Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen

AbstractThe establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli

Induction of sterilizing immunity against West Nile Virus (WNV), by immunization with WNV-like particles produced in insect cells

No specific vaccine for West Nile virus (WNV) is currently available for human use. In the present study, we describe the generation of WNV-like particles (WNV-LPs) in insect cells by use of recombinant baculoviruses expressing the WNV structural proteins prME or CprME. BALB/c mice immunized with purified WNV-LPs developed WNV-specific antibodies that had potent neutralizing activities. Mice immunized with prME-like particles (prME-LPs) showed no morbidity or mortality after challenge with WNV. Immunization with prMELPs can induce sterilizing immunity without producing any evidence of viremia or viral RNA in the spleen or brain. These results suggest that WNV-LPs hold promise as a vaccine candidate for WNV infection

Ribavirin potentiates interferon action by augmenting interferon-stimulated gene induction in hepatitis C virus cell culture models

The combination of pegylated interferon (PEG-IFN) and ribavirin is the standard treatment for chronic hepatitis C. Our recent clinical study suggests that ribavirin augments the induction of interferon-stimulated genes (ISGs) in patients treated for hepatitis C virus (HCV) infection. In order to further characterize the mechanisms of action of ribavirin, we examined the effect of ribavirin treatment on ISG induction in cell culture. In addition, the effect of ribavirin on infectious HCV cell culture systems was studied. Similar to interferon (IFN)-α, ribavirin potently inhibits JFH-1 infection of Huh7.5.1 cells in a dose-dependent manner, which spans the physiological concentration of ribavirin in vivo. Microarray analysis and subsequent quantitative polymerase chain reaction assays demonstrated that ribavirin treatment resulted in the induction of a distinct set of ISGs. These ISGs, including IFN regulatory factors 7 and 9, are known to play an important role in anti-HCV responses. When ribavirin is used in conjunction with IFN-α, induction of specific ISGs is synergistic when compared with either drug applied separately. Direct up-regulation of these antiviral genes by ribavirin is mediated by a novel mechanism different from those associated with IFN signaling and intracellular double-stranded RNA sensing pathways such as RIG-I and MDA5. RNA interference studies excluded the activation of the Toll-like receptor and nuclear factor κB pathways in the action of ribavirin

Hepatitis C virus treatment in the real world: optimising treatment and access to therapies

Chronic HCV infections represent a major worldwide public health problem and are responsible for a large proportion of liver related deaths, mostly because of HCV-associated hepatocellular carcinoma and cirrhosis. The treatment of HCV has undergone a rapid and spectacular revolution. In the past 5 years, the launch of direct acting antiviral drugs has seen sustained virological response rates reach 90% and above for many patient groups. The new treatments are effective, well tolerated, allow for shorter treatment regimens and offer new opportunities for previously excluded groups. This therapeutic revolution has changed the rules for treatment of HCV, moving the field towards an interferon-free era and raising the prospect of HCV eradication. This manuscript addresses the new challenges regarding treatment optimisation in the real world, improvement of antiviral efficacy in `hard-to-treat' groups, the management of patients whose direct acting antiviral drug treatment was unsuccessful, and access to diagnosis and treatment in different parts of the world

Development of an Aryloxazole Class of Hepatitis C Virus Inhibitors Targeting the Entry Stage of the Viral Replication Cycle

Reliance on hepatitis C virus (HCV) replicon systems and protein-based screening assays has led to treatments that target HCV viral replication proteins. The model does not encompass other viral replication cycle steps such as entry, processing, assembly and secretion, or viral host factors. We previously applied a phenotypic high-throughput screening platform based on an infectious HCV system and discovered an aryloxazole-based anti-HCV hit. Structure– activity relationship studies revealed several compounds exhibiting EC50 values below 100 nM. Lead compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in combination with existing HCV drugs. In vivo pharmacokinetics studies of 7ii showed high liver distribution and long half-life without obvious hepatotoxicity. The lead compounds are promising as preclinical candidates for the treatment of HCV infection and as molecular probes to study HCV pathogenesis

High-Throughput Screening, Discovery, and Optimization to Develop a Benzofuran Class of Hepatitis C Virus Inhibitors

Using a high-throughput, cell-based HCV luciferase reporter assay to screen a diverse small-molecule compound collection (~300 000 compounds), we identified a benzofuran compound class of HCV inhibitors. The optimization of the benzofuran scaffold led to the identification of several exemplars with potent inhibition (EC50 25 µM), and excellent selectivity (selective index = CC50/EC50, > 371-fold). The structure–activity studies culminated in the design and synthesis of a 45-compound library to comprehensively explore the anti-HCV activity. The identification, design, synthesis, and biological characterization for this benzofuran series is discussed

Modeling PNPLA3 ‐Associated NAFLD Using Human‐Induced Pluripotent Stem Cells

Funder: NIH Oxford Cambridge Scholars ProgramFunder: Intramural Research Program of NIDDKFunder: European Research Council GrantFunder: Cambridge Hospitals National Institute for Health Research Biomedical Research CelnterFunder: Wellcome Trust and Medical Research Council Cambridge Stem Cell InsituteFunder: National Centre for the Replacement Refinement and Reduction of Animals in Research; Id: http://dx.doi.org/10.13039/501100010757Funder: Wellcome Sanger InstituteBackground and Aims: NAFLD is a growing public health burden. However, the pathogenesis of NAFLD has not yet been fully elucidated, and the importance of genetic factors has only recently been appreciated. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in patatin‐like phospholipase domain‐containing protein 3 (PNPLA3). Nonetheless, very little is known about the mechanisms by which this gene and its variants can influence disease development. To investigate these mechanisms, we have developed an in vitro model that takes advantage of the unique properties of human‐induced pluripotent stem cells (hiPSCs) and the CRISPR/CAS9 gene editing technology. Approach and Results: We used isogenic hiPSC lines with either a knockout (PNPLA3KO) of the PNPLA3 gene or with the I148M variant (PNPLA3I148M) to model PNPLA3‐associated NAFLD. The resulting hiPSCs were differentiated into hepatocytes, treated with either unsaturated or saturated free fatty acids to induce NAFLD‐like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3KO hepatocytes showed higher lipid accumulation as well as an altered pattern of response to lipid‐induced stress. Interestingly, loss of PNPLA3 also caused a reduction in xenobiotic metabolism and predisposed PNPLA3KO cells to be more susceptible to ethanol‐induced and methotrexate‐induced toxicity. The PNPLA3I148M cells exhibited an intermediate phenotype between the wild‐type and PNPLA3KO cells. Conclusions: Together, these results indicate that the I148M variant induces a loss of function predisposing to steatosis and increased susceptibility to hepatotoxins

Hepatic abnormalities in patients with chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare congenital disorder characterized by repeated bacterial and fungal infections. Aside from a high incidence of liver abscess, little is known about hepatic involvement in CGD. The aim of this study was to describe the spectrum of liver abnormalities seen in CGD. The charts of 194 patients with CGD followed at the NIH were reviewed, with a focus on liver abnormalities. Liver enzyme elevations occurred on at least one occasion in 73% of patients during a mean of 8.9 years of follow-up. ALT elevations were generally transient. Although transient alkaline phosphatase (ALP) elevations were also common, persistent ALP elevations lasting up to 17.6 years were seen in 25% of patients. Liver abscess occurred in 35% of patients. Drug-induced hepatotoxicity was documented in 15% of patients but likely occurred more frequently. Hepatomegaly was found in 34% and splenomegaly in 56% of patients. Liver histology showed granulomata in 75% and lobular hepatitis in 90% of specimens. Venopathy of the portal vein was common (80%) and associated with splenomegaly. Venopathy of the central vein was also common (63%) and was associated with the number of abscess episodes. Nodular regenerative hyperplasia (NRH) was seen in 9 patients, including 6 of 12 autopsy specimens. CONCLUSION: Liver enzyme abnormalities occur frequently in patients with CGD. In addition to liver abscesses and granulomata, drug hepatotoxicity is likely underappreciated. Vascular lesions such as venopathy and--to a lesser extent--NRH are common. The cause and clinical consequences of venopathy await prospective evaluation

Defective Hepatic Response to Interferon and Activation of Suppressor of Cytokine Signaling 3 in Chronic Hepatitis C

Approximately half of hepatitis C virus (HCV)-infected patients do not respond to current interferon (IFN)-α combination therapy. To understand IFN-α resistance in vivo, we examined the dynamic responses to both type I and type II IFNs, human IFN (hIFN)- α, -γ, and consensus IFN, in the chimpanzee model