Seurat-1: HepaRG, repeated and single dose exposure for Mitochondrial Health and LipidTox

The purpose of this report is to describe the experimental procedure used in order to detect changes in mitochondrial membrane potential and lipid accumulation following exposure of HepaRG cells to various chemicals both by repeated exposure as single exposure to chemicals. This procedure was created for the SEURAT-1 Project runs 15 to 18 and was developed by using live cell imaging.JRC.I.5-Systems Toxicolog

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells are able to differentiate into various cell types that can be applied in human based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. Here we describe a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. We defined which signalling pathways are regulated and/or activated upon neuronal differentiation. Providing this information is critical in order to be able to apply the cell model for the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept we used rotenone, an inhibitor of mitochondrial respiratory complex I, to assess the activation of Nrf2 signaling pathway, a key regulator of the Antioxidant-Response-Element-(ARE)-driven cellular defense mechanism against oxidative stress.JRC.F.3-Chemicals Safety and Alternative Method

Nrf2 pathway activation upon rotenone treatment in human iPSC-derived neural stem cells undergoing differentiation towards neurons and astrocytes

Activation of Nrf2/ARE signaling pathway occurs ubiquitously in most cell types upon induction of oxidative stress. Rotenone, an inhibitor of mitochondrial complex I, can be used to trigger oxidative stress, stimulate the activation of Nrf2 pathway in neuronal and astrocytic cells and assess neurotoxicity. We have previously demonstrated that an acute treatment with rotenone can induce Nrf2 activation, which leads to astrocyte activation and dopaminergic (DA) neuronal cell death in a mixed neuronal/astrocytic cell model derived from human induced pluripotent stem cells (hiPSCs). In this study, we characterized the effects of a repeated dose treatment with rotenone (14 days) on hiPSC-derived neural stem cells (NSCs) undergoing differentiation, assessing the expression and the activation of the Nrf2 pathway. Our results show that Nrf2 signaling increases during NSC differentiation. Moreover, we observed that rotenone treatment induced a progressive activation of Nrf2 signaling together with a transient induction of astrocyte reactivity, a reduction of neurite length leading to neuronal cell death, in particular of DA neurons. Altogether these data indicate that hiPSC-NSC models are relevant test systems for the evaluation of Nrf2 pathway activation upon induced oxidative stress, allowing further understanding of the molecular mechanisms underlying exposure to (developmental) neurotoxicants.JRC.F.3-Chemicals Safety and Alternative Method

Regenerative toxicology: the role of stem cells in the development of chronic toxicities

Human stem cell lines and their derivatives have been widely discussed as cellular models in predictive toxicology. Huge efforts are currently undertaken to investigate the suitability of stem cells derivatives as unlimited cellular source for the development of in vitro toxicity tests that qualify for a better understanding of the mode of action of hazardous chemicals. Nevertheless, the contribution of stem cells and precursors to the development of chronic toxicities and carcinogenesis have not received so much attention so far and remains a gap in the current research strategy on stem cells as tools for toxicity testing in vitro. The current review describes selected examples of toxicants that are targeting stem cells/progenitor cells which result in the development of long term toxicities.JRC.I.5-Systems Toxicolog