Automated Patch Clamp on mESC-Derived Cardiomyocytes for Cardiotoxicity Prediction

Cardiovascular side effects are critical in drug development and have frequently led to late-stage project terminations or even drug withdrawal from the market. Physiologically relevant and predictive assays for cardiotoxicity are hence strongly demanded by the pharmaceutical industry. To identify a potential impact of test compounds on ventricular repolarization, typically a variety of ion channels in diverse heterologously expressing cells have to be investigated. Similar to primary cells, in vitro–generated stem cell–derived cardiomyocytes simultaneously express cardiac ion channels. Thus, they more accurately represent the native situation compared with cell lines overexpressing only a single type of ion channel. The aim of this study was to determine if stem cell–derived cardiomyocytes are suited for use in an automated patch clamp system. The authors show recordings of cardiac ion currents as well as action potential recordings in readily available stem cell–derived cardiomyocytes. Besides monitoring inhibitory effects of reference compounds on typical cardiac ion currents, the authors revealed for the first time drug-induced modulation of cardiac action potentials in an automated patch clamp system. The combination of an in vitro cardiac cell model with higher throughput patch clamp screening technology allows for a cost-effective cardiotoxicity prediction in a physiologically relevant cell system.

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Novel Automated Patch-clamp Assays on Stem Cell-derived Cardiomyocytes: Will they Standardize In Vitro Pharmacology and Arrhythmia Research?

Journal of Physical Chemistry & Biophysics ◽

10.4172/2161-0398.1000153 ◽

2014 ◽

Vol 4 (4) ◽

Author(s):

Amuzescu B

Keyword(s):

Stem Cell ◽

Patch Clamp ◽

In Vitro Pharmacology ◽

Automated Patch Clamp

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Characterizing Human Ion Channels in Induced Pluripotent Stem Cell–Derived Neurons

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057112457821 ◽

2012 ◽

Vol 17 (9) ◽

pp. 1264-1272 ◽

Cited By ~ 40

Author(s):

Alison Haythornthwaite ◽

Sonja Stoelzle ◽

Alexander Hasler ◽

Andrea Kiss ◽

Johannes Mosbacher ◽

...

Keyword(s):

Stem Cell ◽

Ion Channels ◽

Patch Clamp ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Excitable Cells ◽

Automated Patch Clamp ◽

Induced Pluripotent ◽

Two Populations ◽

A Current

Neurons derived from human-induced pluripotent stem cells were characterized using manual and automated patch-clamp recordings. These cells expressed voltage-gated Na+ (Nav), Ca2+ (Cav), and K+ (Kv) channels as expected from excitable cells. The Nav current was TTX sensitive, IC50 = 12 ± 6 nM ( n = 5). About 50% of the Cav current was blocked by 10 µM of the L-type channel blocker nifedipine. Two populations of the Kv channel were present in different proportions: an inactivating (A-type) and a noninactivating type. The A-type current was sensitive to 4-AP and TEA (IC50 = 163 ± 93 µM; n = 3). Application of γ-aminobutyric acid (GABA) activated a current sensitive to the GABAA receptor antagonist bicuculline, IC50 = 632 ± 149 nM ( n = 5). In both devices, comparable action potentials were generated in the current clamp. With unbiased, automated patch clamp, about 40% of the cells expressed Nav currents, whereas visual guidance in manual patch clamp provided almost a 100% success rate of patching “excitable cells.” These results show high potential for pluripotent stem cell–derived neurons as a useful model for drug discovery, in combination with automated patch-clamp recordings for high-throughput and high-quality drug assessments at human neuronal ion channels in their correct cellular background.

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Screening system for drug-induced arrhythmogenic risk combining a patch clamp and heart simulator

Science Advances ◽

10.1126/sciadv.1400142 ◽

2015 ◽

Vol 1 (4) ◽

pp. e1400142 ◽

Cited By ~ 58

Author(s):

Jun-ichi Okada ◽

Takashi Yoshinaga ◽

Junko Kurokawa ◽

Takumi Washio ◽

Tetsushi Furukawa ◽

...

Keyword(s):

Patch Clamp ◽

Ventricular Arrhythmia ◽

Paradigm Shift ◽

Screening System ◽

Drug Induced ◽

Characteristic Type ◽

Drug Concentrations ◽

Automated Patch Clamp ◽

Electrocardiogram Ecg

To save time and cost for drug discovery, a paradigm shift in cardiotoxicity testing is required. We introduce a novel screening system for drug-induced arrhythmogenic risk that combines in vitro pharmacological assays and a multiscale heart simulator. For 12 drugs reported to have varying cardiotoxicity risks, dose-inhibition curves were determined for six ion channels using automated patch clamp systems. By manipulating the channel models implemented in a heart simulator consisting of more than 20 million myocyte models, we simulated a standard electrocardiogram (ECG) under various doses of drugs. When the drug concentrations were increased from therapeutic levels, each drug induced a concentration-dependent characteristic type of ventricular arrhythmia, whereas no arrhythmias were observed at any dose with drugs known to be safe. We have shown that our system combining in vitro and in silico technologies can predict drug-induced arrhythmogenic risk reliably and efficiently.

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The TGFβ Family in Human Placental Development at the Fetal-Maternal Interface

Biomolecules ◽

10.3390/biom10030453 ◽

2020 ◽

Vol 10 (3) ◽

pp. 453

Author(s):

Susana M. Chuva de Sousa Lopes ◽

Marta S. Alexdottir ◽

Gudrun Valdimarsdottir

Keyword(s):

Stem Cell ◽

Transforming Growth Factor Beta ◽

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Cell Model ◽

Embryonic Stem ◽

Model Systems ◽

Special Focus ◽

Placental Development

Emerging data suggest that a trophoblast stem cell (TSC) population exists in the early human placenta. However, in vitro stem cell culture models are still in development and it remains under debate how well they reflect primary trophoblast (TB) cells. The absence of robust protocols to generate TSCs from humans has resulted in limited knowledge of the molecular mechanisms that regulate human placental development and TB lineage specification when compared to other human embryonic stem cells (hESCs). As placentation in mouse and human differ considerably, it is only with the development of human-based disease models using TSCs that we will be able to understand the various diseases caused by abnormal placentation in humans, such as preeclampsia. In this review, we summarize the knowledge on normal human placental development, the placental disease preeclampsia, and current stem cell model systems used to mimic TB differentiation. A special focus is given to the transforming growth factor-beta (TGFβ) family as it has been shown that the TGFβ family has an important role in human placental development and disease.

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Synthesis and Evaluation of Novel α-Aminoamides Containing Benzoheterocyclic Moiety for the Treatment of Pain

Molecules ◽

10.3390/molecules26061716 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1716

Author(s):

Kun Tong ◽

Ruotian Zhang ◽

Fengzhi Ren ◽

Tao Zhang ◽

Junlin He ◽

...

Keyword(s):

Ion Channels ◽

Patch Clamp ◽

Formalin Test ◽

Sodium Ion ◽

Inhibitory Potency ◽

Voltage Gated ◽

Patch Clamp Electrophysiology ◽

Sodium Ion Channels

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.

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In vitro assessing the risk of drug-induced cardiotoxicity by embryonic stem cell-based biosensor

Sensors and Actuators B Chemical ◽

10.1016/j.snb.2010.11.050 ◽

2011 ◽

Vol 155 (1) ◽

pp. 214-219 ◽

Cited By ~ 23

Author(s):

Qingjun Liu ◽

Hui Yu ◽

Zhou Tan ◽

Hua Cai ◽

Weiwei Ye ◽

...

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Embryonic Stem ◽

Drug Induced

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Use of Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes in Preclinical Cancer Drug Cardiotoxicity Testing: A Scientific Statement From the American Heart Association

Circulation Research ◽

10.1161/res.0000000000000291 ◽

2019 ◽

Vol 125 (10) ◽

Cited By ~ 16

Author(s):

Gary Gintant ◽

Paul Burridge ◽

Lior Gepstein ◽

Sian Harding ◽

Todd Herron ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Cardiac Injury ◽

Induced Pluripotent Stem Cell ◽

Heart Association ◽

Cancer Drug ◽

Great Promise ◽

Drug Induced ◽

Induced Pluripotent

It is now well recognized that many lifesaving oncology drugs may adversely affect the heart and cardiovascular system, including causing irreversible cardiac injury that can result in reduced quality of life. These effects, which may manifest in the short term or long term, are mechanistically not well understood. Research is hampered by the reliance on whole-animal models of cardiotoxicity that may fail to reflect the fundamental biology or cardiotoxic responses of the human myocardium. The emergence of human induced pluripotent stem cell–derived cardiomyocytes as an in vitro research tool holds great promise for understanding drug-induced cardiotoxicity of oncological drugs that may manifest as contractile and electrophysiological dysfunction, as well as structural abnormalities, making it possible to deliver novel drugs free from cardiac liabilities and guide personalized therapy. This article briefly reviews the challenges of cardio-oncology, the strengths and limitations of using human induced pluripotent stem cell–derived cardiomyocytes to represent clinical findings in the nonclinical research space, and future directions for their further use.

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Assessing Drug-Induced Long QT and Proarrhythmic Risk Using Human Stem-Cell-Derived Cardiomyocytes in a Ca2+ Imaging Assay: Evaluation of 28 CiPA Compounds at Three Test Sites

Toxicological Sciences ◽

10.1093/toxsci/kfz102 ◽

2019 ◽

Vol 170 (2) ◽

pp. 345-356 ◽

Cited By ~ 6

Author(s):

Hua Rong Lu ◽

Haoyu Zeng ◽

Ralf Kettenhofen ◽

Liang Guo ◽

Ivan Kopljar ◽

...

Keyword(s):

Stem Cell ◽

Microelectrode Array ◽

Torsade De Pointes ◽

Transient Assay ◽

Potential Drug ◽

Human Stem Cell ◽

Long Qt ◽

Drug Induced ◽

Vitro Assay

Abstract The goal of this research consortium including Janssen, MSD, Ncardia, FNCR/LBR, and Health and Environmental Sciences Institute (HESI) was to evaluate the utility of an additional in vitro assay technology to detect potential drug-induced long QT and torsade de pointes (TdP) risk by monitoring cytosolic free Ca2+ transients in human stem-cell-derived cardiomyocytes (hSC-CMs). The potential proarrhythmic risks of the 28 comprehensive in vitro proarrhythmia assay (CiPA) drugs linked to low, intermediate, and high clinical TdP risk were evaluated in a blinded manner using Ca2+-sensitive fluorescent dye assay recorded from a kinetic plate reader system (Hamamatsu FDSS/µCell and FDSS7000) in 2D cultures of 2 commercially available hSC-CM lines (Cor.4U and CDI iCell Cardiomyocytes) at 3 different test sites. The Ca2+ transient assay, performed at the 3 sites using the 2 different hSC-CMs lines, correctly detected potential drug-induced QT prolongation among the 28 CiPA drugs and detected cellular arrhythmias-like/early afterdepolarization in 7 of 8 high TdP-risk drugs (87.5%), 6 of 11 intermediate TdP-risk drugs (54.5%), and 0 of 9 low/no TdP-risk drugs (0%). The results were comparable among the 3 sites and from 2 hSC-CM cell lines. The Ca2+ transient assay can serve as a user-friendly and higher throughput alternative to complement the microelectrode array and voltage-sensing optical action potential recording assays used in the HESI-CiPA study for in vitro assessment of drug-induced long QT and TdP risk.

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Global transcriptional profiling reveals similarities and differences between human stem cell-derived cardiomyocyte clusters and heart tissue

Physiological Genomics ◽

10.1152/physiolgenomics.00118.2011 ◽

2012 ◽

Vol 44 (4) ◽

pp. 245-258 ◽

Cited By ~ 50

Author(s):

Jane Synnergren ◽

Caroline Améen ◽

Andreas Jansson ◽

Peter Sartipy

Keyword(s):

Stem Cell ◽

Ion Channels ◽

Human Heart ◽

Embryonic Stem ◽

Heart Tissue ◽

Phenotypic Characterization ◽

Adult Heart ◽

Promising Source

It is now well documented that human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes. These cells constitute a promising source of material for use in drug development, toxicity testing, and regenerative medicine. To assess their utility as replacement or complement to existing models, extensive phenotypic characterization of the cells is required. In the present study, we used microarrays and analyzed the global transcription of hESC-derived cardiomyocyte clusters (CMCs) and determined similarities as well as differences compared with reference samples from fetal and adult heart tissue. In addition, we performed a focused analysis of the expression of cardiac ion channels and genes involved in the Ca2+-handling machinery, which in previous studies have been shown to be immature in stem cell-derived cardiomyocytes. Our results show that hESC-derived CMCs, on a global level, have a highly similar gene expression profile compared with human heart tissue, and their transcriptional phenotype was more similar to fetal than to adult heart. Despite the high similarity to heart tissue, a number of significantly differentially expressed genes were identified, providing some clues toward understanding the molecular difference between in vivo sourced tissue and stem cell derivatives generated in vitro. Interestingly, some of the cardiac-related ion channels and Ca2+-handling genes showed differential expression between the CMCs and heart tissues. These genes may represent candidates for future genetic engineering to create hESC-derived CMCs that better mimic the phenotype of the cardiomyocytes present in the adult human heart.

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