95 research outputs found

    Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes

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    The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various invivo and invitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable invitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na+-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasi

    Hepatic differentiation of human pluripotent stem cells on human liver progenitor HepaRG-derived acellular matrix

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    Human hepatocytes are extensively needed in drug discovery and development. Stem cell-derived hepatocytes are expected to be an improved and continuous model of human liver to study drug candidates. Generation of endoderm-derived hepatocytes from human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent stem cells, is a complex, challenging process requiring specific signals from soluble factors and insoluble matrices at each developmental stage. In this study, we used human liver progenitor HepaRG-derived acellular matrix (ACM) as a hepatic progenitor-specific matrix to induce hepatic commitment of hPSC-derived definitive endoderm (DE) cells. The DE cells showed much better attachment to the HepaRG ACM than other matrices tested and then differentiated towards hepatic cells, which expressed hepatocyte-specific makers. We demonstrate that Matrigel overlay induced hepatocyte phenotype and inhibited biliary epithelial differentiation in two hPSC lines studied. In conclusion, our study demonstrates that the HepaRG ACM, a hepatic progenitor-specific matrix, plays an important role in the hepatic differentiation of hPSCs. (C) 2016 Elsevier Inc. All rights reserved.Peer reviewe

    Advances in tetrahydropyrido[1,2-a]isoindolone (valmerins) series: Potent glycogen synthase kinase 3 and cyclin dependent kinase 5 inhibitors.

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    International audienceAn efficient synthetic strategy was developed to modulate the structure of the tetrahydropyridine isoindolone (Valmerin) skeleton. A library of more than 30 novel final structures was generated. Biological activities on CDK5 and GSK3 as well as cellular effects on cancer cell lines were measured for each novel compound. Additionally docking studies were performed to support medicinal chemistry efforts. A strong GSK3/CDK5 dual inhibitor (38, IC50 GSK3/CDK5 32/84 nM) was obtained. A set of highly selective GSK3 inhibitors was synthesized by fine-tuning structural modifications (29 IC50 GSK3/CDK5 32/320 nM). Antiproliferative effects on cells were correlated with the in vitro kinase activities and the best effects were obtained with lung and colon cell lines

    Nanofibrillar cellulose hydrogel promotes three-dimensional liver cell culture

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    Over the recent years, various materials have been introduced as potential 3D cell culture scaffolds. These include protein extracts, peptide amphiphiles, and synthetic polymers. Hydrogel scaffolds without human or animal borne components or added bioactive components are preferred from the immunological point of view. Here we demonstrate that native nanofibrillar cellulose (NFC) hydrogels derived from the abundant plant sources provide the desired functionalities. We show 1) rheological properties that allow formation of a 3D scaffold in-situ after facile injection, 2) cellular biocompatibility without added growth factors, 3) cellular polarization, and 4) differentiation of human hepatic cell lines HepaRG and HepG2. At high shear stress, the aqueous NFC has small viscosity that supports injectability, whereas at low shear stress conditions the material is converted to an elastic gel. Due to the inherent biocompatibility without any additives, we conclude that NFC generates a feasible and sustained microenvironment for 3D cell culture for potential applications, such as drug and chemical testing, tissue engineering, and cell therapy.Peer reviewe

    General review on in vitro hepatocyte models and their applications.

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    International audienceIn vitro hepatocyte models represent very useful systems in both fundamental research and various application areas. Primary hepatocytes appear as the closest model for the liver in vivo. However, they are phenotypically unstable, have a limited life span and in addition, exhibit large interdonor variability when of human origin. Hepatoma cell lines appear as an alternative but only the HepaRG cell line exhibits various functions, including major cytochrome P450 activities, at levels close to those found in primary hepatocytes. In vitro hepatocyte models have brought a substantial contribution to the understanding of the biochemistry, physiology, and cell biology of the normal and diseased liver and in various application domains such as xenobiotic metabolism and toxicity, virology, parasitology, and more generally cell therapies. In the future, new well-differentiated hepatocyte cell lines derived from tumors or from either embryonic or adult stem cells might be expected and although hepatocytes will continue to be used in various fields, these in vitro liver models should allow marked advances, especially in cell-based therapies and predictive and mechanistic hepatotoxicity of new drugs and other chemicals. All models will benefit from new developments in throughput screening based on cell chips coupled with high-content imaging and in toxicogenomics technologies

    Stem cell-derived hepatocytes and their use in toxicology.

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    International audienceBetter prediction of safety risk and understanding of mechanism of action of drug candidates remain a major challenge in order to prevent late stage attrition. Continuous efforts are made to improve and develop new models, especially in some areas such as hepatotoxicity. Besides primary hepatocytes and transformed liver cell lines, stem cells either isolated from embryos or adult tissues or obtained by reprogramming somatic cells are emerging as a new potential source of unlimited numbers of hepatocytes. Presently, only hepatocyte-like cells expressing low levels of liver-specific markers, especially drug metabolizing and detoxifying enzymes, are usually obtained, making them still unsuitable as metabolically competent cells for toxicity studies. The only exceptions are some hepatoma cell lines, particularly the HepaRG cell line that can differentiate from a bipotent progenitor stage to attain the functional capacity of normal adult hepatocytes in primary culture without losing the indefinite growth property of transformed cells. Since the research field on stem cells is growing fast marked advances might be expected in the next future

    Optimization of the HepaRG cell model for drug metabolism and toxicity studies.

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    International audienceThe HepaRG cell line is the first human cell line able to differentiate in vitro into mature hepatocyte-like cells. Our main objective within the framework of the EEC-LIINTOP project was to optimize the use of this cell line for drug metabolism and toxicity studies, especially after repeat treatments. The main results showed that differentiated HepaRG cells: (i) retained their drug metabolism capacity (major CYPs, phase 2 enzymes, transporters and nuclear receptors) and responsiveness to prototypical inducers at relatively stable levels for several weeks at confluence. The levels of several functions, including some CYPs such as CYP3A4, were dependent on the addition of dimethyl sulfoxide in the culture medium; (ii) sustained the different types of chemical-induced hepatotoxicity, including steatosis, phospholipidosis and cholestasis, after acute and/or repeat treatment with reference drugs. In particular, drug-induced vesicular steatosis was demonstrated in vitro for the first time. In conclusion, our results from the LIINTOP project, together with other studies reported concomitantly or more recently in the literature, support the conclusion that the metabolically competent human HepaRG cells represent a surrogate to primary human hepatocytes for investigating drug metabolism parameters and both acute and chronic effects of xenobiotics in human liver

    Early alterations of bile canaliculi dynamics and the rho kinase/myosin light chain kinase pathway are characteristics of drug-induced intrahepatic cholestasis

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    International audienceIntrahepatic cholestasis represents 20%-40%of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells and a set of 12 cholestatic drugs and six noncholestatic drugs. In this study, we analyzed bile canaliculi dynamics, Rho kinase (ROCK)/myosin light chain kinase (MLCK) pathway implication, efflux inhibition of taurocholate [a predominant bile salt export pump (BSEP) substrate], and expression of the major canalicular and basolateral bile acid transporters. We demonstrated that 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both bile canaliculi dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds, by contrast, had no effect. Cotreatment with ROCK inhibitor Y-27632 [4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride] and MLCK activator calmodulin reduced bile canaliculi constriction and dilatation, respectively, confirming the role of these pathways in drug-induced intrahepatic cholestasis. By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles was not observed with all cholestatic drugs; moreover, examples of noncholestatic compounds were reportedly found to inhibit BSEP. Transcripts levels of major bile acid transporters were determined after 24-hour treatment. BSEP, Na+-taurocholate cotransporting polypeptide, and organic anion transporting polypeptide B were downregulated with most cholestatic and some noncholestatic drugs, whereas deregulation of multidrug resistance-associated proteins was more variable, probably mainly reflecting secondary effects. Together, our results show that cholestatic drugs consistently cause an early alteration of bile canaliculi dynamics associated with modulation of ROCK/MLCK and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis

    Progressive and Preferential Cellular Accumulation of Hydrophobic Bile Acids Induced by Cholestatic Drugs Is Associated with Inhibition of Their Amidation and Sulfation

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    International audienceDrug-induced intrahepatic cholestasis is characterized by cellular accumulation of bile acids (BAs), whose mechanisms remain poorly understood. The present study aimed to analyze early and progressive alterations of BA profiles induced by cyclosporine A, chlorpromazine, troglitazone, tolcapone, trovafloxacin, and tacrolimus after 4-hour, 24-hour, and 6-day treatments of differentiated HepaRG cells. In BA-free medium, the potent cholestatic drugs cyclosporine A, chlorpromazine, and troglitazone reduced endogenous BA synthesis after 24 hours, whereas the rarely cholestatic drugs tolcapone, trovafloxacin, and tacrolimus reduced BA synthesis only after 6 days. In the presence of physiologic serum BA concentrations, cyclosporine A, chlorpromazine, and troglitazone induced early and preferential cellular accumulation of unconjugated lithocholic, deoxycholic, and chenodeoxycholic acids that increased 8- to 12-fold and 47- to 50-fold after 24 hours and 6 days, respectively. Accumulation of these hydrophobic BAs resulted from strong inhibition of amidation, and in addition, for lithocholic acid reduction of its sulfoconjugation, and was associated with variable alterations of uptake and efflux transporters. Trovafloxacin also caused BA accumulation, especially after 6 days, whereas tolcapone and tacrolimus were still without effect. However, when exogenous BAs were added to the medium at cholestatic serum concentrations, a 6-day treatment with all drugs resulted in cellular BA accumulation with higher folds of chenodeoxycholic and lithocholic acids. At the tested concentration, tolcapone had the lowest effect. These results bring the first demonstration that major cholestatic drugs can cause preferential and progressive in vitro cellular accumulation of unconjugated toxic hydrophobic BAs and bring new insights into mechanisms involved in drug-induced cellular accumulation of toxic BAs

    Predictive Value of Cellular Accumulation of Hydrophobic Bile Acids as a Marker of Cholestatic Drug Potential

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    International audienceDrug-induced cholestasis is mostly intrahepatic and characterized by alterations of bile canaliculi dynamics and morphology as well as accumulation of bile acids (BAs) in hepatocytes. However, little information exists on first changes in BA content and profile induced by cholestatic drugs in human liver. In the present study we aimed to analyze the effects of a large set of cholestatic and noncholestatic drugs in presence of physiological serum concentrations and 60-fold higher levels of 9 main BAs on cellular accumulation of BAs using HepaRG hepatocytes. BAs were measured in cell layers (cells + bile canaliculi) and culture media using HPLC coupled with tandem MS/MS after 24h-treatment. Comparable changes in total and individual BA levels were observed in cell layers and media from control and noncholestatic drug-treated cultures unconjugated BAs were actively amidated and lithocholic acid (LCA) was entirely sulfated. By contrast, cellular accumulation of LCA and in addition, of the two other hydrophobic BAs, chenodeoxycholic acid and deoxycholic acid, was evidenced only with cholestatic compounds in presence of BA mixtures at normal and 60-fold serum levels, respectively, suggesting that LCA was the first BA to accumulate. Cellular accumulation of hydrophobic BAs was associated with inhibition of their amidation and for LCA, its sulfation. In conclusion, these results demonstrated that cellular accumulation of unconjugated hydrophobic BAs can be caused by various cholestatic drugs in human hepatocytes and suggest that their cellular detection, especially that of LCA, could represent a new strategy for evaluation of cholestatic potential of drugs and other chemicals
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