Abstract
BackgroundWidely accessible small animal models suitable for the study of hepatitis C virus (HCV) in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice.Methodology/Principal FindingsWe report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH) within a vascularized rat collagen type I/human fibronectin (rCI/hFN) gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC) in severe combined immunodeficient X beige (SCID/bg) mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7) mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4α (HNF4α) mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM) components and/or hepatocyte growth factor (HGF)-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM) analyses of gel tissue.Conclusion/SignificanceThe system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.
Highlights
Hepatitis C virus (HCV) is a major cause of morbidity and mortality, resulting in hepatitis, cirrhosis and liver failure, hepatocellular carcinoma and immune complex vasculitis
We report here on the development of a simple model of primary human hepatoblast and hepatoma cell engraftment and hepatitis C virus (HCV) infection in healthy, adult immunodeficient mice
We chose to use human fetal hepatoblasts (HFH), rather than human adult hepatocytes, for several reasons: 1) these cells are routinely available well-suited for scheduled transplantation experiments; 2) HFH may be expanded in vitro by using conditions that maintain typical hepatocytic morphology and gene expression patterns even after several months of culture [15]; 3) HFH may be transduced with lentiviruses in vitro [29,30]; and 4) HFH may be combined with hematopoietic stem cell (HSC) co-engraftment from the same human fetal liver [13,31] to investigate the development of an immunocompetent mouse model for studies of HCV pathogenesis and pre-clinical testing of prophylactic and therapeutic HCV vaccines
Summary
Hepatitis C virus (HCV) is a major cause of morbidity and mortality, resulting in hepatitis, cirrhosis and liver failure, hepatocellular carcinoma and immune complex vasculitis. The most successful mouse model to date uses albumin promoter-urokinase-type plasminogen activator (Alb-uPA)-transgenic mice that overexpress uPA in the liver which results in hypofibrinogenemia, neonatal bleeding and progressive liver degeneration [1]. This defect is lethal in homozygous mice, they can be rescued by transplanting wild-type murine or human hepatocytes within the first several weeks of life [2,3]. Accessible small animal models suitable for the study of hepatitis C virus (HCV) in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not engrafted in immunodeficient mice
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