Fabian Alexander Grimm College of Veterinary Medicine and Biomedical Sciences, Texas A&M University
Yasuhiro Iwata College of Veterinary Medicine and Biomedical Sciences, Texas A&M University
Oksana Sirenko Ph.D. Molecular Devices
Michael Bittner Translational Genomics Research Institute, Texas A&M University
Ivan Rusyn College of Veterinary Medicine and Biomedical Sciences, Texas A&M University
Cell-Based HCS Assays Have Become an Increasingly Attractive Alternative to Traditional In Vitro and In Vivo Testing
Figure 1. Assay plexing for multidimensional toxicity screening of iPSC-derived cardiomyocytes and hepatocytes. In this study, we present a combinatorial approach to comprehensively assess cardiotoxic and hepatotoxic effects of test chemicals in vitro through screening of cardiophysiologic effects (calcium flux and GPCR activity assays, plate 1) and high-content imaging-based determination of cytotoxicity, mitochondrial integrity, ROS formation, cytoskeletal integrity, and lipid accumulation (plates 2–5). GPCR, G-protein-coupled receptor; iPSC, induced pluripotent stem cell; ROS, reactive oxygen species.
To date, toxicity testing of pharmaceutical and industrial chemicals, as well as environmental agents, relies primarily on data derived from animal studies. While in vivo models are still widely regarded as the most acceptable testing systems for regulatory decision making, the low throughput, high costs, regulatory pressure, and the limited predictability of human biological responses have led to the reduction of animal use being a primary goal in toxicology.1–3 However, these challenges welcome new opportunities for novel in vitro and computational technologies as feasible alternatives to traditional animal testing. A promising solution to overcome the limitations of traditional toxicity testing lies in emerging high-throughput screening (HTS) technologies to complement and potentially replace in vivo testing.4 Current federal initiatives to improve acceptance of HTS data in regulatory decision-making include the Tox21 and ToxCast programs.5,6 Likewise, HTS is widely applied in pharmaceutical drug development to improve selection criteria to prioritize lead molecules for animal testing.7
HTS can be broadly divided into two categories: biochemical assays and cell-based assays.8,9 While biochemical assays are easily accessible, data interpretation is usually target specific.8 To date, cell-based HTS assays rely primarily on the use of tumor-derived and primary cell lines and cover relatively narrow biological phenotypes, such as cell proliferation and/or cytotoxicity. Consequently, there is a considerable demand to increase both physiological relevance and multidimensionality of HTS assays.
Recent breakthroughs in stem cell technologies have resulted in the development and widespread availability of induced pluripotent stem cell (iPSC)-derived cell types, organotypic cell culture models that resemble their somatic counterparts both genetically and physiologically.10,11 In fact, human iPSC-derived two- and three-dimensional culture systems are considered to be highly physiologically relevant and hold promise to overcome the limitations associated with traditional cell lines and primary cells.10 A number of studies have indicated the potential for iPSC cardiomyocytes and hepatocytes to replicate cell-specific adverse effects of test chemicals.12–14 iPSC cardiomyocytes are a particularly attractive in vitro model system due to their use for evaluation of cardiac function, a challenging phenotype to model even in animals.15 Likewise, iPSC hepatocytes retain metabolic capacity on par with primary hepatocytes.16
A major challenge for regulatory acceptance of the data from HTS assays is in ensuring that tissue- and pathway-specific effects of chemicals can be captured. For example, cardiotoxicity and hepatotoxicity can be induced by a variety of mechanisms, including reactive oxygen species (ROS) formation, mitochondrial dysfunction, and disorders of lipid metabolism.17–20 Thus, simultaneous detection of various phenotypes through multidimensional combination of high-content screening (HCS) assays can provide valuable orthogonal information on a variety of tissue- and pathway-specific endpoints.21,22
This study used iPSC cardiomyocytes and hepatocytes to demonstrate the potential of a variety of HCS assay combinations for testing the potential toxicity of chemicals and complex substances (Fig. 1). The overall aim was to improve in vitro toxicity testing by reducing the time and cost of the assays while enhancing the mechanistic interpretation of the readouts so that confidence in animal replacement tests is improved. In particular, we demonstrate that intracellular calcium flux measurements to assess effects on cardiomyocyte contractility can be successfully combined with a competitive ELISA to determine G-protein-coupled receptor (GPCR) activation, a mechanism by which cardiotoxic compounds can induce chronotropic effects. Moreover, we applied high-content imaging to simultaneously capture effects on cell viability, mitochondrial integrity, and ROS formation in iPSC-derived cardiomyocytes and hepatocytes. We also demonstrate that imaging can be applied to assess cytoskeletal integrity and lipid accumulation, an indicator of hepatocellular steatosis, in iPSC-derived hepatocytes.
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.
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ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. GEN presents here one article "High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes." Authors of the paper are Grimm Fabian Alexander, Iwata Yasuhiro, Sirenko Oksana, Bittner Michael, and Rusyn Ivan.