Transcriptional, Functional, and Mechanistic Comparisons of Stem Cell–Derived Hepatocytes, HepaRG Cells, and Three-Dimensional Human Hepatocyte Spheroids as Predictive In Vitro Systems for Drug-Induced Liver Injury

Abstract

Reliable and versatile hepatic in vitro systems for the prediction of drug pharmacokinetics and toxicity are essential constituents of preclinical safety assessment pipelines for new medicines. Here, we compared three emerging cell systems—hepatocytes derived from induced pluripotent stem cells, HepaRG cells, and three-dimensional primary human hepatocyte (PHH) spheroids—at transcriptional and functional levels in a multicenter study to evaluate their potential as predictive models for drug-induced hepatotoxicity. Transcriptomic analyses revealed widespread gene expression differences between the three cell models, with 8148 of 17,462 analyzed genes (47%) being differentially expressed. Expression levels of genes involved in the metabolism of endogenous as well as xenobiotic compounds were significantly elevated in PHH spheroids, whereas genes involved in cell division and endocytosis were significantly upregulated in HepaRG cells and hepatocytes derived from induced pluripotent stem cells, respectively. Consequently, PHH spheroids were more sensitive to a panel of drugs with distinctly different toxicity mechanisms, an effect that was amplified by long-term exposure using repeated treatments. Importantly, toxicogenomic analyses revealed that transcriptomic changes in PHH spheroids were in compliance with cholestatic, carcinogenic, or steatogenic in vivo toxicity mechanisms at clinically relevant drug concentrations. Combined, the data reveal important phenotypic differences between the three cell systems and suggest that PHH spheroids can be used for functional investigations of drug-induced liver injury in vivo in humans.

An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D

“…When analyzing the individual compound effects on the 2D and 3D HepaRG cultures, it is important to understand that different cell systems respond slightly differently to liver toxins, dependent on the compound toxicity mechanism. Although primary human hepatocytes, in 2D and 3D, are the gold standard for testing several mechanisms of hepatotoxicity (e.g., cholestasis, steatosis, and carcinogenesis), 39 HepaRG cells can also be utilized for various toxicity and mechanistic testing applications, including steatosis, 40,41 phospholipidosis, 40 cholestasis, 42 genotoxicity, 43 and mitochondrial dysfunction. 44 The compounds evaluated in this experiment are associated with steatosis (i.e., amiodarone and tamoxifen), mitochondrial dysfunction (i.e., amiodarone, rosiglitazone, benzbromarone, and tetracycline), and cholestasis (i.e., rifampicin).…”

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity: Emerging Culture Paradigms and Applications

“…The phenotypes of such systems are however drastically different from the liver in vivo. Specifically, seeding of hepatocytes on stiff, collagen‐coated plastic culture plates entails a rapid loss of hepatic functions, mediated by a burst of different microRNAs that inhibit hepatic gene expression already 30 min after exposure to the substratum . Accordingly, in the last decade, it has become evident that 2D cultures of hepatocytes have important limitations and do not faithfully inform about liver biology and drug response in vivo.…”

Characterization of hepatocyte-based in vitro systems for reliable toxicity testing

“…The induced pluripotent stem cell technique has undoubtedly gained most attention in this respect. However, despite being promising, this procedure could still benefit from improvement, as the resulting hepatocyte-like cells are not able to pick up all types of liver insults triggered by chemical compounds (Bell et al 2017).…”