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Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential.

ABSTRACT: In the past few years, the use of specific cell types derived from induced pluripotent stem cells (iPSCs) has developed into a powerful approach to investigate the cellular pathophysiology of numerous diseases. Despite advances in therapy, heart disease continues to be one of the leading causes of death in the developed world. A major difficulty in unravelling the underlying cellular processes of heart disease is the extremely limited availability of viable human cardiac cells reflecting the pathological phenotype of the disease at various stages. Thus, the development of methods for directed differentiation of iPSCs to cardiomyocytes (iPSC-CMs) has provided an intriguing option for the generation of patient-specific cardiac cells. In this review, a comprehensive overview of the currently published iPSC-CM models for hereditary heart disease is compiled and analysed. Besides the major findings of individual studies, detailed methodological information on iPSC generation, iPSC-CM differentiation, characterization, and maturation is included. Both, current advances in the field and challenges yet to overcome emphasize the potential of using patient-derived cell models to mimic genetic cardiac diseases.

SUBMITTER: van Mil A

PROVIDER: S-EPMC6887927 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential.

van Mil Alain A Balk Geerthe Margriet GM Neef Klaus K Buikema Jan Willem JW Asselbergs Folkert W FW Wu Sean M SM Doevendans Pieter A PA Sluijter Joost P G JPG

Cardiovascular research 20181201 14

In the past few years, the use of specific cell types derived from induced pluripotent stem cells (iPSCs) has developed into a powerful approach to investigate the cellular pathophysiology of numerous diseases. Despite advances in therapy, heart disease continues to be one of the leading causes of death in the developed world. A major difficulty in unravelling the underlying cellular processes of heart disease is the extremely limited availability of viable human cardiac cells reflecting the pat ...[more]

PMID: 30169602

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Project description:Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients' genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with iPSC.

Project description:Stem cell therapy has the potential to regenerate cardiac function after myocardial infarction. In this study, we sought to examine if fibrin microthread technology could be leveraged to develop a contractile fiber from human pluripotent stem cell derived cardiomyocytes (hPS-CM). hPS-CM seeded onto fibrin microthreads were able to adhere to the microthread and began to contract seven days after initial seeding. A digital speckle tracking algorithm was applied to high speed video data (>60 fps) to determine contraction behaviour including beat frequency, average and maximum contractile strain, and the principal angle of contraction of hPS-CM contracting on the microthreads over 21 days. At day 7, cells seeded on tissue culture plastic beat at 0.83 ± 0.25 beats/sec with an average contractile strain of 4.23±0.23%, which was significantly different from a beat frequency of 1.11 ± 0.45 beats/sec and an average contractile strain of 3.08±0.19% at day 21 (n = 18, p < 0.05). hPS-CM seeded on microthreads beat at 0.84 ± 0.15 beats/sec with an average contractile strain of 3.56±0.22%, which significantly increased to 1.03 ± 0.19 beats/sec and 4.47±0.29%, respectively, at 21 days (n = 18, p < 0.05). At day 7, 27% of the cells had a principle angle of contraction within 20 degrees of the microthread, whereas at day 21, 65% of hPS-CM were contracting within 20 degrees of the microthread (n = 17). Utilizing high speed calcium transient data (>300 fps) of Fluo-4AM loaded hPS-CM seeded microthreads, conduction velocities significantly increased from 3.69 ± 1.76 cm/s at day 7 to 24.26 ± 8.42 cm/s at day 21 (n = 5-6, p < 0.05). hPS-CM seeded microthreads exhibited positive expression for connexin 43, a gap junction protein, between cells. These data suggest that the fibrin microthread is a suitable scaffold for hPS-CM attachment and contraction. In addition, extended culture allows cells to contract in the direction of the thread, suggesting alignment of the cells in the microthread direction.

Project description:AimsDiastolic dysfunction (DD) is common among hypertrophic cardiomyopathy (HCM) patients, causing major morbidity and mortality. However, its cellular mechanisms are not fully understood, and presently there is no effective treatment. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) hold great potential for investigating the mechanisms underlying DD in HCM and as a platform for drug discovery.Methods and resultsIn the present study, beating iPSC-CMs were generated from healthy controls and HCM patients with DD. Micropatterned iPSC-CMs from HCM patients showed impaired diastolic function, as evidenced by prolonged relaxation time, decreased relaxation rate, and shortened diastolic sarcomere length. Ratiometric Ca2+ imaging indicated elevated diastolic [Ca2+]i and abnormal Ca2+ handling in HCM iPSC-CMs, which were exacerbated by β-adrenergic challenge. Combining Ca2+ imaging and traction force microscopy, we observed enhanced myofilament Ca2+ sensitivity (measured as dF/Δ[Ca2+]i) in HCM iPSC-CMs. These results were confirmed with genome-edited isogenic iPSC lines that carry HCM mutations, indicating that cytosolic diastolic Ca2+ overload, slowed [Ca2+]i recycling, and increased myofilament Ca2+ sensitivity, collectively impairing the relaxation of HCM iPSC-CMs. Treatment with partial blockade of Ca2+ or late Na+ current reset diastolic Ca2+ homeostasis, restored diastolic function, and improved long-term survival, suggesting that disturbed Ca2+ signalling is an important cellular pathological mechanism of DD. Further investigation showed increased expression of L-type Ca2+channel (LTCC) and transient receptor potential cation channels (TRPC) in HCM iPSC-CMs compared with control iPSC-CMs, which likely contributed to diastolic [Ca2+]i overload.ConclusionIn summary, this study recapitulated DD in HCM at the single-cell level, and revealed novel cellular mechanisms and potential therapeutic targets of DD using iPSC-CMs.

Project description:Ion channels and cytoskeletal proteins in the cardiac dyad play a critical role in maintaining excitation-contraction (E-C) coupling and provide cardiac homeostasis. Functional changes in these dyad proteins, whether induced by genetic, epigenetic, metabolic, therapeutic, or environmental factors, can disrupt normal cardiac electrophysiology, leading to abnormal E-C coupling and arrhythmias. Animal models and heterologous cell cultures provide platforms to elucidate the pathogenesis of arrhythmias for basic cardiac research; however, these traditional systems do not truly reflect human cardiac electro-pathophysiology. Notably, patients with the same genetic variants of inherited channelopathies (ICC) often exhibit incomplete penetrance and variable expressivity which underscores the need to establish patient-specific disease models to comprehend the mechanistic pathways of arrhythmias and determine personalized therapies. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) inherit the genetic background of the patient and reflect the electrophysiological characteristics of the native cardiomyocytes. Thus, iPSC-CMs provide an innovative and translational pivotal platform in cardiac disease modeling and therapeutic screening. In this review, we will examine how patient-specific iPSC-CMs historically evolved to model arrhythmia syndromes in a dish, and their utility in understanding the role of specific ion channels and their functional characteristics in causing arrhythmias. We will also examine how CRISPR/Cas9 have enabled the establishment of patient-independent and variant-induced iPSC-CMs-based arrhythmia models. Next, we will examine the limitations of using human iPSC-CMs with respect to in vitro arrhythmia modeling that stems from variations in iPSCs or toxicity due to gene editing on iPSC or iPSC-CMs and explore how such hurdles are being addressed. Importantly, we will also discuss how novel 3D iPSC-CM models can better capture in vitro characteristics and how all-optical platforms provide non-invasive and high- throughput electrophysiological data that is useful for stratification of emerging arrhythmogenic variants and drug discovery. Finally, we will examine strategies to improve iPSC-CM maturity, including powerful gene editing and optogenetic tools that can introduce/modify specific ion channels in iPSC-CMs and tailor cellular and functional characteristics. We anticipate that an elegant synergy of iPSCs, novel gene editing, 3D- culture models, and all-optical platforms will offer a high-throughput template to faithfully recapitulate in vitro arrhythmogenic events necessary for personalized arrhythmia monitoring and drug screening process.

Dataset Information

Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential.

Publications

Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential.

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