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Published in final edited form as:

Circ Res. 2015 June 19; 117(1): 80–88. doi:10.1161/CIRCRESAHA.117.305365.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes:

Insights into Molecular, Cellular, and Functional Phenotypes
Ioannis Karakikes1,2, Mohamed Ameen1, Vittavat Termglinchan1,2, and Joseph C. Wu1,2,3
1Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA
2Department of Medicine, Division of Cardiovascular Medicine, Stanford University School of
Medicine, Stanford, CA
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3Institute
of Stem Cell Biology and Regenerative Medicine, Stanford University School of
Medicine, Stanford, CA

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
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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.

Keywords
induced pluripotent stem cells; cardiovascular disease modeling; precision medicine;
cardiomyocytes

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
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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

Correspondence: Joseph C. Wu, MD, PhD, 265 Campus Drive, Room G1120B, Stanford, CA 94305. joewu@stanford.edu or Ioannis
Karakikes, PhD, ioannis1@stanford.edu.
DISCLOSURES
JCW is a co-founder of Stem Cell Theranostics
Non-standard Abbreviations and Acronyms:
None.
 Karakikes et al. Page 2

differences among people that may influence their responses to therapies. However,
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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
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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
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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
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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).

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In this review article, we will highlight the current state of iPSC-CMs, focusing on their
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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
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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 acid35, 36 and Wnt signaling
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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
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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),

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and triadin (TRDN). However, their relative expression differs from that of the human adult
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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).

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
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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
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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

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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

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heterogeneity, and disease status, it is well documented that iPSC-CMs display mixed AP
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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

Multiple ionic currents have been characterized in single iPSC-CMs, including the sodium
(INa), the L- and T-type calcium (ICa,L and ICa,T), the hyperpolarization-activated pacemaker
(If), the transient outward potassium (Ito), the inward rectifier potassium (IK1), and the rapid
and slow activating components of the delayed rectifier potassium currents (IKr and IKs,
respectively). However, the functional properties of additional currents, such as the ATP-
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sensitive K+ current (IK,ATP), and the Na+-Ca+ exchange current (INCX) have not yet been
reported in iPSC-CMs, whereas the atrial-selective ion current, acetylcholine-activated K+
(IK,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

ICa,L and ICa,T

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

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

IK
Three K+ currents (Ito, IKr, and IKs) 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 IK1 is either absent or significantly smaller than
that reported for native ventricular CMs. Importantly, IKr contributes to repolarization of the
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cardiac AP, and block of IKr 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

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healthy versus diseased individuals, suggesting that adverse drug responses could be
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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
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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+
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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
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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

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adipogenic cocktail19 or glucose-free medium60 and tissue engineering approaches61. These

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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
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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
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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
methylation67, 68 have emerged as critical modulators of cardiac gene expression in
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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

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relatively immature phenotype of the iPSC-CMs is not necessarily a disadvantage for certain
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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
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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
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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.
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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.

Acknowledgements
The authors gratefully acknowledge Joseph Gold, Ian Chen and Blake Wu for critical reading, and Varachaya
Khwanjaipanich for preparing the illustrations. Due to space limitations, we are unable to include all of the
important citation relevant to this subject; we apologize to those investigators whose work was omitted here.

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SOURCES OF FUNDING
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Funding support from National Institute of Health (NIH) R01 HL113006, NIH R01 HL123968, NIH R24
HL117756, and California Institute of Regenerative Medicine (CIRM) IT1-06596 and DR2A-05394 (J.C.W), NIH
K99 HL104002, AHA 15BGIA22730027 and Stanford CVI Seed Grant (I.K.), and Prince Mahidol Award
Foundation, Thailand (V.T).

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

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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+

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is extruded by the Na2+/Ca2+ exchanger (NCX1) and returned to the SR by the

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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
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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.
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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 (IK1) and the presence of prominent
pacemaker currents that contribute to their automaticity.
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