High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent

*These authors contributed equally to this work. Cell-based high-content screening (HCS) assays have become an increasingly attractive alternative to traditional in vitro and in vivo testing in pharmaceutical drug development and toxicological safety assessment. The time- and cost-effectiveness of HCS assays, combined with the organotypic nature of human induced pluripo-tent stem cell (iPSC)-derived cells, open new opportunities to employ physiologically relevant in vitromodel systems to improve screening for potential chemical hazards. In this study, we used two human iPSC types, cardiomyocytes and hepatocytes, to test various high-content andmolecular assay combinations for their applicability in a multiparametric screening format. Effects on cardiomyocyte beat frequency were characterized by calcium flux measurements for up to 90min. Subsequent correlation with intracellular cAMP levels was used to determine if the effects on cardiac physiology were G-protein-coupled receptor dependent. In addition, we utilized high-content cell imaging to simultaneously determine cell viability, mi-tochondrial integrity, and reactive oxygen species (ROS) formation in both cell types. Kinetic analysis indicated that ROS formation is best detectable 30min following initial treatment, whereas cytotoxic effects were most stable after 24 h. For hepatocytes, high-content imaging was also used to evaluate cytotoxicity and cytoskeletal integrity, as well as mitochondrial integrity and the potential for lipid accumulation. Lipid accumulation, a marker for hepatic steatosis, was most reliably detected 48 h following treatment with test compounds. Overall, our results demonstrate how a compendium of assays can be utilized for quantitative screening of chemical effects in iPSC cardiomyocytes and hepatocytes and enable rapid and cost-efficient multidimensional biological profiling of toxicity