Repopulation of mouse liver with human hepatocytes and in vivo infection with hepatitis B virus

Abstract

Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver diseases and hepatotropic viruses, for which no permissive cell lines exist. Here, we report partial repopulation of the liver of immunodeficient urokinase-type plasminogen activator (uPA)/recombinant activation gene-2 (RAG-2) mice with normal human hepatocytes isolated from the adult liver. In the transplanted mice, the production of human albumin was demonstrated, indicating that human hepatocytes remained functional in the mouse liver for at least 2 months after transplantation. Inoculation of transplanted mice with human hepatitis B virus (HBV) led to the establishment of productive HBV infection. According to human-specific genomic DNA analysis and immunostaining of cryostat liver sections, human hepatocytes were estimated to constitute up to 15% of the uPA/RAG-2 mouse liver. This is proof that normal human hepatocytes can integrate into the mouse hepatic parenchyma, undergo multiple cell divisions, and remain permissive for a human hepatotropic virus in a xenogenic liver. This system will provide new opportunities for studies on etiology and therapy of viral and nonviral human liver diseases, as well as on hepatocyte biology and hepatocellular transplantation. Persistent infection with hepatitis B virus (HBV) is a major worldwide health problem, and chronically infected individuals are at high risk for developing cirrhosis and hepatocellular carcinoma. 1,2 Despite the availability of an HBV vaccine, there are still more than 350 million chronically infected people worldwide, and the few antiviral treatments currently available have a limited rate of efficacy. The narrow host range of HBV and the lack of both in vitro systems and of convenient animal models have greatly hampered our understanding of the complete virus life cycle, as well as the development of more effective antiviral drugs aimed at eradicating the virus from chronic carriers. 3 Chimpanzees are the only animal species infectable with HBV, 4,5 but studies with these animals and evaluation of antiviral therapies are severely restricted because of their limited availability and high costs. Animal models based on HBV-related hepadnaviruses, such as woodchuck and Pekin duck hepatitis B viruses, are often used for assessment of antiviral drugs 6-8 and have provided important information about factors involved in establishment of virus infection, viral persistence, and hepatocarcinogenesis. 9-14 However, woodchucks are relatively large animals of outbred origins that are difficult to handle in many laboratories, and chronic hepadnavirus infection in birds does not lead to cancer. The development of HBV-expressing transgenic mice has also provided important insights regarding viral pathobiology and the role of HBV gene products in hepatocellular injury. 12,[15][16][17][18][19] Although infectious virus can be produced in transgenic mice, their hepatocytes are not permissive for infection. Therefore, the still-unknown early step...

Quantitative Gene Expression Analysis Reveals Transition of Fetal Liver Progenitor Cells to Mature Hepatocytes after Transplantation in uPA/RAG-2 Mice

“…In the heterozygous state some host cells can delete the transgene and form large regeneration nodules. 19,20 The transplanted EGFP-transgenic liver progenitor cells participated in tissue regeneration and formed cohesive cell clusters within the host liver of the uPA mice. The high frequency of small-and medium-sized EGFP-positive regeneration nodules in our experiments suggests efficient initial integration of fetal liver progenitor cells in the host liver and subsequent clonal expansion.…”

“…The uPA/ RAG-2 mice were generated by crossbreeding of uPAtransgenic mice originally described by Sandgren and colleagues 18 with the RAG-2 mouse as described elsewhere. 19,20 All animals were maintained and handled in accordance with institutional guidelines. For preparing fetal liver progenitor cells from EGFP embryos (embryonic day 13.5) the livers were removed under the binocular microscope.…”

Morphological and biochemical characterization of a human liver in a uPA-SCID mouse chimera

“…An environment that favors hepatocyte engraftment is encountered in animals with severe, chronic liver disease caused by the overexpression of a "noxious" protein, as in the urokinase plasminogen activator (uPA)-transgenic mouse. 4 uPA transgenic mice backcrossed with "severe immune deficient" mice, such as Swiss athymic nude (nu/nu) mice, 5 recombination activation gene 2 (RAG-2) knockout mice, 6,7 or SCID/beige mice, 8 allow the engraftment and repopulation by xenogeneic hepatocytes.…”

Generation of the Adenovirus Vector-Mediated CRISPR/Cpf1 System and the Application for Primary Human Hepatocytes Prepared from Humanized Mice with Chimeric Liver

“…However, primary human hepatocytes do not proliferate in culture. To overcome the problem, chimeric mice with humanized livers, [9][10][11] in which the liver has been repopulated with functional human hepatocytes, have been developed and could serve as a source of human liver cells.…”