Persistent hepatic IFN system activation in HBV-HDV infection determines viral replication dynamics and therapeutic response.

Hepatitis delta virus (HDV), a satellite virus of HBV, is regarded as the most severe type of hepatitis virus because of the substantial morbidity and mortality. The IFN system is the first line of defense against viral infections and an essential element of antiviral immunity; however, the role of the hepatic IFN system in controlling HBV-HDV infection remains poorly understood. Herein, we showed that HDV infection of human hepatocytes induced a potent and persistent activation of the IFN system whereas HBV was inert in triggering hepatic antiviral response.

Functional changes of cocultured hepatocyte sheets subjected to continuous liver regeneration stimulation in cDNA-uPA/SCID mouse: Differences in transplantation sites.

Aim: The formation of a secondary liver is expected in ectopic transplants in liver therapy. It is reported that the transplantation of hepatocyte sheets constitutes one of the techniques used to form a secondary liver. Accordingly, we established a subcutaneous transplant for hepatocyte/fibroblast sheets in previous studies.

Humanized liver mouse model with transplanted human hepatocytes from patients with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency (OTCD) is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. To develop new drugs or therapies for OTCD, it is ideal to use models that are more closely related to human metabolism and pathology. Primary human hepatocytes (HHs) isolated from two patients (a 6-month-old boy and a 5-year-old girl) and a healthy donor were transplanted into host mice (hemi-, hetero-OTCD mice, and control mice, respectively).

Detection of acute toxicity of aflatoxin B1 to human hepatocytes in vitro and in vivo using chimeric mice with humanized livers.

Aflatoxin B1 (AFB1), a mycotoxin, is acutely hepatotoxic to many animals including humans. However, there are marked interspecies differences in sensitivity to AFB1-induced toxicity depending on bioactivation by cytochrome P450s (CYPs). In the present study, we examined the applicability of chimeric mice with humanized livers and derived fresh human hepatocytes for in vivo and vitro studies on AFB1 cytotoxicity to human hepatocytes.

Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: Novel, long-term, functional two-dimensional in vitro tool for developing new drugs.

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.

Prediction of Human Hepatic Clearance for Cytochrome P450 Substrates via a New Culture Method Using the Collagen Vitrigel Membrane Chamber and Fresh Hepatocytes Isolated from Liver Humanized Mice.

In drug discovery, hepatocytes have been widely utilized as in vitro tools for predicting the in vivo hepatic clearance (CL) of drug candidates. However, conventional hepatocyte models do not always reproduce in vivo physiological function, and CYP activities in particular decrease quite rapidly during culture. Furthermore, conventional in vitro assays have limitations in their ability to predict hepatic CL of metabolically stable drug candidates.

A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers.

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells®, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice®).

Transmission of HBV DNA Mediated by Ceramide-Triggered Extracellular Vesicles.

Background & Aims: An extracellular vesicle (EV) is a nanovesicle that shuttles proteins, nucleic acids, and lipids, thereby influencing cell behavior. A recent crop of reports have shown that EVs are involved in infectious biology, influencing host immunity and playing a role in the viral life cycle. In the present work, we investigated the EV-mediated transmission of hepatitis B virus (HBV) infection.

Novel robust in vitro hepatitis B virus infection model using fresh human hepatocytes isolated from humanized mice.

The molecular mechanisms underlying the hepatitis B virus (HBV) life cycle are poorly understood because of the lack of appropriate in vitro infection models. Herein, we report a highly effective in vitro HBV infection system using fresh human hepatocytes (HHs) isolated from chimeric mice with humanized livers. After the inoculation of sera collected from HBV-infected chimeric mice or patients to HHs, we measured levels of HBV DNA, mRNA, covalently closed circular DNA, and viral protein expression in HHs.

Chimeric mice with hepatocyte-humanized liver as an appropriate model to study human peroxisome proliferator-activated receptor-α.

Peroxisome proliferator (PP)-activated receptor-α (PPARα) agonists exhibit species-specific effects on livers of the rodent and human (h), which has been considered to reside in the difference of PPARα gene structures. However, the contribution of h-hepatocytes (heps) to the species-specificity remains to be clarified. In this study, the effects of fenofibrate were investigated using a hepatocyte-humanized chimeric mouse (m) model whose livers were replaced with h-heps at >70%.

Chimeric mice with humanized livers: a unique tool for in vivo and in vitro enzyme induction studies.

We performed in vivo and in vitro studies to determine the induction of human cytochrome P450 (CYP) using chimeric mice with humanized liver (PXB-mice®) and human hepatocytes isolated from the PXB-mice (PXB-cells), which were derived from the same donor. For the in vivo study, PXB-mice were injected with 3-methylcholanthrene (3-MC, 2 or 20 mg/kg) or rifampicin (0.1 or 10 mg/kg) for four days.

Morphological and microarray analyses of human hepatocytes from xenogeneic host livers.

We previously produced mice with human hepatocyte (h-hep) chimeric livers by transplanting h-heps into albumin enhancer/promoter-driven urokinase-type plasminogen activator-transgenic severe combined immunodeficient (SCID) mice with liver disease. The chimeric livers were constructed with h-heps, mouse hepatocytes, and mouse hepatic sinusoidal cells (m-HSCs). Here, we investigated the morphological features of the chimeric livers and the h-hep gene expression profiles in the xenogeneic animal body.

Therapeutic potential of propagated hepatocyte transplantation in liver failure.

Background: This study aimed to evaluate the therapeutic potential of intrasplenic transplantation of culture-propagated homologous hepatocytes in rats suffering from acute liver failure (ALF).

Methods: ALF was induced in dipeptidyl peptidase IV-negative (DPPIV(-)) Fischer 344 rats by totally removing the two anterior liver lobes (68% of the liver) and ligating the pedicle of the right lobe (24% of the liver). Hepatocytes isolated from DPPIV(+) Fischer 344 rats were cultured for 11 d to propagate 3-fold, and the resulting hepatocytes were dubbed "culture-propagated hepatocytes (CPHEPs)".

In vitro evaluation of cytochrome P450 and glucuronidation activities in hepatocytes isolated from liver-humanized mice.

Cryopreserved human (h-) hepatocytes are currently regarded as the best in vitro model for predicting human intrinsic clearance of xenobiotics. Although fresh h-hepatocytes have greater plating efficiency on dishes and greater metabolic activities than cryopreserved cells, performing reproducible studies using fresh hepatocytes from the same donor and having an "on demand" supply of fresh hepatocytes are not possible. In this study, cryopreserved h-hepatocytes were transplanted into albumin enhancer/promoter-driven, urokinase-type plasminogen activator, transgenic/severe combined immunodeficient (uPA/SCID) mice to produce chimeric mice, the livers of which were largely replaced with h-hepatocytes.

Hepatic hyperplasia associated with discordant xenogeneic parenchymal-nonparenchymal interactions in human hepatocyte-repopulated mice.

Liver mass is optimized in relation to body mass. Rat (r) and human (h) hepatocytes were transplanted into liver-injured immunodeficient mice and allowed to proliferate for 3 or 11 weeks, respectively, when the transplants stopped proliferating. Liver/body weight ratio was normal throughout in r-hepatocyte-bearing mice (r-hep-mice), but increased continuously in h-hepatocyte-bearing mice (h-hep-mice), until reaching approximately three times the normal m-liver size, which was considered to be hyperplasia of h-hepatocytes because there were no significant differences in cell size among host (mouse [m-]) and donor (r- and h-) hepatocytes.

The mechanism by which mouse spermatozoa are injured during freezing.

To improve the cryopreservation protocol for mouse sperm, we attempted to estimate the type and extent of cryoinjury at various steps of the process. First, we demonstrated that mouse sperm are sensitive to chilling at -15 C and that the sensitivity is dependent on the length of exposure. To estimate cryoinjuries, sperm suspensions were ice-seeded at -5 or -15 C, frozen with liquid nitrogen (LN(2)) gas and then frozen in LN(2).

Susceptibility of chimeric mice with livers repopulated by serially subcultured human hepatocytes to hepatitis B virus.

Unlabelled: We previously identified a small population of replicative hepatocytes in long-term cultures of human adult parenchymal hepatocytes (PHs) at a frequency of 0.01%-0.09%.

Multiplicative mononuclear small hepatocytes in adult rat liver: their isolation as a homogeneous population and localization to periportal zone.

Adult rat liver contains a minor population of hepatocytes called small hepatocytes (SHs) that are smaller in size and show a higher replicative potential than conventional parenchymal hepatocytes (PHs). However, SHs have been hitherto characterized using a "SH-fraction" that was contaminated with PHs. In the present study, we isolated a PH-free SH-fraction from the adult rat liver using fluorescence-activated cell sorter combined with centrifugal elutriation and characterized the hepatocytes in the fraction.

Growth and differentiation of colony-forming human hepatocytes in vitro.

Background/aims: Parenchymal hepatocytes (PHs) of rat contain colony-forming parenchymal hepatocytes (CF-PHs) as a small fraction. We aimed to demonstrate the presence of CF-PHs in humans and characterize them with respect to growth and differentiation potential.

Methods: Human PHs were co-cultured with Swiss 3T3 cells in the medium containing human serum, EGF, nicontinamide, and ascorbic acid 2-phosphate.

Near completely humanized liver in mice shows human-type metabolic responses to drugs.

Human hepatocytes were transplanted into urokinase-type plasminogen activator-transgenic SCID mice (uPA/SCID mice), which are immunodeficient and undergo liver failure. The transplanted cells were characterized in terms of their in vivo growth potential and functions. The human hepatocytes progressively repopulated the murine host liver.

Pleiotrophin/heparin-binding growth-associated molecule as a mitogen of rat hepatocytes and its role in regeneration and development of liver.

Previously pleiotrophin (PTN) was identified among proteins secreted by Swiss 3T3 cells as a mitogen for cultured adult rat hepatocytes. The present study showed that the growth of rat hepatocytes was enhanced when cultured with rat hepatic stellate cells (HSCs). HSCs expressed PTN mRNA and secreted its protein in the co-cultures.