Ion channel expression and electrophysiology of singular human (primary and induced pluripotent stem cell derived) cardiomyocytes

Schmid, Christina; Abi‐Gerges, Najah; Zellner, Dietmar; Rast, Georg

doi:10.1101/2021.03.04.433834

Cited by 4 publications

(3 citation statements)

References 54 publications

(145 reference statements)

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“…To investigate single cells, the singularization of monolayer hiPSC-CM cultures was performed by splitting the confluent cells of a single well into 4–6 new wells of a 24-well plate, as described previously [ 19 , 20 ]. Between day 1 and 3 after singularizing, the hiPSC-CMs were used for experiments.…”

Section: Methodsmentioning

confidence: 99%

“…All the procedures were performed ideally under ribonuclease (RNase)-free conditions. For the technical details and solutions, see [ 20 ]. The randomized selection of single cardiomyocytes was accomplished by picking the central viable cell of an arbitrarily chosen field of view under an inverted CKX41 microscope (Olympus, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

“…Single-cell mRNA expression was determined in hiPSC-CMs and adult human primary cardiomyocytes, utilizing Ambion’s Single Cell-to-CT kit (Thermo Fisher Scientific, Waltham, MA, USA) in combination with sequence-specific TaqMan gene expression assays (Thermo Fisher Scientific, Waltham, MA, USA, Supplementary Table S7 ). The experiments were performed according to the manufacturer´s instructions (for details see [ 20 ]). The qPCR of every target gene was performed in single reactions as technical duplicates using the Roche Lightcycler 480 (Hoffmann-La Roche, Basel, Switzerland).…”

Section: Methodsmentioning

confidence: 99%

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Ion Channel Expression and Electrophysiology of Singular Human (Primary and Induced Pluripotent Stem Cell-Derived) Cardiomyocytes

Schmid

Abi‐Gerges

Leitner

et al. 2021

Cells

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Subtype-specific human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are promising tools, e.g., to assess the potential of drugs to cause chronotropic effects (nodal hiPSC-CMs), atrial fibrillation (atrial hiPSC-CMs), or ventricular arrhythmias (ventricular hiPSC-CMs). We used single-cell patch-clamp reverse transcriptase-quantitative polymerase chain reaction to clarify the composition of the iCell cardiomyocyte population (Fujifilm Cellular Dynamics, Madison, WI, USA) and to compare it with atrial and ventricular Pluricytes (Ncardia, Charleroi, Belgium) and primary human atrial and ventricular cardiomyocytes. The comparison of beating and non-beating iCell cardiomyocytes did not support the presence of true nodal, atrial, and ventricular cells in this hiPSC-CM population. The comparison of atrial and ventricular Pluricytes with primary human cardiomyocytes showed trends, indicating the potential to derive more subtype-specific hiPSC-CM models using appropriate differentiation protocols. Nevertheless, the single-cell phenotypes of the majority of the hiPSC-CMs showed a combination of attributes which may be interpreted as a mixture of traits of adult cardiomyocyte subtypes: (i) nodal: spontaneous action potentials and high HCN4 expression and (ii) non-nodal: prominent INa-driven fast inward current and high expression of SCN5A. This may hamper the interpretation of the drug effects on parameters depending on a combination of ionic currents, such as beat rate. However, the proven expression of specific ion channels supports the evaluation of the drug effects on ionic currents in a more realistic cardiomyocyte environment than in recombinant non-cardiomyocyte systems.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Ion Channel Expression and Electrophysiology of Singular Human (Primary and Induced Pluripotent Stem Cell-Derived) Cardiomyocytes

Schmid

Abi‐Gerges

Leitner

et al. 2021

Cells

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Rapid ionic current phenotyping (RICP) identifies mechanistic underpinnings of iPSC-CM AP heterogeneity

Clark,

Wei,

Fullerton

et al. 2023

Preprint

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As a renewable, easily accessible, human-derived in vitro model, human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) are a promising tool for studying arrhythmia-related factors, including cardiotoxicity and congenital proarrhythmia risks. An oft-mentioned limitation of iPSC-CMs is the abundant cell-to-cell variability in recordings of their electrical activity. Here, we develop a new method, rapid ionic current phenotyping (RICP), that utilizes a short (10 s) voltage clamp protocol to quantify cell-to-cell heterogeneity in key ionic currents. We correlate these ionic current dynamics to action potential recordings from the same cells and produce mechanistic insights into cellular heterogeneity. We present evidence that the L-type calcium current is the main determinant of upstroke velocity, rapid delayed rectifier K+ current is the main determinant of the maximal diastolic potential, and an outward current in the excitable range of slow delayed rectifier K+ is the main determinant of action potential duration. We measure an unidentified outward current in several cells at 6 mV that is not recapitulated by iPSC-CM mathematical models but contributes to determining action potential duration. In this way, our study both quantifies cell-to-cell variability in membrane potential and ionic currents, and demonstrates how the ionic current variability gives rise to action potential heterogeneity. Based on these results, we argue that iPSC-CM heterogeneity should not be viewed simply as a problem to be solved but as a model system to understand the mechanistic underpinnings of cellular variability.

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Does Enhanced Structural Maturity of hiPSC-Cardiomyocytes Better for the Detection of Drug-Induced Cardiotoxicity?

et al. 2023

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Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are currently used following the Comprehensive in vitro Proarrhythmic Assay (CiPA) initiative and subsequent recommendations in the International Council for Harmonization (ICH) guidelines S7B and E14 Q&A, to detect drug-induced cardiotoxicity. Monocultures of hiPSC-CMs are immature compared to adult ventricular cardiomyocytes and might lack the native heterogeneous nature. We investigated whether hiPSC-CMs, treated to enhance structural maturity, are superior in detecting drug-induced changes in electrophysiology and contraction. This was achieved by comparing hiPSC-CMs cultured in 2D monolayers on the current standard (fibronectin matrix, FM), to monolayers on a coating known to promote structural maturity (CELLvo™ Matrix Plus, MM). Functional assessment of electrophysiology and contractility was made using a high-throughput screening approach involving the use of both voltage-sensitive fluorescent dyes for electrophysiology and video technology for contractility. Using 11 reference drugs, the response of the monolayer of hiPSC-CMs was comparable in the two experimental settings (FM and MM). The data showed no functionally relevant differences in electrophysiology between hiPSC-CMs in standard FM and MM, while contractility read-outs indicated an altered amplitude of contraction but not changes in time course. RNA profiling for cardiac proteins shows similarity of the RNA expression across the two forms of 2D culture, suggesting that cell-to-matrix adhesion differences may explain account for differences in contraction amplitude. The results support the view that hiPSC-CMs in both 2D monolayer FM and MM that promote structural maturity are equally effective in detecting drug-induced electrophysiological effects in functional safety studies.

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Ion channel expression and electrophysiology of singular human (primary and induced pluripotent stem cell derived) cardiomyocytes

Cited by 4 publications

References 54 publications

Ion Channel Expression and Electrophysiology of Singular Human (Primary and Induced Pluripotent Stem Cell-Derived) Cardiomyocytes

Ion Channel Expression and Electrophysiology of Singular Human (Primary and Induced Pluripotent Stem Cell-Derived) Cardiomyocytes

Rapid ionic current phenotyping (RICP) identifies mechanistic underpinnings of iPSC-CM AP heterogeneity

Does Enhanced Structural Maturity of hiPSC-Cardiomyocytes Better for the Detection of Drug-Induced Cardiotoxicity?

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