Defining optimal enzyme and matrix combination for replating of human induced pluripotent stem cell-derived cardiomyocytes at different levels of maturity

Koc, Arzuhan; Goktas, Sevilay Sahoglu; Caglar, Tuba Akgul; Çağavı, Esra

doi:10.1016/j.yexcr.2021.112599

Cited by 8 publications

(8 citation statements)

References 46 publications

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“…To generate iPSC lines, PBMCs isolated from P1 and P2 (Figure 1c) and two unrelated healthy controls (C1 and C2) were reprogrammed by transduction with Sendai virus carrying the pluripotency genes OCT4 , SOX2 , KLF4 , and MYC . The C1 healthy control line used in this study was reported previously (Koc et al, 2021). The iPSC lines were characterized by the expression of pluripotency‐related markers Tra1‐60 and SOX2 proteins and the significantly increased mRNA expression of OCT4, NANOG, and SOX2 similar to the internal control of the hESC line (Figure 1e,f).…”

Section: Resultsmentioning

confidence: 99%

“…iPSCs were directed to differentiate into cardiomyocytes by using established protocols modulating WNT signals by small molecules (Koc et al, 2021; Lian et al, 2013). Starting from differentiation day 8, contracting cardiomyocytes were observed under the light microscope (Zeiss).…”

Section: Methodsmentioning

confidence: 99%

“…iPSC lines were compared to the hESC (WA07) line that was commercially purchased from the WiCell organization. The C1 healthy control line used in this study was generated and reported previously (Koc et al, 2021).…”

Section: Generation Of Induced Pluripotent Stem Cellsmentioning

confidence: 99%

“…Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) to analyze gene expression of pluripotency, cardiomyocyte-specific, 3germ layer genes, and housekeeping genes based on published or designed primer sets (Table S2). cDNA was synthesized from 100 ng RNA using an iScript Advanced cDNA Synthesis Kit (Bio-Rad) and qRT-PCR was performed as previously published (Koc et al, 2021).…”

Section: Gene Expression Analysismentioning

confidence: 99%

“…Cultures were plated as single-cell layers onto the Matrigel-coated 8-chamber slides or 35-mm glass-bottom culture dishes and were fixed with 4% paraformaldehyde for 10 min at RT. For immunocytochemical analysis, the described protocols (Koc et al, 2021) and manufacturers' guidelines for supplied antibodies were used (Table S3). Confocal or spinning disk confocal microscopes (Zeiss) were used for image acquisition and data processing.…”

Section: Immunocytochemistrymentioning

confidence: 99%

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Functional evaluation of the tachycardia patient‐derived iPSC cardiomyocytes carrying a novel pathogenic SCN5A variant

Sahoglu

Kazci

Tuncay

et al. 2022

Journal Cellular Physiology

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Tachycardia is characterized by high beating rates that can lead to life‐threatening fibrillations. Mutations in several ion‐channel genes were implicated with tachycardia; however, the complex genetic contributors and their modes of action are still unclear. Here, we investigated the influence of an SCN5A gene variant on tachycardia phenotype by deriving patient‐specific iPSCs and cardiomyocytes (iPSC‐CM). Two tachycardia patients were genetically analyzed and revealed to inherit a heterozygous p.F1465L variant in the SCN5A gene. Gene expression and immunocytochemical analysis in iPSC‐CMs generated from patients did not show any significant changes in mRNA levels of SCN5A or gross NaV1.5 cellular mislocalization, compared to healthy‐derived iPSC‐CMs. Electrophysiological and contraction imaging analysis in patient iPSC‐CMs revealed intermittent fibrillation‐like states, occasional arrhythmic events, and sustained high‐paced contractions that could be selectively reduced by flecainide treatment. The patch‐clamp analysis demonstrated a negative shift in the voltage‐dependent activation at the patient‐derived iPSC‐CMs compared to the healthy control line, suggestive of a gain‐of‐function activity associated with the SCN5A+/p.F1465L variant. Our patient‐derived iPSC‐CM model recapitulated the clinically relevant characteristics of tachycardia associated with a novel pathogenic SCN5A+/p.F1465L variant leading to altered Na+ channel kinetics as the likely mechanism underlying high excitability and tachycardia phenotype.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Generation Of Induced Pluripotent Stem Cellsmentioning

confidence: 99%

Section: Gene Expression Analysismentioning

confidence: 99%

Section: Immunocytochemistrymentioning

confidence: 99%

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Functional evaluation of the tachycardia patient‐derived iPSC cardiomyocytes carrying a novel pathogenic SCN5A variant

Sahoglu

Kazci

Tuncay

et al. 2022

Journal Cellular Physiology

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Software Tool for Automatic Quantification of Sarcomere Length and Organization in Fixed and Live 2D and 3D Muscle Cell Cultures In Vitro

et al. 2022

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Sarcomeres are the structural units of the contractile apparatus in cardiac and skeletal muscle cells. Changes in sarcomere characteristics are indicative of changes in the sarcomeric proteins and function during development and disease. Assessment of sarcomere length, alignment, and organization provides insight into disease and drug responses in striated muscle cells and models, ranging from cardiomyocytes and skeletal muscle cells derived from human pluripotent stem cells to adult muscle cells isolated from animals or humans. However, quantification of sarcomere length is typically time consuming and prone to user‐specific selection bias. Automated analysis pipelines exist but these often require either specialized software or programming experience. In addition, these pipelines are often designed for only one type of cell model in vitro. Here, we present an easy‐to‐implement protocol and software tool for automated sarcomere length and organization quantification in a variety of striated muscle in vitro models: Two dimensional (2D) cardiomyocytes, three dimensional (3D) cardiac microtissues, isolated adult cardiomyocytes, and 3D tissue engineered skeletal muscles. Based on an existing mathematical algorithm, this image analysis software (SotaTool) automatically detects the direction in which the sarcomere organization is highest over the entire image and outputs the length and organization of sarcomeres. We also analyzed videos of live cells during contraction, thereby allowing measurement of contraction parameters like fractional shortening, contraction time, relaxation time, and beating frequency. In this protocol, we give a step‐by‐step guide on how to prepare, image, and automatically quantify sarcomere and contraction characteristics in different types of in vitro models and we provide basic validation and discussion of the limitations of the software tool. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Staining and analyzing static hiPSC‐CMs with SotaTool Alternate Protocol: Sample preparation, acquisition, and quantification of fractional shortening in live reporter hiPSC lines Support Protocol 1: Finding the image resolution Support Protocol 2: Advanced analysis settings Support Protocol 3: Finding sarcomere length in non‐aligned cells

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Replating Protocol for Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Koc

Çağavı

2021

Embryonic Stem Cell Protocols

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Defining optimal enzyme and matrix combination for replating of human induced pluripotent stem cell-derived cardiomyocytes at different levels of maturity

Cited by 8 publications

References 46 publications

Functional evaluation of the tachycardia patient‐derived iPSC cardiomyocytes carrying a novel pathogenic SCN5A variant

Functional evaluation of the tachycardia patient‐derived iPSC cardiomyocytes carrying a novel pathogenic SCN5A variant

Software Tool for Automatic Quantification of Sarcomere Length and Organization in Fixed and Live 2D and 3D Muscle Cell Cultures In Vitro

Replating Protocol for Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes

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