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Specific induction and long-term maintenance of high purity ventricular cardiomyocytes from human induced pluripotent stem cells.

ABSTRACT: Currently, cardiomyocyte (CM) differentiation methods require a purification step after CM induction to ensure the high purity of the cell population. Here we show an improved human CM differentiation protocol with which high-purity ventricular-type CMs can be obtained and maintained without any CM purification process. We induced and collected a mesodermal cell population (platelet-derived growth factor receptor-? (PDGFR?)-positive cells) that can respond to CM differentiation cues, and then stimulated CM differentiation by means of Wnt inhibition. This method reproducibly generated CMs with purities above 95% in several human pluripotent stem cell lines. Furthermore, these CM populations were maintained in culture at such high purity without any further CM purification step for over 200 days. The majority of these CMs (>95%) exhibited a ventricular-like phenotype with a tendency to structural and electrophysiological maturation, including T-tubule-like structure formation and the ability to respond to QT prolongation drugs. This is a simple and valuable method to stably generate CM populations suitable for cardiac toxicology testing, disease modeling and regenerative medicine.

SUBMITTER: Fukushima H

PROVIDER: S-EPMC7605685 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Specific induction and long-term maintenance of high purity ventricular cardiomyocytes from human induced pluripotent stem cells.

Fukushima Hiroyuki H Yoshioka Miki M Kawatou Masahide M López-Dávila Víctor V Takeda Masafumi M Kanda Yasunari Y Sekino Yuko Y Yoshida Yoshinori Y Yamashita Jun K JK

PloS one 20201102 11

Currently, cardiomyocyte (CM) differentiation methods require a purification step after CM induction to ensure the high purity of the cell population. Here we show an improved human CM differentiation protocol with which high-purity ventricular-type CMs can be obtained and maintained without any CM purification process. We induced and collected a mesodermal cell population (platelet-derived growth factor receptor-α (PDGFRα)-positive cells) that can respond to CM differentiation cues, and then st ...[more]

PMID: 33137106

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Project description:BackgroundWhile mutations in the cardiac type 2 ryanodine receptor (RyR2) have been linked to exercise-induced or catecholaminergic polymorphic ventricular tachycardia (CPVT), its association with polymorphic ventricular tachycardia (PMVT) occurring at rest is unclear. We aimed at constructing a patient-specific human-induced pluripotent stem cell (hiPSC) model of PMVT occurring at rest linked to a single point mutation in RyR2.MethodsBlood samples were obtained from a patient with PMVT at rest due to a heterozygous RyR2-H29D mutation. Patient-specific hiPSCs were generated from the blood samples, and the hiPSC-derived cardiomyocytes (CMs) were generated via directed differentiation. Using CRIPSR/Cas9 technology, isogenic controls were generated by correcting the RyR2-H29D mutation. Using patch-clamp, fluorescent confocal microscopy and video-image-based analysis, the molecular and functional properties of RyR2-H29D hiPSCCMs and control hiPSCCMs were compared.FindingsRyR2-H29D hiPSCCMs exhibit intracellular sarcoplasmic reticulum (SR) Ca2+ leak through RyR2 under physiological pacing. RyR2-H29D enhances the contribution of inositol 1,4,5-trisphosphate receptors to excitation-contraction coupling (ECC) that exacerbates abnormal Ca2+ release in RyR2-H29D hiPSCCMs. RyR2-H29D hiPSCCMs exhibit shorter action potentials, delayed afterdepolarizations, arrhythmias and aberrant contractile properties compared to isogenic controls. The RyR2-H29D mutation causes post-translational remodeling that is fully reversed with isogenic controls.InterpretationTo conclude, in a model based on a RyR2 point mutation that is associated with short-coupled PMVT at rest, RyR2-H29D hiPSCCMs exhibited aberrant intracellular Ca2+ homeostasis, shortened action potentials, arrhythmias and abnormal contractile properties.FundingFrench Muscular Dystrophy Association (AFM; project 16,073, MNM2 2012 and 20,225), "Fondation de la Recherche Médicale" (FRM; SPF20130526710), "Institut National pour la Santé et la Recherche Médicale" (INSERM), National Institutes of Health (ARM; R01 HL145473) and New York State Department of Health (NYSTEM C029156).

Project description:The SGLT2 inhibitor empagliflozin improved cardiovascular outcomes in patients with diabetes. As the cardiac mechanisms remain elusive, we investigated the long-term effects (up to 2 months) of empagliflozin on excitation-contraction (EC)-coupling in human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) in a blinded manner. IPSC from 3 donors, differentiated into pure iPSC-CM (4 differentiations), were treated with a clinically relevant concentration of empagliflozin (0.5 ?mol/l) or vehicle control. Treatment, data acquisition, and analysis were conducted externally blinded. Epifluorescence microscopy measurements in iPSC-CM showed that empagliflozin has neutral effects on Ca2+ transient amplitude, diastolic Ca2+ levels, Ca2+ transient kinetics, or sarcoplasmic Ca2+ load after 2 weeks or 8 weeks of treatment. Confocal microscopy determining possible effects on proarrhythmogenic diastolic Ca2+ release events showed that in iPSC-CM, Ca2+ spark frequency and leak was not altered after chronic treatment with empagliflozin. Finally, in patch-clamp experiments, empagliflozin did not change action potential duration, amplitude, or resting membrane potential compared with vehicle control after long-term treatment. Next-generation RNA sequencing (NGS) and mapped transcriptome profiles of iPSC-CMs untreated and treated with empagliflozin for 8 weeks showed no differentially expressed EC-coupling genes. In line with NGS data, Western blots indicate that empagliflozin has negligible effects on key EC-coupling proteins. In this blinded study, direct treatment of iPSC-CM with empagliflozin for a clinically relevant duration of 2 months did not influence cardiomyocyte EC-coupling and electrophysiology. Therefore, it is likely that other mechanisms independent of cardiomyocyte EC-coupling are responsible for the beneficial treatment effect of empagliflozin. KEY MESSAGES: This blinded study investigated the clinically relevant long-term effects (up to 2 months) of empagliflozin on cardiomyocyte excitation-contraction (EC)-coupling. Human cardiomyocytes derived from induced pluripotent stem cells (iPSC-CM) were used to study a human model including a high repetition number of experiments. Empagliflozin has neutral effects on cardiomyocyte Ca2+ transients, sarcoplasmic Ca2+ load, and diastolic sarcoplasmic Ca2+ leak. In patch-clamp experiments, empagliflozin did not change the action potential. Next-generation RNA sequencing, mapped transcriptome profiles, and Western blots of iPSC-CM untreated and treated with empagliflozin showed no differentially expressed EC-coupling candidates.

Dataset Information

Specific induction and long-term maintenance of high purity ventricular cardiomyocytes from human induced pluripotent stem cells.

Publications

Specific induction and long-term maintenance of high purity ventricular cardiomyocytes from human induced pluripotent stem cells.

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