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. 2015 Nov;13(9):529-46.
doi: 10.1089/adt.2015.659. Epub 2015 Nov 5.

High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes

Fabian Alexander Grimm  1 , Yasuhiro Iwata  1 , Oksana Sirenko  2 , Michael Bittner  3 , Ivan Rusyn  1
Affiliations

High-Content Assay Multiplexing for Toxicity Screening in Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Hepatocytes

Fabian Alexander Grimm et al. Assay Drug Dev Technol. 2015 Nov.

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.

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Figures

<b>Fig. 1.</b>
Fig. 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.
<b>Fig. 2.</b>
Fig. 2.
Assessment of cardiophysiologic effects of isoproterenol (green circle), TAB (red square), cisapride (blue triangle), propranolol (orange triangle), sotalol (gray diamond), doxorubicin (open black circle), menadione (open purple square), sunitinib (open green triangle), crizotinib (open blue triangle) by combined calcium flux monitoring, and cAMP formation. (A) Basal cardiomyocyte beat frequencies after 0 (preread), 10, 30, 60, and 90 min following addition of 1:5 (v/v) medium or vehicle (1% DMSO in medium). (B, C) Concentration–response plots showing effects of drugs on cardiomyocyte beat frequencies after 30 and 90 min. The potential for GPCR activation was assessed by measuring intracellular cAMP levels by competitive ELISA. (D) Evaluation of GPCR assay controls: background fluorescence at 590 nm was determined using cAMP-free assay buffer and omitting Rabbit anti-cAMP antibody. Treatment with cell medium and vehicle was used to determine basal cAMP levels in cardiomyocytes; stimulation of cells with 20 μM forskolin was used as a positive control, resulting in maximum cAMP formation and competitive repression of the cAMP-horseradish peroxidase signal. (E) Concentration–response plots of drugs that did not, or only slightly, affect GPCR activity in cardiomyocytes. (F) Concentration–response plots of three drugs that significantly (twofold or more) increased cAMP formation in cardiomyocytes. Data points in all plots represent mean ± SEM of at least three replicates. DMSO, dimethyl sulfoxide; TAB, tetraoctylammonium bromide.
<b>Fig. 3.</b>
Fig. 3.
Combined assessment of ROS formation, cytotoxicity, and mitochondrial integrity in iPSC cardiomyocytes. The upper row summarizes results on ROS formation, the second and third rows depict data derived from nuclear and mitochondrial imaging for evaluation of cytotoxicity and mitochondrial integrity. The left column includes plots showing basal levels (percentages of cells with elevated levels of ROS, viable cells, and cells with intact mitochondria) of medium and vehicle (1% DMSO in medium)-treated cardiomyocytes. The second and third columns show concentration–response plots for TAB (red square), doxorubicin (open black circle), menadione (open purple square), and crizotinib (open blue triangle) after 30 min and 24 h of incubation. All data points represent mean ± SEM.
<b>Fig. 4.</b>
Fig. 4.
Combined assessment of cytotoxicity (column 1), cytoskeletal integrity (column 2), mitochondrial integrity (column 3), and lipid accumulation (column 4) in iPSC hepatocytes. Upper row: Basal percentages of control cells with intact nuclei, cytoskeletal integrity, mitochondria, and lipid accumulation 48 h following treatment with medium and vehicle (1% DMSO in medium). Lower row: Concentration–response plots of hepatocytes treated with TAB (red square), aflatoxin B1 (brown hexagon), amiodarone (black and white diamond), menadione (open purple square), crizotinib (open blue triangle), and sunitinib (open green triangle) for 48 h. Data points represent mean ± SEM.
<b>Fig. 5.</b>
Fig. 5.
Combined assessment of ROS formation, cytotoxicity, and mitochondrial integrity in iPSC hepatocytes. The first row summarizes results on ROS formation, the second and third rows depict data derived from nuclear and mitochondrial imaging for evaluation of cytotoxicity and mitochondrial integrity. The left column includes plots summarizing basal levels (percentages of cells with elevated levels of ROS, viable cells, and cells with intact mitochondria) of medium and vehicle (1% DMSO in medium)-treated cardiomyocytes. The second and third columns show concentration–response plots for TAB (red square), doxorubicin (open black circle), menadione (open purple square), and crizotinib (open blue triangle) after 30 min and 24 h of incubation. All data points represent mean ± SEM. Combined assessment of ROS formation, cytotoxicity, and mitochondrial integrity in iPSC hepatocytes treated with TAB (red square), doxorubicin (open black circle), menadione (open purple square), crizotinib (open blue triangle), and sunitinib (open green triangle) for up to 24 h. Plots show comparisons between untreated and vehicle (1% DMSO)-treated cells and high-content imaging-derived concentration–response profiles. Data points represent mean ± SEM.
<b>Fig. 6.</b>
Fig. 6.
Application of combinatorial high-content screening assays for descriptive and mechanistic toxicity profiling of complex mixtures. Plots show (A–B) cardiomyocyte beat frequency (beats per minute, BPM) and (C–D) cAMP formation concentration–response profiles for two (A and C; B and D) DMSO extracts of commercial gas oils. Individual data points and mean ± SEM are indicated for each concentration. Cardiomyocyte beat frequency profiles demonstrate initial chronotropic effects (≤ 1% dilution), followed by total inhibition of cardiomyocyte beating, an indicator of cell death. Moreover, cardiac chronotropic effects correlate well with cAMP formation, a cellular indicator of GPCR stimulation.
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