Host and viral determinants for engraftment of virus permissive human hepatocytes into chimeric immunodeficient mice
Authors: Eric Robinet, Thomas F. Baumert
Journal: Journal of Hepatology (2010)

Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID miceJournal of HepatologyVol. 53Issue 3PreviewThe human liver-uPA+/+-SCID mouse is currently the best small animal model available for viral hepatitis infection studies. Full-Text PDF Editorial on Vanwolleghem et al. “Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.” J. Hepatol. 2010, September issue.Hepatitis C virus (HCV) infection is a major cause of chronic liver disease including liver cirrhosis and hepatocellular carcinoma [[1]Georgel P, Schuster C, Zeisel MB, Stoll-Keller F, Berg T, Bahram B, et al. Virus–host interactions in hepatitis C virus infection – implications for molecular pathogenesis and antiviral strategies. Trends Mol Med, in press.Google Scholar]. The current antiviral therapy is characterized by limited efficacy, high costs, and substantial side effects. A vaccine for the prevention of HCV infection is not available. Thus, novel strategies for the prevention and treatment of HCV infection are urgently needed (for a review see [1Georgel P, Schuster C, Zeisel MB, Stoll-Keller F, Berg T, Bahram B, et al. Virus–host interactions in hepatitis C virus infection – implications for molecular pathogenesis and antiviral strategies. Trends Mol Med, in press.Google Scholar, 2Zeisel M.B. Cosset F.L. Baumert T.F. Host neutralizing responses and pathogenesis of hepatitis C virus infection.Hepatology. 2008; 48: 299-307Crossref PubMed Scopus (44) Google Scholar]). The development of efficient antiviral treatments and vaccines has been hampered by the lack of a convenient small animal model for HCV infection.Although HCV has been shown to enter into mouse cell lines expressing human HCV receptors [[3]Ploss A. Evans M.J. Gaysinskaya V.A. Panis M. You H. de Jong Y.P. et al.Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.Nature. 2009; 457: 882-886Crossref PubMed Scopus (742) Google Scholar], mouse hepatocytes are unable to produce infectious HCV infection [[4]Ploss A. Rice C.M. Towards a small animal model for hepatitis C.EMBO Rep. 2009; 10: 1220-1227Crossref PubMed Scopus (65) Google Scholar]. Over the past two decades the chimpanzee has been commonly used to study mechanisms of acute and chronic HCV infection (for review see [[21]Bukh J. A critical role for the chimpanzee model in the study of hepatitis C.Hepatology. 2004; 39: 1469-1475Crossref PubMed Scopus (152) Google Scholar]). These studies have enormously contributed to our current understanding of HCV infection [[5]Yanagi M. Purcell R.H. Emerson S.U. Bukh J. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.Proc Natl Acad Sci USA. 1997; 94: 8738-8743Crossref PubMed Scopus (457) Google Scholar] as well as the preclinical evaluation of antivirals [[6]Lanford R.E. Hildebrandt-Eriksen E.S. Petri A. Persson R. Lindow M. Munk M.E. et al.Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.Science. 2010; 327: 198-201Crossref PubMed Scopus (1443) Google Scholar]. However, the chimpanzee model is characterized by limited availability, high costs, and important ethical restrictions. Furthermore, hepatocytes of the tree shrew Tupaia belangeri are susceptible to HCV infection [[8]Zhao X. Tang Z.Y. Klumpp B. Wolff-Vorbeck G. Barth H. Levy S. et al.Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection.J Clin Invest. 2002; 109: 221-232Crossref PubMed Scopus (76) Google Scholar]. However, viral load in infected animals is low [[9]Amako Y. Tsukiyama-Kohara K. Katsume A. Hirata Y. Sekiguchi S. Tobita Y. et al.Pathogenesis of hepatitis C virus infection in Tupaia belangeri.J Virol. 2010; 84: 303-311Crossref PubMed Scopus (103) Google Scholar].The discovery of a hepatocyte-lethal phenotype in mice carrying a urokinase-type plasminogen activator transgene controlled by an albumin promotor (Alb-uPA) with the reconstitution of livers of those mice with xenografted rat hepatocytes [[10]Sandgren E.P. Palmiter R.D. Heckel J.L. Daugherty C.C. Brinster R.L. Degen J.L. Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene.Cell. 1991; 66: 245-256Abstract Full Text PDF PubMed Scopus (320) Google Scholar] has laid the foundation for the development of small animal models of infection with hepatitis B and C viruses using xenografted primary human hepatocytes (PHH) (for review see [[7]Barth H. Robinet E. Liang T.J. Baumert T.F. Mouse models for the study of HCV infection and virus–host interactions.J Hepatol. 2008; 49: 134-142Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]).The uPA-xenograft model had been first established for the development of a hepatitis B mouse model by transplanting woodchuck or PHH into Alb-uPA mice on an immunodeficient recombination activating gene 2 knock-out (Rag-2−/−) background. Repopulating woodchuck hepatocytes in Alb-uPA/rag-2 mice, which lack mature B and T lymphocytes, allowed productive infection with woodchuck hepatitis B virus [[11]Petersen J. Dandri M. Gupta S. Rogler C.E. Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma.Proc Natl Acad Sci USA. 1998; 95: 310-315Crossref PubMed Scopus (109) Google Scholar]. Subsequently, Mercer and colleagues demonstrated that the severe combined immunodeficiency disorder (SCID-beige)/Alb-uPA mouse engrafted with PHH can be infected with HCV in vivo [[12]Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (746) Google Scholar]. A further development of this model is a system that can be regulated for repopulating the liver of immunodeficient mice, based on mice lacking the fumaryl acetoacetate hydrolase [Fah], recombination activating gene 2 [Rag-2−/−], and the gamma-chain of the receptor for IL-2 [Il-2rgamma or γc] (Fah/Rag-2−/−/γc−/− model). Oral administration of a small molecule (NTBC) prevents the accumulation of hepatotoxic metabolites resulting from the Fah deficiency. By regulating hepatotoxicity using NTBC, this model allows for transplantation of PHH at any age, which may improve robustness of engraftment and mortality [[13]Azuma H. Paulk N. Ranade A. Dorrell C. Al-Dhalimy M. Ellis E. et al.Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.Nat Biotechnol. 2007; 25: 903-910Crossref PubMed Scopus (606) Google Scholar].Although all chimeric mouse models are characterized by the absence of a functional immune system [[7]Barth H. Robinet E. Liang T.J. Baumert T.F. Mouse models for the study of HCV infection and virus–host interactions.J Hepatol. 2008; 49: 134-142Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar], they are very useful for the study of HBV and HCV infection in vivo as well as for the evaluation of antivirals targeting the virus or essential host factors present in transplanted hepatocytes [14Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (277) Google Scholar, 15Meuleman P. Hesselgesser J. Paulson M. Vanwolleghem T. Desombere I. Reiser H. et al.Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo.Hepatology. 2008; 48: 1761-1768Crossref PubMed Scopus (188) Google Scholar]. The hepatocyte-lethal phenotype of these models allows for transplanting immunodeficient mice with PHH by intrasplenic injection. Successful engraftment of hepatocytes is monitored by quantification of human albumin concentrations in the plasma or serum. For uPA-transgene models, the transplantation is usually performed within the first two weeks of life and human albumin levels are determined at least three weeks after transplantation. If the liver repopulation reaches a threshold level, human liver chimeric mice can then be infected by HBV [[16]Dandri M. Burda M.R. Torok E. Pollok J.M. Iwanska A. Sommer G. et al.Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (323) Google Scholar] or HCV [[12]Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (746) Google Scholar].Nevertheless, the wide-spread use and further development of these model systems are hampered by the complexity of their set-up, essentially for technical reasons. One limitation is the availability of high quality PHH, but the major obstacles are the technical challenges to achieve high repopulation of the chimeric mouse liver with human hepatocytes. Interestingly, several independent studies have shown that the level of liver repopulation required for HCV infection is significantly higher than that required for HBV infection: while 15% liver parenchyma replacement by PHH is sufficient to support a HBV infection [[16]Dandri M. Burda M.R. Torok E. Pollok J.M. Iwanska A. Sommer G. et al.Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (323) Google Scholar], at least 50% replacement is needed to support a HCV infection [[12]Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (746) Google Scholar]. Thus, the identification of technical or biological parameters allowing efficient engraftment of permissive PHH is of crucial importance for the improvement of the model, in particular for robust infection with HCV.To identify essential viral and host factors for successful engraftment of HCV permissive hepatocytes, the Leroux–Roels laboratory at Ghent University, Belgium, has performed a comprehensive functional analysis of a large series of animals which is published in this issue of the Journal [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. In this study, performed by one of the most experienced laboratories in the field, animal survival, engraftment efficacy, kinetics of liver repopulation, and viral infection was analyzed in 400 transplanted chimeric upA-SCD mice.First, Vanwolleghem and colleagues [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar] demonstrated that the first 7 days after transplantation are the most critical period for survival. This period is characterized by frequent identification of necrotic donor hepatocytes in portal vein branches and periportal sinusoids. The necrosis tended to be more extensive in mice transplanted with PHH than with murine hepatocytes, the latter repopulating the liver approximately four-times faster than the former donor cells after this critical period.Peri-operative problems and the use of fresh PHH were identified as independent negative risk factors for survival of the host. Since fresh and frozen PHH led to similar engraftment levels, both in terms of fraction of engrafted mice and human albumin levels, cryopreserved PHH are superior to fresh PHH regarding the final outcome of permissive chimeric mice. Engraftment level seemed to be mainly donor-dependent, with commercially available frozen PHH again being the best source of PHH in this series. Interestingly, donor age and PHH viability were not predictors of successful engraftment (Fig. 1).The quality of the viral inoculum is another important factor for successful infection. The use of serum-derived HCV has been reported to provide higher HCV plasma titers than infection with cell culture-derived recombinant HCV [[14]Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (277) Google Scholar]. Vanwolleghem and colleagues demonstrate that viral load and proper conservation and storage of the inoculum are crucial for successful infection [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. Serial freezing/thawing cycles clearly impair the infectivity of the inoculum. In this regard, it is of interest to note, that the same laboratory has characterized and titrated reference genotypes 1–6 HCV batches from pooled sera of chronically infected chimpanzees in uPA-SCID mice in collaboration with the Laboratory of Infectious Diseases, National Institutes of Health, Bethesda [[18]Bukh J, Meuleman P, Tellier R, Engle RE, Feinstone SM, Eder G, et al. Challenge pools of hepatitis C virus genotypes 1–6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models. J Infect Dis 201:1381–89.Google Scholar].Most importantly, this comprehensive analysis allowed the identification of criteria for HCV infection with 100% success rate (Fig. 1). These criteria include (i) serum human albumin level >1 mg/ml at time of HCV injection and during the course of the experiment and (ii) a HCV inoculum containing >104 IU HCV RNA with no more than one freeze–thaw cycle [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. The human albumin threshold of 1 mg/ml defined in the present study is similar to the one suggested by Mercer et al. [[12]Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (746) Google Scholar] using the same model. Higher levels have been reported to be required by another laboratory using the uPA-SCID model [[19]Kamiya N, Iwao E, Hiraga N, Tsuge M, Imamura M, Takahashi S, et al. Practical evaluation of a mouse with chimeric human liver model for hepatitis C virus infection using an NS3-4A protease inhibitor. J Gen Virol. 2010 Feb 17. [Epub ahead of print].Google Scholar] or in the Fah/Rag-2−/−/γc−/− model [[14]Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (277) Google Scholar]. Standardization of the sampling method (plasma versus serum) and human albumin quantification method and a more accurate correlation between human albumin concentrations and liver replacement index could help in clarifying these apparent differences. Interestingly, improved PHH engraftment was obtained in the Fah/Rag-2−/−/γc−/− model by increasing the PHH dose up to 3–5 × 106 injected cells [[14]Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (277) Google Scholar], instead of 0.5–1 × 106 PHH in the uPA-SCID model. Whether increasing the PHH number in the uPA-SCID model would also improve the engraftment level remains to be determined. An important difference between the two models is the fact that the uPA-SCID mice requires transplantation at a young age (at approximately three weeks of age), while adult Fah/Rag-2−/−/γc−/− mice can be transplanted at a later age, probably injecting a higher number of PHH. Further differences between the inducible (Fah/Rag-2−/−/γc−/−) and the uPA-SCID model are related to the increased expression of uPA in the latter. uPA transforms proHGF to its active form HGF, activates plasminogen, and promotes liver matrix proteolysis during repair, a series of processes that may promote the successful engraftment and proliferation of PHH [[20]Naldini L. Vigna E. Bardelli A. Follenzi A. Galimi F. Comoglio P.M. Biological activation of pro-HGF (hepatocyte growth factor) by urokinase is controlled by a stoichiometric reaction.J Biol Chem. 1995; 270: 603-611Crossref PubMed Scopus (231) Google Scholar].Although additional studies are required to better understand the biochemical and biological mechanisms involved in PHH engraftment, the identification of host and viral factors required for engraftment of permissive hepatocytes by Vanwolleghem and colleagues [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar] will be very helpful to apply and further develop humanized mouse models for HBV and HCV infections. Thus, the results of this thorough and comprehensive study will be very useful for laboratories using and advancing the current humanized mouse models e.g. by approaches to reconstitute a human immune system. Furthermore, this study may be also of interest for teams aiming to set-up this important in vivo model for the study of HBV and HCV infection. The further development as well as a more wide spread use of humanized mouse models will advance our knowledge of virus–host interactions and the development of urgently needed novel efficient antivirals.Conflict of interestsThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Editorial on Vanwolleghem et al. “Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.” J. Hepatol. 2010, September issue. Hepatitis C virus (HCV) infection is a major cause of chronic liver disease including liver cirrhosis and hepatocellular carcinoma [[1]Georgel P, Schuster C, Zeisel MB, Stoll-Keller F, Berg T, Bahram B, et al. Virus–host interactions in hepatitis C virus infection – implications for molecular pathogenesis and antiviral strategies. Trends Mol Med, in press.Google Scholar]. The current antiviral therapy is characterized by limited efficacy, high costs, and substantial side effects. A vaccine for the prevention of HCV infection is not available. Thus, novel strategies for the prevention and treatment of HCV infection are urgently needed (for a review see [1Georgel P, Schuster C, Zeisel MB, Stoll-Keller F, Berg T, Bahram B, et al. Virus–host interactions in hepatitis C virus infection – implications for molecular pathogenesis and antiviral strategies. Trends Mol Med, in press.Google Scholar, 2Zeisel M.B. Cosset F.L. Baumert T.F. Host neutralizing responses and pathogenesis of hepatitis C virus infection.Hepatology. 2008; 48: 299-307Crossref PubMed Scopus (44) Google Scholar]). The development of efficient antiviral treatments and vaccines has been hampered by the lack of a convenient small animal model for HCV infection. Although HCV has been shown to enter into mouse cell lines expressing human HCV receptors [[3]Ploss A. Evans M.J. Gaysinskaya V.A. Panis M. You H. de Jong Y.P. et al.Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.Nature. 2009; 457: 882-886Crossref PubMed Scopus (742) Google Scholar], mouse hepatocytes are unable to produce infectious HCV infection [[4]Ploss A. Rice C.M. Towards a small animal model for hepatitis C.EMBO Rep. 2009; 10: 1220-1227Crossref PubMed Scopus (65) Google Scholar]. Over the past two decades the chimpanzee has been commonly used to study mechanisms of acute and chronic HCV infection (for review see [[21]Bukh J. A critical role for the chimpanzee model in the study of hepatitis C.Hepatology. 2004; 39: 1469-1475Crossref PubMed Scopus (152) Google Scholar]). These studies have enormously contributed to our current understanding of HCV infection [[5]Yanagi M. Purcell R.H. Emerson S.U. Bukh J. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee.Proc Natl Acad Sci USA. 1997; 94: 8738-8743Crossref PubMed Scopus (457) Google Scholar] as well as the preclinical evaluation of antivirals [[6]Lanford R.E. Hildebrandt-Eriksen E.S. Petri A. Persson R. Lindow M. Munk M.E. et al.Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.Science. 2010; 327: 198-201Crossref PubMed Scopus (1443) Google Scholar]. However, the chimpanzee model is characterized by limited availability, high costs, and important ethical restrictions. Furthermore, hepatocytes of the tree shrew Tupaia belangeri are susceptible to HCV infection [[8]Zhao X. Tang Z.Y. Klumpp B. Wolff-Vorbeck G. Barth H. Levy S. et al.Primary hepatocytes of Tupaia belangeri as a potential model for hepatitis C virus infection.J Clin Invest. 2002; 109: 221-232Crossref PubMed Scopus (76) Google Scholar]. However, viral load in infected animals is low [[9]Amako Y. Tsukiyama-Kohara K. Katsume A. Hirata Y. Sekiguchi S. Tobita Y. et al.Pathogenesis of hepatitis C virus infection in Tupaia belangeri.J Virol. 2010; 84: 303-311Crossref PubMed Scopus (103) Google Scholar]. The discovery of a hepatocyte-lethal phenotype in mice carrying a urokinase-type plasminogen activator transgene controlled by an albumin promotor (Alb-uPA) with the reconstitution of livers of those mice with xenografted rat hepatocytes [[10]Sandgren E.P. Palmiter R.D. Heckel J.L. Daugherty C.C. Brinster R.L. Degen J.L. Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene.Cell. 1991; 66: 245-256Abstract Full Text PDF PubMed Scopus (320) Google Scholar] has laid the foundation for the development of small animal models of infection with hepatitis B and C viruses using xenografted primary human hepatocytes (PHH) (for review see [[7]Barth H. Robinet E. Liang T.J. Baumert T.F. Mouse models for the study of HCV infection and virus–host interactions.J Hepatol. 2008; 49: 134-142Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]). The uPA-xenograft model had been first established for the development of a hepatitis B mouse model by transplanting woodchuck or PHH into Alb-uPA mice on an immunodeficient recombination activating gene 2 knock-out (Rag-2−/−) background. Repopulating woodchuck hepatocytes in Alb-uPA/rag-2 mice, which lack mature B and T lymphocytes, allowed productive infection with woodchuck hepatitis B virus [[11]Petersen J. Dandri M. Gupta S. Rogler C.E. Liver repopulation with xenogenic hepatocytes in B and T cell-deficient mice leads to chronic hepadnavirus infection and clonal growth of hepatocellular carcinoma.Proc Natl Acad Sci USA. 1998; 95: 310-315Crossref PubMed Scopus (109) Google Scholar]. Subsequently, Mercer and colleagues demonstrated that the severe combined immunodeficiency disorder (SCID-beige)/Alb-uPA mouse engrafted with PHH can be infected with HCV in vivo [[12]Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (746) Google Scholar]. A further development of this model is a system that can be regulated for repopulating the liver of immunodeficient mice, based on mice lacking the fumaryl acetoacetate hydrolase [Fah], recombination activating gene 2 [Rag-2−/−], and the gamma-chain of the receptor for IL-2 [Il-2rgamma or γc] (Fah/Rag-2−/−/γc−/− model). Oral administration of a small molecule (NTBC) prevents the accumulation of hepatotoxic metabolites resulting from the Fah deficiency. By regulating hepatotoxicity using NTBC, this model allows for transplantation of PHH at any age, which may improve robustness of engraftment and mortality [[13]Azuma H. Paulk N. Ranade A. Dorrell C. Al-Dhalimy M. Ellis E. et al.Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.Nat Biotechnol. 2007; 25: 903-910Crossref PubMed Scopus (606) Google Scholar]. Although all chimeric mouse models are characterized by the absence of a functional immune system [[7]Barth H. Robinet E. Liang T.J. Baumert T.F. Mouse models for the study of HCV infection and virus–host interactions.J Hepatol. 2008; 49: 134-142Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar], they are very useful for the study of HBV and HCV infection in vivo as well as for the evaluation of antivirals targeting the virus or essential host factors present in transplanted hepatocytes [14Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (277) Google Scholar, 15Meuleman P. Hesselgesser J. Paulson M. Vanwolleghem T. Desombere I. Reiser H. et al.Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo.Hepatology. 2008; 48: 1761-1768Crossref PubMed Scopus (188) Google Scholar]. The hepatocyte-lethal phenotype of these models allows for transplanting immunodeficient mice with PHH by intrasplenic injection. Successful engraftment of hepatocytes is monitored by quantification of human albumin concentrations in the plasma or serum. For uPA-transgene models, the transplantation is usually performed within the first two weeks of life and human albumin levels are determined at least three weeks after transplantation. If the liver repopulation reaches a threshold level, human liver chimeric mice can then be infected by HBV [[16]Dandri M. Burda M.R. Torok E. Pollok J.M. Iwanska A. Sommer G. et al.Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (323) Google Scholar] or HCV [[12]Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (746) Google Scholar]. Nevertheless, the wide-spread use and further development of these model systems are hampered by the complexity of their set-up, essentially for technical reasons. One limitation is the availability of high quality PHH, but the major obstacles are the technical challenges to achieve high repopulation of the chimeric mouse liver with human hepatocytes. Interestingly, several independent studies have shown that the level of liver repopulation required for HCV infection is significantly higher than that required for HBV infection: while 15% liver parenchyma replacement by PHH is sufficient to support a HBV infection [[16]Dandri M. Burda M.R. Torok E. Pollok J.M. Iwanska A. Sommer G. et al.Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (323) Google Scholar], at least 50% replacement is needed to support a HCV infection [[12]Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (746) Google Scholar]. Thus, the identification of technical or biological parameters allowing efficient engraftment of permissive PHH is of crucial importance for the improvement of the model, in particular for robust infection with HCV. To identify essential viral and host factors for successful engraftment of HCV permissive hepatocytes, the Leroux–Roels laboratory at Ghent University, Belgium, has performed a comprehensive functional analysis of a large series of animals which is published in this issue of the Journal [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. In this study, performed by one of the most experienced laboratories in the field, animal survival, engraftment efficacy, kinetics of liver repopulation, and viral infection was analyzed in 400 transplanted chimeric upA-SCD mice. First, Vanwolleghem and colleagues [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar] demonstrated that the first 7 days after transplantation are the most critical period for survival. This period is characterized by frequent identification of necrotic donor hepatocytes in portal vein branches and periportal sinusoids. The necrosis tended to be more extensive in mice transplanted with PHH than with murine hepatocytes, the latter repopulating the liver approximately four-times faster than the former donor cells after this critical period. Peri-operative problems and the use of fresh PHH were identified as independent negative risk factors for survival of the host. Since fresh and frozen PHH led to similar engraftment levels, both in terms of fraction of engrafted mice and human albumin levels, cryopreserved PHH are superior to fresh PHH regarding the final outcome of permissive chimeric mice. Engraftment level seemed to be mainly donor-dependent, with commercially available frozen PHH again being the best source of PHH in this series. Interestingly, donor age and PHH viability were not predictors of successful engraftment (Fig. 1). The quality of the viral inoculum is another important factor for successful infection. The use of serum-derived HCV has been reported to provide higher HCV plasma titers than infection with cell culture-derived recombinant HCV [[14]Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (277) Google Scholar]. Vanwolleghem and colleagues demonstrate that viral load and proper conservation and storage of the inoculum are crucial for successful infection [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. Serial freezing/thawing cycles clearly impair the infectivity of the inoculum. In this regard, it is of interest to note, that the same laboratory has characterized and titrated reference genotypes 1–6 HCV batches from pooled sera of chronically infected chimpanzees in uPA-SCID mice in collaboration with the Laboratory of Infectious Diseases, National Institutes of Health, Bethesda [[18]Bukh J, Meuleman P, Tellier R, Engle RE, Feinstone SM, Eder G, et al. Challenge pools of hepatitis C virus genotypes 1–6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models. J Infect Dis 201:1381–89.Google Scholar]. Most importantly, this comprehensive analysis allowed the identification of criteria for HCV infection with 100% success rate (Fig. 1). These criteria include (i) serum human albumin level >1 mg/ml at time of HCV injection and during the course of the experiment and (ii) a HCV inoculum containing >104 IU HCV RNA with no more than one freeze–thaw cycle [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar]. The human albumin threshold of 1 mg/ml defined in the present study is similar to the one suggested by Mercer et al. [[12]Mercer D.F. Schiller D.E. Elliott J.F. Douglas D.N. Hao C. Rinfret A. et al.Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (746) Google Scholar] using the same model. Higher levels have been reported to be required by another laboratory using the uPA-SCID model [[19]Kamiya N, Iwao E, Hiraga N, Tsuge M, Imamura M, Takahashi S, et al. Practical evaluation of a mouse with chimeric human liver model for hepatitis C virus infection using an NS3-4A protease inhibitor. J Gen Virol. 2010 Feb 17. [Epub ahead of print].Google Scholar] or in the Fah/Rag-2−/−/γc−/− model [[14]Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (277) Google Scholar]. Standardization of the sampling method (plasma versus serum) and human albumin quantification method and a more accurate correlation between human albumin concentrations and liver replacement index could help in clarifying these apparent differences. Interestingly, improved PHH engraftment was obtained in the Fah/Rag-2−/−/γc−/− model by increasing the PHH dose up to 3–5 × 106 injected cells [[14]Bissig K.D. Wieland S.F. Tran P. Isogawa M. Le T.T. Chisari F.V. et al.Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.J Clin Invest. 2010; 120: 924-930Crossref PubMed Scopus (277) Google Scholar], instead of 0.5–1 × 106 PHH in the uPA-SCID model. Whether increasing the PHH number in the uPA-SCID model would also improve the engraftment level remains to be determined. An important difference between the two models is the fact that the uPA-SCID mice requires transplantation at a young age (at approximately three weeks of age), while adult Fah/Rag-2−/−/γc−/− mice can be transplanted at a later age, probably injecting a higher number of PHH. Further differences between the inducible (Fah/Rag-2−/−/γc−/−) and the uPA-SCID model are related to the increased expression of uPA in the latter. uPA transforms proHGF to its active form HGF, activates plasminogen, and promotes liver matrix proteolysis during repair, a series of processes that may promote the successful engraftment and proliferation of PHH [[20]Naldini L. Vigna E. Bardelli A. Follenzi A. Galimi F. Comoglio P.M. Biological activation of pro-HGF (hepatocyte growth factor) by urokinase is controlled by a stoichiometric reaction.J Biol Chem. 1995; 270: 603-611Crossref PubMed Scopus (231) Google Scholar]. Although additional studies are required to better understand the biochemical and biological mechanisms involved in PHH engraftment, the identification of host and viral factors required for engraftment of permissive hepatocytes by Vanwolleghem and colleagues [[17]Vanwolleghem T. Libbrecht L. Hansen B.E. Desombere I. Roskams T. Meuleman P. et al.Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice.J Hepatol. 2010; 53: 468-476Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar] will be very helpful to apply and further develop humanized mouse models for HBV and HCV infections. Thus, the results of this thorough and comprehensive study will be very useful for laboratories using and advancing the current humanized mouse models e.g. by approaches to reconstitute a human immune system. Furthermore, this study may be also of interest for teams aiming to set-up this important in vivo model for the study of HBV and HCV infection. The further development as well as a more wide spread use of humanized mouse models will advance our knowledge of virus–host interactions and the development of urgently needed novel efficient antivirals. Conflict of interestsThe authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The authors acknowledge financial support of their work by the European Union (ERC-2008-AdG-233130-HEPCENT and INTERREG-IV-2009-FEDER-Hepato-Regio-Net), ANRS ( 2007/306 and 2008/354 ), the Région Alsace ( 2007/09 ), the Else Kröner-Fresenius Foundation (EKFS P17//07//A83/06 ), the Ligue Contre le Cancer ( CA 06/12 ), Inserm, University of Strasbourg, and the Strasbourg University Hospitals, France .