Liver Xeno-Repopulation with Human Hepatocytes in Fah−/−Rag2−/− Mice after Pharmacological Immunosuppression
2010; Elsevier BV; Volume: 177; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2010.091154
ISSN1525-2191
AutoresZhiying He, Haibin Zhang, Xin Zhang, Dong‐Fu Xie, Yixin Chen, Kirk J. Wangensteen, Stephen C. Ekker, Meri T. Firpo, Changcheng Liu, Dao Xiang, Xiaoyuan Zi, Lijian Hui, Guang-Shun Yang, Xiaoyan Ding, Yiping Hu, Xin Wang,
Tópico(s)Organ Transplantation Techniques and Outcomes
ResumoFunctional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury–induced donor hepatocyte selection: the uPA/SCID mice and Fah−/− Rag2−/−Il2rg−/− mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah−/−Rag2−/− mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah−/−Rag2−/− mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines. Functional human hepatocytes xeno-engrafted in mouse liver can be used as a model system to study hepatitis virus infection and vaccine efficacy. Significant liver xeno-repopulation has been reported in two kinds of genetically modified mice that have both immune deficiency and liver injury–induced donor hepatocyte selection: the uPA/SCID mice and Fah−/− Rag2−/−Il2rg−/− mice. The lack of hardy breeding and the overly elaborated technique in these two models may hinder the potential future application of these models to hepatitis virus infection and vaccination studies. Improving the transplantation protocol for liver xeno-repopulation from human hepatocytes will increase the model efficiency and application. In this study, we successfully apply immunosuppressive drug treatments of anti-asialo GM1 and FK506 in Fah−/−Rag2−/− mice, resulting in significant liver xeno-repopulation from human hepatocytes and human fetal liver cells. This methodology decreases the risk of animal mortality during breeding and surgery. When infected with hepatitis B virus (HBV) sera, Fah−/−Rag2−/− mice with liver xeno-repopulation from human hepatocytes accumulate significant levels of HBV DNA and HBV proteins. Our new protocol for humanized liver could be applied in the study of human hepatitis virus infection in vivo, as well as the pharmacokinetics and efficacy of potential vaccines. Human hepatocytes xeno-engrafted into the liver of immunodeficient mice could be used as a model to study human hepatitis virus infection in vivo as well as the efficacy of potential vaccines.1Dandri M Burda MR Török E Pollok JM Iwanska A Sommer G Rogiers X Rogler CE Gupta S Will H Greten H Petersen J Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (324) Google Scholar, 2Meuleman P Libbrecht L De Vos R de Hemptinne B Gevaert K Vandekerckhove J Roskams T Leroux-Roels G Morphological and biochemical characterization of a human liver in an uPA-SCID mouse chimera.Hepatology. 2005; 41: 847-856Crossref PubMed Scopus (312) Google Scholar, 3Mercer DF Schiller DE Elliott JF Douglas DN Hao C Rinfret A Addison WR Fischer KP Churchill TA Lakey JR Tyrrell DL Kneteman NM Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (748) Google Scholar, 4Vanwolleghem T Bukh J Meuleman P Desombere I Meunier JC Alter H Purcell RH Leroux-Roels G Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain.Hepatology. 2008; 47: 1846-1855Crossref PubMed Scopus (115) Google Scholar, 5Dandri M Murray JM Lutgehetmann M Volz T Lohse AW Petersen J Virion half-life in chronic hepatitis B infection is strongly correlated with levels of viremia.Hepatology. 2008; 48: 1079-1086Crossref PubMed Scopus (52) Google Scholar, 6Sugiyama M Tanaka Y Kurbanov F Maruyama I Shimada T Takahashi S Shirai T Hino K Sakaida I Mizokami M Direct cytopathic effects of particular hepatitis B virus genotypes in severe combined immunodeficiency transgenic with urokinase-type plasminogen activator mouse with human hepatocytes.Gastroenterology. 2009; 136 (e3): 652-662Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar Engrafted human hepatocytes can be serially transplanted from primary mice into secondary mice without losing hepatic function.7Azuma H Paulk N Ranade A Dorrell C Al-Dhalimy M Ellis E Strom S Kay MA Finegold M Grompe M Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.Nature Biotechnol. 2007; 25: 903-910Crossref Scopus (613) Google Scholar Mouse recipients of human liver cells must have two capabilities: robust liver repopulation and immune tolerance for human hepatocytes. Liver xeno-repopulation from human hepatocytes was first reported in uPA/Rag2−/− mice1Dandri M Burda MR Török E Pollok JM Iwanska A Sommer G Rogiers X Rogler CE Gupta S Will H Greten H Petersen J Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (324) Google Scholar and uPA/SCID mice.2Meuleman P Libbrecht L De Vos R de Hemptinne B Gevaert K Vandekerckhove J Roskams T Leroux-Roels G Morphological and biochemical characterization of a human liver in an uPA-SCID mouse chimera.Hepatology. 2005; 41: 847-856Crossref PubMed Scopus (312) Google Scholar, 3Mercer DF Schiller DE Elliott JF Douglas DN Hao C Rinfret A Addison WR Fischer KP Churchill TA Lakey JR Tyrrell DL Kneteman NM Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (748) Google Scholar, 8Tateno C Yoshizane Y Saito N Kataoka M Utoh R Yamasaki C Tachibana A Soeno Y Asahina K Hino H Asahara T Yokoi T Furukawa T Yoshizato K Near completely humanized liver in mice shows human-type metabolic responses to drugs.Am J Pathol. 2004; : 165901-165912Google Scholar The levels of liver xeno-repopulation varies in several reports, ranging from 10% to as high as 90%.1Dandri M Burda MR Török E Pollok JM Iwanska A Sommer G Rogiers X Rogler CE Gupta S Will H Greten H Petersen J Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (324) Google Scholar, 8Tateno C Yoshizane Y Saito N Kataoka M Utoh R Yamasaki C Tachibana A Soeno Y Asahina K Hino H Asahara T Yokoi T Furukawa T Yoshizato K Near completely humanized liver in mice shows human-type metabolic responses to drugs.Am J Pathol. 2004; : 165901-165912Google Scholar Humanized livers in uPA/SCID mice are susceptible to hepatitis B virus (HBV)1Dandri M Burda MR Török E Pollok JM Iwanska A Sommer G Rogiers X Rogler CE Gupta S Will H Greten H Petersen J Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (324) Google Scholar, 2Meuleman P Libbrecht L De Vos R de Hemptinne B Gevaert K Vandekerckhove J Roskams T Leroux-Roels G Morphological and biochemical characterization of a human liver in an uPA-SCID mouse chimera.Hepatology. 2005; 41: 847-856Crossref PubMed Scopus (312) Google Scholar and HCV3Mercer DF Schiller DE Elliott JF Douglas DN Hao C Rinfret A Addison WR Fischer KP Churchill TA Lakey JR Tyrrell DL Kneteman NM Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (748) Google Scholar, 4Vanwolleghem T Bukh J Meuleman P Desombere I Meunier JC Alter H Purcell RH Leroux-Roels G Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain.Hepatology. 2008; 47: 1846-1855Crossref PubMed Scopus (115) Google Scholar infection. However, uPA mice have several disadvantages: i) neonatal death during colony breeding; ii) transplantation of hepatocytes into newborn mice (within the second week of life) is technically difficult due to a bleeding disorder in the mice; iii) there is uncontrollable selection for donor cells; iv) there is autoreversion of endogenous hepatocytes; and v) kidney damage is induced by the human complement system.1Dandri M Burda MR Török E Pollok JM Iwanska A Sommer G Rogiers X Rogler CE Gupta S Will H Greten H Petersen J Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (324) Google Scholar, 2Meuleman P Libbrecht L De Vos R de Hemptinne B Gevaert K Vandekerckhove J Roskams T Leroux-Roels G Morphological and biochemical characterization of a human liver in an uPA-SCID mouse chimera.Hepatology. 2005; 41: 847-856Crossref PubMed Scopus (312) Google Scholar, 8Tateno C Yoshizane Y Saito N Kataoka M Utoh R Yamasaki C Tachibana A Soeno Y Asahina K Hino H Asahara T Yokoi T Furukawa T Yoshizato K Near completely humanized liver in mice shows human-type metabolic responses to drugs.Am J Pathol. 2004; : 165901-165912Google Scholar, 9Rhim JA Sandgren EP Degen JL Palmiter RD Brinster RL Replacement of diseased mouse liver by hepatic cell transplantation.Science. 1994; 263: 1149-1152Crossref PubMed Scopus (509) Google Scholar Recently, robust liver xeno-repopulation from human hepatocytes was found in Fah−/−Rag2−/−Il2rg−/− mice, cross-bred from Fah−/− mice and Rag2−/−Il2rg−/− mice.7Azuma H Paulk N Ranade A Dorrell C Al-Dhalimy M Ellis E Strom S Kay MA Finegold M Grompe M Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.Nature Biotechnol. 2007; 25: 903-910Crossref Scopus (613) Google Scholar Fah−/−Rag2−/−Il2rg−/− mice have advantages over previous immunodeficient uPA models.7Azuma H Paulk N Ranade A Dorrell C Al-Dhalimy M Ellis E Strom S Kay MA Finegold M Grompe M Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.Nature Biotechnol. 2007; 25: 903-910Crossref Scopus (613) Google Scholar First, Rag2−/−Il2rg−/− mice lack B, T, and NK cells, rendering more complete immunodeficiency compared with either Rag2−/− or SCID mice.10Traggiai E Chicha L Mazzucchelli L Bronz L Piffaretti JC Lanzavecchia A Manz MG Development of a human adaptive immune system in cord blood cell-transplanted mice.Science. 2004; 304: 104-107Crossref PubMed Scopus (796) Google Scholar Second, liver injury in Fah−/− mice is controllable by switching on and off 2-(2-nitro-4-trifluoro-methyl-benzoyl)-1, 3 cyclohexanedione (NTBC) administration.11Grompe M Lindstedt S al-Dhalimy M Kennaway NG Papaconstantinou J Torres-Ramos CA Ou CN Finegold M Pharmacological correction of neonatal lethal hepatic dysfunction in a murine model of hereditary tyrosinaemia type I.Nat Genet. 1995; 10: 453-460Crossref PubMed Scopus (264) Google Scholar NTBC inhibits accumulation of toxic metabolites in hepatocytes to maintain Fah−/− mice in a healthy state. When the NTBC is removed, a powerful selection for fumaryl acetoacetate hydrolase (Fah) expressing cells is induced in the liver.12Overturf K al-Dhalimy M Ou CN Finegold M Grompe M Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.Am J Pathol. 1997; 151: 1273-1280PubMed Google Scholar However, maintenance of Fah−/−Rag2−/−Il2rg−/− mice during colony breeding, animal growing, and cell transplantation surgery are with high mortality in our experiments. The genotyping of animal offspring is overly elaborate. These concerns have not been discussed in previous publications7Azuma H Paulk N Ranade A Dorrell C Al-Dhalimy M Ellis E Strom S Kay MA Finegold M Grompe M Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.Nature Biotechnol. 2007; 25: 903-910Crossref Scopus (613) Google Scholar, 13Bissig KD Le TT Woods NB Verma IM Repopulation of adult and neonatal mice with human hepatocytes: a chimeric animal model.Proc Natl Acad Sci USA. 2007; 104: 20507-20511Crossref PubMed Scopus (157) Google Scholar and may present a barrier to larger scale research projects. In comparison, Fah−/−Rag2−/− mice were much more tolerant of breeding and surgery procedures. However, Fah−/−Rag2−/− mice were thought to have no capacity for liver xeno-repopulation, because their NK cells are intact.7Azuma H Paulk N Ranade A Dorrell C Al-Dhalimy M Ellis E Strom S Kay MA Finegold M Grompe M Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.Nature Biotechnol. 2007; 25: 903-910Crossref Scopus (613) Google Scholar We hypothesized that treatment of Fah−/− Rag2−/− mice with anti-asialo GM1 could result in complete depletion of NK cells as seen in Fah−/−Rag2−/− Il2rg−/− mice.14Wang M Ellison CA Gartner JG HayGlass KT Natural killer cell depletion fails to influence initial CD4 T cell commitment in vivo in exogenous antigen-stimulated cytokine and antibody responses.J Immunol. 1998; 160: 1098-1105PubMed Google Scholar, 15Sherwood ER Lin CY Tao W Hartmann CA Dujon JE French AJ Varma TK Beta 2 microglobulin knockout mice are resistant to lethal intraabdominal sepsis.Am J Respir Crit Care Med. 2003; 167: 1641-1649Crossref PubMed Scopus (43) Google Scholar We further tested the combined treatments of both anti-asialo GM1 and the immunosuppressor tacrolimus (FK506) to Fah−/−Rag2−/− mice.16Masri MA The mosaic of immunosuppressive drugs.Mol Immunol. 2003; 39: 1073-1077Crossref PubMed Scopus (53) Google Scholar, 17Escribano O Fernández-Moreno MD Piña MJ Fueyo J Menor C Román ID Guijarro LG Pretreatment with FK506 up-regulates insulin receptors in regenerating rat liver.Hepatology. 2002; 36: 555-561Crossref PubMed Scopus (12) Google Scholar The results indicated that the combined treatments enabled Fah−/−Rag2−/− recipients to have a high level of liver xeno-repopulation by human hepatocytes as seen in Fah−/−Rag2−/−Il2rg−/− mice. Our results revealed a new and easily controlled mouse model with humanized liver. Using the same treatments, liver xeno-repopulation with human fetal liver progenitor cells was also achieved in Fah−/−Rag2−/− mice. Finally, for the first time, we were able to prove that human HBV actively replicated in the humanized Fah−/−Rag2−/− mice and that viral proteins were released in the serum of humanized Fah−/−Rag2−/− mice, which showed no significant difference with previous reports of human HBV infection in humanized uPA/SCID mice.1Dandri M Burda MR Török E Pollok JM Iwanska A Sommer G Rogiers X Rogler CE Gupta S Will H Greten H Petersen J Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (324) Google Scholar, 2Meuleman P Libbrecht L De Vos R de Hemptinne B Gevaert K Vandekerckhove J Roskams T Leroux-Roels G Morphological and biochemical characterization of a human liver in an uPA-SCID mouse chimera.Hepatology. 2005; 41: 847-856Crossref PubMed Scopus (312) Google Scholar, 5Dandri M Murray JM Lutgehetmann M Volz T Lohse AW Petersen J Virion half-life in chronic hepatitis B infection is strongly correlated with levels of viremia.Hepatology. 2008; 48: 1079-1086Crossref PubMed Scopus (52) Google Scholar Fah−/− mice were crossed with Rag2−/−Il2rg−/− mice (Taconic). Strains of both Fah−/−Rag2−/− mice and Fah−/−Rag2−/−Il2rg−/− mice were obtained by gradual cross-breeding. PCR-based genotyping with primers for Fah,18Grompe M al-Dhalimy M Finegold M Ou CN Burlingame T Kennaway NG Soriano P Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice.Genes Dev. 1993; 7: 2298-2307Crossref PubMed Scopus (294) Google Scholar Rag2, and Il2rg genes10Traggiai E Chicha L Mazzucchelli L Bronz L Piffaretti JC Lanzavecchia A Manz MG Development of a human adaptive immune system in cord blood cell-transplanted mice.Science. 2004; 304: 104-107Crossref PubMed Scopus (796) Google Scholar were used to determine genotypes of offspring. Animals were maintained with drinking water containing NTBC at a concentration of 7.5 μg/ml. All mice were housed in individually ventilated cage (IVC) system under special pathogen-free (SPF) facility with barrier conditions, and animal care was in accordance with institutional guidelines. Anti-asialo GM1 (αAsGM1, 50 mg in 200 μl saline, Wako) was i.p. injected into Fah−/−Rag2−/− mice 24 hours before cell transplantation and then at 7-day intervals after transplantation. Control mice were treated with nonspecific IgG (eBioscience, San Diego, CA). Mice were sacrificed after anesthetization, and bone marrow cells from the femur were collected. Bone marrow cells (1 × 106/tube) were incubated for 10 minutes at 4°C with Fc blocker to prevent nonspecific binding. The cells were then stained with FITC-conjugated anti-mouse CD3 mAb, FITC-conjugated anti-mouse CD19 mAb, PE-conjugated anti-NK1.1, and IgM mAb (all Abs from eBioscience). Percentage of fluorescence-positive cells was analyzed using FACSCalibur (Becton-Dickinson, Franklin Lakes, NJ). Human liver tissue was provided by University of Minnesota Medical Center, Fairview (Minnesota) and Eastern Hepatobiliary Surgery Hospital, Second Military Medical University (Shanghai, China) from donor livers that had been reduced in size for allotransplantation. The procedure for preparation of human hepatocytes for transplantation into mice was performed under institutional guidelines. Human fetal liver tissues were derived from the first- and second-trimester fetuses between 98 and 116 days of gestation from Changhai hospital, Second Military Medical University (Shanghai, China). All human tissues were negative for HBV infection. Patients gave written, informed consent. Experiments were approved by Ethical Committee on Ethics of Biomedicine Research, Second Military Medical University. The approved IRB numbers are 0608M91366 (University of Minnesota) and 2007LL006 (Second Military Medical University). Human hepatocytes were isolated as described previously.2Meuleman P Libbrecht L De Vos R de Hemptinne B Gevaert K Vandekerckhove J Roskams T Leroux-Roels G Morphological and biochemical characterization of a human liver in an uPA-SCID mouse chimera.Hepatology. 2005; 41: 847-856Crossref PubMed Scopus (312) Google Scholar Human fetal liver tissues were cut and digested by collagenase D (2.5 mg/ml, Roche, Basel, Switzerland) for 20 minutes at 37°C. Digested cells were filtered through a 70 μm nylon mesh. E-cadherin-positive (E-Cad+) cells were enriched as previously.19Nierhoff D Ogawa A Oertel M Chen YQ Shafritz DA Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity.Hepatology. 2005; 42: 130-139Crossref PubMed Scopus (96) Google Scholar The cells positive for E-cadherin and high SSC were sorted by FACSVantage (Becton-Dickinson) using Rat anti-human E-cadherin (eBioscience). Human hepatocytes (3 × 105) or human fetal cells were injected into the spleens of mice recipients. Seven days before cell transplantation, NTBC in drinking water was reduced to 50% of the original level for three days and further reduced to 25% for two days. Then, NTBC was totally discontinued two days before cell transplantation. In serial transplantation, the engrafted human hepatocytes in chimeric liver of recipients were isolated after liver perfusion.7Azuma H Paulk N Ranade A Dorrell C Al-Dhalimy M Ellis E Strom S Kay MA Finegold M Grompe M Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.Nature Biotechnol. 2007; 25: 903-910Crossref Scopus (613) Google Scholar, 12Overturf K al-Dhalimy M Ou CN Finegold M Grompe M Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.Am J Pathol. 1997; 151: 1273-1280PubMed Google Scholar The recipients were first examined for liver repopulation by biopsy of liver tissue. Harvested liver sections were stained for FAH expression by immunohistochemical analysis. Recipients with the highest level of liver repopulation were selected for collection of donor cells for secondary transplantation. The percentage of FAH-positive hepatocytes was used as a reference to estimate the actual number of human hepatocytes in the prepared cell suspension. FK506 (Astellas, Dublin, Ireland) dissolved in the drinking water at 7.5 μg/ml was administered to adult mouse to achieve a dose of 1 μg/g body weight per day. A separate group of control mice received only sterilized water. FK506 blood concentration was measured by microparticle enzyme immunoassay (MEIA, Abbott Laboratories, Alameda, CA). Blood was collected from the retro orbital vein of anesthetized mice. Kits of Human albumin (hAlb) ELISA Quantitation (Bethyl, Montgomery, TX) and Human α1-Antitrypsin (hAAT) ELISA Quantitation (GenWay, San Diego, CA) were used to measure human proteins according manufacturer's protocols. For molecular assay, Human Alu sequences in liver of chimeric mice were amplified by PCR as described previously,1Dandri M Burda MR Török E Pollok JM Iwanska A Sommer G Rogiers X Rogler CE Gupta S Will H Greten H Petersen J Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus.Hepatology. 2001; 33: 981-988Crossref PubMed Scopus (324) Google Scholar and real-time PCR of human Alu and AAT gene were conducted as described in previous publication.3Mercer DF Schiller DE Elliott JF Douglas DN Hao C Rinfret A Addison WR Fischer KP Churchill TA Lakey JR Tyrrell DL Kneteman NM Hepatitis C virus replication in mice with chimeric human livers.Nat Med. 2001; 7: 927-933Crossref PubMed Scopus (748) Google Scholar, 20Igarashi Y Tateno C Tanaka Y Tachibana A Utoh R Kataoka M Ohdan H Asahara T Yoshizato K Engraftment of human hepatocytes in the livers of rats bearing bone marrow reconstructed with immunodeficient mouse bone marrow cells.Xenotransplantation. 2008; 15: 235-245Crossref PubMed Scopus (17) Google Scholar Human serum containing a high HBV DNA content (108 IU/ml) was obtained from a HBV chronic carrier. Six weeks after human hepatocyte transplantation, chimeric Fah−/−Rag2−/− mice were infected with HBV by i.p. injection of 100 μl of above serum. Viral DNA was extracted from HBV-infected chimeric mice sera using the QIAamp Blood Kit (Qiagen, Hilden, Germany), and quantification of HBV-DNA was analyzed using real-time PCR (LightCycler, Basel, Switzerland). Known amounts of cloned HBV DNA were amplified in parallel to establish a standard bar for quantification. HBsAg and HBeAg in sera of HBV-infected chimeric mice were determined by Electrical chemiluminescence immunoassay analysis method (MODULAR ANALYTICS E170, Roche). Associated reagents for HBsAg and HBeAg determination are also manufactured by Roche. HBsAg and HBeAg were interpreted using the ratio of the sample signal to the cutoff signal (S/CO), and S/CO ≥1.00 was positive. Five-μm-thick sections were examined by immunohistochemistry with rabbit anti FAH antibody (AbboMax, San Jose, CA). Human cells within the mice livers were analyzed with a polyclonal antibody against human-specific albumin (Bethyl) and α1-Antitrypsin (Thermo, Freemont, CA). Expression of HBV proteins was assessed using a polyclonal rabbit antibody against HBcAg (Dako, Copenhagen, Denmark) and a polyclonal goat antibody against HBsAg (Dako). Positive controls from clinical biopsies were stained with these antibodies, and normal liver biopsies as negative controls. Blood was collected from the retro-orbital sinus of test animals. Plasma was prepared using Microtainer plasma separator tubes (Becton-Dickinson) and stored at −80°C. Biochemical evaluation of liver function was performed as previously described.21Wilber A Wangensteen KJ Chen Y Zhuo L Frandsen JL Bell JB Chen ZJ Ekker SC McIvor RS Wang X Messenger RNA as a source of transposase for sleeping beauty transposon-mediated correction of hereditary tyrosinemia type I.Mol Ther. 2007; 15: 1280-1287Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Calculations of sample size, cell numbers, and percentage of repopulation were performed as previously described.22Wang X Montini E Al-Dhalimy M Lagasse E Finegold M Grompe M Kinetics of liver repopulation after bone marrow transplantation.Am J Pathol. 2002; 161: 565-574Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar Excel was used to calculate average ± SD. The statistical significance of difference between sample groups was calculated by Student's t-test. P values <0.05 were regarded as statistically significant. We determined the capacities of repopulation from human hepatocytes in Fah−/−Rag2−/−Ilr2g−/− mice. Human hepatocytes (3 × 105) were injected into spleen, and total NTBC withdrawal lasted for 12 weeks. Recipients were sacrificed, and liver samples were harvested for detection of human hepatocyte engraftment. Fah−/− Rag2−/−Ilr2g−/− recipients on NTBC were as controls. FAH antibody immunostaining indicated that liver samples from 5 of 8 Fah−/−Rag2−/−Ilr2g−/− recipients had FAH-positive hepatocytes at levels from 3.1% to 71.3% of total hepatocytes (Figure 1A, Supplemental Table 1 at http://ajp.amjpathol.org). However, the Fah−/−Rag2−/− Il2rg−/− mouse breeders have a very small litter size in our colony breeding process (Supplemental Figure 1 at http://ajp.amjpathol.org). For this reason, Fah−/−Rag2+/-Il2rg+/− mice could be used as breeders to generate Fah−/−Rag2−/−Il2rg−/− mice. Number of Fah−/−Rag2−/− Il2rg−/− mice was much lower than expected, and only between 1 in 30 and 1 in 20 offspring had the triple homozygous mutant genotype. The pups with genotype of Fah−/−Rag2−/−Il2rg−/− were also less fit than Fah−/− and Fah−/−Rag2−/− littermates. They had about 60% more death during colony breeding and after surgery for cell transplantation (Supplemental Figure 2 at http://ajp.amjpathol.org). Because of the significantly low numbers of Fah−/−Rag2−/−Il2rg−/− mice during colony breeding and the significant numbers of Fah−/−Rag2−/−Il2rg−/− recipients lost after cell transplantation, Fah−/−Rag2−/− Il2rg−/− mice could not be used for efficient generation of humanized mice. Thus, we chose the Fah−/−Rag2−/− mouse to study the capacity for human hepatocyte repopulation after pharmacological treatment to deplete NK cells. Using the same experimental protocol and under same conditions, we determined the capacities of repopulation from human hepatocytes in Fah−/−Rag2−/− mice. Fah−/− Rag2−/− recipients on NTBC were used as controls that were free of induced liver injury. Three of 17 Fah−/− Rag2−/− recipients were found with detectable FAH-positive hepatocytes, existing in single cells and small nodules (Figure 1B; Supplemental Table 1 at http://ajp.amjpathol.org). Xeno-engraftment of human hepatocytes in Fah−/−Rag2−/− recipients was not successful in a previous report.7Azuma H Paulk N Ranade A Dorrell C Al-Dhalimy M Ellis E Strom S Kay MA Finegold M Grompe M Robust expansion of human hepatocytes in Fah−/−/Rag2−/−/Il2rg−/− mice.Nature Biotechnol. 2007; 25: 903-910Crossref Scopus (613) Google Scholar Without treatment of gradual withdrawal of NTBC before cell transplantation, but instead with abrupt removal of NTBC after cells were transplanted, none of eight Fah−/−Rag2−/−Ilr2g−/− and eight Fah−/−Rag2−/− recipients had any detectable FAH positive hepatocytes (Supplemental Table 1 at http://ajp.amjpathol.org). In addition, eight Fah−/−Rag2−/−Ilr2g−/− and eight Fah−/−Rag2−/− recipients that were always kept on NTBC had no detectable FAH positive hepatocytes (Supplemental Table 1 at http://ajp.amjpathol.org). In comparison with Fah−/−Rag2−/−Ilr2g−/− mice, Fah−/−Rag2−/− mice have significant levels of NK cells (Figure 1D). Anti-asialo GM1 was known from previous reports to deplete NK cells.15Sherwood ER Lin CY Tao W Hartmann CA Dujon JE French AJ Varma TK Beta 2 microglobulin knockout mice are resistant to lethal intraabdominal sepsis.Am J Respir Crit Care Med. 2003; 167: 1641-1649Crossref PubMed Scopus (43) Google Scholar, 16Masri MA The mosaic of immunosuppressive drugs.Mol Immunol. 2003; 39: 1073-1077Crossref PubMed Scopus (53) Google Scholar At 24 hours after i.p. injection of anti-asialo GM1, the level of NK cells labeled by NK1.1+CD3− was reduced to 5.6% of the original level in Fah−/−Rag2−/− mice (Figure 1D). After anti-asialo GM1 treatment, Fah−/−Rag2−/− mice were transplanted by human hepatocytes (3 × 105) and selection was performed, sample harvest and FAH immuno-assay performed as before. Results indicated that samples from 10 of 18 Fah−/−Rag2−/− recipients had FAH-positive hepatocytes ranging from 0.1% to 16.2% of total hepatocytes at six weeks and from 3.4% to 31.7% at twelve weeks (Figures 1C and 2, A, B, and E). Fah−/−Rag2−/− mice after treatment with anti-asialo GM1 gained capacity for liver xeno-repopulation with human hepatocytes. However, the levels of xeno-liver repopulation in Fah−/− Rag2−/− recipients were still lower than those in Fah−/− Rag2−/−Ilr2g−/− recipients. FK506 is well known for its effects on immunosuppression.16Masri MA The mosaic of immunosuppressive drugs.Mol Immunol. 2003; 39: 1073-1077Crossref PubMed Scopus (53) Google Scholar FK506 was also found to induce hepatocyte proliferation and promote liver regeneration in partial hepatectomized rats.17Escribano O Fernández-Moreno MD Piña MJ Fueyo J Menor C Román ID Guijarro LG Pretreatment with FK506 up-regulates insulin receptors in regenerating rat liver.Hepatology. 2002; 36: 555-561Crossref PubMed Scopus (12) Google Scholar We tested the potential effect of FK506 on promoti
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