Regina Stöber

Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells

Graphical abstract

Transcriptomics of Hepatocytes Treated with Toxicants for Investigating Molecular Mechanisms Underlying Hepatotoxicity

Transcriptomics is a powerful tool for high-throughput gene expression profiling. Transcriptome microarray experiments conducted with RNA isolated from hepatocytes after exposure to toxicants enable a deep insight into the molecular mechanisms of hepatotoxicity. This understanding, along with structure-activity relationships underlying hepatotoxicity, will provide a novel strategy to design cost-effective and safer therapeutics. Transcriptomics studies conducted with established hepatotoxic drugs in various in vitro and in vivo hepatotoxicity test systems have contributed to the elucidation of the mechanistic basis of liver insults, which were later on substantiated at the proteomics and metabolomics levels. The present chapter is focused on comprehensive transcriptomics of cultured primary hepatocytes treated with chemicals by applying Affymetrix microarray technology. It also describes the detailed protocol for culturing of hepatocytes, their exposure to toxicants as well as sample collection, including RNA isolation, RNA target preparation and finally the hybridization to gene chips for microarray expression analysis.

“Watching the Detectives” report of the general assembly of the EU project DETECTIVE Brussels, 24–25 November 2015

Abstract SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and “-omics” technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich “-omics” databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.

Highlight report: False positives in genotoxicity testing

Genotoxicity testing represents a central topic in toxicology (Oka et al. 2018; Barcham et al. 2018; Ireno et al. 2014; Zrnzevic et al. 2017). One current challenge is the relatively high number of false positive results. A second problem is that in vivo mutagenicity tests, such as the mouse micronucleus assay, are no longer allowed for testing of cosmetics. To improve the situation, Gamze Ates and colleagues from Brussels University have established a novel assay based on gene expression in Hepa RG cells (Ates et al. 2018). The authors tested a set of 84 genes by qRT-PCR and successfully differentiated five known positive and five negative test compounds. Currently, much effort is invested to improve in vitro (Leist et al. 2017; Cipriano et al. 2017; Deharde et al. 2016; Luckert et al. 2017; Reif et al. 2015) and in silico (Hoehme et al. 2018; Schenk et al. 2017; Ghallab et al. 2016) systems. Although the current approach of Ates and colleagues is of high interest it still requires larger numbers of test compounds and blinded testing to study whether the accuracy of the novel assay is sufficient. Moreover, a systematic comparison to the currently applied test battery to the results obtained with human hepatocytes would be highly welcome in future.

Intracellular lysophosphatidic acid influences cell migration

Altered cellular metabolism has been intensively studied in relation to carcinogenesis and tumor development (Currie et al. 2013; Pavlova and Thompson 2016; Santos and Schulze 2012). However, relatively, little is known about the mechanisms how choline metabolism influences the tumor phenotype (Okazaki et al. 2010; Glunde et al. 2015; Granata et al. 2014, 2015; Marchan et al. 2012; Ghallab 2014; Hassan 2017). In the September issue of Cancer Research, a new concept has been presented how choline metabolism influences the phenotype of tumor cells (Marchan et al. 2017): the intracellular concentration of the signaling lipid lysophosphatidic acid (LPA) is critical for tumor cell migration. In the first step, the authors overexpressed and knocked down glycerol-3-phosphate acetyltransferase 1 (GPAM) in tumor cell lines. This influenced intracellular concentrations of LPA, which was associated with higher or lower migration activity. However, the casual relationship between intracellular LPA and migration was established by an elegant method. This technique allows the direct introduction of LPA into cells by a cationic transfection reagent (Marchan et al. 2017). This experiment formally proved that tumor cell migration is stimulated by increased intracellular LPA concentrations. This finding is conceptually new because so far LPA was only known to act via extracellular receptors. In the past decades, numerous factors have been shown to influence prognosis of carcinomas, such as the humoral and cellular immune system (Schmidt et al. 2008; Heimes et al. 2017a, b; Godoy et al. 2014), cytoskeleton (Hellwig et al. 2016; Stock et al. 2015), redox system (Cadenas et al. 2010), and circulation clock-associated factors (Cadenas et al. 2014). The observation that intracellular choline metabolites influence tumor cell migration and adhesion is relatively new and the responsible mechanisms have not yet been fully elucidated (Stewart et al. 2012; Marchan et al. 2012; Lesjak et al. 2014). The recently discovered mechanism of intracellular LPA adds a new aspect of how choline metabolism contributes to the phenotype of tumor cells.

Corrigendum to “Gene networks and transcription factor motifs defining the differentiation of human stem cells into hepatocyte-like cells” [J Hepatol 2015;63:934–942]

IfADo-Leibniz Research Centre for Working Environment and Human Factors at the Technical University Dortmund, Dortmund, Germany; Leibniz Institute for Natural Product Research and Infection Biology eV-Hans-Knöll Institute, Jena, Germany; University of Cologne, Institute of Neurophysiology and Center for Molecular Medicine Cologne (CMMC), Robert-Koch-Str. 39, 50931 Cologne, Germany; MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh EH16 4UU, United Kingdom; Takara Bio Europe AB (former Cellartis AB), Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden; Systems Biology Research Center, School of Bioscience, University of Skövde, Sweden; NovaHep AB, Arvid Wallgrens Backe 20, 41346 Gothenburg, Sweden; Charité University Medicine Berlin, Department of General-, Visceraland Transplantation Surgery, D13353 Berlin, Germany; Center for Liver Cell Research, Department of Pediatrics and Juvenile Medicine, University of Regensburg Hospital, Regensburg, Germany; Eberhard Karls University Tübingen, BG Trauma Center, Siegfried Weller Institut, D72076 Tübingen, Germany; Department of Physiology, Faculty of Biological Sciences, University of Concepción, Chile