Review

Human Induced Pluripotent Stem

CellDerived Cardiomyocytes
Insights Into Molecular, Cellular, and Functional Phenotypes
Ioannis Karakikes, Mohamed Ameen, Vittavat Termglinchan, Joseph C. Wu

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 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-derived cardiomyocytes could eliminate confounding species-specific
and interpersonal variations and ultimately pave the way for the development of personalized medicine for
cardiovascular diseases. However, the predictive power of iPSC-derived cardiomyocytes as a valuable model is
contingent on comprehensive and rigorous molecular and functional characterization.(Circ Res. 2015;117:80-88.
DOI: 10.1161/CIRCRESAHA.117.305365.)
Key Words: cardiovascular diseases cardiovascular disease modeling induced pluripotent stem cells
myocytes, cardiac precision medicine

Induced Pluripotent Stem CellDerived
Cardiomyocytes: A New and Versatile Human
In Vitro Cardiomyocyte Model
Recent advances in genomics and molecular medicine prom-
ise to revolutionize human health by enabling more precise
prediction, prevention, and treatment of cardiovascular diseas-
es on an individual level.1 This precision medicine approach
is based on the ability to diagnose and stratify patients into
different treatment groups by correlating a patients genotype
with their cellular phenotype, and uncover how the genetic
differences among people could influence their responses to
therapies. However, realizing this potential requires the devel-
opment of accurate disease models. Models that recapitulate
individual patients disease at the molecular and cellular level
could lead to a better understanding of the disease progression
and pathogenesis and ultimately enable the prediction of indi-
vidual patients responses to targeted treatments.
Disease models have been and will continue to be in-
strumental in providing important insights into the molecu-
lar 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 hu-
man cardiomyocytes. Particularly, considerable differences
exist between cardiomyocytes from small animal models and
human cardiomyocytes, including beating rates, energetics,
Ca2+ cycling, myofilament composition, expression of key ion
channels, and cellular electrophysiology. These differences in
physiology are substantially less between humans and large
animal models such as nonhuman 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 provides an unprecedent-
ed opportunity for the generation of human patient-specific
cells for use in disease modeling, personalized drug screen-
ing, and regenerative approaches toward precision medi-
cine.6,7 Implementation of this unique and clinically relevant
model system presents a significant advantage in cardiovas-
cular research as it can circumvent complications in translat-
ing 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 tis-
sue, and primary cultures of nonhuman animal origin. First,
the derivation of iPSC-CMs is at most minimally invasive

Original received March 1, 2015; revision received May 20, 2015; accepted May 22, 2015. In April 2015, the average time from submission to first
decision for all original research papers submitted to Circulation Research was 13.84 days.
From the Stanford Cardiovascular Institute (I.K., M.A., V.T., J.C.W.), Department of Medicine, Division of Cardiovascular Medicine (I.K., V.T., J.C.W.),
and Institute of Stem Cell Biology and Regenerative Medicine (J.C.W.), Stanford University School of Medicine, CA.
Correspondence to Ioannis Karakikes, PhD, or Joseph C. Wu, MD, PhD, Stanford Cardiovascular Institute, Stanford University School of Medicine, 265
Campus Dr, Room G1120B, Stanford, CA 94305. E-mail ioannis1@stanford.edu or joewu@stanford.edu
2015 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.117.305365

80
 Karakikes et al Current Status of iPSC-Derived Cardiomyocytes 81
Nonstandard Abbreviations and Acronyms
AP action potential
BTHS Barth syndrome
DCM dilated cardiomyopathy
iPSC induced pluripotent stem cell
iPSC-CM induced pluripotent stem cellderived cardiomyocytes
LQT long QT
SR sarcoplasmic reticulum
(typically via skin biopsy or blood draws) and can theoreti-
cally 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, allow-
ing for the assessment of genotypephenotype associations.
During 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 ex-
ample, iPSC models have been recently used to describe car-
diac channelopathies such as long-QT syndromes (LQT1,810
LQT2,1113 LQT3/Brugada syndrome,14 and LQT8/Timothy
syndrome15), catecholaminergic polymorphic ventricular
tachycardia (CPVT),1618 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 (Figure1).
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 cardiovascu-
lar 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/transforming
growth factor-, Wnt, and bone morphogenetic protein, play
pivotal roles in establishing the cardiovascular system.2325 By
mimicking endogenous developmental signaling cues, direct-
ed differentiation methodologies for generating iPSC-CMs
have been developed.2628 Several permutations of growth
factors and small molecules have recently been reported to
improve reproducibility and efficiency of iPSC-CM differen-
tiation protocols in both adherent and suspension cultures.5
Further purification of CMs from a mixed population of iPSC-
derived cells can be accomplished by nongenetic methods, in-
cluding cell-surface markers,29,30 mitochondria-specific dyes,31
fluorescent probes,32 and glucose deprivation.33 Although
iPSC lines seem to respond differently to developmental sig-
nals because of the intrinsic differences in their genetic back-
ground, these differentiation protocols have been successfully
applied to iPSCs derived from distinct sources of somatic
cells and reprogramming methods.2628,34,35 However, the resul-
tant iPSC-CM population is a heterogeneous pool of atrial-,
ventricular-, and nodal-like cells. As native atrial, nodal, and
ventricular myocytes possess distinct molecular and func-
tional 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 acid36,37 and Wnt signaling pathways.38 However, the
elucidation of the molecular mechanism(s) underlying the car-
diomyocyte subtype specification would be essential to fur-
ther refine the current differentiation protocols and improve
our understanding of lineage-specific development.
Molecular Profiling
At the molecular level, the differentiation of iPSCs toward the
cardiomyocyte lineage is orchestrated by the sequential ex-
pression of distinct sets of genes in specific stages in a pattern
consistent with normal cardiac development: mesoderm for-
mation (T and MIXL1), cardiogenic mesoderm (MESP1, ISL1,
and KDR), cardiac-specific progenitors (NKX2.5, GATA4,
TBX5, MEF2C, and HAND1/2), and structural genes encod-
ing for sarcomeric-related proteins of terminal differentiated
cardiomyocytes (MYL2, MYL7, MYH6, and TNNT2).26,34 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 chan-
nels (KCNH2 and KCNQ1).39 Furthermore, iPSC-CMs ex-
press genes that encode critical components of the Ca2+
cycling machinery such as inositol trisphosphate receptor
(IPTR3), ryanodine receptor 2 (RYR2), sarcoplasmic reticu-
lum (SR) Ca2+-ATPase (SERCA2), calsequestrin 2 (CASQ2),
calreticulin (CALR), junctophilin 2 (JPH2), phospholam-
ban (PLN), sodium calcium exchanger (NCX1), and triadin
(TRDN). However, their relative expression differs from that
of the human adult ventricular tissue.17,40,41 Mitochondrial
complexes IV and genes involved in cholesterol metabolism
(PRKAG1 and PRKAG2) as well as genes that confer protec-
tion against apoptotic and oxidative stress processes (BCL2L1
and SOD1, respectively) are also expressed in iPSC-CMs.42
Taken together, these studies demonstrated that the gene ex-
pression in iPSC-CMs closely mirrors the patterns observed in
human cardiomyocytes (Figure2).
From a disease modeling perspective, the faithful expres-
sion of disease-associated alleles is a prerequisite for proper
manifestation of the disease phenotypes in iPSC-CMs. For ex-
ample, mutations in genes encoding sarcomeric components,
including TNNT2 and MYH7, have been implicated in the 2
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 man-
ner. Recently, iPSC-CMs have been derived from patients
82Circulation ResearchJune 19, 2015

Figure 1. Current applications of patient-specific induced pluripotent stem cellderived cardiomyocyte (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.

harboring deleterious mutation that recapitulated key disease
aspects of HCM, DCM, and LQT syndromes.8,9,1115,21,22 Such
models are currently being used to decipher the complex geno-
typephenotype relationships and to determine disease effects
of specific genetic variants.
Ultrastructural Features
To maximize the potential applications of iPSC-CMs in car-
diovascular medicine, it is essential for these cells to recapitu-
late the ultrastructural properties of adult cardiomyocytes. It
has been reported that early stage iPSC-CMs are small mor-
phologically and exhibit an immature ultrastructure closely
resembling that of fetal CMs (ie, absence of T-tubules and
underdeveloped contractile machinery).4345 However, on
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.44,45
Similarly, by combining bioengineering approaches with
electric stimulation, Nunes et al46 developed a platform called
biowire that enables the generation of iPSC-CMs with ultra-
structural properties similar to those seen in the native CMs. The
progressive lengthening of 3-dimensional iPSC-CM tissues
that mimics heart growth during development further im-
proved cell alignment and increased sarcomeric ultrastructural
organization.47 Despite these advances, the contractile proper-
ties of iPSC-CMs and engineered tissues remain at rudimen-
tary levels.4750
Electrophysiological Phenotypes and Ion
Channel Function
Comprehensive analyses of the electrophysiological proper-
ties of iPSC-CMs have been reported. Based on the action
potential (AP) phenotypes recorded in isolated cells, iPSC-
CMs consist of a heterogeneous population categorized as
atrial-, nodal-, or ventricular-like.15,34,5153 However, cell cul-
ture conditions and differentiation protocols may influence
these AP properties, possibly undermining the correctness of
this classification.54,55 Although the direct electrophysiologi-
cal comparison between iPSC-CMs and human adult CMs
is challenging because of 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 and slower
 Karakikes et al Current Status of iPSC-Derived Cardiomyocytes 83

Figure 2. Expression of key structural and functional genes in induced pluripotent stem cellderived cardiomyocytes (iPSC-CMs).
A, Schematic of the major structural and functional features of iPSC-CMs. In adult CMs, on membrane depolarization a small amount of Ca2+
influx induced by activation of voltage-dependent L-type Ca2+ channels (CACNAC1) triggers the release of Ca2+ from the sarcoplasmic reticulum
(SR) through the ryanodine receptors (ryanodine receptor 2 [RYR2]), termed Ca2+-induced Ca2+-release 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 (TNNI3), 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 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
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 -sarcomeric actinin in iPSC-CMs. C, Line-scan
images and spontaneous Ca2+ transients in iPSC-CMs. ACTC1 indicates actin, , cardiac muscle 1; CACNA1C, calcium channel, voltage-
dependent, L type, 1C subunit; IPTR3, inositol 1,4,5-trisphosphate receptor, type 3; KCNH2, potassium voltage-gated channel, subfamily H
(eag-related), member 2; KCNIP2, potassium channelinteracting protein 2; KCNJ2, potassium inwardly rectifying channel, subfamily J, member
2; KCNQ1, potassium voltage-gated channel, KQT-like subfamily, member 1; MYBPC3, myosin binding protein C; MYH6, myosin, heavy chain 6,
; MYH7, myosin, heavy chain 7, ; MYL2, myosin, light chain 2; MYL3, myosin, light chain 3; SCN5A, sodium channel, voltage-gated, type V, -
subunit; SERCA2, SR Ca2+-ATPase 2; TNNI3, troponin I type 3; TNNT2, troponin T type 2; TTN, titin; and TPM1, tropomyosin 1 ().
84Circulation ResearchJune 19, 2015
upstroke velocity when compared with the human native
counterparts.53 Moreover, recent studies suggest that the
ventricular-like iPSC-CM subtype exhibits many key car-
diac 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 (Figure3A).
Multiple ionic currents have been characterized in single
iPSC-CMs, including the sodium (INa), the L- and T-type calci-
um (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, respec-
tively; Figure 3B). However, the functional properties of ad-
ditional currents, such as the ATP-sensitive K+ current (IK,ATP)
and the Na+Ca+ exchange current (INCX), have not yet been re-
ported in iPSC-CMs, whereas the atrial-selective ion current,
acetylcholine-activated K+ (IK,ACh), has recently been reported
in human embryonic stem cell-derived atrial-like CMs.36 We
briefly summarize some of these major channels below:
INa
iPSC-CMs have prominent Na+ currents with activation and
inactivation gating characteristics that are analogous to those
of native human ventricular CMs.14,53
ICa,L and ICa,T
The activation and inactivation gating properties of ICa in iP-
SC-CMs are similar to those obtained from human ventricular
myocytes.15,53 By contrast, although the ICa,T current has been
found in a subset of cells, its properties have not defined.56
If
In ventricular-like iPSC-CMs, the presence of hyperpolar-
ization-activated If promotes phase 4 depolarization and thus
contribute to automaticity.53
IK
Three K+ currents (Ito, IKr, and IKs) have been recorded in
iPSC-CMs with maximum densities and activation proper-
ties comparable with the values reported for human cardiac
myocytes.8,53 By contrast, the density of IK1 is either absent
or significantly smaller than that reported for native ven-
tricular CMs. Importantly, IKr contributes to repolarization
of the cardiac AP, and block of IKr prolongs the ventricular
AP, which is manifested by QT prolongation on the surface
ECG. Prolongation of the AP and associated increased QT
interval can lead to early afterdepolarizations on the cellu-
lar level and trigger the ventricular arrhythmia Torsades de
pointes. Notably, by measuring AP duration and quantifying
drug-induced arrhythmias, such as early afterdepolarizations
and delayed afterdepolarizations, drug-induced cardiotoxic-
ity profiles for healthy subjects, LQT syndrome, HCM, and
DCM patients were recapitulated at the single cell level us-
ing the iPSC-CM technology.39,57 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 use of
iPSC-CMs in screening for proarrhythmic potential of cur-
rent and new drug entities in conjunction with in silico mod-
eling is a major focus of the Food and Drug Administration
Comprehensive In Vitro Proarrhythmia Assay initiative.58
In summary, multiple voltage-gated ion channels are simi-
larly present in iPSC-CMs and adult CMs leading to charac-
teristic cardiac APs (Figure3B). 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 ob-
served 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
Figure 3. Comparison of action
potentials (APs) of ventricular-like
induced pluripotent stem cellderived
cardiomyocytes (iPSC-CMs) and adult
ventricular cardiomyocytes. A, Schematic
of ventricular APs. Phases 0 to 4 are the
rapid upstroke, early repolarization,
plateau, late repolarization, and diastole,
respectively. B, The ionic currents and
the genes that generate the currents with
schematics of the current trajectories
are shown above and below the APs.
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
the inward rectifier K+ currents (IK1) and the
presence of prominent pacemaker currents
that contribute to their automaticity. hiPSC
indicates human iPSC.
 Karakikes et al Current Status of iPSC-Derived Cardiomyocytes 85
have been focused on using electric or mechanical stimulation
that seems to promote electrophysiological maturation.46-50
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, SR, and the sarcomere through excitation
contraction coupling.59 Extensive characterization of sponta-
neous whole-cell [Ca2+]i transients in iPSC-CMs suggests the
presence of functional excitationcontraction coupling that
resembles the native myocardium.41 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 SR receptors, recapitu-
lating the Ca2+-induced Ca2+-release phenomenon in iPSC-
CMs,41,46 a key mechanism underlying excitationcontraction
coupling.59 Of note, iPSC-CM cultured on microgrooved sub-
strates 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.40 In catecholaminergic polymorphic ventricular
tachycardia, an inherited disease characterized by stress-
induced ventricular arrhythmias, iPSC-CMbased modeling
also supports the presence of functional SR and ryanodine
receptor Ca2+ transients.17 However, the Ca2+ handling kinet-
ics in iPSC-CMs seem to be relatively slow and characterized
by a U-shape Ca2+ waveform, suggesting that iPSC-CMs have
an immature Ca2+-induced Ca2+-release mechanism.60 Indeed,
iPSC-CMs exhibit a poorly developed SR and absence of
T-tubules that likely affect their Ca2+ handling properties.43,44
Metabolic Profile
Various studies have demonstrated that iPSC-CMs have a
metabolic phenotype that resembles embryonic cardiomyo-
cytes, which mostly rely on glycolysis for energy production
instead of lipid oxidation as seen in adult ventricular myo-
cytes. Strategies to facilitate metabolic maturation have been
developed, including culturing iPSC-CMs with an adipogenic
cocktail19 or glucose-free medium61 and tissue engineering
approaches.62 These approaches were able to yield advanced
levels of metabolic phenotype maturation, as evidenced by
significant increases of fatty acid -oxidation.19,61,62
By enhancing iPSC-CM metabolic maturation, iPSC-CMs
have been used to recapitulate key features of mitochondrial
disorders. In a recent study, Drawnel et al61 showed that expos-
ing normal iPSC-CMs in diabetic-like conditions (high glu-
cose, endothelin-1, and cortisol) could induce an increment of
lipid accumulation, oxidative stress, and sarcomeric disarray,
phenocopying diabetic cardiomyopathy. Intriguingly, the iP-
SC-CMs derived from patients with type 2 diabetes mellitus,
a disease with complex and multifactorial pathogenesis, also
recapitulated key pathophysiological phenotypes in vitro that
corresponded to the clinical status of the original donor. A phe-
notypic drug screening in this model revealed molecules and
pathways that may provide therapeutic relevance consistent
with the clinical type 2 diabetes mellitus subtype.61 However,
the genetic and the epigenetic basis underlying the observed
cellular phenotypes and differential response to treatments
were not examined. Similarly, by combining bioengineer-
ing and gene editing approaches, Wang et al62 modeled Barth
syndrome (BTHS), an X-linked cardiac and skeletal mito-
chondrial myopathy caused by mutation of the gene encod-
ing Tafazzin (TAZ). BTHS iPSC-CMs displayed an impaired
biogenesis of cardiolipin, a major phospholipid of the mito-
chondria. Subsequently, a novel mechanism was discovered
showing that the TAZ deficiency in BTHS markedly increased
reactive oxygen species production, and that suppression of
reactive oxygen species reversed the BTHS cardiomyopathic
phenotype in 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 trans-
lated 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 re-
vealed that they share molecular, electrophysiological, meta-
bolic, mechanical, and ultrastructural properties with primary
human cardiomyocytes, but also exhibit diverse functional
characteristics resembling fetal rather than adult cardiomyo-
cytes. The structure and function of iPSC-CMs can be further
enhanced by prolonged culture and bioengineering approach-
es, but the factors and signaling pathways affecting maturation
are incompletely understood. Diverse epigenetic processes,
including long-noncoding RNA,63 microRNAs,64,65 chroma-
tin and histone proteins,66,67 and DNA methylation,68,69 have
emerged as critical modulators of cardiac gene expression in
development and disease. Hence, future studies using high-
throughput omic technologies63 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 develop-
ment 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 pheno-
type resembling the adult cardiomyocytes might not be attain-
able 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 cellderived cardiomyocytes.70
With the launching of the Precision on Medicine Initiative,1
the iPSC-based models of cardiovascular disease are well po-
sitioned to provide a powerful tool for studying genotype
phenotype association and for predicting individual patient
responses to therapies. However, relating gene variations
86Circulation ResearchJune 19, 2015
among individuals to clinical phenotypes may not be straight-
forward. 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 as-
sess their predictive power.71 The recent initiatives by the
National Institutes of Health and the California Institute of
Regenerative Medicine 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 sequencing72
and nuclease-mediated genome editing,73 are rapidly advanc-
ing the application of the iPSC-CM technology toward future
precision medicine approaches. Targeted gene editing using
site-specific nucleases, such as zinc finger nucleases (ZNF),
transcription activatorlike effector nucleases (TALENs), and
the clustered regularly interspaced short palindromic repeat/
clustered regularly interspaced short palindromic repeatas-
sociated protein 9 systems (CRISPR/Cas9), is a powerful tool
that allows for reverse genetics, genome engineering, and tar-
geted transgene integration experiments to be performed in a
precise and predictable manner. Indeed, genes have been add-
ed 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 to date has been to correct mutations that cause
monogenic cardiomyopathies, including DCM74 and BTHS,62
and LQT syndrome.75 These studies have provided a proof
of principle that the observed phenotypes are caused by spe-
cific 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, in-
cluding specificity and off-target effects, efficiency, selection
of targeted sites, and delivery methods.76
Overall, the iPSC-CMs present a new and rapidly devel-
oping technology with exciting applications, and with further
refinements it could pave the way for the development of per-
sonalized medicine for cardiovascular diseases.
Acknowledgments
We gratefully acknowledge Joseph Gold, Ian Chen, and Blake Wu
for critical reading, and Varachaya Khwanjaipanich for preparing the
illustrations. Because of 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.
Sources of Funding
This work was support from the National Institutes of Health (NIH)
R01 HL113006, NIH R01 HL123968, NIH R24 HL117756, and
California Institute of Regenerative Medicine IT1-06596 and DR2A-
05394 (J.C. Wu), NIH K99 HL104002, AHA 15BGIA22730027 and
Stanford CVI Seed Grant (I. Karakikes), and Prince Mahidol Award
Foundation, Thailand (V. Termglinchan).
Disclosures
J.C. Wu is a cofounder of Stem Cell Theranostics. The other authors
report no conflicts.
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