The Embryonic Stem Cell Test as Tool to Assess Structure-Dependent Teratogenicity: The Case of Valproic Acid

Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition

Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop

The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) organised a workshop to discuss the state-of-the-art research on noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and as analytical and therapeutic agents. There was agreement that ncRNA expression profiling data requires careful evaluation to determine the utility of specific ncRNAs as biomarkers. To advance the use of ncRNA in regulatory toxicology, the following research priorities were identified: (1) Conduct comprehensive literature reviews to identify possibly suitable ncRNAs and areas of toxicology where ncRNA expression profiling could address prevailing scientific deficiencies. (2) Develop consensus on how to conduct ncRNA expression profiling in a toxicological context. (3) Conduct experimental projects, including, e.g., rat (90-day) oral toxicity studies, to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Thereby, physiological ncRNA expression profiles should be established, including the biological variability of healthy individuals. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, molecular events should be dose-dependently linked with substance-induced apical effects. Applying a holistic approach, knowledge on ncRNAs, 'omics and epigenetics technologies should be integrated into adverse outcome pathways to improve the understanding of the functional roles of ncRNAs within a regulatory context

Present state and future perspectives of using pluripotent stem cells in toxicology research

The use of novel drugs and chemicals requires reliable data on their potential toxic effects on humans. Current test systems are mainly based on animals or in vitro–cultured animal-derived cells and do not or not sufficiently mirror the situation in humans. Therefore, in vitro models based on human pluripotent stem cells (hPSCs) have become an attractive alternative. The article summarizes the characteristics of pluripotent stem cells, including embryonic carcinoma and embryonic germ cells, and discusses the potential of pluripotent stem cells for safety pharmacology and toxicology. Special attention is directed to the potential application of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for the assessment of developmental toxicology as well as cardio- and hepatotoxicology. With respect to embryotoxicology, recent achievements of the embryonic stem cell test (EST) are described and current limitations as well as prospects of embryotoxicity studies using pluripotent stem cells are discussed. Furthermore, recent efforts to establish hPSC-based cell models for testing cardio- and hepatotoxicity are presented. In this context, methods for differentiation and selection of cardiac and hepatic cells from hPSCs are summarized, requirements and implications with respect to the use of these cells in safety pharmacology and toxicology are presented, and future challenges and perspectives of using hPSCs are discussed

Development of a numerical pump testing framework

It has been shown that left ventricular assist devices (LVADs) increase the survival rate in end-stage heart failure patients. However, there is an ongoing demand for an increased quality of life, fewer adverse events, and more physiological devices. These challenges necessitate new approaches during the design process. In this study, computational fluid dynamics (CFD), lumped parameter (LP) modeling, mock circulatory loops (MCLs), and particle image velocimetry (PIV) are combined to develop a numerical Pump Testing Framework (nPTF) capable of analyzing local flow patterns and the systemic response of LVADs. The nPTF was created by connecting a CFD model of the aortic arch, including an LVAD outflow graft to an LP model of the circulatory system. Based on the same geometry, a three-dimensional silicone model was crafted using rapid prototyping and connected to an MCL. PIV studies of this setup were performed to validate the local flow fields (PIV) and the systemic response (MCL) of the nPTF. After validation, different outflow graft positions were compared using the nPTF. Both the numerical and the experimental setup were able to generate physiological responses by adjusting resistances and systemic compliance, with mean aortic pressures of 72.2–132.6 mm Hg for rotational speeds of 2200–3050 rpm. During LVAD support, an average flow to the distal branches (cerebral and subclavian) of 24% was found in the experiments and the nPTF. The flow fields from PIV and CFD were in good agreement. Numerical and experimental tools were combined to develop and validate the nPTF, which can be used to analyze local flow fields and the systemic response of LVADs during the design process. This allows analysis of physiological control parameters at early development stages and may, therefore, help to improve patient outcomes