 2 [Rag2], and the γ-chain of the receptor for IL-2 [Il-2rγ]) with human hepatocytes. Here we have shown that a high transplantation dose (3 × 106 to 5 × 106 human hepatocytes/mouse) generates a higher rate of liver chimerism than was previously obtained in these mice, up to 95% human hepatocyte chimerism. Mice with a high level of human liver chimerism propagated both HBV and HCV, and the HCV-infected mice were responsive to antiviral treatment. This human liver chimeric mouse model will expand the experimental possibilities for studying HBV and HCV infection, and possibly other human hepatotropic pathogens, and prove useful for antiviral drug testing.
Advances in our understanding of two major human pathogens, hepatitis B virus (HBV) and hepatitis C virus (HCV), have been limited by a lack of suitable model systems for their study. Surrogate systems such as HBV recombinant baculoviruses have been developed to allow in vitro studies of HBV biology in the face of a lack of cell lines permissive to infection by this agent. The generation of infectious HCV culture systems has also allowed progress in the study of HCV virology. However, restriction of tropism of the available cell culture infectious HCV strains to hepatocellular carcinoma cell lines has constrained the general applicability of findings from these systems to events occurring in infected primary hepatocytes. The recent development of a model in which primary human hepatocytes have been shown to be rendered susceptible to persistent HCV infection when supported in an in vitro culture by stromal elements1 may allow significant advances in in vitro modeling of HCV biology.
Despite such recent advances in in vitro options for the study of hepatotropic viruses, in vitro cell culture systems do not necessarily recapitulate in vivo cell differentiation and function or virus-cell interactions in the infected host. Progress in the in vivo study of hepatotropic viruses has been constrained by the narrow host range of HBV and HCV. Productive infection by HBV and HCV is limited to humans and chimpanzees, and although important advances in this field have been made by analyses of infected chimpanzees, sizable studies are limited by ethical considerations, high cost, and limitations on availability. Studies of the Pekin duck and woodchuck models have led to advances in our understanding of hepadnaviruses; however, we are hampered by the outbred nature of these models and by a lack of available data on the immunobiology of these hosts and the restricted availability of suitable reagents. Attempts to develop small animal models for HCV have been impeded somewhat by similar limitations, and attempts to infect primates other than chimpanzees with HCV have not been successful. The development of HBV transgenic mice has been critical in revealing the mechanisms of control of HBV replication,2 but although transgenic animals produce infectious virus, murine hepatocytes are not susceptible to HBV infection.
Largely because of the restricted tropism of HBV and HCV, attempts to develop small animal models for the study of human hepatotropic virus have recently centered on the creation of human liver chimeric immunodeficient mice. The first developed and best characterized of these systems is the one based on transgenic mice expressing urokinase plasminogen activator (uPA) in hepatocytes under the albumin promoter (Alb-uPA mice). uPA, produced within the livers of these mice, is hepatotoxic, and when they are crossed onto immunodeficient backgrounds, transplanted xenogeneic hepatocytes thus possess a selective growth advantage over endogenous hepatocytes and are able to populate and expand within the liver. Immunodeficient Alb-uPA mice reconstituted with human hepatocytes, have been demonstrated to be susceptible to productive infections with both HBV and HCV3, 4 and this model has been used for a variety of investigations, including drug metabolism studies,5 the assessment of antiviral compounds,6 the demonstration of viral neutralization,7 and the analysis of mechanisms of control of viral replication.8 However, because of a number of technical challenges associated with this model, wide adoption of this system has not occurred, with its use essentially limited to a few specialized centers. The breeding of homozygous Alb-uPA immunodeficient mice is hampered by infertility; in addition, there is relatively high perinatal mortality in this lineage.4 Furthermore, this lineage has a bleeding diathesis, consistent with the development of Alb-uPA mice as a model for exploring coagulation, that can be associated with the death of transplanted animals from diffuse hemorrhaging.3 Hepatocyte transplantation in this model must be carried out at an early age, and there is a short window of opportunity for successful repopulation, with the optimal age range for this procedure being 5 to 14 days.4 Somatic mutations leading to deletion of the uPA transgene can also develop and lead to the presence of wild-type mouse hepatocytes that can compete with transplanted xenogeneic hepatocytes; this limits the success of repopulation with human hepatocytes in those animals in which this occurs. Animals successfully repopulated with human hepatocytes have also been reported to remain somewhat unhealthy,9 and renal disease has been observed in this model.5
In order to develop a more robust chimeric human liver mouse model, two groups have recently harnessed the fumaryl acetoacetate hydrolase (FAH) knockout mouse lineage. This enzyme is the terminal factor in the tyrosine catabolism pathway, and the accumulation of toxic tyrosine metabolites resulting from its deficiency leads to fulminant hepatic failure in FAH-deficient mice. However, the administration of 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC) blocks the activity of an enzyme upstream of FAH and thus abrogates the accumulation of toxic metabolites that mediate hepatotoxicity. Thus, the timing of the toxic insult applied to native murine hepatocytes to provide a selective advantage to transferred human hepatocytes in the repopulation of the liver can be manipulated by the administration and withdrawal of NTBC. Grompe and colleagues10 crossed FAH knockout mice with recombination activating gene 2 knockout/interleukin-2R gamma chain knockout mice deficient in T, B, and natural killer cells; this triple knockout model has been termed the FRG lineage. After the administration of an adenoviral vector encoding uPA, up to 90% engraftment of transplanted human hepatocytes was noted.10 Using FRG mice, Verma and coworkers11 were able to create human chimeric liver mice without the additional step of administering a vector encoding an hepatotoxic transgene, albeit at lower levels of repopulation of 10% to 20%.
In recent work, Verma and colleagues12 demonstrated robust repopulation of FAH mouse livers with human hepatocytes simply by increasing the number of transplanted human hepatocytes to 3 to 5 × 106 hepatocytes per mouse versus an inoculum of 0.2 to 1 × 106 hepatocytes administered in their previous study.11 An average rate of chimerism with human hepatocytes of approximately 40% was achieved without any requirements for additional manipulation, with some animals demonstrating over 80% human hepatocyte chimerism.12 In addition, a strong correlation between serum levels of human albumin and the level of human hepatocyte chimerism was shown, and thus yielding a relatively noninvasive measure of reconstitution of chimeric animals for use in this model. Further experiments demonstrated FRG human hepatocyte chimeric mice to have a capacity for productive infection by both HBV and HCV, and preliminary experiments demonstrated the feasibility of studying antiviral therapies for both HBV and HCV in this model.12
The FRG human hepatocyte chimeric model appears to hold a number of advantages over previously developed models. As noted above, treatment with NTBC can maintain the viability of FRG mice and therefore make the timeframe for hepatocyte reconstitution in these animals less critical. Furthermore, in contrast to previous studies using the Alb-uPA lineage, in which human hepatocyte repopulation appeared to require fresh hepatocytes, the livers of FRG mice have been successfully reconstituted with hepatocytes up to 48 hours after harvest, and success has also been reported with cryopreserved hepatocytes10, 11; this increases the potential for more widespread use of this model. Successful serial transplantation of human hepatocytes from one FRG mouse to another FRG mouse has also been demonstrated,10 and yielding the potential for an inbred mouse lineage reconstituted with hepatocytes from a single individual in which the biology of a range of viral variants or the efficacy of a range of treatments may be assessed.
The development of the FRG human hepatocyte chimeric model, an apparently robust and reproducible system, has exciting potential for the further study of the biology and therapeutics of HBV and HCV. Furthermore, the advent of immunodeficient mouse models reconstituted with human adaptive immune systems via the transfer of CD34-positive progenitors13 raises the possibility of this model being combined with such systems; a small animal model may result in which the immunopathogenesis of HBV and HCV14 and the potential immunomodulatory effects of candidate therapies can be studied. The model additionally has potential for advancing studies in a range of hepatology-related fields, including the study of hepatocyte differentiation from stem cells and induced pluripotent stem cells.14 Especially because of the variability in species-specific hepatocyte tropism for candidate gene therapy vectors, such models also provide a useful platform for the exploration of directed gene therapy of the liver.15 Thus, although the extent to which this new model can be harnessed by the hepatological research community remains to be seen, a wide range of areas will potentially be advanced by successfully utilization of this experimental system.