Cellular Dynamics at the SOT 55th Annual Meeting and ToxExpo
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Assessment of the Electromechanical Window of Human IPSC-derived Cardiomyocytes and its Modulation by Compounds Affecting Cardiac Function
X Zhang, B Xi, X Wang, Y A Abassi. Biology, ACEA Biosciences Inc, San Diego, CA
The heart is an electromechanical pump which is regulated by electrical signals emanating from the SA node controlling the extent and timing of cardiac output. The intricate relationship between electrical signals and mechanical output or contraction is also preserved in cardiomyocytes, the individual functional units of the heart. We sought to investigate and characterize the precise timing of the electrical and contractile activity of human iPSC-derived cardiomyocytes and its modulation by reference compounds using the xCELLigence RTCA CardioECR System. The CardioECR system utilizes specialized plates containing both impedance and field potential electrodes. The field potential electrodes measure the integrated ion channel activity of cardiomyocyts while the impedance electrodes simultaneously measure the contractile activity. First, we optimized assay conditions in terms of cell seeding density and culture conditions to obtain stable field potential and impedance signals. Using the field potential and contraction waveforms, we derived a parameter termed Electromechanical Window (EMW) which is the timing difference between integrated ion channel activity and contractile activity. Under normal assay conditions and once electrical and mechanical connections are formed, the EMW becomes stable. Treatment of the cells with compounds which modulate ion channels such as sodium and calcium significantly affects the timing of the field potential duration as well as contractile signals to the same extent which does not significantly affect the EMW. IKr inhibitors such as E4031 also does not affect the EMW at sub EAD doses. However, concentrations which cause EAD can significantly change the EMW. Furthermore, compounds which only affect the contractile machinery of the cardiomyocytes do significantly affect the EMW by either increasing or decreasing the EMW depending on mechanism of action. In summary, simultaneous measurement of electrical and contractile activity of cardiomyocytes and assessment of the timing of these events can provide further insight into mechanism of compounds modulating cardiac function.
Evaluation of Acute and Chronic Effects of Kinase Inhibitors on Electrical and Contractile Activity of Human iPSC-derived Cardiomyocytes
X Zhang, B Xi, YA Abassi. Biology, ACEA Biosciences Inc, San Diego, CA
Tyosine kinase inhibitors (TKi) have revolutionized treatment of multiple types of cancer and can induce tumor shrinkage and prolong patient survival. However, they may also have certain degree of on target or off-target cardiovascular side effects in a subset of treated patients. In many cases, existing preclinical safety evaluation of the compound, which mainly focuses on animal toxicity studies and blockade of human Ether-a-go-go related channel (hERG), were not able to anticipate adverse cardiac effects. Therefore, there is an immediate need to develop new predictive preclinical assays and screens to test the potential adverse effects of kinase inhibitors. Here, we report a new in vitro screening assay to evaluate potential acute and chronic cardiac liability of TKi (sunitinib,dasatinib, nilotinib, sorafenib, lapatinib, erlotinib and imatinib) . We treated human induced pluripotent stem cell-derived cardiomyocytes (hiPS-C) with a panel of FDA-approved TKis for up to 2 days. Acute and chronic drug effects on attachment, viability, contraction and electrophysiology of iPSC were simultaneously examined and quantified using the xCELLigence RTCA CardioECR system. Tested drugs displayed differential effects acute and chronic effects on integrated ion channel activity as well as contractility. Furthermore, a number of kinase inhibitors which appeared to be safe based on acute affects, displayed longer term chronic effects on hiPSC. Taken together, this multi-parameter assay using hiPSC in conjunction with simultaneous measurement of ion channel activity, contractility and viability can provide incisive information on TKi-induced cardiotoxicity.
Identification of the Cellular Effects of Sofosbuvir and Amiodarone Using hiPSC-derived Cardiomyocytes
DC Millard1, CJ Strock2, S Stoelzle-Feix3, N Becker3, K Juhasz3,4, N Fertig3, CT January5, B Anson6, JD Ross1.
1. Axion Biosystems, Inc, Atlanta, GA, 2. Cyprotex, Watertown, MA, 3. Nanion Technologies GmbH, Munich, Germany, 4. Technische Universität München, Munich, Germany, 5. School of Medicine and Public Health, University of Wisconsin, Madison, WI, 6. Fujifilm Cellular Dynamics , Madison, WI
Sofosbuvir-based drugs have significantly advanced care for hepatitis C virus (HCV)-infected patients. While exhibiting a clean safety profile throughout development, recent post-marketing reports indicate that severe symptomatic bradycardia can occur through combined administration of sofosbuvir and amiodarone. The underlying cellular mechanism of this drug-drug interaction remains unknown. Here, we present a mechanistic interrogation of the cellular effects of sofosbuvir and amiodarone using human stem cell derived cardiomyocytes (hSC-CMs), multiwell microelectrode array (MEA), automated patch clamp (APC), and impedance measurements. MEA recordings demonstrated that sofosbuvir alone had no effect on hSC-CM electrophysiology, but reversed the effects of amiodarone when added in combination, leading to a 53% decrease in field potential duration and 100% increase in spontaneous beat rate relative to vehicle controls. APC assays demonstrated that the drug combination did not block potassium, sodium, or calcium currents beyond the assay controls. Simultaneous MEA and impedance measurements revealed differential responses on electrical and contractile activity; hSC-CMs remained electrically active while contractile activity ceased within ~2 hours of application with the drug combination. The impact on cardiomyocyte electrophysiology and contractility without a direct effect on ion channel activity suggests that the combination of amiodarone and sofosbuvir may impair intracellular calcium handling. These results demonstrate the importance of measuring multiple endpoints within the functional excitation-contraction cascade and, more generally, reinforce the utility of hSC-CMs as an integrated assessment of cardiac safety liability in vitro.
Understanding the Role of the Integrated Stress Response in iPSC Derived Human Cardiac Myocytes: From Progenitor Cell to Differentiated Cardiac Myocyte
GH Searfoss, JA Willy, TK Baker. Investigative Toxicology, Eli Company, Indianapolis, IN
Disruptions of normal cellular protein synthesis/processing, endoplasmic reticulum function, and nutrient starvation can lead to the induction of the integrated stress response (ISR) program in an attempt to preserve and restore cellular function by activation of EIF2 kinases, resulting in phosphorylation of eukaryotic initiation factor 2 on the alpha subunit (eIF2α~P) and resulting activating transcription factor 4 (ATF4). If the stress is unresolved, prolonged activation of the ISR will result in elevated levels of CHOP, and resulting cell death. A cell’s ability to respond to these stressors likely depends on the overall differentiation state of the cell. Differentiated cells derived from induced pluripotent stem cells (iPSC) provide a useful model for examining a cells ability to mount an ISR depending on its state of differentiation, as all the cells derive from a single initial iPSC and cells along the differentiation lineage will share the same genetic and epigenetic background. Using iPSC derived human cardiac myocytes and their proximal progenitor cells, we show that the cardiac myocyte progenitor cells display a more robust response to inducers of cell stress with increased levels of ISR stress response mediators ATF4, and CHOP compared to differentiated cardiac myocytes. Consequent with increased activation of the ISR, the progenitor cells are more sensitive to cellular stress and show increased cytolethality relative to fully differentiated cardiac myocytes. These data provide insight into possible differential susceptibility of early progenitor cell pools to integrated stress response inducing treatments in vivo, and provides a possible screening tool for identifying molecules that may differentially impact progenitor cell pools.
Use of Human iPSC-derived Cells as a Means to Investigate the Relationship Between Genes and Disease
B Anson. Fujifilm Cellular Dynamics, Madison, WI
Induced pluripotent stem cells have changed the fundamental fabric of what is possible in cell biology. From over 60 different diseases, induced pluripotent stem cell (iPSC) derived tissues demonstrate a relevant phenotypic in vitro to in vivo correlation with pathology. In addition, large banks of iPSC lines from diseased individuals are being created to further connect genetics to phenotypic biology. Further, genetic engineering techniques have improved in efficiency and cost such that isogenetic controls can be made from diseased lines. These iPSC lines enable a better understanding of biological and toxicological consequences of disease and create a means to screen for novel therapeutic modalities. Specifically, iPSC-derived cardiomyocytes from diabetic individuals show a phenotype with the hallmarks of diabetic cardiomyopathy. This model was used to better understand cardiac disease as well as screen libraries for compounds to improve this phenotype. Further, iPSC cardiomyocytes from 250 patients with left ventricular hypertrophy have phenotypic hallmarks of disease that correlate with specific single nucleotide polymorphisms from a larger genome wide association study. This effort, in collaboration with NHLBI and the Medical College of Wisconsin, was one of the largest efforts to demonstrate the functional impact of genetic diversity. This presentation will focus on toxicological utility of iPSC derived tissues using exemplary cases including genetics of diabetes, left ventricular hypertrophy, and Central Nervous System (CNS) diseases.
A Four-organ-on-a-chip Microfluidic System: Towards a Tool for Long-term Systemic Toxicity Assessment
A Lavado1, C Oleaga1, S Rothemund1, Y Cai1, L Kumanchik1, LR Bridges1, C Martin1, M Jackson1, CW McAleer1, CJ Long1, J Langer2, A Riu2, R Note2, S Teissier3, J Cotovio3, L Breton3, ML Shuler4, JJ Hickman1.
1. NanoScience Technology Center, University of Central Florida, Orlando, FL, 2. L’Oreal Research and Innovation division, Clark, NJ, 3. L’Oreal Research and Innovation division, Aulnay-sous-Bois, France, 4. Biomedical Engineering, Cornell University, Ithaca, NY
Concerns about long-term repeat-dose toxicity assessment have been raised since the ban on animal testing for the cosmetic industry. Hence, the generation and characterization of alternatives in vitro systems capable of reproducing the functionality of specific human organs in a quantifiable manner is currently a focal point of intensive research and development. However, the long-standing inadequacies inherent to the use of conventional techniques for predicting human tissue behavior encourage the development of more complex and integrative approaches. Body-on-a-chip systems utilize function-based cell models that accurately capture and predict multi organ complexity within correctly scaled and physiologically relevant platforms. We demonstrate a functional human 4-organ system under continuous flow conditions in a serum-free defined medium utilizing a pumpless platform for 28 days. This system, with extended functional viability, incorporated human primary liver cells to produce a more accurate human body-on-a-chip than cell lines. Computer simulations of the platform established flow rates and resultant shear stress within accepted ranges. Viability of the system was demonstrated for 28 days as well as functional activity of cardiac, muscle, neuronal and liver modules. The presented phenotypic culture model exhibits function over 28 days, representing the next generation of in vitro systems for repeat-dose systemic toxicity screening. Further steps involve the integration of a skin module, mimicking a cosmetic application via topical exposure.
A Human Heart-liver Platform to Study Drug Metabolism and Toxicity
C Oleaga1, G Legters1, L Kumanchik1, V Platt1, LR Bridges1, C Martin1, M Jackson1, CW McAleer1, CJ Long1, J Langer2, A Riu3, R Note3, JJ Hickman1, S Teissier3, J Cotovio3, L Breton3.
1. NanoScience Technology Center, University of Central Florida, Orlando, FL, 2. L’Oreal Research and Innovation division, Clark, NJ, 3. L’Oreal Research and Innovation division, Aulnay-sous-Bois, France
The regulatory context of March 2013 requires efforts to strengthen the development of alternative methods, in the field of cosmetic ingredients, to assess systemic toxicity. However, conventional in vitro assays do not fully represent physiological conditions. Xenobiotic metabolism is the missing piece of the puzzle in non-hepatic toxicology assays. Thus, we suggest connecting the metabolic engine of the liver to an organ susceptible of xenobiotic damage for the generation of an in vitro toxicity system to bridge the gap that exists or for physiologic toxicity prediction. We have developed a human heart-liver system to study drug metabolism dependent toxicity. This platform combines cardiac and hepatic modules that maintain full functionality in serum-free medium, under flow, for 14 days. Upon drug treatment, changes in (i) cardiac beating frequency and contractile force, and (ii) hepatic p450 activities, albumin and urea productions were studied. The drugs, cyclophosphamide and terfenadine, used for the validation of this system, represent both case scenarios; a non-cardiotoxic pre-drug that converts into a cardiotoxic metabolite, and the opposite, a cardiotoxic pre-drug that converts into a non-cardiotoxic metabolite. It is commonly accepted that the integration of a metabolic function in future toxicology models can improve adverse effects prediction in humans. Our system integrates this concept and enables a functional readout of cardio- and hepatotoxicity under serum-free conditions for acute and chronic exposure of drugs and their metabolites. The next steps will consist of better characterizing the model and screening a larger set of reference compounds.
Categorization of UVCBs Using Chemical-Biological Read Across
F Grimm1, Y Iwata1, O Sirenko2, WK Russell1, Y Luo1, C Crittenden2, FA Wright3, DM Reif3, J Yeakley4, B Seligmann4, P Shepard4, T Roy5, PJ Boogaard6, H Ketelslegers7, AM Rohde7, I Rusyn1.
1. Texas A&M University, College Station, TX, 2. Molecular Devices LLC., Sunnyvale, CA, 3. North Carolina State University, Raleigh, NC, 4. Biospyder Technologies Inc., Carlsbad, CA, 5. University of South Carolina, Beaufort, SC, 6. SHELL International BV, The Hague, Netherlands, 7. Concawe, Brussels, Belgium
Chemicals of Unknown or Variable composition, Complex reaction products, and Biological materials (UVCBs) present a major challenge for registrations under the REACH and US High Production Volume regulatory programs. In addition to frequent variations in their chemical composition, many gaps in available toxicity data preclude confident groupings of these substances for read across applications. Here, we present a comprehensive experimental and computational approach to categorize UVCBs according to global similarities in (1) their chemical composition using Ion Mobility Mass Spectrometry (IMMS) and (2) their bioactivities using a suite of in vitro models. For chemical read across, we analysed 20 petroleum substances from four distinct product groups by IMMS to determine substance-specific quantitative parameters including m/z distribution, drift time, carbon numbers, and double bond equivalents. For biological read across, we exposed induced pluripotent stem cell-derived cardiomyocytes and hepatocytes to a DMSO-soluble extract series of 26 petroleum substances comprising six product groups for up to 72 hours. Dose-response profiles for live cell cardiophysiology assessments (calcium-flux), high-content cell imaging (cytotoxicity and mitochondrial integrity), and targeted transcriptomics (TempO-seq) revealed product group-specific similarities. Data integration in ToxPi software and subsequent correlation analysis revealed group-specific clustering and a high degree of correlation between biological and chemical data sets. Altogether, we demonstrate how novel analytical chemistry and in vitro screening approaches can be effectively utilized to categorize UVCBs thereby indicating their potential applicability in regulatory submissions.
Comparison of In Vitro and Clinical In Vivo Effects of Pimobendan A Canine Heart Failure Drug
C Obejero-Paz1, N Sadekova2, J Kramer1, A Bruening-Wright1, K Norton2, AM Brown1.
1. Charles River Laboratories, Cleveland, OH, 2. Charles River Laboratories, Montreal, QC, Canada
Heart failure is a common disease for which new drugs are constantly sought. Critical to the process are higher throughput in vitro systems that screen inotropic and lusitropic effects. We have validated the CardioECR instrument (ACEA Biosciences) by comparing contractile effects of pimobendan in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) with well-known contractile effects in clinical canine studies. Pimobendan exerts inotropic effects by acting as a calcium sensitizer and phosphodiesterase inhibitor. Impedance was measured using iCELL cardiomyocytes2 paced at 0.67 Hz. As many as 12 drugs per day at two replicates/drug can be screened. For analysis we developed custom macros to calculate several contractile parameters including first derivatives (dΩ/dt). For the canine study we instrumented four naïve male Beagle dogs with Data Sciences International (DSI) Physiotel Digital L21 implants equipped with blood pressure, LVP catheters and ECG leads fixed in Lead 2 configuration. DSI Ponemah software was used to collect heart rate, dP/dt max dP/dt min, LVEDP, systemic arterial pressure, QT interval duration and ECG parameters for baseline (2 hours predose) and up to 24 hours post dose. We used 0.1-10 μM pimobendan for the impedance study and 0.1, 0.3 and 1 mg/kg pimobendan for the animal study. The total Cmax obtained with a single oral 0.25 mg/kg dose was 0.012 μM. At 90% PB the free Cmax was 0.0012 μM. Impedance results: at 10 μM pimobendan induced an increase in twitch amplitude (12 ± 1%, mean ± sem), dΩ/dt max (7 ± 2%), twitch area (25 ± 3%) and relaxation to 50% (6 ± 3%) after one hour exposure. The trend was apparent at 1 μM and became statistically significant between 3 -10 μM. Telemetered conscious dog model results: at 1 mg/kg pimobendan produced a mean increase of LV dP/dt max of 83% over a 7-hour period. The changes were statistically significant. The change in LVP dP/dt max correlated with a decrease in the QA interval. No changes were observed in LVEDP. These results support the impedance results. In conclusion, in vitro impedance screening of hiPSC-CMs at customary screening concentrations detected the contractility effects of pimobendan.
Dietary Supplement Ingredients Alter Beating Parameters of iCell Cardiomyocytes
R Calvert, S Vohra, M Ferguson, P Wiesenfeld. US FDA CFSAN, Laurel, MD
Dietary supplements may contain ingredients that are cardiac stimulants and potential cardiotoxins. In vitro studies might help identify ingredients of potential concern. iCell cardiomyocytes, a beating human heart cell line, were used to evaluate the effects of phenylethylamine (PEA), higenamine, ephedrine, and caffeine on heart cell function. For PEA and higenamine, levels in cell media were based on published blood levels in humans or animals after intravenous administration. Ephedrine and caffeine levels were based on published blood levels following various oral doses in humans. In addition, at low to midrange levels, each chemical was examined either with or without added caffeine. Caffeine levels (50 uM) approximated human blood levels after consumption of caffeine-enriched dietary supplements. To obtain values for beats per minute (BPM), peak width, etc., rhythmic rise and fall in intracellular calcium levels following 30 min of treatment were measured using a FLIPR Tetra instrument. Experiments were done in triplicate. Higenamine 31.3, 31.3 + caffeine and 313 ng/ml significantly increased BPM in an escalating manner. Peak width narrowed with increasing BPM. PEA caused increases in BPM at 0.8 and 8 ug/ml, along with narrower peak widths. In contrast, 80 ug/ml PEA greatly reduced BPM and widened peak width. This may be a toxic effect of PEA at 80 ug/ml. Adding caffeine to PEA 8 ug/ml or higenamine 31.3 ng/ml further increased BPM. Ephedrine produced a significant BPM dose response for concentrations from 0.5 to 5.0 uM. Caffeine alone resulted in an increased BPM only at a toxic level of 250 uM, while 10 and 50 uMhad no effect. There was no significant enhancing effect of caffeine when combined with ephedrine versus ephedrine alone. These in vitro results would suggest that further testing might be warranted in vivo to evaluate cardiovascular effects.
Diglycolic Acid Treatment Affects iPSC Cardiomyocyte Beating
K Bailey, H Toomer, R Sprando, R Calvert. US FDA CFSAN, Laurel, MD
Renal toxicity of diglycolic acid (DGA) has been described, but the effect of DGA on other organs has not been closely examined. An in house in vivostudy detected elevated creatine kinase levels in rats treated with DGA, suggesting that damage may be occurring in the heart or skeletal muscle. This research focused on evaluating cardiac effects of DGA by treating human iPSC beating cardiomyocytes with increasing concentrations of DGA in media at pH 7.8 for 30 minutes. Cardiomyocyte beat rate (BPM) as well as estimates of relative cytoplasmic Ca2+ concentrations were measured using a fluorescent dye that detects Ca2+ in the cytoplasm during myocyte contraction. After 30 minutes of DGA treatment, BPM remained near control rates at concentrations of 2.5 mM and 5 mM. BPM decreased significantly at concentrations of 7.5 mM DGA and higher. No beats were detected in cells treated with 50 mM DGA. Peak amplitude was significantly decreased in all groups treated with DGA for 30 minutes, including concentrations below 5 mM where BPM did not change. Interestingly, BPM returned to control levels in cells previously treated with up to 25 mM DGA after replacing DGA containing media with fresh control media for 30 minutes. Though BPM returned to control levels, amplitude remained significantly lower in all cells except for those treated with 2.5 mM DGA. No beats were detected after fresh media replacement in cells exposed to 50 mM DGA. These in vitrofindings indicating DGA cardiotoxicity in iPSCs correlate with the preliminary findings in our in vivo study where rats treated with 300 mg/kg of DGA developed abnormal cardiac pathology. The changes in beat rate and Ca2+signal amplitude of the beating iPSC suggest that DGA affects Ca2+ flux in cardiac cells and that acute toxic effects of DGA are partially reversible.
Evaluation of Batch Variations in Induced Pluripotent Stem Cell Derived- Human Cardiomyocytes
L Pang1, J Hua Huo1,2, A Kamalakar1, B Word1, B Lyn-Cook1, X Yang3, N Stockbridge4.
1. Division of Biochemical Toxicology, NCTR/US FDA, Jefferson, AR, 2. Department of Cardiovascular Medicine, First Hospital of Xi’an Jiaotong University, Xi’an, China, 3. Division of Systems Biology, NCTR/US FDA, Jefferson, AR, 4. Division of Cardiovascular and Renal Products, Office of New Drugs, CDER/US FDA, Silver Spring, MD
Drug-induced proarrhythmia is a major safety issue in drug development. Sensitive in vitro assays that can predict drug-induced cardiotoxicity in humans have been a focus of toxicology research for the past decade. Recently, induced pluripotent stem cell derived-human cardiomyocytes (iPSC-hCMs) have become a popular model because they largely replicate the electrophysiological behavior of human ventricular cardiomyocytes. “The Heart in the Petri Dish” has been proposed for personalized cardiac drug selection and adverse drug response prediction; however, many procedures are involved in the cardiomyocyte differentiation and purification process, and iPSC-hCMs are in a continuing state of maturation, which may result in large batch-to-batch variations. In this study, we examined the cardiac ion channel gene expression profile and electrophysiological response of three different batches of iCells from Cellular Dynamics International. We found that, based on field potential duration (FPD) data, the three batches of cells had similar sensitivities: the minimal concentration of hERG, sodium, and calcium channel blockers that elicited ± 10% changes in FPD compared to the vehicle control were identical among the three batches of cells; with an Iks blocker, there was a three-fold difference among the batches. Rate correction of FPD with either Bazett’s or Fridericia’s formula increased the variability. Most of the cardiac ion channel genes were expressed uniformly among three batches of cells, with the exception of CACNA1C, the expression of which was high when the FPD value was low. Careful evaluation of the performance of iPSC-hCMs and data analysis methods is warranted for the use of these cells in regulatory toxicity testing.
Functional Integrated Human Cardiac System for Toxicological and Pharmaceutical Studies
CJ Long1, C Oleaga1, M Stancescu1, P Molnar1, CW McAleer1, W McLamb1, G Legters1, J Prot2, JJ Hickman1.
1. NanoScience Technology Center, University of Central Florida, Orlando, FL, 2. Biomedical Engineering, Cornell University, Ithaca, NY
The pharmaceutical industry invests decades on research for drug development together with billions of dollars to launch them into the market to improve the human condition, but failures still occur in a large percentage of cases. Cardiotoxicity is the number one adverse effect that causes new market releases to be withdrawn. This is indicative of the fact that current regulatory tests fail to accurately predict human cardiotoxicity during the preclinical evaluations. We have developed an in vitro human cardiac system that features the two main predictors of cardiac toxicity, conduction velocity and contractile force . Human stem cell derived cardiomyocytes were integrated with a BioMicroelectromechanical system construct for force determination and a microelectrode array for electrical conduction, both in a serum-free medium. Sotalol, norepinephrine and verapamil were used to validate the platform for toxicity and to attempt to differentiate mechanisms of toxicity without the complexity of the whole heart using the two functional readouts. This cardiac system, utilizing a multivariant functional profile, has the ability to reproduce the toxic events that affect the heart as well as address mechanistic aspects, examples of which will be discussed. This novel technology is currently being adapted as a regulatory cardiotoxic platform.  Stancescu et al., Biomaterials 60:20-30, 2105.
Multi-Spheroid Imaging Analysis of Human Induced Pluripotent Stem Cell-derived Cardiomyocyte by Cellvoyager CV7000 System for the Assessment of In Vitro Cardiotoxicity of Anti-Cancer Kinase Inhibitors
T Nagakura1, T Matsubara2, K Sawada1.
1. Eisai Co, Ltd., Tsukuba, Japan, 2. Yokogawa Denki Co, Ltd., Kanazawa, Japan
Introduction: Throughput screening is extremely important for early lead optimization process. Human induced pluripotent stem cell-derived cardiomyocyte (iPS-CM) are especially attractive because they express ion channels and demonstrate spontaneous mechanical and electrical activity. Actually, many publications showed that electrophysiological assessment with multi-electrode array system has excellent correlations between field potential duration and QTc prolongation. In the present study, we would like to introduce new in vitro cardiotoxicity assay method in iPS-CM by live-cell imaging analysis and present assay results with several anti-cancer kinase inhibitors.
Methods: Human iPS-CM (iCell® Cardiomyocytes) was seeded into 96-half well plate which has 250 of fibronectin-coated spots in each well and spheroids were formed on all spots after 7 days culture in a plate. Calcium (Ca) sensing fluorescence dye and tested kinase inhibitor solutions were added and the dynamic changes in fluorescent intensity of all spheroids were measured at 24 hour after compounds incubation using Cellvoyager CV7000 system. From 30 seconds live-Ca image capture for each well, beat rate, minimum and maximum cellular Ca levels and peak Ca amplitude were analyzed. Spheroid size, nuclear intensicty and mitochondoria function were also checked at the same time.
Results: Human iPS-CM was incubated 24 hour with a concentration of 0.01, 0.1, 1.0 and 10 μmol/L of each kinase inhibitors, such as sunitinib, sorafenib, erlotinib, nilotinib, pazopanib and afatinib, respectively. Spontanious beating of iPS-CM spheroids were reduced by addition of sunitinib at 0.1 μmol/L and above. At 10 μmol/L, arrest of cardiomyocyte spheroids was observed by sunitinib. Sorafenib and nilotinib also slowed beat rate and sorafenib increased maximum cellular Ca levels. On the other hand, erlotinib, pazopanib and afatinib were not found significant effect on iPS-CM beating upto 10 μmol/L. More detail analyzed results including changes in nuclear intensicty and mitochondoria function will be presented.
Sunitinib-induced Cardiotoxicity Is Associated with Changes in Expression Levels of Cardiac MicroRNAs in Human Induced Pluripotent Stem Cell (iPSC)-derived Cardiomyocytes
MC White, L Ren, X Yang. Systems Biology, US FDA National Center for Toxicological Research, Jefferson, AR
Sunitinib (Sutent®) is an FDA-approved multi-kinase inhibitor with activity against vascular endothelial growth factor receptor (VEGFR) 1 & 2, platelet-derived growth factor receptor (PDGFR) α & β, and multiple other receptor kinases. Sunitinib is used to treat gastrointestinal stromal tumors (GIST), advanced renal cell carcinoma (RCC), and pancreatic neuroendocrine tumors (pNET). Despite clinical efficacy, a major concern associated with sunitinib therapy remains the development of cardiac dysfunction, which may include arrhythmia, decreased left ventricle ejection fraction (LVEF), and congestive heart failure. A better understanding of the molecular mechanisms underlying sunitinib-induced cardiotoxicity is needed. Here, we screened human induced pluripotent stem cell (iPSC)-derived cardiomyocytes with clinically-relevant concentrations of sunitinib and measured beating patterns, beating amplitude, and cytotoxicity. Because recent evidence has highlighted the role of microRNAs (miRNAs) in cardiomyocyte function, we then profiled changes in the miRNA transcriptome at corresponding concentrations and timepoints in order to explore potential biomarkers of functional toxicity. Sunitinib caused significant changes (fold-change ≥ 2; p ≤ 0.05) in over 150 miRNAs, including a subset of miRNAs associated with cardiac function and cardiovascular disease (e.g. miR-133a/b, miR-320a/b, miR-1, miR-125a/b-5p). These data provide clues as to how the miRNA transcriptome reflects cardiomyocyte function following sunitinib exposure, and support further evaluation of miRNA changes as biomarkers for cardiotoxicity.
The Cardio-Specific Effects of VX Exposure on Human-Induced Pluripotent Stem Cell-derived Cardiomyocytes
D Carmany1, C Phillps2, R Dorsey2, J Madren-Whalley2, R Kristovich2, H Salem2.
1. US Army ECBC, Excet Inc, Aberdeen Proving Grounds, MD, 2. US Army ECBC, Aberdeen Proving Grounds, MD
Organophosphates are well known inhibitors of cholinesterases. Many of these are used for general pesticide and agricultural purposes, but several have also been developed for use as Chemical Warfare Agents (CWAs). These include compounds such as Sarin, Soman, and VX. Although the cholinergic nervous system effects have been well characterized for these agents. Many signs and symptoms unrelated to the nervous system are also observed. To better understand these secondary effects, efforts are ongoing to develop cell based models of exposure using human cell lines to better correlate toxicity. These models will add in the identification and characterization of secondary toxic effects which leading to better countermeasures and care for those exposed. To better understand cardio-specific symptoms, a human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) model has been developed and is being used to study the toxic effects of VX. The model evaluates hiPSC-CMs on both the impedance based xCELLigence System and High-Content Analyzer (HCA) platforms. Cell lines and media from several manufactures were tested to create a model that would meet the program goals for functionality and repeatability. The iCell Cardiomyocytes2 manufactured by CDI using the GE cell line recommended RPMI with the B27 supplement media gave the most repeatable results on the xCELLigence system while permitting the testing of compounds that are unstable in serum based media. Our models showed that VX has an immediate effect of increasing the beat rate will simultaneously decreasing the beat strength of the cells. This result was dose dependent with higher concentrations increasing and lower concentrations decreasing the toxic effect on both the beat rate and beat strength. These changes were not due to cell death. The HCA confirmed that cell death was not responsible for the effects, but that an immediate decrease in intracellular calcium levels may play a possible role. Calcium is well known to be responsible for cardiomyocyte signaling. With our models being able to both identify cardio function and measure calcium levels, we are next planning to test various drugs involved with calcium regulation in the heart to determine if any currently available compound can counteract the VX induced toxicity.
Toxicological Categorization of P- and E-Series Glycol Ethers Using High-Content Screening of Human Induced Pluripotent Stem Cell (iPSC)-derived Cells
Y Iwata1, F Grimm1, M Wilson1, M Bittner2, O Sirenko3, J Rowlands4, N Ball4, I Rusyn1.
1. Veterinary Integrative Biosciences, Texas A&M University, College Station, TX, 2. Engineering Experiment Station, Texas A&M University, College Station, TX, 3. Veterinary Integrative Biosciences, Molecular Devices, LLC, Sunnyvale, CA, 4. The Dow Chemical Company, Midland, MI
High-content screening (HCS) assays utilizing novel organotypic cell culture models are an attractive approach for predictive safety assessments of chemicals through biological data-based read across. To test the hypothesis that HCS represents a feasible approach to categorize chemicals based on similarities in their in vitro toxicity profiles, we screened eight propylene (P-series) and twelve ethylene (E-series) glycol ethers, structurally related yet toxicologically diverse group of prototypical industrial high production volume chemicals. We used two human induced pluripotent stem cell (iPSC)-derived cell types, cardiomyocytes and hepatocytes. Cells were exposed to glycol ethers in concentration-response over five (semi-)logs for up to 48 hours. Toxicity endpoints included effects on cardiomyocyte beating patterns. Cytotoxicity, mitochondrial and cytoskeletal integrity, and (in hepatocytes) reactive oxygen species formation and lipid accumulation were also assessed. Data were fit to a concentration-response to derive point-of-departure values that were used as quantitative descriptors for categorization in Toxicological Prioritization Index (ToxPi). We found that there is a correlation between the length of the alcohol group and induced effects such that glycol ethers can be categorized based on simple glycols, methyl-, ethyl-, propyl-, butyl-, and hexyl ethers. Within an alcohol group based category, in general there was increasing cytotoxicity between mono-, di-, and tri-substituted glycol ethers. A trend was observed with integrative ToxPi evaluation that combined all endpoints and cell-types, which was more prominent than when the data was expressed by individual cell-type. However, the trends in in vitro data did not align with the data trends in in vivo toxicity for these substances as there was no separation between E- and P- series glycol ethers in terms of biological activity. Future efforts should be directed towards correlating biological data and physico-chemical data sets and gene expression analysis as additional means to define biological similarity.
Combining xCELLigence® CardioECR Technology and iPSC Cardiomyocytes for Relevant Frontline and Mechanistic Cardiotoxicity Screening
High-throughput Imaging and Kinetic Endpoints for Toxicity Testing in iPSC-derived (iCell) Cardiomyocytes and Hepatocytes in 2D and 3D Spheroid Cultures
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