title: High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes.
abstract: 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 pluripotent stem cell (iPSC)-derived cells, open new opportunities to employ physiologically relevant in vitro model 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 and molecular assay combinations for their applicability in a multiparametric screening format. Effects on cardiomyocyte beat frequency were characterized by calcium flux measurements for up to 90 min. 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, mitochondrial integrity, and reactive oxygen species (ROS) formation in both cell types. Kinetic analysis indicated that ROS formation is best detectable 30 min 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.
authors.affiliation: 1 Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University , College Station, Texas.
authors.forename: Fabian Alexander
authors.initials: FA
authors.lastname: Grimm
authors.affiliation: 1 Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University , College Station, Texas.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Iwata
authors.affiliation: 2 Molecular Devices, LLC , Sunnyvale, California.
authors.forename: Oksana
authors.initials: O
authors.lastname: Sirenko
authors.affiliation: 3 Translational Genomics Research Institute, Texas A&M University , College Station, Texas.
authors.forename: Michael
authors.initials: M
authors.lastname: Bittner
authors.affiliation: 1 Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University , College Station, Texas.
authors.forename: Ivan
authors.initials: I
authors.lastname: Rusyn
mesh_terms: D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000633:toxicity; D004353:Drug Evaluation, Preclinical / Q000379:methods; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D017382:Reactive Oxygen Species; D043562:Receptors, G-Protein-Coupled; D004317:Doxorubicin