An Implantable Vascularized Protein Gel Construct That Supports Human Fetal Hepatoblast Survival and Infection by Hepatitis C Virus in Mice

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.

Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens

“…We have also shown that other classic primary human hepatocyte culture models – such as hepatocytes on rigid collagen, collagen gel sandwich, Matrigel spheroids, and even randomly distributed co-cultures of the same two cell types used in MPCCs – do not sustain HBV or HCV infection as robustly as MPCCs 15,28 . Other groups have developed primary human hepatocyte models that permit HBV infection 56 and, more recently, HCV infection 57–59 . Nonetheless, while they do offer initial exciting results, it remains to be determined whether these models offer the stability in hepatic phenotype, reproducibility, and robustness that is routinely observed with MPCCs.…”

Mice engrafted with human hematopoietic stem cells support a human myeloid cell inflammatory response in vivo

“…For several years now, the HIV/AIDS research community has used human bone marrow grafts in immuno‐deficient mice to study HIV/AIDS because HIV tropism for human cells made it impossible to study the biology of HIV infectivity in mice . More recently, and in large part because of their success, these human–mouse chimeric models have been adapted to study human responses in diabetes, sepsis and burn injury . Here we build on the development of these chimeric mouse technologies to show that human myeloid cells can be analyzed in an in vivo model of cutaneous inflammation .…”

“…For example, when the HIV/AIDS research community encountered problems modeling the disease progression in standard mouse models because of the human‐specific tropism of the HIV virus, they created chimeric mice with human CD34 + ‐HSPCs grafts to study infection . Since then, the wider availability of these models has led to their widespread use to study other human diseases including diabetes, sepsis, and burn injury …”

New Methods in Tissue Engineering: Improved Models for Viral Infection

“…Similarly, long-term cultures of primary human fetal hepatocytes were developed by plating a hepatic cell mixture at low density, allowing outgrowth of nonparenchymal cells and formation of hepatic islands that were subsequently cultured under DMSO-containing conditions to maintain characteristic hepatocytic features; these cultures were susceptible to HBV infection for up to 10 weeks in culture (151). In 3D engineered tissues, Bcl-2-transduced HUVECs have been used to modulate fetal hepatocyte phenotype and permit HCV replication (151a). …”