Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Insights into Molecular, Cellular, and Functional Phenotypes

Abstract

Disease models are essential for understanding cardiovascular disease pathogenesis and developing new therapeutics. The human induced pluripotent stem cell (iPSC) technology has generated significant enthusiasm for its potential application in basic and translational cardiac research. Patient-specific iPSC-derived cardiomyocytes (iPSC-CMs) offer an attractive experimental platform to model cardiovascular diseases, study the earliest stages of human development, accelerate predictive drug toxicology tests, and advance potential regenerative therapies. Harnessing the power of iPSC-CMs could eliminate confounding species-specific and inter-personal variations, and ultimately pave the way for the development of personalized medicine for cardiovascular diseases. However, the predictive power of iPSC-CMs as a valuable model is contingent on comprehensive and rigorous molecular and functional characterization.

iPSC-CMs: a new and versatile human in vitro cardiomyocyte model

Recent advances in genomics and molecular medicine promise to revolutionize human health by enabling much more precise prediction, prevention, and treatment of cardiovascular diseases on an individual level.¹ This precision medicine approach is based on the ability to better diagnose and stratify patients into different treatment groups by correlating a patient's genotype with their cellular phenotype and uncovering the genetic differences among people that may influence their responses to therapies. However, realizing this potential requires the development of accurate disease models. Models that recapitulate individual patients' diseases at the molecular and cellular level could lead to a better understanding of the disease progression and pathogenesis, and ultimately enable the prediction of individual patient's responses to targeted treatments.

Disease models have been and will continue to be instrumental in providing important insights into the molecular basis of cardiovascular development and disease.² The knowledge gained from studying transgenic animals and transformed cell lines has already been successfully applied to understand human cardiovascular disease. Nevertheless, this translation would be significantly strengthened by the availability of patient-specific in vitro models. Human based models are particularly important for cardiovascular research because the physiology of animal models is different from human cardiomyocytes. Particularly, considerable differences exist between cardiomyocytes from small animal models and human cardiomyocytes, including beating rates, energetics, myofilament composition, expression of key ion channels and electrophysiology as well as Ca²⁺ cycling. These differences in physiology are substantially less between humans and large animal models such as non-human primates, pigs, and dogs.³

The recent advent of the human induced pluripotent stem cell (iPSC) technology,⁴ and an increasingly refined capacity to differentiate iPSCs into disease-relevant cell types such as cardiomyocytes (iPSC-CMs),⁵ provide an unprecedented opportunity for the generation of human patient-specific cells for use in disease modeling, personalized drug screening, and regenerative approaches toward precision medicine.^{6, 7} Implementation of this unique and clinically relevant model system presents a significant advantage in cardiovascular research as it can circumvent complications in translating data from models across different species and biological characteristics.

iPSC-CMs offer several advantages over current in vitro models such as immortalized cell lines, human cadaveric tissue, and primary cultures of nonhuman animal origin. First, the derivation of iPSC-CMs is at most minimally invasive (typically via skin biopsy or blood draws) and can theoretically provide an unlimited supply of human cardiomyocytes. Second, iPSC-CMs can be functionally characterized in vitro to model the complex cellular physiology of cardiomyocytes.. Third, iPSC-CMs recapitulate the genome of a subject, allowing for the assessment of genotype-phenotype associations.

Over the past few years, there has been considerable progress in the iPSC-CM technology and its contributions to cardiovascular research are already well-recognized. For example, iPSC models have been recently used to describe cardiac channelopathies such as long QT syndromes (LQT1,^8-10 LQT2,^11-13 LQT3/Brugada syndrome,¹⁴ and LQT8 Timothy syndrome¹⁵), catecholaminergic polymorphic ventricular tachycardia (CPVT),^16-18 arrhythmogenic right ventricular dysplasia (ARVD),^{19, 20} familial hypertrophic cardiomyopathy (HCM),²¹ and familial dilated cardiomyopathy (DCM).²² As the iPSC-CM technology continues to evolve, it will greatly facilitate the study of inherited and acquired cardiovascular diseases, infectious diseases, cardiovascular development, drug discovery, toxicology screening, and personalized cell therapy (Figure 1).

Figure 1. Current applications of patient-specific iPSC-CM technology.

iPSC-CMs have been used for disease modeling of inherited cardiomyopathies and channelopathies, regenerative therapies, drug discovery and cardiotoxicity testing, as well as for studying metabolic abnormalities and cardiac development.

In this review article, we will highlight the current state of iPSC-CMs, focusing on their phenotype and function. We will discuss the molecular phenotypes, electrophysiological and calcium handling properties, and bioenergetics. We will also explore what the future may hold for their use in cardiovascular research, pharmacology, and regenerative medicine toward precision medicine.

Generation of iPSC-CMs

Studies with different model organisms have demonstrated that signaling pathways, such as Activin/Nodal/TGF-β, Wnt, and BMP, play pivotal roles in establishing the cardiovascular system.^23-25 By mimicking endogenous developmental signaling cues, directed differentiation methodologies for generating iPSC-CMs have been developed.^26-28 Several permutations of growth factors and small molecules have recently been reported to improve reproducibility and efficiency of iPSC-CM differentiation protocols in both adherent and suspension cultures.⁵ Further purification of CMs from a mixed population of iPSC-derived cells can be accomplished by non-genetic methods, including cell-surface markers,^{29, 30} mitochondria specific dyes,³⁰ fluorescent probes,³¹ and glucose deprivation.³² Although iPSC lines appear to respond differently to developmental signals due to intrinsic differences in their genetic background, these differentiation protocols have been successfully applied to iPSCs derived from distinct sources of somatic cells and reprogramming methods.^{26-28, 33, 34} However, the resultant iPSC-CMs population is a heterogeneous pool of atrial-, ventricular- and nodal-like cells. As native atrial, nodal and ventricular myocytes possess distinct molecular and functional properties, coaxing human iPSC differentiation toward specific cardiomyocyte subtypes remains challenging for the current methodologies. In this respect, recent reports suggest that pluripotent stem cells could be directed either to atrial-like or to ventricular-like cardiomyocytes by modulating the retinoic acid^{35, 36} and Wnt signaling pathways.³⁷ However, the elucidation of the molecular mechanism(s) underlying the cardiomyocyte subtype specification would be essential to further refine the current differentiation protocols and improve our understanding of lineage-specific development.

Molecular profiling

At the molecular level, the differentiation of iPSCs towards the cardiomyocyte lineage is orchestrated by the sequential expression of distinct sets of genes in specific stages in a pattern consistent with normal cardiac development: mesoderm formation (BRY and MIXL1), cardiogenic mesoderm (MESP1, ISL1, and KDR), cardiac specific progenitors (NKX2.5, GATA4, TBX5, MEF2C, and HAND1/2), and structural genes encoding for sarcomeric-related proteins of terminal differentiated cardiomyocytes (MYL2, MYL7, MYH6, and TNNT2).^{26, 33} Gene expression analysis has revealed that normal iPSC-CMs and disease-specific iPSC-CMs expressed all the major cardiac ion channel genes found in the adult left ventricular cardiac tissue, including sodium channel (SCN5A), L-type calcium channels (CACNA1C and CACNA1D), and potassium channels (KCNH2 and KCNQ1).³⁸ Furthermore, iPSC-CMs express genes that encode critical components of the Ca²⁺ cycling machinery such as inositol trisphosphate receptor (IP3R), ryanodine receptor (RYR2), sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a), calsequestrin 2 (CASQ2), calreticulin (CALR), junctophilin 2 (JPH2), phospholamban (PLN), sodium calcium exchanger (NCX), and triadin (TRDN). However, their relative expression differs from that of the human adult ventricular tissue.^{17, 39, 40} Mitochondrial complexes I–V and genes involved in cholesterol metabolism (PRKAG1 and PRKAG2) as well as genes that confer protection against apoptotic and oxidative stress processes (BCL2L1 and SOD1, respectively) are also expressed in iPSC-CMs.⁴¹ Taken together, these studies demonstrated that the gene expression in iPSC-CMs closely mirrors the patterns observed in human cardiomyocytes (Figure 2).

Figure 2. Expression of key structural and functional genes in iPSC-CMs.

A) Schematic of the major structural and functional features of iPSC-CMs. In adult CMs, upon membrane depolarization a small amount of Ca²⁺ influx induced by activation of voltage-dependent L-type Ca2+ channels (CACNAC1) triggers the release of Ca²⁺ through the ryanodine receptors (RYR2) SR, termed Ca²⁺-induced Ca²⁺-release (CICR) mechanism. The released Ca²⁺ ions diffuse through the cytosolic space and bind to troponin C (TNNC1), resulting in the release of inhibition induced by troponin I (TNNI1), which activates the sliding of thin and thick filaments, and lead to cardiac contraction. Recovery occurs as Ca²⁺ is extruded by the Na²⁺/Ca²⁺ exchanger (NCX1) and returned to the SR by the sarco(endo)plasmic Ca²⁺-ATPase (ATP2A2) pumps on the nonjunctional region of the SR that are regulated by phospholamban (PLN). This process is conserved in iPSC-CMs, but major differences exist, such as a nascent SR, the presence of an Inositol 1,4,5-trisphosphate (IP₃)-releasable Ca²⁺ pool, and the complete absence of T-tubules. The genes encoding the major transmembrane ion channels involved in the generation of action potential are also shown. B) Immunofluorescence staining of cardiac troponin T and a-sarcomeric actinin in iPSC-CMs. C) Line-scan images and spontaneous Ca²⁺ transients in iPSC-CMs. KCNIP2: Potassium channel-interacting protein 2; KCNH2: Potassium voltage-gated channel, subfamily H (eag-related), member 2; KCNQ1: Potassium voltage-gated channel, KQT-like subfamily, member 1; NCX1: Na⁺/Ca²⁺ exchanger; KCNJ2: Potassium inwardly-rectifying channel, subfamily J, member 2; RYR2: Ryanodine receptor 2; SERCA2: ATPase, Ca²⁺ transporting, cardiac muscle, slow twitch 2; PLN: Phospholamban; CACNA1C; Calcium channel, voltage-dependent, L type, alpha 1C subunit; SCN5A: Sodium channel, voltage-gated, type V, alpha subunit; TNNT2: Troponin T type 2; MYL2: Myosin, light chain 2; MYL3: Myosin, light chain 3; TNNI3: Troponin I type 3; TNNC: Troponin C type 1; TTN: Titin; MYH6: Myosin, heavy chain 6, alpha; MYH7: Myosin, heavy chain 7, beta; MYBPC3: Myosin binding protein C; TPM1: Tropomyosin 1 (alpha); ACTC1: Actin, alpha, cardiac muscle 1.

From a disease modeling perspective, the faithful expression of disease-associated alleles is a prerequisite for proper manifestation of the disease phenotypes in iPSC-CMs. For example, mutations in genes encoding sarcomeric components, including TNNT2 and MYH7, have been implicated in the two most common forms of inherited cardiomyopathies, HCM and DCM. Mutations in the genes encoding potassium (KCNQ1 and KCNH2), sodium (SCN5A), and calcium (CACNA1C) channels are the most common cause of the LQT syndromes, which is usually inherited in an autosomal dominant manner. Recently, iPSC-CMs have been derived from patients harboring deleterious mutation that recapitulated key disease aspects of HCM, DCM, and LQTS.^{8, 9, 11-15, 21, 22} Such models are currently being utilized to decipher the complex genotype-phenotype relationships and to determine disease effects of specific genetic variants.

Ultrastructural features

To maximize the potential applications of iPSC-CMs in cardiovascular medicine, it is essential for these cells to recapitulate the ultrastructural properties of adult cardiomyocytes. It has been reported that early stage iPSC-CMs are small morphologically and exhibit an immature ultrastructure closely resembling that of fetal CMs (i.e., absence of T-tubules and underdeveloped contractile machinery).^42-44 However, upon prolonged culture, there were significant improvements in myofibril alignment, density and morphology showing adult-like appearance of Z disks, A-bands, I-bands and H-zones, although no clear M-bands or T-tubules were observed.^{43, 44} Similarly, by combining bioengineering approaches with electrical stimulation, Nunes et al⁴⁵ developed a platform called ‘biowire’ that enables the generation of iPSC-CMs with ultrastructural properties similar to those seen in the native CMs. The progressive lengthening of 3-D iPSC-CM tissues that mimics heart growth during development further improved cell alignment and increased sarcomeric ultrastructural organization.⁴⁶ Despite these advances, the contractile properties of iPSC-CMs and engineered tissues remain at rudimentary levels.^46-49

Electrophysiological phenotypes and ion channel function

Comprehensive analyses of the electrophysiological properties of iPSC-CMs have been reported. Based on the action potential (AP) phenotypes recorded in isolated cells, iPSC-CMs are consist of a heterogeneous population categorized as atrial-, nodal-, or ventricular-like.^{15, 33, 50-52} However, cell culture conditions and differentiation protocols used may influence these AP properties, possibly undermining the correctness of this classification.^{53, 54} Although the direct electrophysiological comparison between iPSC-CMs and human adult CMs is challenging due to experimental discrepancies, tissue heterogeneity, and disease status, it is well documented that iPSC-CMs display mixed AP phenotypes characterized by relatively positive maximum diastolic potential (MDP) and slower upstroke velocity when compared to the human native counterparts.⁵² Moreover, recent studies suggest that the ventricular-like iPSC-CM subtype exhibits many key cardiac electrophysiological properties analogous to those of human CMs. The ventricular-like APs display properties of more mature human CMs, including a distinct plateau phase (phase 2) after which repolarization accelerates (phase 3), with AP durations that are within the normal range of the human electrocardiographic QT interval (Figure 3a).⁵²

Figure 3. Comparison of action potentials of ventricular-like iPSC-CMs and adult ventricular cardiomyocytes.

Schematic of ventricular action potentials. Phases 0–4 are the rapid upstroke, early repolarization, plateau, late repolarization, and diastole, respectively. The ionic currents and the genes that generate the currents with schematics of the current trajectories are shown above and below the action potentials. For individual ion channel currents, the voltage dependence of channel-gating properties of ventricular-like iPSC-CMs is remarkably analogous to adult ventricular cardiomyocytes, but significant differences also exist, such as reduced or absence of inward rectifier K⁺ currents (I_K1) and the presence of prominent pacemaker currents that contribute to their automaticity.

Multiple ionic currents have been characterized in single iPSC-CMs, including the sodium (I_Na), the L- and T-type calcium (I_Ca,L and I_Ca,T), the hyperpolarization-activated pacemaker (I_f), the transient outward potassium (I_to), the inward rectifier potassium (I_K1), and the rapid and slow activating components of the delayed rectifier potassium currents (I_Kr and I_Ks, respectively). However, the functional properties of additional currents, such as the ATP-sensitive K⁺ current (I_K,ATP), and the Na⁺-Ca⁺ exchange current (I_NCX) have not yet been reported in iPSC-CMs, whereas the atrial-selective ion current, acetylcholine-activated K⁺ (I_K,ACh), has recently been reported in hESC-derived atrial like CMs³⁵. Here we briefly summarize some of these major channels below.

I _Na

iPSC-CMs have prominent Na⁺ currents with activation and inactivation gating characteristics that are analogous to those of native human ventricular CMs.^{14, 52}

I_Ca,L and I_Ca,T

The activation and inactivation gating properties of I_Ca in iPSC-CMs are similar to those obtained from human ventricular myocytes.^{15, 52} By contrast, although the I_Ca,T current has been found in a subset of cells, its properties have not defined.⁵⁵

I _f

In ventricular-like iPSC-CMs, the presence of hyperpolarization-activated If promotes phase 4 depolarization and thus contribute to automaticity.⁵²

I _K

Three K⁺ currents (I_to, I_Kr, and I_Ks) have been recorded in iPSC-CMs with maximum densities and activation properties comparable to values reported for human cardiac myocytes.^{8, 52} By contrast, the density of I_K1 is either absent or significantly smaller than that reported for native ventricular CMs. Importantly, I_Kr contributes to repolarization of the cardiac AP, and block of I_Kr prolongs the ventricular AP, which is manifest by QT prolongation on the surface ECG. Prolongation of the AP and associated increased QT interval can lead to early afterdepolarizations on the cellular level and trigger the ventricular arrhythmia Torsades de Pointes (TdP). Notably, by measuring AP duration and quantifying drug-induced arrhythmias, such as EADs and delayed afterdepolarizations (DADs), drug-induced cardiotoxicity profiles for healthy subjects, long QT, HCM, and DCM patients were recapitulated at the single cell level by utilizing the iPSC-CM technology.^{38, 56} Interestingly, the iPSC-CMs exhibited distinct responses to known cardiotoxic drugs when derived from healthy versus diseased individuals, suggesting that adverse drug responses could be accurately predicted in individual patients. This raises the prospect of proarrhythmic drugs being readily identified early in a development program, and those individuals at high risk for proarrhythmia could be identified before deleterious drug exposures. The utility of iPSC-CMs in screening for proarrhythmic potential of current and new drug entities in conjunction with in silico modeling, is a major focus of the FDA Comprehensive In Vitro Proarrythmia Assay (CiPA) initiative.⁵⁷

In summary, multiple voltage-gated ion channels are similarly present in iPSC-CMs and adult CMs leading to characteristic cardiac action potentials (Figure 3b).⁵² But, significant differences do exist, such as reduced inward rectifier K⁺ currents and the presence of prominent pacemaker currents. As a result, the iPSC-CMs exhibit spontaneous automaticity, which is not observed in healthy human ventricular myocytes. At the tissue level, these properties may be significantly altered when the cells are coupled in a functional syncytium and recent efforts have been focused on utilizing electrical or mechanical stimulation that appear to promote electrophysiological maturation.^{45, 49} Future studies are clearly needed to characterize in detail the electrophysiological properties of iPSC-CMs at both single-cell and tissue levels.

Excitation contraction coupling and calcium handling

Myocardial contraction and relaxation are coordinated on a beat-to-beat basis by the orchestrated cycling of calcium from the cytoplasm, sarcoplasmic reticulum (SR) and the sarcomere through excitation-contraction coupling (E-C coupling).⁵⁸ Extensive characterization of spontaneous whole-cell [Ca²⁺]_i transients in iPSC-CMs suggests the presence of functional E-C coupling that resembles the native myocardium.⁴⁰ Specifically, it has been demonstrated that Ca²⁺ influx via the depolarization-activated L-type Ca²⁺ channels triggered a marked release of the SR Ca²⁺ stores via the Ca²⁺ sensitive ryanodine sarcoplasmic reticulum receptors (RYRs), recapitulating the “Ca²⁺ induced Ca²⁺ release” (CICR) phenomenon in iPSC-CMs,^{40, 45} a key mechanism underlying E-C coupling.⁵⁸ Of note, iPSC-CM cultured on micro-grooved substrates displayed significantly improved Ca²⁺ cycling and more organized SR Ca²⁺ release in response to caffeine, suggesting that SR Ca²⁺ cycling properties can be influenced by culture conditions.³⁹ In CPVT, an inherited disease characterized by stress-induced ventricular arrhythmias, iPSC-CM based model also support the presence of functional SR and RyR Ca²⁺ transients.¹⁷ However, the Ca²⁺ handling kinetics in iPSC-CMs appear to be relatively slow and characterized by a U-shape Ca²⁺ waveform, suggesting that iPSC-CMs have an immature CICR mechanism.⁵⁹ Indeed, iPSC-CMs exhibit a poorly developed SR and absence of T- tubules that likely affect their Ca²⁺ handling properties.^{42, 43}

Metabolic profile

Various studies have demonstrated that iPSC-CMs have a metabolic phenotype that resembles embryonic ventricular myocytes, which mostly rely on glycolysis for energy production instead of lipid oxidation as seen in adult ventricular myocytes. Strategies to facilitate metabolic maturation have been developed, including culturing iPSC-CMs with an adipogenic cocktail¹⁹ or glucose-free medium⁶⁰ and tissue engineering approaches⁶¹. These approaches were able to yield advanced levels of metabolic phenotype maturation, as evidenced by significant increases of fatty acid beta oxidation.^{19, 60, 61}

By enhancing iPSC-CM metabolic maturation, iPSC-CMs have been used to recapitulate key features of mitochondrial disorders. In a recent study Drawnel et al.⁶⁰ showed that exposing normal iPSC-CMs in diabetic-like conditions (high glucose, endothelin-1, and cortisol) could induce an increment of lipid accumulation, oxidative stress, and sarcomeric disarray, phenocopying diabetic cardiomyopathy. Intriguingly, iPSC-CMs derived from type 2 diabetic patients (T2DM), a disease with complex and multifactorial etiology, also recapitulated key pathophysiological phenotypes in vitro that corresponded to the clinical status of the original donor. Phenotypic drug screening in this model revealed molecules and pathways that may provide therapeutic relevance consistent with the clinical T2DM subtype.⁶⁰ However, the genetic and the epigenetic basis underlying the observed cellular phenotypes and differential response to treatments were not examined. Similarly, by combining bioengineering and gene editing approaches Wang et al.⁶¹ modeled Barth syndrome (BTHS), an X-linked cardiac and skeletal mitochondrial myopathy caused by mutation of the gene encoding Tafazzin (TAZ). BTHS iPSC-CMs displayed an impaired biogenesis of cardiolipin, a major phospholipid of the mitochondria. Subsequently, a novel mechanism was discovered showing that a TAZ deficiency in BTHS markedly increased reactive oxygen species (ROS) production and that suppression of ROS rescued the aberrant metabolic, sarcomeric, and contractile phenotypes of BTHS iPSC-CMs.

Conclusions and Future Perspectives

The recent advent of iPSC-CM technology has enabled the modeling of human cardiovascular disease phenotypes, drug screening, and the development of regenerative approaches, representing a technological breakthrough that could be translated into revolutionary diagnostic and therapeutic modalities for individual patients. Patient-specific iPSC-CMs provide a novel human-based experimental platform to recapitulate key features of human cardiomyocyte biology. To date, the detailed characterization of patient-specific iPSC-CMs has revealed that they share molecular, electrophysiological, metabolic, mechanical, and ultrastructural properties with primary human cardiomyocytes, but also exhibit diverse functional characteristics resembling fetal rather than adult cardiomyocytes. The structure and function of iPSC-CMs can be further enhanced by prolonged culture and bioengineering approaches, but the factors and signaling pathways affecting maturation are incompletely understood. Diverse epigenetic processes, including long-noncoding RNA (lncRNA),⁶² microRNAs,^{63, 64} chromatin and histone proteins,^{65, 66} and DNA methylation^{67, 68} have emerged as critical modulators of cardiac gene expression in development and disease. Hence, future studies utilizing high-throughput ‘omic’ technologies⁶² will be essential to unravel the genetic and epigenetic mechanisms involved in shaping the phenotype of iPSC-CMs. A better understanding of the underlying mechanisms could potentially lead to the development of strategies to create cardiomyocytes with mature-like phenotypes. The functional maturation is indeed a desirable phenotype, but it is important to acknowledge that a phenotype resembling the adult cardiomyocytes might not be attainable in in vitro cell culture conditions. It is also important to recognize that the relatively immature phenotype of the iPSC-CMs is not necessarily a disadvantage for certain application. For example, immature cells could potentially be better suited for cell therapy applications, as they can become mature after integration into the host myocardium, as recently observed for embryonic stem cell-derived cardiomyocytes (ESC-CMs).⁶⁹

With the launching of the Precision on Medicine Initiative,¹ the iPSC-based models of cardiovascular disease are well-positioned to provide a powerful tool for studying genotype-phenotype association and for predicting individual patient responses to therapies. However, relating gene variations among individuals to clinical phenotypes may not be straightforward. Ultimately, we will need to evaluate and validate the iPSC-CM technology using larger numbers of patient-specific cell lines. To this end, the availability of well-characterized, disease-relevant iPSC biobanks will be indispensable to assess their predictive power.⁷⁰ The recent initiatives by the National Institute of Health (NIH) and the California Institute of Regenerative Medicine (CIRM) to establish state-of-the-art human iPSC biorepositories will address the increasing need for quality-controlled, disease-relevant, and research-grade iPSC lines. Eventually, these efforts will provide researchers across academia and industry with access to high-quality iPSC lines from diverse genetic backgrounds and cardiovascular diseases to conduct prospective studies to establish causal associations between genetic variations and drug response.

New technologies, including next-generation sequencing⁷¹ and nuclease-mediated genome editing⁷², are rapidly advancing the application of the iPSC-CM technology towards future precision medicine approaches. Targeted gene editing using site-specific nucleases, such as Zinc finger nucleases (ZFNs), Transcription activator–like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems, is a powerful tool that allows for reverse genetics, genome engineering and targeted transgene integration experiments to be performed in a precise and predictable manner. Indeed, genes have been added into specific loci and gene mutations have been introduced or used to correct disease-causing mutations in iPSCs-based cardiovascular disease models in vitro. The most common application so far has been to correct mutations that cause monogenic cardiomyopathies, including DCM⁷³ and BTHS,⁶¹ and LQT syndrome.⁷⁴ These studies have provided a proof of principle that the observed phenotypes are caused by specific mutations, suggesting that this approach could be used to uncover the underlying pathological disease mechanism. It should be noted, however, that gene editing with engineered nucleases is problematic. Significant challenges remain, including specificity and off-target effects, efficiency, selection of targeted sites and delivery methods⁷⁵.

Overall, the iPSC-CMs present a new and rapidly developing technology with exciting applications, and with further refinements it could pave the way for the development of personalized medicine for cardiovascular diseases.