This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.

Human adult stem cells (hASCs) have become an attractive source for autologous cell transplantation, tissue engineering, developmental biology, and the generation of human-based alternative in vitro models. Among the 3 germ cell layers, the mesoderm is the origin of today's most widely used and characterized hASC populations. A variety of isolated nonhematopoietic mesoderm-derived stem cell populations exist, and all of them show important differences in terms of function, efficacy, and differentiation potential both in vivo and in vitro. To better understand whether the intrinsic properties of these cells contribute to the overall differentiation potential of hASCs, we compared the global gene expression profiles of 4 mesoderm-derived stem cell populations: human adipose tissue-derived stromal cells, human bone marrow-derived stromal cells (hBMSCs), human (fore)skin-derived precursor cells (hSKPs), and human Wharton's jelly-derived mesenchymal stem cells (hWJs). Significant differences in gene expression profiles were detected between distinct stem cell types. hSKPs predominantly expressed genes involved in neurogenesis, skin, and bone development, whereas hWJs and, to some extent, hBMSCs showed an increased expression of genes involved in cardiovascular and liver development. Interestingly, the observed differential gene expression of distinct hASCs could be linked to existing differentiation data in which hASCs were differentiated toward specific cell types. As such, our data suggest that the intrinsic gene expression of the undifferentiated stem cells has an important impact on their overall differentiation potential as well as their application in stem cell-based research. Yet, the factors that define these intrinsic properties remain to be determined.

In the last decade, microRNAs have emerged as key factors that negatively regulate mRNA expression. It has been estimated that more than 50% of protein-coding genes are under microRNA control and each microRNA is predicted to repress several mRNA targets. In this respect, it is recognized that microRNAs play a vital role in various cellular and molecular processes and that, depending on the biological pathways in which they intervene, distorted expression of microRNAs can have serious consequences. It has recently been shown that specific microRNA species are also correlated with toxic responses induced by xenobiotics. Since the latter are primarily linked to the extent of detoxification in the liver by phase I and phase II biotransformation enzymes and influx and efflux drug transporters, the regulation of the mRNA levels of this particular set of genes through microRNAs is of great importance for the overall toxicological outcome. Consequently, in this paper, an overview of the current knowledge with respect to the complex interplay between microRNAs and the expression of biotransformation enzymes and drug transporters in the liver is provided. Nuclear receptors and transcription factors, known to be involved in the transcriptional regulation of these genes, are also discussed.

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