Physiol Rev 96: 1093–1126, 2016

Published June 22, 2016; doi:10.1152/physrev.00036.2015

HUMAN INDUCED PLURIPOTENT STEM CELLS AS

A PLATFORM FOR PERSONALIZED AND
PRECISION CARDIOVASCULAR MEDICINE
Elena Matsa, John H. Ahrens, and Joseph C. Wu

Stanford Cardiovascular Institute, Department of Medicine, Division of Cardiology, and Institute of Stem Cell
Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California

Matsa E, Ahrens JH, Wu JC. Human Induced Pluripotent Stem Cells as a Platform for

L
Personalized and Precision Cardiovascular Medicine. Physiol Rev 96: 1093–1126,
2016. Published June 22, 2016; doi:10.1152/physrev.00036.2015.—Human in-
duced pluripotent stem cells (hiPSCs) have revolutionized the field of human disease
modeling, with an enormous potential to serve as paradigm shifting platforms for
preclinical trials, personalized clinical diagnosis, and drug treatment. In this review, we describe
how hiPSCs could transition cardiac healthcare away from simple disease diagnosis to prediction
and prevention, bridging the gap between basic and clinical research to bring the best science to
every patient.

I. INTRODUCTION 1093 vidual variability (11, 45). Progress in genetic and genomic
II. TOOLS FOR REPROGRAMMING... 1094 research has highlighted some disease-associated genomic
III. GENERATIONS OF FUNCTIONAL... 1097 DNA (gDNA) polymorphisms [e.g., single nucleotide poly-
IV. BIOMEDICAL DATA SCIENCE 1099 morphisms (SNPs) or insertions/deletions (indels)] that pro-
V. hiPSC-BASED MODELS OF HUMAN... 1101 vide causal relationships among genotypes, disease suscep-
VI. ENGINEERED DISEASE MODELS... 1107 tibility, and pharmacokinetics (i.e., drug absorption, distri-
VII. INDUCED MODELS OF CARDIAC... 1110 bution, metabolism, and excretion) or pharmacodynamics
VIII. PATIENT RECRUITMENT VERSUS... 1111 (i.e., the physiological effects of the drug). Such relation-
IX. PHARMACOGENOMICS 1112 ships account for phenotypic variations of clinical impor-
X. POTENTIAL APPLICATIONS OF hiPSCs... 1113 tance in drug therapy and disease progression (173). How-
XI. CONCLUSIONS AND FUTURE... 1116 ever, large-scale studies to establish causal associations be-
tween genetic variations and clinical indications have been
difficult to conduct. The lack of robust methods to experi-
I. INTRODUCTION
mentally test the functional relevance of naturally occurring
polymorphisms in human cells has delayed progress, leav-
The ability to reprogram terminally differentiated human ing the determination of the molecular and cellular basis of
cells into induced pluripotent stem cells (hiPSCs) has fun- human phenotypic variation an unresolved challenge.
damentally altered the previous belief that cellular differen- However, revolutionary and rapidly evolving genome edit-
tiation is a unidirectional, nonreversible developmental
ing technologies such as clustered regularly interspaced
process (84, 257, 259). The discovery of cellular repro-
short palindromic repeats (CRISPR/Cas9), transcription ac-
gramming has opened the possibility to create in vitro mod-
tivator-like effector nucleases (TALEN), and zinc finger nu-
els of human diseases by isolating and reprogramming so-
cleases (ZFN), can significantly decrease the time and effort
matic cells from patients carrying familial disease-associ-
ated mutations. These patient-specific platforms have been it takes to generate human cell lines carrying specific gDNA
used extensively to understand the molecular mechanisms mutations or polymorphisms (46). Thanks to their abilities
leading to pathophysiological perturbations in humans, to undergo indefinite proliferation and single-cell clonal ex-
and test the pathways via which several drugs may amelio- pansion, hiPSCs serve as a particularly useful platform for
rate a disease phenotype or cause cytotoxicity (181, 182). genome editing (82, 97, 308). Therefore, hiPSCs can facili-
tate identification of exactly how disease mutations and
The capacity of hiPSCs to retain patient-specific genomic, polymorphisms result in cellular phenotypes, providing an
transcriptomic, proteomic, metabolomic, and other indi- experimental platform for precision medicine. By advanc-
vidualized big data information makes it possible to extend ing a new model of patient-powered research combined
their application beyond disease modeling, and into the with a patient-focused drug development program, preci-
field of precision medicine which encompasses the adoption sion medicine promises to accelerate biomedical discoveries
of novel prevention and treatment strategies based on indi- and offer clinicians novel tools, knowledge, and therapies

0031-9333/16 Copyright © 2016 the American Physiological Society 1093

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 MATSA ET AL.

that will enable them to select optimized treatments for each
individual patient (36a, 233a).
In this review, we focus on the ability of hiPSCs to facilitate
precision medicine in relation to heart disease. Heart disease,
whether familial, congenital, or acquired, remains the leading
cause of mortality in men and women worldwide (150, 198).
Currently, over 17 million deaths per year are attributed to
heart disease, and this number is projected to rise to 30 million
over the following 15 years (300a). Despite decades of exten-
sive and expensive research, few interventions have signifi-
cantly improved patients’ survival in heart failure (34). Even
the most commonly prescribed treatments (e.g., ␤-adrenore-
ceptor blockers) act primarily by delaying disease progression,
and can have adverse side effects such as fainting, seizures, or
bradycardia (71, 78, 143). These facts point to the need for
enhancing our understanding of the molecular mechanisms
leading to heart failure, and for developing improved predic-
tive, preventive, and reparative therapies. As detailed later on
in this review, small and large animal models ranging from
fruit flies to primates have been utilized extensively to model
the pathophysiology of human heart disease (192). However,
species-specific variation has hindered significant progress,
making any therapeutic advancements disproportional to the
amount of time and money invested (226). Thus cutting-edge
21st century heart failure research is required to use novel
human-based and human-relevant research methodologies to
efficiently address the lack of effective therapies against heart
failure.
We hereby show examples of how hiPSC-derived cardiomy-
ocytes (hiPSC-CMs) can be used to generate human models
of heart diseases in a dish, which faithfully recapitulate the
human heart’s pathophysiology and respond appropriately
to clinically relevant pharmacological intervention. We also
outline how, in the relatively short time since their discov-
ery, hiPSCs have been married with next generation se-
quencing (NGS) and genome editing technologies to high-
light molecular mechanisms leading to heart disease, en-
abling us to link disease-associated mutations or
polymorphisms to disease outcome, severity, and response
to therapy. Finally, we highlight the future prospects of
hiPSCs to ably address the critical need for incorporating a
human-based platform into drug discovery, disease diagno-
sis, and therapeutic pipelines.
II. TOOLS FOR REPROGRAMING SOMATIC
CELLS TO hiPSCs
The first reprograming methods published by the Ya-
manaka (257) and Thomson (319) labs utilized retroviral
and lentiviral vectors to generate hiPSCs from human skin
fibroblasts. They were able to demonstrate that transgenic
overexpression of OCT4, SOX2, KLF4, and c-MYC
(OSKM) or OCT4, SOX2, NANOG, and LIN28 (OSNL)
was necessary and sufficient to induce pluripotency in fibro-
blasts. Subsequent interrogation of reprogramming mecha-
nisms identified that the OSKM or OSNL factors synergis-
tically activate the endogenous pluripotency machinery fol-
lowing a series of consecutive events (FIGURE 1). The first
step to reprogramming is initiation, and it is characterized
by loss of somatic cell gene expression, metabolic changes,
increased cellular proliferation rate, inhibition of apoptosis
and cellular senescence pathways, and initiation of mesen-
chymal-to-epithelial transition (MET) (48). These changes
are followed by maturation, a stage when cells acquire ex-
pression of pluripotency-associated genes, but are still de-
pendent on transgene overexpression to become fully repro-
grammed. The final stage of reprogramming, known as the

Initiation Maturation Stabilization

↓ somatic cell gene expression ↑ pluripotency gene expression Epigenetic remodeling

≠ in metabolism (e.g., OCT4, NANOG) ↑ telomere length

↑ proliferation rate X-chromosome reactivation

of apoptosis

of cellular senescence pathways

Mesenchymal-to-epithelial transition Transgene dependence

0 4 8 12 Time (days)
FIGURE 1. Stages of hiPSC reprogramming. Mechanistic analyses have demonstrated that reprogramming
of somatic cells to hiPSCs consists of three major steps. The first step is “initiation,” and it is characterized by
loss of somatic cell gene expression, metabolic changes, increase in cellular proliferation rate, inhibition of
apoptosis and cellular senescence pathways, and initiation of mesenchymal-to-epithelial transition (MET).
These changes are followed by “maturation,” a stage when cells acquire expression of pluripotency-associated
genes, but are still dependent on transgene overexpression to become fully reprogrammed. The final stage of
reprogramming is “stabilization,” and it is achieved when cells undergo X-chromosome reactivation and
telomere elongation, acquire epigenetic characteristics of pluripotent cells, and are transgene independent.
1, Increased; 2, decreased; ⫽, change; ;, inhibition.

1094 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 HUMAN INDUCED PLURIPOTENT STEM CELLS
stabilization phase, is achieved when cells undergo X-chromo-
some reactivation and telomere elongation, acquire the epige-
netic characteristics of pluripotent cells, and become transgene
independent. hiPSCs are thought to continue stabilization and
to acquire genetic and epigenetic signatures that more closely
resemble those of embryonic stem cells (ESCs) (270) at late
passages, typically over passage 16 (14, 221).
Reprogramming efficiency (i.e., the number of starting so-
matic cells which transition to hiPSCs) has been reported to
be significantly enhanced by addition of supplementary plu-
ripotency factors (12) or epigenetic modifiers (160) to the
reprogramming mix. Rais et al. (225) and Lujan et al. (168)
also demonstrated in both human and mouse cells that al-
most 100% of cells in a differentiated population are capa-
ble of reprogramming following depletion of Mbd3, a core
member of the Mbd3/NuRD (nucleosome remodeling and
deacetylation) repressor complex. Even though some con-
troversy exists around this topic, likely due to due technical
differences (64), these studies potentially demonstrate that
while iPSC reprogramming is typically a stochastic process,
it can become deterministic upon epigenetic manipulation.
Beyond the investigation of hiPSC reprogramming mecha-
nisms, clinically relevant advances have been achieved by
the development of automated, high-throughput derivation
and characterization protocols for hiPSCs (215, 245), use of
chemically defined media (24), and substitution of retrovi-
ral and lentiviral transgene overexpression vectors by inte-
gration-free systems that circumvent the risk of spontane-
ous tumor formation due to insertional mutagenesis. Such
methods include the use of excisable viral vectors, noninte-
grating viruses (250, 251), and even virus-independent ap-
proaches such as plasmid, episomal, DNA, protein, or
mRNA mediated transgene overexpression (114, 293,
324). The advantages and disadvantages of the most prom-
inently used nonintegrating reprogramming techniques are
outlined in TABLE 1 and are discussed in detail below.
A. Nonintegrating Sendai Virus
One of the most common currently used techniques to in-
duce pluripotency is based on F-deficient Sendai virus (SeV/
⌬F), a nontransmissible negative-sense single-stranded
RNA virus that replicates in the cytoplasm of infected cells
without DNA intermediates or genomic integration into the
target cell genome. Despite being one of the most reliably
integration-free reprogramming methods currently avail-
able (72), the use of Sendai virus is expensive and requires
more stringent biosafety containment measures than non-
viral reprogramming methods. Moreover, residual viral
material persists for an extended period of cell culture (up
to 10-20 passages), thus delaying the establishment of virus-
free hiPSC lines for downstream analysis and differentia-
tion experiments (80). Recently, temperature-sensitive Sen-
dai virus particles have been developed by engineering spe-
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
cific backbone mutations that affect key viral proteins, and
allow faster and more efficient removal of viral material
from the cytoplasm of host cells by a temperature shift (10).
This technology decreases the time and increases the effi-
ciency of establishing desired hiPSC lines. With the use of
this method, various cell types, including fibroblasts and
peripheral blood mononuclear cells (PBMCs), have been
effectively reprogrammed (41, 60, 72).
B. Reprogramming With Episomal Plasmids
As an alternative to retroviral or lentiviral-mediated induc-
tion of pluripotency, several DNA-based reprogramming
methods have also been developed using either nonreplicat-
ing (56, 114, 209) or replicating episomal vectors (317).
These techniques are attractive because they reduce biosafety
concerns involved in the production and use of viral particles,
as well as the risk of tumorigenesis due to potential reactiva-
tion of the KLF4 and c-MYC oncogenes, or due to insertional
mutagenesis. Nevertheless, the reprogramming efficiency
achieved using nonreplicating vectors is relatively low and re-
quires multiple transfections of the target cells (114, 209).
Potential explanations for this phenomenon include the low
transfection efficiencies of large polycistronic plasmids and
enhanced transgene silencing mechanisms operating on plas-
mid-based vectors in mammalian cells (167).
To overcome these limitations, the Epstein-Barr virus
(EBV)-based reprogramming technology was developed.
This potent methodology takes advantage of the EBV-en-
coded nuclear antigen-1 (EBNA-1) trans-acting element,
and EBV-encoded oriP cis-acting element to enable plas-
mids to persist by dividing within human cells as multicopy
episomes that attach to chromosomes during mitosis (104,
209). Therefore, the use of episomal vector systems signifi-
cantly increases reprogramming efficiency through pro-
longed transgene expression within transfected cells (209).
Following multiple cell divisions, oriP/EBNA-based vectors
progressively diminish from the targeted cells, thus elimi-
nating potential tumorigenesis or differentiation deficien-
cies due to transgene reactivation.
Minicircle vectors were also developed as an improvement
to classical episomal vectors (114). Minicircles are episomal
vectors devoid of any bacterial plasmid and are therefore
smaller in size than standard plasmids. This attractive fea-
ture enhances their transfection and expression rates (38).
However, the reprogramming efficiency achieved using
minicircle vectors is low, and their production and purifi-
cation methodology is relatively complex and time consum-
ing (202). To address the aforementioned drawbacks of this
system, a novel, single plasmid reprogramming system
called 4-in-1 codon optimized mini intronic plasmid
(CoMiP) was developed, which carries codon-optimized se-
quences of the canonical OKSM reprogramming factors to
achieve more accurate and faster translation of transgenes.
1095
 MATSA ET AL.

Table 1. Tools for reprogramming somatic cells to hiPSCs

Reference
Tool Subtype Vector Transgenes Efficiency, % ⴙ ⴚ Nos.

Viral Integrating Retrovirus OSKC, OSK, OS, 0.001–0.1 1Efficiency, robust Risk for random 58, 213,
OK, O integration into 257
somatic
genome
Lentivirus OSKC, OSNL, 0.0001–0.1 Can infect nondividing Same as above 103, 201
OSN, OSL cells
Excisable Cre-LoxP Lentivirus OSKC, OSK 0.005–0.01 2Risk for IM Labor intensive, 246
may leave
footprint
Nonintegrating Adenovirus OSKC 0.001 No risk for IM 2Efficiency 325
Sendai virus OSKC 1 Same as above 1Cost 72
Nonviral, DNA Plasmids EBV Episome OSKCNL & 0.00003–0.02 2Risk for IM 2Efficiency, need 316, 318
based SV40LT for additional
factors
Minicircle OSNL 0.0005–0.005 Inexpensive, easy to use, 2Efficiency, rapid 114
2Risk for IM transgene
silencing may
require
multiple
transfections
CoMIP OSKC 0.02–0.03 Same as above Same as above 56, 57
Transposons PiggyBac OSKC 0.1 1Efficiency May leave 123
footprint, risk
for random
insertion
Sleeping Beauty OSKC 0.02 2Risk for IM Same as above 50
HAC OSKC & p53 0.00007–0.00014 Built-in safeguard system 2Efficiency 94
shRNA based on herpes
simplex virus
Nanoparticle OSKC, SKN & 0.001–0.049 Stable transgene source Labor intensive 29, 151
NR5A2
Nonviral, non-DNA CPP OSKC 0.001 No risk of IM 2Efficiency 135
based
RNA replicon VEE OSKC, OSKG 0.25 1Efficiency, easy, 2risk Possibility of RT 315
for IM to cDNA,
leading to IM
Synthetic Cationic lipid OSKC & VPA, & 1.4–4.4 Same as above Expensive, labor 293
mRNAs hypoxia intensive
miRNAs Cationic lipid miR-200c, 302, 0.1 Same as above Same as above 194
369 s
Small molecules N/A O & VC6T 0.00005 Minimal genetic Greatly 158
(mouse) modification, easy to 2efficiency,
use not
demonstrated
in humans
VC6TFZ 0.2 No genetic modification, Not 98
no risk for IM, easy to demonstrated
use in human cells

A summary of the plethora of methods available for the reprogramming of somatic cells to hiPSCs, with
reference to their individual efficiencies, advantages (⫹), and disadvantages (⫺). O, OCT4; S, SOX2; K, KLF4;
C, c-MYC; N, NANOG; L, LIN28; SV40LT, simian vacuolating virus 40 large T antigen; IM, insertional
mutagenesis; VPA, valproic acid; EBV, Epstein Barr virus; CoMIP, codon optimized mini intronic plasmid; RT,
reverse transcription; VEE, Venezuelan equine encephalitis self-replicating RNA virus; HAC, human artificial
chromosome; CPP, cell penetrating peptides; VC6T; VPA, CHIR99021, 616452, and tranylcypromine;
VC6TFZ, VC6T, forskolin and 3-deazaneplanocin A; 1, high; 2, low.

Similar to minicircles, the CoMiP vector system overcomes
transgene silencing limitations that are often observed with
regular plasmids, and provides at least 2–10 times higher
levels of transgene expression, as demonstrated in vitro
with HEK293 cells and in vivo with mouse liver cells (166).
Furthermore, the 4-in-1 CoMiP vector is a highly efficient,
integration-free, and cost-effective methodology applicable
for hiPSC reprograming in a wide variety of cell types (57).
potential to integrate into the host genome. Careful down-
stream screening methods must be used to confirm the der-
ivation of integration-free pluripotent cells to guarantee the
safety of these systems (80).
C. Non-DNA Approaches for the Induction of
Pluripotency

One area of potential concern when using episomal DNA- One of the first non-DNA tools used for the generation of
based reprogramming methods is that they maintain a small hiPSCs was transient transfection of modified mRNAs. This

1096 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 HUMAN INDUCED PLURIPOTENT STEM CELLS
approach not only guarantees the derivation of integration-
free hiPSCs due to the lack of any DNA intermediates, but
also obviates further time-consuming screening experi-
ments (177, 293). Initial reprogramming experiments using
mRNA transfection required a laborious series of consecu-
tive mRNA transfections for 14 days, as well as pretreat-
ment of target cells with the expensive interferon-␣ antag-
onist B18R (177) to prevent cell death from repeated
mRNA transfections (44). Another limitation of initial pro-
tocols for mRNA-mediated reprogramming for clinical ap-
proaches was the requirement of mouse embryonic fibro-
blast (MEF) feeder cells and conditioned media, which po-
tentially increases the risk of transmitting undetected
animal pathogens to human cells (4). Recent optimizations
of the established mRNA reprogramming factors addressed
some of the aforementioned problems, allowing the deriva-
tion of footprint-free hiPSCs from human fibroblasts with-
out the use of feeder cells or other potentially xeno-contam-
inated reagents (294), using only a single mRNA transfec-
tion (315). Still, mRNA-based reprogramming has thus far
only allowed reprogramming of adherent cells, which limits
the types of differentiated cells that can be reprogrammed.
Another successful DNA-free method for hiPSC generation
involves the use of transmembrane-permeable fusion pro-
teins (135, 152). However, due to slow kinetics, low effi-
ciency, and high cost (80), this technique has not been
widely adopted. In addition, an exciting alternative ap-
proach has been the elimination of classical reprogramming
factors by exclusively using small-molecule compounds to
reprogram mouse somatic cells (98). Reproducing this tech-
nique using human somatic cells would greatly broaden its
clinical interest.
D. Selecting the Right Reprogramming Tools
for Clinical Applications
Recent evidence suggests that the number and size of karyo-
type abnormalities, such as copy number variations
(CNVs), are significantly higher in isogenic hiPSC lines gen-
erated using integrating reprogramming methods compared
with nonintegrating methods (126). This confirms the need
for adopting safe reprogramming methods for clinical ap-
plications. It is important to note that not all reprogram-
ming routes may lead to the same pluripotency state. Al-
though genetic background and cell type of origin contrib-
ute to heterogeneity among hiPSC lines (231), the type of
reprogramming strategy used can also affect the stoichiom-
etry and expression levels of individual transgenes, thus
leading to transcriptional diversity amongst reprogrammed
populations (327). Carefully assessing the quality of the
reprogrammed state is vital as new technologies emerge.
Quality control (QC) testing and release assays that ensure
the identity, safety, purity, and viability of new hiPSCs lines
include the expression of pluripotency-associated markers
(e.g., OCT4, SSEA4, TRA-1-81, TRA-1-160) in over 70%
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
of the population, normal 46 XX or 46 XY karyotype,
identical short tandem repeat (STR) profiles between paren-
tal and hiPSC lines, sterility, absence of mycoplasma or
endotoxin (⬍0.5 EU/ml) contamination, lack of episomal
or other vector integration into gDNA, viral panel clearance
(e.g., retroviruses, adeno-associated viruses, HIV, EBV,
ETC, and bovine or porcine viruses), and over 50% cell
viability per cryopreserved vial (9). These assays should be
conducted prior to the use of hiPSCs for clinical applica-
tions.
III. GENERATION OF FUNCTIONAL HUMAN
CARDIOMYOCYTES FROM hiPSCs
A. Genetic and Epigenetic Control of Cardiac
Specification
Cardiac specification during development is thought to be a
four-stage process tightly controlled by transcription factor
networks, signaling pathways, and epigenetic modifications
(25). On the basis primarily of mouse studies, it is thought
that the first stage of cardiac specification during develop-
ment consists of cardiac mesoderm formation around the
time of gastrulation (261). This process is facilitated by
upregulation of NODAL and BMP4 signaling (6). Subse-
quent induction of WNT signaling by BMP4 activates the
expression of essential mesoderm transcription factors,
such as brachyury (T), which in turn stimulate Mesp1, a
master regulator and marker of cardiogenic mesoderm for-
mation (15). During the second stage of cardiac specifica-
tion, Mesp1⫹ cardiac mesoderm progenitors give rise to
two distinct progenitor cell populations known as the first
heart field (FHF) and second heart field (SHF) progenitors
(154). This process is controlled by intrinsic signaling as
well as complex extrinsic signaling by BMP4 from the un-
derlying anterior ectoderm, and BMP4, FGF, and WNT
signaling from the overlying anterior endoderm (247).
In the FHF, WNT signaling inhibition through the Mesp1-
activated gene Dkk1 leads to upregulation of the cardiac
progenitor marker Nkx2-5 and the FHF-specific marker
T-box transcription factor Tbx5 (70). This incorporates the
third stage of cardiac specification, known as patterning of
the heart fields, which is followed by the fourth and final
stage of terminal cardiac differentiation. The latter is
brought about by Nkx2.5 and Tbx5-mediated activation of
connexin 40 (Gja5), Nppa, and sarcomere-specific genes
(18) which induce the formation of atrial cardiomyocytes
(CMs), left ventricular CMs, and cardiac bundle of His
pacemaker cells from the FHF (95). Nkx2.5 and Tbx5
physically interact to coregulate their downstream targets
during cardiac differentiation and morphogenesis (18),
whereas their independent binding activity also serves to
prevent transcription factors from distributing to ectopic
loci and activate lineage-inappropriate genes (169). Positive
1097
 MATSA ET AL.
regulation of Nkx2.5 by polycomb repressive complex 1
(PRC1)-mediated chromatin remodeling (241) and negative
regulation by histone 3 trimethylation (205) add an addi-
tional level of complexity to these temporally and spatially
controlled gene regulatory pathways.
In the SHF, Isl1 and Mef2c act downstream of Mesp1 (125)
to activate cardiac-specific factors such as Smyd1, Hand1,
Irx4, and Fgf10 (217, 295) and ultimately give rise to atrial
CMs, right ventricular CMs, and sinoatrial node pace-
maker cells. Mef2c is known to be regulated by DNA meth-
ylation and histone acetylation (33, 260), further support-
ing the importance of epigenetic modifications during car-
diac specification. Even though many details of this process
are yet to be untangled, our current understanding of car-
diac specification during development has greatly facili-
tated in vitro cardiac differentiation of hiPSCs, as described
below.
B. Protocols for Cardiac Differentiation
Cardiac differentiation of pluripotent stem cells has pro-
gressed from an original efficiency of 5–10% to over 90%
efficiency within the past 15 years (23). Original protocols
were based on random differentiation of three-dimensional
pluripotent stem cell aggregates, known as embryoid bod-
ies, to CMs. Techniques including the enrichment with Per-
coll separation gradients (305), flow cytometry sorting us-
ing the mitochondrial dye tetramethylrhodamine methyl es-
ter perchloratethe (TMRM) (92), and the use of the surface
markers EMILIN2 (280), SIRPA (62), or VCAM1 (277)
have been shown to selectively enrich CM populations.
However, the most widely reproduced method for CM en-
richment has been based on the distinct metabolic differ-
ences in glucose and lactate metabolism between CMs and
other differentiated or undifferentiated cell types, where
glucose-deprived culture media containing abundant lac-
tate was found to favor the survival of CMs over other cell
types (272).
Advances have also been made in cardiac specification of
hiPSCs based on the observation that this process is highly
dependent on precise modulation of both endogenous (131,
212) and exogenous BMP and WNT signaling pathways
(122, 307). Targeted protocols that use key signaling
growth factors such as BMP4 and Activin A enabled con-
version of pluripotent stem cells to CMs with an improved
efficiency (26, 321). This discovery also emphasized the
importance of using hiPSC reprogramming strategies that
do not allow for continued overexpression of reprogram-
ming factors, because OCT4, SOX2, and NANOG each
have known roles in inhibiting differentiation by modulat-
ing BMP4-mediated mesendoderm induction (188, 292).
In protocols developed more recently, growth factors were
also replaced by small molecules (e.g., CHIR99021 and
1098 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
IWR1) that modulate BMP and WNT signaling (22, 24, 81,
159). Small molecules are advantageous compared with
growth factors in that they are cheaper to produce, simpler
to store, more stable, and more amenable to quality control
(137). Finally, the labor intensive process of embryoid body
formation has been replaced by hiPSC differentiation in
monolayer (145) or suspension cultures (37) that generate
over 90% pure CM populations, and are amenable to au-
tomation and scale-up of 1.5–2 billion cells per liter of
culture. These remarkable advancements in cardiac specifi-
cation of hiPSCs have greatly enhanced their potential for
clinical application.
C. Generation and Characterization of
hiPSC-CMs at a High-Throughput Scale
The clinical application of hiPSC-CMs necessitates their
production and functional characterization in large scale
(52). In parallel to the evolution of cardiac differentiation
protocols in suspension bioreactors as mentioned above
(66, 132, 235, 252), automated liquid handling platforms
(e.g., Tecan Freedom Evo 100, Sartotious CompacT SelecT,
and Beckman Coulter Biomek FXP) for robotic scale-up of
mouse and human pluripotent stem cells and their differen-
tiated cardiac derivatives have been evaluated and demon-
strated to improve the consistency and quality of cell cul-
tures (105, 139, 215, 267).
Industrial-scale phenotyping of hiPSC-CM properties is
also advancing. High content imaging (HCI) platforms
have been used to perform 96, 384, or 1,536-well screening
for small molecules to improve reprogramming efficiency,
hiPSC culture, and cardiac differentiation and maturation
(52). Importantly, HCI has been used to assess drug-in-
duced cardiotoxicity in hiPSC-CMs. For example, Sirenko
et al. (242) have used HCI platforms to perform Calcei-
nAM, Hoechst, and MitoTracker imaging in hiPSC-CMs to
evaluate the cardiotoxicity of 131 drugs affecting Na⫹, K⫹,
and Ca2⫹ channels, as well as adreno-, dopamine, and his-
tamine receptors. Mioulane et al. (193) also used the
Thermo Scientific Arrayscan imaging platform to study cel-
lular stress induced by chelerythrine, a cell-permeable pro-
tein kinase C inhibitor, by performing combined TMRM,
caspase-3, and BOBO-1 staining to measure mitochondrial
content, apoptosis, and cell death, respectively. More de-
tailed high content assessment of hiPSC-CM mitochondrial
function has been performed by measuring glycolysis and
oxidative phosphorylation (OXPHOS) using the Seahorse
Extracellular Flux Analyzer (63, 289).
Importantly, the development of 96-well noninvasive
multi-electrode array (MEA) systems (e.g., Multichannel
Systems and Axion Biosystems Maestro) and contractility
analysis platforms (e.g., Cellogy Pulse and SONY SI8000)
enable recording of field potentials and contraction profiles
of hiPSC-CMs in an automated fashion. Besides enabling
 HUMAN INDUCED PLURIPOTENT STEM CELLS
high-throughput functional analysis of drug-induced effects
in hiPSCs, such platforms also enable monitoring of chronic
toxicity using long-term recordings over several hours,
days, or weeks. For example, Gilchrist et al. (79) performed
high-content MEA recordings at 0.5, 1, 2, and 4 h post
exposure to compounds with known proarrhythmic prop-
erties (e.g., verapamil, ranolazine, flecainide, amiodarone,
ouabain, cisapride, and terfenadine) and found that cardio-
toxicity could be recapitulated in vitro using hiPSC-CMs.
Similar data were presented using video-edge contractility
analysis by Maddah et al. (174).
The evolution of hiPSC-CM technologies which enable
high-throughput culture and integration of these cells into
high-content industrial platforms that assess structure, mi-
tochondrial function, electrophysiology, calcium tran-
sients, and contractility are paramount towards their poten-
tial applications in precision medicine.
IV. BIOMEDICAL DATA SCIENCE
Our ability to generate pure hiPSC-CM populations in large
scale has opened the route towards performing in vitro
functional characterization assays (e.g., structural, electro-
physiological, contractile, and metabolic profiling) using
automated high-throughput screening platforms (52, 128).
The combination of these platforms with next generation
biomedical data science is transforming hiPSC-based explo-
ration of molecular mechanisms that underlie cellular func-
tion and contribute to human health. High-throughout se-
quencing information is becoming increasingly important
in biomedical research (286), and it has gradually become
more cost effective and easier to perform thanks to contin-
uous improvement in related technologies (178, 228, 269).
The most prominent NGS technologies are compared and
contrasted in TABLE 2 and are outlined in detail below.
A. Prominent High-Throughput Sequencing
Technologies
A key vendor for NGS platforms is Illumina (http://
www.illumina.com), whose bridge amplification tech-
nology allows billions of reads to be sequenced per run.
The bridge amplification method relies on segregation of
identical amplified sequences into millions of small clus-
ters, which are immobilized on a flow cell surface to
facilitate enzymatic access to the DNA. Amplified se-
quences are then detected using fluorescence-labeled po-
lymerization terminator dNTPs that allow parallel se-
quencing of millions of clusters by detecting the fluores-
cence emission following addition of each dNTP. A
different detection chemistry is employed by the SOLiD
System, which is currently marketed by Thermo Fisher Sci-
entific (http://www.thermofisher.com). In this platform, a
set of four fluorescently labeled di-base probes compete for
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
ligation to the sequencing primer, and the specificity of
ligation is analyzed by interrogating the first and second
bases that are incorporated in each ligation reaction. Mul-
tiple cycles of ligation and detection finally enable reading
of the full fragment. Another platform marketed by Ther-
moFisher is the Ion Torrent, which encompasses the Ion
PGM, Ion Proton, and Ion S5 systems. This platform uti-
lizes yet another unique detection technology, which relies
on identification of proton release when a nucleotide is
incorporated into a DNA fragment by the polymerase en-
zyme, resulting in a detectable local shift in pH for each
dNTP introduced. The Ion Torrent also uses a water-in-oil
emulsified droplet method for initial PCR-based fragment
amplification, which is a methodology shared with Roche’s
454 platforms (http://www.454.com). However, in the 454
platforms, reads are detected using a method known as
sequencing-by-synthesis or pyrosequencing, which relies on
the recognition of pyrophosphate release on nucleotide in-
corporation, rather than chain termination with dNTPs or a
change in pH. Revolutionary technology is also incorpo-
rated into Pacific Biosciences’ RS and Sequel tools (http://
www.pacb.com) which utilize a proprietary Single Mole-
cule, Real-Time (SMRT) technology to deliver sequencing
of the longest reads on the market without the requirement
of a PCR amplification step. Increased read length can im-
prove the alignment process, especially in stretches of highly
repetitive elements. SMRT is based on zero-mode wave-
guides that permit light to specifically illuminate the bottom
of a well in which a DNA polymerase/template complex is
immobilized. Phospholinked nucleotides then enable detec-
tion of the immobilized complex as the DNA polymerase
enzyme produces a natural DNA strand based on the im-
mobilized template. Other NGS platforms include SeqLL’s
HeliScope (http://seqll.com), the first microscope-based
NGS tool that eliminates the need for a PCR amplification
step and, therefore, avoids any associated amplification
bias. The advantages and disadvantages of each technology
are outlined in TABLE 2.
Despite their differences in sequencing chemistries, most
platforms rely on a common experimental workflow, which
consists of random fragmentation of gDNA or RNA, and
ligation of platform-specific adapters to the flanking ends of
the fragments generated to produce a library. Libraries are
then amplified through platform-specific PCR (e.g., on glass
or beads) and undergo coupled DNA synthesis and detec-
tion processes. As multiple sequencing reactions are run
simultaneously, this process is also known as Massively
Parallel Sequencing (42). Desynchronization of reads dur-
ing a sequencing and detection cycle is a source of NGS
errors, which can range from base substitutions to inser-
tions or deletions, depending on the sequencing platform
used (286). The average number of times a given region is
sequenced by independent reads in a sequencing experiment
(known as read “depth”) can be increased to boost confi-
dence in the accuracy of sequencing information (3).
1099
 MATSA ET AL.

Table 2. Overview of high-throughput platforms available for biomedical science applications

Reads/ Time/ Reference
Vendor Chemistry Platform run run Applications ⴙ ⴚ Nos.

Illumina Reversible termination MiSeq 15 M 4–55 h A 2Equipment cost 1Cost per sample 222
MiSeq FGx Same as above Same as above
NextSeq 500 130–400 M 11–29 B Same as above Same as above
h
HiSeq 2500 300 M-4 B 7 h-6 C 2Cost per sample, 1Instrument cost, 165
days 1scale and 1time, 2read
throughput length
HiSeq 3000 2.5 B 1–3.5 Same as Same as above Same as above
days above
HiSeq 4000 2.5–5 B Same as Same as above Same as above
above
Thermo Fisher Proton detection Ion PGM 400 K-6 M 2.3– A 2Cost and time 2Read no. and 222
7.3 h sample
throughput
Ion Proton I 60–80 M 2–4 h B Same as above Same as above 190
Ion S5 3–80 M 2.5–4 Same as Same as above Same as above
h above
Ion S5 XL Same as 1Sample 1Equipment cost
above throughput
Ligation SOLiD 1.4–2.8 B 8h C 2Error rate, 2cost, 2Read length, 1run 165
5500/5500xl 1throughput time
SOLiD 2h Same as 2Time 1Instrument cost
5500W/5500xlW above
PacBio Real-time sequencing RS I 23 K 1–12 h B Longest read length, 1Error rate 222, 229
no PCR step,
simple
preparation
RS II 62 K Same as Same as above 1Cost 229
above
Sequel 400 K 1–6 h A Same as above Same as above
Roche Pyro-sequencing 454 GS Junior 20–100 K 3.5 h A 1Read length 1Error rate, 1cost, 165
2throughput
454 GS FLX⫹ 20 K-1 M 6h Same as above Same as above
SeqLL Single molecule HeliScope 600–800 M 8 days C Nonbiased 1Run time, cost, 268
representation of and error rate
reads
Oxford Nanopore Exonuclease gridION* 4–10 M Flexible C Greatly 1read 1Error rate 88
sequencing length, 2run time

A summarized cross-comparison of the sequencing abilities, applications, advantages (⫹) and disadvantages
(⫺) of next generation sequencing instruments commercialized by leading vendors in the field. WG, whole
genome; K, thousand; M, million; B, billion; PacBio; Pacific Biosciences; PGM, Personal Genome Machine; 1,
high; 2, low; no., number; A, targeted gene panels; B, exome and RNA sequencing and targeted gene panels;
C, de novo sequencing; WG, exome, RNA, ChIP, and methyl sequencing, and targeted gene panels; *Instru-
ment is under development.

B. Biological Information Obtained Via High-
Throughput Technologies
NGS platforms enable the deciphering of complex yet valu-
able biological information. For example, whole genome
(WG) or exome sequencing can be used to identify muta-
tions that may cause genetic disorders, or genomic poly-
morphisms that may underlie disease progression, disease
severity, and response to pharmacotherapy. A read depth of
30 –100⫻ is recommended to enable accurate detection of
gDNA variation (146). However, as the read depth in-
creases, so does the sequencing cost (279). Using targeted
capture arrays that focus on a subgroup of target genes
involved in a specific pathway or disease can provide a
balance between performance and cost. Notably, in a recent
publication, Wilson et al. (298a) used a padlock probe ap-
proach to create a cost-effective and rapid clinical-grade
NGS platform that enables detection of thousands of mu-
tations in over 50 genes (e.g., TNNT2, MYH7, and
LMNA/C) that are associated with familial cardiomyopa-
thies. Such platforms promise to facilitate the identification
of underlying disease mutations, and gather personalized
information that will expedite precision medicine endeav-
ours.
RNA-sequencing (RNA-seq) is a widely used method to
generate dynamic and quantitative information regarding
transcribed mRNA present in a tissue or cell population at
the time of sample collection. Its high sensitivity allows
detection of low abundance transcripts and splice variants,
which can contribute towards unraveling the molecular
mechanisms of cellular processes (144). As mentioned

1100 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 HUMAN INDUCED PLURIPOTENT STEM CELLS
above for gDNA sequencing, several gene targeted plat-
forms (e.g., Ion AmpliSeq panels for genes involved in de-
velopmental, heart, or metabolic disease) are available for
RNA-seq, all of which are designed to reduce the cost of
experimentation, and increase sample throughput. RNA-
seq is also not limited to the quantification of mRNA tran-
scripts; depending on the methods used for RNA isolation
and library preparation, it can sequence other RNA popu-
lations such as miRNA, tRNA, or ribosome-bound RNA to
detect mRNA actively being translated into proteins (108).
The ability to measure the abundance of different RNAs has
in recent years refined the investigation of a variety of bio-
logical problems, including optimizing experimental condi-
tions (e.g., drug dose and timing), time-course experiments
(e.g., to monitor developmental processes, or disease pro-
gression), population-specific response profiling (e.g., in
healthy and diseased individuals), studying response to
treatment (e.g., to several different drugs), and performing
large-scale RNA interference (RNAi) studies (249).
Because gene expression can also be greatly influenced by
epigenetic modifications such as histone methylation or
acetylation, as well as gDNA methylation of cytosine gua-
nine dinucleotides (CpGs) (112), chromatin immunopre-
cipitation sequencing (ChIP-seq) and DNA methylation se-
quencing (Methyl-Seq) can also provide valuable informa-
tion regarding biological processes. ChIP-seq involves
cross-linking of histone proteins to gDNA (e.g., using form-
aldehyde), followed by an immunoprecipitation reaction
with antibodies against histone modifications associated
with actively transcribed genes (e.g., H3K4Me3 and
H3K9ac) or silenced genes (e.g., H3K27Me3). The result-
ing immunoprecipitated DNA is then sequenced in an NGS
platform to identify genes associated with each type of his-
tone epigenetic modification (224). On the other hand,
Methyl-seq relies on the comparison of native gDNA to
bisulfite-treated gDNA, in which non-CpG cytidine nucle-
otides are converted to uracil nucleotides (141). Regions
lacking bisulfite conversion are thought to be methylated
and silenced, since DNA methylation is strongly correlated
with the suppression of gene expression (278). Epigenetic
modifications influence developmental processes (107), and
a variety of disorders, including heart disease (1, 47), and
their study helps us understand the physiological and path-
ological mechanisms involved.
Large international projects aim to capture genome-wide
relationships using large population samples and a variety
of platforms and sequencing approaches. These include the
1000 Genomes Project Consortium and the 100,000 Ge-
nome Project (100kGP) (282). De-identified genomic data are
freely available and publicly accessible through resources such
as the ClinVar (http://www.nh.gov/clinvar/cbi.nlm.ni) or the
ClinGen (http://www.clinicalgenome.org) databases. In addi-
tion to the collection of samples, large efforts are underway to
integrate different classes of omic information. These include
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
the interactive personal omics profiling (iPOP) initiative (155),
which is intended to assemble an individual’s exome, tran-
scriptome, proteome, metabolome, microbiome, and other
omes to create a dynamic profile of the physiology and
health state of the individual. Such collectives can become
stepping stones towards precision health by improving risk
prediction as well as disease diagnosis, monitoring, and
treatment.
C. The Combination of hiPSC and NGS
Technologies
In hiPSCs, transcriptional and epigenetic variation originat-
ing from genetic background have been shown to dominate
over cell type of origin or derivation method in regards to
the variation observed among lines from different patients
(40). This demonstrates that hiPSCs preserve patient-spe-
cific omic information and, therefore, provide a useful ex-
perimental platform to study the pathophysiological effects
of individualized biomedical data. An interesting study re-
lated to hypertrophic cardiomyopathy (HCM) was pub-
lished by Zhi et al. (323), who performed whole exome
sequencing in seven sibling trios to identify missense or
nonsense mutations that were associated with increased left
ventricular (LV) mass. Functional correlation of these mu-
tations to hypertrophy-related gene signaling changes in
hiPSC-CMs led to the conclusion that a predicted conserved
and damaging nonsense variant in exon 13 (A2099G) of the
THBS1 gene was significantly associated with pathologi-
cally increased LV mass. It is anticipated that, in combina-
tion with NGS tools, hiPSC technology will serve as a next-
generation biomedical interface enabling us to establish
strong phenotype-to-genotype correlations and understand
complex biological systems (90). Further examples of stud-
ies which provide proof-of-principle that hiPSCs can be
used to understand how complex gene networks and poly-
genic modifications may lead to heart disease are discussed
in more detail in the following section.
V. hiPSC-BASED MODELS OF HUMAN
INNATE DISEASE
The most widely studied hiPSC-based models of human
cardiovascular disease are those caused by familial or con-
genital mutations. Following clinical consultation and ob-
taining informed consent, patients carrying familial muta-
tions associated with cardiovascular disease are recruited
for hiPSC generation (FIGURE 2). Subsequent cardiac spec-
ification of hiPSCs generates beating cells (24) which, de-
spite their relatively immature structural and electrophysi-
ological state (25, 184), have been shown to recapitulate the
clinical phenotypes observed in the donors. Disease model
derivation has also been achieved in integration-free chem-
ically defined conditions, making them more clinically rel-
evant (20). The growing body of research regarding innate
1101
 MATSA ET AL.

Innate Engineered Induced

Healthy donor Diseased donor Healthy donor Healthy donor

Reprogramming
to hiPSCs Genome Disease-
Healthy inducing
editing
conditions conditions

hiPSC
differentiation

Phenotypic
& functional
characterization

Structure Protein assay Electrophysiology Molecular pathway interrogation

FIGURE 2. hiPSC-based disease modeling approaches. hiPSC modeling of cardiovascular disease has mainly
been based on three approaches: 1) the generation of patient-specific disease lines using somatic cells from
patients with familial mutations associated with heart disease (innate model); 2) the introduction of pathogenic
mutations in healthy hiPSC-CMs via genome editing approaches; and 3) the induction of heart disease in
hiPSC-CMs from healthy individuals, either via drug treatment, pathogen infection, media supplementation, or
exposure to stress-inducing conditions (induced model).

models of cardiac diseases is summarized in TABLE 3
below.
A. Cardiac Channelopathies
Among the most extensively researched diseases using
hiPSC-based models are arrhythmic disorders caused by
mutations in cardiac ion channels, which are known as
cardiac channelopathies. This category of disorders in-
cludes congenital long QT (LQT) syndrome, catecholamin-
ergic polymorphic ventricular tachycardia (CPVT), Timo-
thy syndrome, and Brugada syndrome, which are clinically
characterized by arrhythmias, ventricular fibrillation, sei-
zures, and even sudden death (248). As summarized in
TABLE 3, characterization via MEA and patch-clamp elec-
trophysiological techniques has enabled identification of
prolonged field potential duration (FPD) and action poten-
tial duration (APD) in hiPSC-CMs from patients with LQT
syndrome type 1 and type 2 loss-of-function autosomal
dominant mutations in potassium ion channels (184, 197).
These abnormalities were found to be due to diminished IKs
or IKr currents resulting from trafficking defects in the pro-
teins responsible for the respective ion channel formation.
␤-Adrenergic stimulation or pharmacological blockade of
cardiac repolarization currents (e.g., with E4031) caused
LQT syndrome hiPSC-CMs to develop arrhythmias in the
form of early afterdepolarizations (EADs), which could be
corrected by treatment with ␤-blockers (e.g., propranolol
and nadolol). Novel therapeutic avenues, such as small mol-
ecule-mediated modulation of chaperone proteins with N-
[N-(N-acetyl-L-leucyl)-L-leucyl]-L-norleucine (ALLN) (187)
and and allele-specific RNA interference (183), were also able to
ameliorate the disease phenotypes. These findings were
among the first to provide proof-of-principle that hiPSC-
CMs can recapitulate the human pathophysiology of LQT
syndrome.
Other arrhythmia syndromes have also been studied, in-
cluding LQT syndrome type 3 (LQT3), which is caused by
gain-of-function mutations in the sodium ion channel-en-
coding gene SCN5A. These hiPSC-CMs showed markedly
prolonged APD (due to decreased voltage-dependent inac-
tivation of Na⫹ channels) and late or persistent Na⫹ cur-
rents. These phenomena were reversed by a combination of
increased pacing rate and NaV1.5 sodium channel block-
ade (e.g., with mexiletine). This treatment regimen closely
mirrors clinical management of LQT3 (172, 175, 209,
264). Similarly, hiPSC-CMs generated from patients carry-
ing mutations in the L-type calcium channel Ca(v)1.2 that
are associated with LQT syndrome type 8, also known as
Timothy syndrome, showed pro-arrhythmia, prolonged
APD, excess Ca2⫹ influx, and abnormal Ca2⫹ transients.
Roscovitine, a Ca(v)1.2 activator, ameliorated the electro-
physiological and Ca2⫹ handling perturbations observed in
these hiPSC-CMs (311).
Finally, hiPSC-CMs from patients with CPVT types 1 and
2, carrying mutations in the ryanodine receptor (RYR2) or
cardiac calsequestrin (CASQ2) genes, showed pro-arrhyth-
mia in the form of delayed afterdepolarizations (DADs),
which were reversed following administration of flecainide,
a Nav1.5 sodium channel and RYR2 blocker that is clini-

1102 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 HUMAN INDUCED PLURIPOTENT STEM CELLS

Table 3. Innate and engineered models of cardiac disease

Model Class Disorder Gene Mutation Reference Nos. Key Phenotypes

Innate Arrhythmia LQT1 KCNQ1 R190Q 197 Protein trafficking defects

syndromes
Exon 7 del 171 1FPD and APD in atrial
and ventricular CMs
LQT2 KCNH2 A561T 184 Serve 2 or absence of Ik,
(LQT1) or Ik (LQT2)
17 Arrhythmias charactrized
by EADs
A614V 110 1Sensitivity to
proarrhythmic drugs
A561P 120 2repolarzation reserve
LQT3 SCN5A F1473C 264 Defective Na⫹ channel
caused by deficiency in
open-state inactivation
of the Na⫹ channel
V1763M 172
V240M 67 Enhanced late Na⫹
channel current (INaL)
R535Q
1644H 175 Delayed repolarization
and prolonged APD
CPVT1 RYR2 F2483I 68 1Diastolic [Ca2⫹]
322 2SR Ca2⫹content,
indicating Ca2⫹ leakage
S406L 121 1Frequency and duraton
of elemantary Ca2⫹
release events (sparks)
M4109R 109
P2328S 142 Arrhy thmias
characterized by
DADs, broad and
double-humped
transists
Q2311D 54 1Senstivity to
proarrhythmic drugs
R420Q 206 Immature ultrastructural
features
CPVT2 CASQ2 D307H 206 1Diastolic [Ca2⫹]
Oscillatory arrhythmic
prepotentials, DADs
207 Myofibrillar disarray,
1SR cisternae size,
1caveolae number
TS CACNA1C G406R 312 Irregular contractility and
electrical activity,
1APD
1Ca2⫹ influx
Cardiomyopathies DCM TNNT2 R173W 254 Abnormal Ca2⫹
regulation,
2contractility
301 Myofibrillar disarray-
RBM20 R636S 304
LMNA/C R225X 243 1Nuclear blebbing and
micronucleation
1Nuclear senescence
and apoptosis
DES A285V 275 Abnormal DES
aggregations
2 Max rate of Ca2⫹ re-
uptake and 2beating
rate
2Tolerance to inotropic
stress
HCM MYH7 R663H 147 1Cellular size and
sarcomere
disorganization
R442G 89 Contractile arrhythmias
Continued

Physiol Rev • VOL 96 • JULY 2016 • www.prv.org 1103

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 MATSA ET AL.

Table 3.—Continued
Model Class Disorder Gene Mutation Reference Nos. Key Phenotypes

MYBPC3 G999-Q1004del 262 Dysregulation of Ca2⫹

cycling and 1[Ca2⫹]i
FXN Silencing 153 FRDA-associated HCM
1Rate of iron
accumulation
2Ca2⫹ uptake and
release kinetics
Disorganized and
fragmented
mitochondrial network
1ROS accumulation
LS PTPN11 T468M- 31 1Nuclear NFATC4
localization
163 1Expression of genes
associated with cardiac
hypertrophy
A324fs335X 32 1Lipogenesis and
apoptosis
ARVD PKP2 c.2484C⬎T 134 2PKP2 and 1PPAR-␥
gene expression
c.2013delC 2PKP2, plakoglobin, and
Cx43 protein density
c.1841T⬎C 170 2␤-Catenin activity
Desmosome
disorganization
DMD DMD exon 45-52del 164 Dystrophin deficiency
1Levels of resting Ca2⫹
Mitochondrial damage
and 1apoptosis
Structural HLHS Not identified Not identified 115 2Ability for cardiac
differentiation
73 2Myofibrillar organization
1Fetal gene expression
pattern
Calcium transient and
electrophysiological
abnormalities
Metabolic ALHD deficiency ALDH2 E487K 63 1Levels of ROS and toxic
aldehydes
1Cell cycle arrest and
activation of apoptotic
signaling
BTHS TAZ c.517delG c.517ins 289 1Levels of ROS,
2mitochondrial
function
1Sparse and irregular
sarcomeres, weak
contractility
Pompe disease GAA D645E 101
exon 18del 227 1Levels of glycogen,
1441delT defective respiration
Deficit of Golgi-based
protein glycosylation
c.796C⬎T 233 Ultrastructural
c.1316T⬎A aberrances, lysosomal
enlargement
Engineered Arrhythmias LQT1 KCNQ1 R190Q 290 Protein trafficking defects
G269S 1APD in atrial and
ventricular CMs
G345E Severe 2 or absence of
IKs (LQT1) or IKr (LQT2)
LQT2 KCNH2 A614V Arrhythmias
characterized by EADs
N996I 13
Continued

1104 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 HUMAN INDUCED PLURIPOTENT STEM CELLS

Table 3.—Continued
Model Class Disorder Gene Mutation Reference Nos. Key Phenotypes

Cardiomyopathies DCM PLN R14del corrected to 130 Correction of:

WT
Ca2⫹ handling
abnormalities
Electrical instability
Abnormal cytoplasmic
distribution of PLN
protein
1Expression cardiac
hypertrophy molecular
markers
TTN W976Rfs 93 2Contractile
A22352fs performance due to
P22582fs A-band truncations
Sarcomere insufficiency
Impaired responses to
mechanical and ␤-
adrenergic stress
Attenuated growth factor
and cell signaling
activation
Metabolic BTHS TAZ c.517delG 289 1Levels of ROS,
c.517ins 2mitochondrial
function
Sparse and irregular
sarcomeres, weak
contractility

Together, Tables 3 and 4 provide a detailed outline of hiPSC-based models of cardiac disease published to date,
with correlation to the gDNA mutations studies, and the key phenotypes observed for each disorder. APD,
action potential duration; FPD; field potential duration; 1, increased; 2, decreased; EAD, early afterdepolar-
ization; DAD, delayed afterdepolarization; hiPSC, human induced pluripotent stem cell; CMs, cardiomyocytes;
SM, small molecule; GF, growth factor; SR, sarcoplasmic reticulum; VPA, valproic acid; ET-1, ETA-b, endo-
thelin receptor type A blocker; ROS, reactive oxygen species; CDI, Cellular Dynamics International; LQT1, 2, 3,
long-QT syndrome type 1, 2, 3; JLNS, Jervell and Lange-Nielsen syndrome;CPVT1, 2, catecholaminergic
polymorphic ventricular tachycardia type 1, 2; TS, Timothy syndrome; DCM, dilated cardiomyopathy; HCM,
hypertrophic cardiomyopathy; FRDA, Friedreich’s ataxia; DFP, deferiprone; LS, LEOPARD syndrome; ARVD,
arrhythmogenic right ventricular dysplasia; DMD, Duchenne muscular dystrophy; HLHS, hypoplastic left heart
syndrome; BTHS, Barth syndrome.

cally used against CPVT (109). Thapsigargin, a sarcoplas-
mic reticulum calcium ATPase pump inhibitor, also amelio-
rated the CPVT phenotype in hiPSC-CMs by depleting in-
ternal Ca2⫹ stores. This supported the mechanistic
explanation that increased diastolic Ca2⫹ can lead to the
development of arrhythmias. In a separate study, ultra-
structural characterization demonstrated that hiPSC-CMs
from CPVT1 patients also have enlarged mitochondrial,
glycogen, and lysosome clusters, as well as thinner Z-bands,
reduced number of sarcomeres per cell, and increased sar-
comere disarray (206). These phenotypes were more pro-
nounced in CPVT2 hiPSC-CMs, demonstrating that hiPSCs
provide a setting for the investigation of the similarities and
differences in the pathophysiological consequences of dif-
ferent mutations.
B. Cardiomyopathies
A second class of cardiac disorders that have been widely
studied using the hiPSC in vitro platform are cardiomyop-
athies. These include familial HCM, dilated cardiomyopa-
thy (DCM), arrhythmogenic right ventricular cardiomyop-
athy (ARVC), LV noncompaction (LVNC), and restrictive
cardiomyopathy (RCM) (91). Most cardiomyopathies are
characterized by disrupted sarcomeric alignment, a phe-
nomenon known as myocardial disarray that is linked to
deterioration in myocardial function, heart failure, and car-
diac sudden death (111). However, each cardiomyopathy
has its own distinct pathophysiological features: DCM is
identified by ventricular dilation and systolic dysfunction;
HCM manifests as thickening primarily of the left ventric-
ular wall; ARVC is distinguished by cardiac enlargement,
aneurysm formation, and fatty infiltration of the ventricles;
and RCM manifests as atrial arrhythmias, abnormal right-
and left-sided filling pressures, and other clinical signs and
symptoms characteristic of right ventricular failure (210).
hiPSC-CMs derived from patients with familial cardiomy-
opathy have been shown to recapitulate the human heart
pathophysiology. For example, hiPSC-CMs carrying a
DCM-related cardiac troponin T mutation (TNNT2
p.R173W) showed characteristic sarcomeric disarray and
other ultrastructural abnormalities such as scattered distri-
bution pattern of Z bodies, as well as reduction in contrac-

Physiol Rev • VOL 96 • JULY 2016 • www.prv.org 1105

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 MATSA ET AL.
tile force, altered regulation of Ca2⫹, and decreased toler-
ance to ␤1-adrenergic challenge (254, 301). These pheno-
types were attenuated by clinically relevant ␤1-blockade
(e.g., with metoprolol), or more experimental pharmaco-
logical inhibition of PDE2A and PDE3A (e.g., with Bay-60-
7550 and milrinone), or transgenic overexpression of sar-
coplasmic reticulum Ca2⫹ adenosine triphosphatase
(SERCA2a). Microarray analysis in hiPSC-CMs that had
undergone SERCA2a gene therapy showed that Ca2⫹ sig-
naling, protein kinase A (PKA) signaling, and G protein-
coupled receptor (GPCR) signaling were mainly responsible
for the phenotypic rescue. It should be noted that the
R173W mutation identified in these DCM patients had not
been previously reported, and was discovered in this family
cohort following exome sequencing of the skin fibroblasts
used to generate hiPSCs. In separate studies, hiPSC-CMs
carrying DCM-associated mutations in the spliceosome
RNA-binding motif protein 20 (RBM20), lamin A/C
(LMNA/C), or desmin (DES) genes (243, 275, 304) also
demonstrated abnormalities that closely recapitulate the
morphological and functional phenotypes of the affected
human heart, as outlined in TABLE 3.
hiPSC models of HCM associated with thick myofilament
myosin heavy chain 7 (MYH7) (89, 148) or myosin binding
protein C3 (MYBPC3) (262) mutations showed that in vitro-
derived hiPSC-CMs are significantly enlarged, as seen in the
diseased human myocardium. hiPSC-CMs also demonstrated
increased myofibril content, contractile arrhythmias (DADs),
Ca2⫹ cycling perturbations, and intracellular Ca2⫹ elevation,
which were worsened by adrenergic stimulation (e.g., with
isoproterenol) or environmental exposure to the potent vaso-
constrictor endothelin-1 (ET-1) (310). These disease pheno-
types were reversed either by ␤-blockade (e.g., with propran-
olol or metoprolol), treatment with endothelin receptor type A
blockers (e.g., BQ-123), or treatment with L-type Ca2⫹ chan-
nel blockers (e.g., verapamil). The latter observation provided
mechanistic validation that Ca2⫹ handling defects and ele-
vated intracellular Ca2⫹ levels are underlying disease mecha-
nisms for HCM. Whole transcriptome sequencing and path-
way enrichment analysis in HCM hiPSC-CMs also showed a
widespread enrichment of genes associated with cellular pro-
liferation (e.g., WNT1), Notch signaling (e.g., DLL1/4), and
FGF signaling (e.g., FGF8/FGFR4) which could serve as po-
tential therapeutic targets (89). HCM manifestations associ-
ated with the multi-organ condition known as LEOPARD
syndrome were likewise studied in a hiPSC-CM platform,
showing that irregularities in RAS-MAPK signal transduction
are implicated in development of disease pathology (31). In
addition, hiPSC-CMs from patients with HCM associated
with the recessive neurodegenerative disorder Friedreich’s
ataxia (FRDA) showed an increase in reactive oxygen species
(ROS) levels caused by silencing of the mitochondrial gene
frataxin (FXN) (153). Additional disease phenotypes, includ-
ing disorganization of the mitochondrial network, intracellu-
lar iron accumulation, and abnormal Ca2⫹ handling kinetics
1106 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
were evident following iron loading-induced stress. Treatment
with molecules that are currently under clinical trial for
FRDA, such as the iron chelator deferiprone (DFP), signifi-
cantly suppressed ROS synthesis, modulated mitochondrial
stress, and improved electrical coupling during hiPSC-CM
contraction.
Finally, hiPSC-CMs carrying ARVC-associated mutations
in desmosome proteins have also recapitulated disease pa-
thologies (e.g., increased lipogenesis and apoptosis, and
desmosome disorganization) following induction of adult-
like metabolic energetics from glycolysis to fatty acid oxi-
dation, therefore demonstrating the ability of hiPSC-CMs
to model adult-onset cardiac disorders (134, 170).
Collectively, these studies not only demonstrate that hiPSC-
CMs can serve as disease modeling and drug discovery plat-
forms, but can also be used to assess the unique interactions
between individual patients’ genetic backgrounds and envi-
ronmental factors, and serve as biomedical data interfaces
to unravel complex molecular pathways implicated in cel-
lular dysfunction and human cardiac or syndromic disease.
C. Structural Defects
Developmental heart defects such as hypoplastic left heart
syndrome (HLHS), characterized by severe underdevelop-
ment of the left ventricle, are another class of cardiac disor-
ders that have been modeled in hiPSC-CMs. hiPSCs from
HLHS patients were found to have a reduced ability to form
beating CMs in vitro, whereas hiPSC-CMs also demon-
strated myofilament disorganization, induction of fetal
gene expression profiles (e.g., HIF1␣), Ca2⫹ handling ab-
normalities, and increased susceptibility to ␤-adrenergic
stimulation and caffeine treatment (73, 116). Further stud-
ies in hiPSC-CMs revealed that disease pathologies could be
associated with alternative Ca2⫹ release mechanisms from
the sarcoplasmic reticulum via the inositol trisphosphate
receptor rather than the ryanodine receptor, thus providing
evidence that hiPSC-CMs can be used for mechanistic in-
vestigations of developmental heart defects. Although no
known mutations linked to HLHS were identified in these
studies, hiPSC-CMs offer an opportunity for further re-
search regarding causative gDNA alterations.
D. Metabolic Disorders
Familial metabolic disorders, also known as inborn errors of
metabolism (IEMs), represent more than 15% of monogenic
disorders (8). Initiating during early infancy or childhood,
over 40% of IEMs patients manifest a cardiovascular pheno-
type that includes limited exercise capacity, HCM, DCM,
LVNC, RCM, and fatal arrhythmias (162). Due to the high
dependence of cardiac muscle on aerobic metabolism (up to
70% of energy in the heart muscle stems from oxidative me-
 HUMAN INDUCED PLURIPOTENT STEM CELLS
tabolism) (85), defects in mitochondrial oxidative phosphor-
ylation (e.g., complex I, II, III, IV, and V disorders, Barth,
Leigh, and MELAS syndromes) or fatty acid ␤-oxidation (e.g.,
CPT2, VLCAD, and CTD syndromes) are more frequently
associated with cardiac findings (19). Other major groups of
metabolic disorders associated with cardiomyopathy include
lysosomal diseases (e.g., Fabry disease), glycogenesis-related
disorders (e.g., Pompe and Danon diseases), organic acidurias,
and glycosylation disorders. Although metabolic disorders ac-
count for a minority of cardiomyopathy cases, identifying an
IEM as the underlying cause of disease has significant prog-
nostic and therapeutic implications (297).
Regardless of the clinical avenues used for therapy, the mech-
anisms by which IEMs lead to cardiomyopathy remain poorly
understood. To bridge this gap, hiPSCs have been used as
platforms to study the mechanisms and physiological pertur-
bations underlying cardiomyopathy related to metabolic dis-
eases. Infantile-onset Pompe disease, an autosomal recessive
IEM caused by mutations of the lysosomal glycogen-hydrolyz-
ing enzyme acid ␣-glucosidase (GAA), is one of the disorders
studied in a hiPSC-CM setting. Pompe and control hiPSC-
CMs demonstrated no significant differences in contractile
strength, kinetics, or clearance rates of autophagosomes.
However, similar to the patients’ myocardium, Pompe hiPSC-
CMs had diminished GAA activity, lysosomal glycogen accu-
mulation, and lysosome enlargement (227). MALDI-TOF-MS
analysis of N-linked glycans revealed that Pompe cardiomy-
opathy could be caused by a glycan-processing abnormality,
as often seen in congenital glycosylation disorders with an
HCM phenotype, therefore providing mechanistic insight into
the disease pathological mechanisms. Gene therapy by lentivi-
ral GAA transfer was able to reverse the disease-specific
hiPSC-CM phenotypes (233).
Another metabolic disorder to be studied in a hiPSC setting is
Barth syndrome (BTHS), a mitochondrial disorder caused by
mutations in the tafazzin (TAZ) gene. Engineered muscular
thin film (MTF) chips (276) constructed from BTHS hiPSC-
CMs had defined metabolic (e.g., excess accumulation of
ROS), structural (e.g., sparse and irregular sarcomeres), and
functional (e.g., weak contractility of engineered heart muscle)
abnormalities (289). These were reversed either by gene re-
placement therapy or genome editing, demonstrating that the
TAZ mutation under investigation was necessary and suffi-
cient for these phenotypes. Therefore, the combination of tis-
sue engineering, genome editing, and hiPSC technologies in
this study provided new insights into the pathogenesis of
BTHS, delivering a biomedical platform to confirm the phe-
notype-to-genotype correlation of a gDNA mutation.
Taking a step further to demonstrate the association of
gDNA alterations to disease manifestation, Ebert et al. (63)
showed that the aldehyde dehydrogenase 2 (ALDH2) ge-
netic polymorphism E487K, which is found in nearly 8% of
the human population and is linked to increased suscepti-
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
bility towards ischemic heart damage and coronary artery
disease (CAD), was associated with elevated accumulation
of ROS and toxic aldehydes (e.g., 4-hydroxynonenal;
4HNE) in heterozygous hiPSC-CMs. These pathological
phenotypes were connected with the induction of cell cycle
arrest and activation of apoptotic signaling genes (e.g.,
JUN) and were aggravated during exposure to an ischemia-
mimetic solution and hypoxic conditions. Alda-1-mediated
activation of ALDH2, treatment with the independent ROS
scavenger catalase, or inhibition of the Jun regulator Jun
NH2-terminal kinase (JNK) were all able to decrease path-
ological ROS levels. Therefore, the authors were able to
shed light onto the underlying molecular bases of a genetic
polymorphism often found in East Asians that is associated
with adverse cardiac function in response to ischemia.
VI. ENGINEERED DISEASE MODELS
THROUGH TARGETED GENOME
EDITING
Targeted gene editing has been historically achieved by homol-
ogous recombination (HR) (157); however, the low efficiency
of HR events in human cells impeded extended application of
this approach. Recent advances in endonuclease-based gene
editing systems have transformed the field of genome engineer-
ing (46). Following discovery that induction of DNA double-
strand breaks (DSBs) increases the frequency of HR events by
several orders of magnitude, targeted nucleases such as ZFN,
TALEN, and CRISPR/Cas9 have emerged as the preferred
methods for improving the efficiency of HR-mediated gene
modification (74). Once endonuclease enzymes induce a DSB
in the DNA, the cell’s natural repair mechanism initiates one
of two different processes: 1) the more likely but also more
error prone nonhomologous end joining (NHEJ) pathway,
whereby the two broken strands are modified by either adding
or removing sheared nucleotides and then physically ligating
the two strands back together; or 2) the more precise homol-
ogy directed repair (HDR) pathway, kinetically favored over
NHEJ only if a DNA strand with homology to the cut gDNA
is present (199). This process can utilize an exogenously pro-
vided homologous sequence or plasmid as a template from
which a complementary strand is synthesized. The newly syn-
thesized strand then ligates with the original, resulting in a
repaired double strand. Endonuclease-based techniques take
advantage of this repair system by providing a template of
semi-homologous sequence with specific and intended muta-
tions that will be copied into the cell’s DNA.
The next section of the review provides more details regard-
ing novel genome editing technologies, by comparing the
ZFN, TALEN, and CRISPR/Cas9 systems, and highlighting
the future research applications of these technologies.
A. DNA Binding Designs
In addition to the induction of DSBs, current nuclease-based
approaches distinguish themselves from previous gene editing
1107
 MATSA ET AL.
systems by the ease with which their DNA recognition do-
mains are customized, a feature that ultimately improves ge-
nome editing efficiency (214). ZFN and TALEN technologies
share a mechanism where repeating domains in their protein-
based DNA binding modules recognize individual nucleotides.
The zinc finger domain, first discovered in 1997, is the core
component of a zinc finger protein (ZFP), which recognizes a
three nucleotide sequence. ZFNs typically comprise 3– 6 zinc
finger proteins and bind 9 –18 nucleotides of the target DNA.
ZFNs may be combined through a modular assembly to rec-
ognize nearly any genetic sequence; however, the codon-based
gene editing limits flexibility and the ability to potentially tar-
get any gDNA sequence. Additionally, even though ZFNs rely
on a very modular assembly, their production remains labor
intensive and time consuming (136).
Similar to the role of the ZFPs, the core TALEN domain
binds to DNA based on a protein-DNA interaction. The
TALEN binding domain is a sequence of 33–35 repeating
amino acids that forms a repeat-variable di residue (RVD).
The 12th and 13th amino acids in each RVD, known as
hypervariable positions, are responsible for binding differ-
ent nucleotides. Based on the affinity of A, T, G, and C to
the respective RVDs NI, HD, NN, and NG, TALENs may
be utilized to recognize any sequence of DNA. This one-to-
one interaction is a large advantage over ZFN in that it
provides increased specificity and ease of customization
(83). However, like ZFNs, to target a new DNA sequence
one must reengineer the primary binding domains. Thanks
to intensive research in recent years, customized TALENS
can be constructed in ⬍1 mo, following minimally labor
intensive procedures that also reduce or eliminate the num-
ber of cuts and ligations required, thus reducing the possi-
bility of orientation error (59, 234).
The most prominent technology for modern genome editing is
the CRISPR/Cas9 system, first reported in 1987. A form of the
CRISPR/Cas9 system can be found throughout bacteria and
prokaryotes. However, the most thoroughly researched and
most applicable to DNA modification is the modular type 2
CRISPR/Cas9 system found only in bacteria. Cas9 is an endo-
nuclease specific to the bacteria Streptococcus pyogenes and is
the only nuclease involved in silencing of foreign DNA (117).
Within bacteria, different CRISPR systems exhibit slight mod-
ifications in length and DNA recognition, but all can be engi-
neered through modular assembly of three main components:
1) the nuclease that cuts DNA; 2) a specificity determining
RNA strand, known as the CRISPR RNA (crRNA); and 3) the
trans-activating crRNA (tracrRNA) (118). Unlike the ZFN
and TALEN recognition of DNA via protein interactions in-
volving complex ␣-helix recognition of nucleotide triplets or
repeating amino acids with hypervariable regions, the
CRISPR-Cas9 system binds to DNA in the same fashion as an
RNA oligo (302). Twenty nucleotides on the crRNA bind with
the 20 nucleotides of the target DNA, recruiting the Cas9
endonuclease, which on its own has no sequence-specific bind-
1108 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
ing. crRNA sequences are easy to design, making the CRISPR/
Cas9 more versatile than either ZFN or TELEN tools.
While it is tempting to further stratify ZFN, TALEN, and
CRISPR/Cas9 by ranking them based on binding efficien-
cies, it would be potentially misleading to do so. A clear and
fixed ranking system is impossible to construct because each
system binds differently to different targets. Currently no
molecular basis explains this discrepancy. However, it has
been suggested that DNA methylation, chromatin state,
genomic position, and DNA sequence may affect DNA
binding and thus explain why different editing efficiencies
are reported for different targets (100).
B. Off-Target Binding
Recent analysis of whole genome sequencing applied to
hiPSCs reveals minimal differences in off-target activity
among ZFN, TALEN, and CRISPR/Cas9 technologies
(244, 255, 281). In addition, the frequency of off-target
activity is typically lower than the rate of naturally accruing
mutations that result from culture maintenance (51). Stud-
ies specific to TALEN and CRISPR/Cas9 identified signifi-
cantly more mutations as a result of cell maintenance than
those causally linked to the application of gene editing
(106). Although some reports indicate high rates of off-
target mutations, these numbers stem from analysis of
transgenic mammalian cell lines such as HEK293, and thus
cannot be put into context relevant to hiPSC culture and the
development of disease models (274). Nevertheless, off-tar-
get binding remains a significant hurdle to clinical adoption
of gene editing techniques. More research is required to
develop improved computational algorithms able to iden-
tify sequences with low risk of off-target binding, and to
predict off-target binding even when DSBs do not occur
(291).
Recently, research has expanded beyond the optimization
of binding domains to explore other creative approaches for
reducing off target mutations. For example, engineered nu-
clease mutations that induce DNA “nicks” or single-
stranded breaks (SSBs) are advantageous because SSB cel-
lular repair circumvents NHEJ repair mechanisms, which
would normally increase the probability of mutagenesis.
Multiple reports identify that both ZFNickases and
TALENickases induce fewer off-target mutations while
demonstrating equal if not better on-target mutation rates
(303). Likewise, nickase-based CRISPR systems (CRISPR/
Cas9n) result in negligible off-target effects without a sac-
rifice of on-target mutation frequency (240).
C. Genome Editing in hiPSC-Based Cardiac
Disease Modeling
In hiPSCs, genome editing has thus far been utilized for the
correction of genetic disorders as mentioned earlier for
 HUMAN INDUCED PLURIPOTENT STEM CELLS
BTHS (289). Karakikes et al. (130) also used genome edit-
ing to correct the PLN gene mutation R14del which has
been associated with ventricular dilation, ventricular ar-
rhythmias, and heart failure. In this study, hiPSC-CMs
heterozygous for the R14del mutation exhibited Ca2⫹ han-
dling abnormalities, electrical instability, abnormal cyto-
plasmic distribution of PLN protein, and increased expres-
sion of molecular markers of cardiac hypertrophy, which
were ameliorated following TALEN-based mutation cor-
rection.
Similarly, genome editing has been utilized for the gener-
ation of hiPSC-based disease models, such as LQT syn-
drome. In two studies by Wang et al. (290) and Bellin et
al. (13), hiPSCs from healthy donors were engineered to
introduce dominant negative mutations in the ion chan-
nel genes KCNQ1 (p.R190Q, p.G269S, and p.G345E)
and KCNH2 (p.A614V and p.N996I) into the safe har-
bor AAVS1 locus. Compared with isogenic controls, ge-
nome-edited lines displayed characteristic LQT syn-
drome phenotypes, which included significant APD pro-
longation, as outlined in TABLE 3. These disease
phenotypes were corrected by drug treatment with the
L-type calcium channel blocker nifedipine, or the KATP
channel opener pinacidil, therefore providing validation
that engineered isogenic hiPSC lines can be used for dis-
ease modeling and drug testing.
In combination with genome editing, hiPSC technology has
also been utilized in functional genomic screening for iden-
tification of the roles essential genes play in specific cellular
process. For example, Hinson et al. (93) were able to gen-
erate cardiac microtissues using hiPSC-CMs to evaluate the
pathogenic mechanism of truncating titin gene variants
(TTNtvs), which are commonly associated with DCM.
TTNtvs engineered into healthy hiPSCs resulted in micro-
tissues with diminished contractile abilities (e.g., reduced
contractile force) and impaired responses to mechanical
and ␤-adrenergic stress. The investigators also found re-
duced expression of growth factors (e.g., TGF␤1, VEGF,
HGF, EGF, and FGF2), hypoxia regulators (e.g., HIF1A
and EPAS1 or HIF2A), and MAP kinases (e.g., MEK and
ERK). VEGF supplementation of hiPSC-CM culture media
ameliorated these phenotypes. The pathogenic mechanisms
of TTN-induced DCM were identified as the disruption of
critical linkages between sarcomerogenesis and adaptive re-
modeling when TTNtvs are present in the A-band domain
of this protein that is responsible for thick filament-binding.
Another interesting finding of this study was that hiPSC-
CMs carrying engineered A-band TTNtvs showed less se-
vere phenotypes compared with patient-specific hiPSCs car-
rying the same mutations, thereby raising the possibility
that genetic background can influence the functional sever-
ity of TTNtvs. This observation has important functional
implications for precision medicine.
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
D. Future Directions of Genome Editing
Technology
The last decade of research has seen rapid progress in the
field of genome editing. It is clear that off-target effects
exist, and resolving them will be important for future clin-
ical applications of this technology. As research continues
to optimize current protocols, more creative approaches
such as the use of recombinant fusion proteins provide new
opportunities (75, 76, 189). Fusing nuclease DNA-binding
domains to alternate protein complexes can create genome-
editing systems with reporter and therapeutic applications.
Depending on which protein complex is used to replace the
normal endonuclease domain, ZFN and TALEN systems
can transcriptionally upregulate or downregulate genes to
alter entire genetic pathways, remodel chromatin networks,
or even act as reporters for gene activity.
A wide range of successful experiments have validated these
supplementary additions to the genetic toolbox and proto-
cols that are readily available through publicly available
repositories (e.g., Addgene). In the case of both ZFNs and
TALENs, the Folk1 endonuclease domain is replaced by
transcriptional activators or repressor domains. Similar
lists of protocols and gene regulatory activities are possible
for CRISPR applications, where the Cas9 must be rendered
catalytically inactive (CRISPR/Cas9d). This allows other
protein complexes to be fused with the CRISPR/Cas9d,
which is guided to the target sequence via the guide RNA,
and can consequently provide targeted gene inhibition
(CRISPRi) (149), activation (CRISPRa) (77), as well as spa-
tiotemporal (204) or conditional (306) gene regulation with
a higher specificity and reproducibility compared with
other technologies such as RNA interference (RNAi) or
Cre-LoxP systems. Modified CRISPR/Cas9 systems can
also inflict alterations of single or multiple genes at a time.
Notably, some groups have synthesized CRISPR/Cas9 li-
braries consisting of 70,290 guides targeting all human Ref-
Seq coding isoforms (138), demonstrating that this technol-
ogy constitutes a powerful genetic perturbation tool, with
wide applications in precision medicine.
VII. INDUCED MODELS OF CARDIAC
DISEASE
Aside from genetic composition, several environmental
factors (e.g., diet, stress, drugs, or pathogens) as well as
secondary factors that occur due to failure in other or-
gans can lead to cardiac disease, or affect disease progres-
sion and response to pharmacotherapy in heart failure
patients (179) (see TABLE 4). Factoring in the effects of all
these parameters is an important part of precision medi-
cine. Very few studies to date, however, have established
direct mechanistic links regarding the complex effects of
environmental and secondary factors on heart failure.
hiPSC-CMs that lack known familial or engineered
1109
 MATSA ET AL.

Table 4. Induced models of cardiac disease

Reference
Model Class Disorder Stimulus Nos. Key Phenotypes

Induced Drug Myopathy Doxorubicin 21, 96 1Contractile ability

Cardiac troponin T and lactate
dehydrogenase leakage
Pathogen Myocarditis CSV-B3 virus 239 Irregular or ceased beating
Calcium handling
abnormalities
1Cell death
Secondary Hypertension ET-1 2 Investigation of differentially
expressed miRNAs and
mRNAs upon induction of
hypertrophy
T2D Glucose, ET-1, cortisol 61 Myofibrillar disarray
Lipid accumulation
1Oxidative stress
Hypertrophy
1Cytosolic [Ca2⫹]

Together, Tables 3 and 4 provide a detailed outline of hiPSC-based models of cardiac disease published to date,
with correlation to the gDNA mutations studies, and the key phenotypes observed for each disorder. ET-1,
endothelin-1; CSV-B3, Coxsackievirus B3; T2D, type 2 diabetes.

gDNA mutations can be exposed in vitro to a variety of B. Drug Induced

environmental conditions (e.g., ischemic injury, hypoxic
conditions, dietary supplements, electromechanical
stress, and infectious agents) to enable determination of
their effects on CM function. Existing studies of induced
hiPSC-models are discussed below.
A. Pathogen Induced
hiPSC-CMs can provide a suitable platform for the investi-
gation of pathological mechanisms and therapeutic targets
for life-threatening infectious diseases affecting the heart,
such as Chagas cardiomyopathy caused by the T. cruzi try-
panosome (176), or viral myocarditis caused by the B3
strain of coxsackievirus (CVB3) (5). In a recent study,
Sharma et al. (239) demonstrated that hiPSC-CMs lacking
any known mutations associated with cardiac disease can
be infected by CVB3 particles and undergo pathological
phenotypic changes such as increasingly irregular beating
patterns, increase in Ca2⫹ transient duration and time to
transient peak, and eventual beating arrest (239). The au-
thors also showed that these phenotypes were ameliorated
by treatment with antiviral drugs (e.g., interferon-␤1, riba-
virin, pyrrolidine dithiocarbamate, and fluoxetine), which
acted by triggering viral RNA and protein clearance path-
ways and reducing the proliferation capacity of the CVB3
virus. This study demonstrated that hiPSC-CMs can be used
to predict antiviral drug efficacy and serve as a sensitive
platform to screen novel antiviral therapeutics for their ef-
fectiveness in a high-throughput fashion.
Pluripotent stem cell-derived CMs have been shown to re-
spond appropriately to a wide variety of drug classes, in-
cluding ␤-adrenergic receptor agonists and class I, II, III,
and IV anti-arrhythmic agents (186, 314). As discussed in
this review, clinically relevant and experimental drugs have
also been used in vitro for the recovery of diseased pheno-
types in models of cardiovascular disease. However, only a
few studies have investigated the effects of known cardio-
toxic drugs in a pluripotent stem cell-based platform that
lacks known mutations. One example was given by Hol-
mgren et al. (96), who found severe in vitro abnormalities
following a 2-day treatment with varying doses of doxoru-
bicin, a chemotherapeutic agent indicated for the treatment
of a variety of cancer types (e.g., leukemia, lymphomas, and
solid tumors) that is also associated with severe cardiotox-
icity, especially in children and adolescent patients (36).
CMs treated with the drug exhibited concentration-depen-
dent alterations in cellular morphology, reduced contractile
ability, lactate dehydrogenase leakage, cardiac troponin T
release, and increased apoptosis. Transcriptome profiling
performed by RNA-seq revealed that differentially ex-
pressed genes due to doxorubicin exposure were associated
with cellular defense mechanisms such as p53 signaling
(e.g., TP53I3), DNA damage response (e.g., GADD45A),
and cellular senescence and response to stress (e.g.,
HIST1H4H). These genes could, therefore, serve as sensi-
tive novel biomarkers for doxorubicin-induced toxicity in
human CMs. In a separate study, Burridge et al. (21) dem-
onstrated that doxorubicin treatment caused higher suscep-
tibility to cell death, metabolic dysfunction, and calcium

1110 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 HUMAN INDUCED PLURIPOTENT STEM CELLS
handling abnormalities in hiPSC-CMs from breast cancer
patients who experienced doxorubicin-induced cardiotox-
icity (DIC) in the clinic compared with hiPSC-CMs from
breast cancer patients who had not experienced DIC. These
data support that hiPSC-CMs are a suitable platform for
identifying patient-specific predilection to drug-induced
cardiotoxicity.
C. Secondary Cardiomyopathy
Cardiomyopathy arising as a secondary feature to medical
conditions initiated at a different organ has also been the
subject of hiPSC-based studies. For example, induced car-
diac hypertrophy was developed in hiPSC-CMs following
5-day exposure to endothelin 1 (ET-1), a potent vasocon-
strictor peptide secreted by endothelial cells which leads to
pulmonary hypertension (39) and also has known implica-
tions in biochemical and structural remodeling of the heart
(30). Mechanistic investigation performed by high-
throughput transcriptome analysis identified a pathological
increase in the expression of fetal genes (e.g., ACTA1,
NPPA, and NPPB). Pathological upregulation of NPPB
was also confirmed at the protein level, and served as the
basis for development of a 384-well HCI phenotypic screen-
ing tool for the assessment of compounds suspected to tar-
get the NPPB pathway, and therefore modulate the hyper-
trophic response. Several compounds, including the Ca2⫹
channel blocker verapamil, were shown to reverse the ef-
fects of cellular hypertrophy induced by ET-1. Other bene-
ficial compounds included BEZ-235 (a dual PI3K-mTOR
inhibitor), fenofibrate (a PPAR␣ activator), and SAHA (a
broad-spectrum histone deacetylase inhibitor).
In a separate study, microRNA (miRNA) sequencing
showed that ET-1 treatment in hiPSC-CMs leads to differ-
ential expression of 250 known (e.g., miR-23a-3p and miR-
208a-3p) and 34 novel miRNAs, the majority of which
were associated with corresponding mRNA expression
changes (2). Some of the complex RNA regulatory mecha-
nisms associated with cardiac hypertrophy were involved in
cardiovascular development (e.g., MEF2C), cell prolifera-
tion and transformation (e.g., MYC, FOS, TGFB2, and
TGFBR3), and mitogen-activated protein kinase signal
transduction (e.g., MAP3K9 and MAP2K6). Differentially
expressed miRNA-mRNA pairs were consistent with sev-
eral animal models of cardiac hypertrophy (28, 102). In
addition, bioinformatics principal component analysis
(PCA) comparing in vitro hiPSC-based findings to human
myocardial biopsies from unrelated patients with left ven-
tricular hypertrophy detected overlapping expression
changes between the two groups.
Induced type II diabetic cardiomyopathy was also modeled
in hiPSC-CMs following prolonged exposure to “diabetic
milieu” consisting of a combination of glucose, ET-1, and
cortisol (61). Gene set enrichment analysis (GSEA) using
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
RNA-sequencing gene expression data showed significant
upregulation in metabolic genes involved in the tricarbox-
ylic acid (TCA) cycle (e.g., PDK1), mitochondrial electron
transport chain (e.g., CYCS), glucose metabolism (e.g.,
ENO2), cell adhesion (e.g., integrins), and extracellular
matrix deposition (e.g., collagens), whereas downregula-
tion was observed in genes related to protein synthesis and
the cellular response to dysfunctional protein production
(e.g., ATF4 and CHOP10). This secondary cardiomyopa-
thy model was consistent with one generated using hiPSC-
CMs from patients with type II diabetes (T2D), therefore
lending support to the hypothesis that clinical chemistry can
induce a phenotypic surrogate of diabetic cardiomyopathy.
The use of NPPB-based high-throughput phenotypic assays
enabled the investigation of therapeutic targets by screening
a library of 480 compounds. Beneficial molecules included
regulators of Ca2⫹ homeostasis, such as inhibitors of volt-
age-gated Ca2⫹ channels (e.g., fluspirilene), molecules that
deplete intracellular Ca2⫹ stores (e.g., thapsigargin), and
inhibitors of Ca2⫹-regulated proteins such as calmodulin
(e.g., W7). The use of Na⫹ and K⫹ channel blockers (e.g.,
phenamil and penitrem A), which reduced the frequency of
CM Ca2⫹ transients, also showed a therapeutic benefit.
Overall, by combining the hiPSC technology with high-
throughput screening assays, these studies demonstrate a
close correlation between hiPSC-CMs and human heart or
animal models, provide mechanistic insight into the patho-
logical gene regulatory pathways of HCM and T2D devel-
opment, and demonstrate nicely that hiPSCs are valuable
platforms in predictive toxicology and safety pharmacology
applications that can benefit the early phases of drug dis-
covery and development.
VIII. PATIENT RECRUITMENT VERSUS
GENE EDITING
Recent improvements in gene editing may supplement the
traditional system of patient recruitment that identifies and
compares a group of diseased individuals to a control group
that lacks disease phenotypes (FIGURE 2). This cause-and-
effect based discovery is hampered by multiple issues, such
as the possibility that phenotypic differences between pa-
tient and control groups are not due to disease-causing mu-
tations, but arise due to other differences in genetic back-
ground (TABLE 5). To reduce the genetic background vari-
ability between patient and control cells, hiPSC lines
generated from family members of the patient unaffected by
the disease have been used (148, 184, 254). However, this
approach can limit, but not abolish, genetic background
variability, given that even siblings or parent-child pairs
only have 50% genetic overlap. Gene editing techniques
circumvent this limitation, but present other challenges
such as off-target effects and clonal heterogeneity (180),
which need to be sufficiently addressed to allow confirma-
tion of casual relationships between the underlying genetics
1111
 MATSA ET AL.

Table 5. Comparison of hiPSC-based disease modeling approaches

Innate Engineered Induced

⫹ Disease modeled against patients’ genetic
background
⫺ Patient recruitment difficult
Nonisogenic disease and control lines
2Need for patient recruitment Enables modeling of noncongenital
conditions
Isogenic disease and control lines
Time consuming Induced conditions may not accurately
recapitulate disease
Costly
Potential risk for genomic instability

The advantages (⫹) and disadvantages (⫺) of innate, induced, and engineered hiPSC-based disease models. 2,
Reduced.

and disease phenotypes. To limit the effects of clonal vari-
ation, some studies have attempted to generate iPSCs in
bulk culture (55, 298). More recently, it has also been
shown that underlying genetic background variation is re-
sponsible for most heterogeneity between hiPSC lines, while
cell type of origin and clonality have minor effects on hiPSC
gene expression and methylation profiles (27, 231, 232a).
Another problem that restricts the paradigm of patient recruit-
ment involves patient and family consent. Not only must a
patient fit a correct disease profile, but the individual and their
family must consent to donate human tissue. As a way to
circumvent these problems, ZFNs, TALENs, and CRISPR/
Cas9 genome editing tools may be applied to create control
groups from diseased cell lines, and vice versa. This reduces the
effort needed to recruit additional patients. Nevertheless, even
this approach does not account for the possibility that multiple
polymorphisms may contribute towards a disease phenotype,
or address the fact that some disorders and clinical abnormal-
ities are not yet correlated to any known mutations.
drugs. Human genetic variations can result from SNPs, in-
dels, or duplication of gDNA sequences. SNPs are by far the
most common type of variation, with more than 14 million
SNPs identified in the human genome (173). Over 60,000 of
these SNPs are located within gene coding regions, and it esti-
mated that more than 90% of human genes contain at least
one SNP. Genetic polymorphisms of proteins involved in
pharmacodynamics and pharmacokinetics are known to be
important determinants of individual variability in drug effi-
cacy. Over 30 drugs are currently prescribed only under ful-
fillment of a genetic testing prerequisite due to their associa-
tion with deleterious gDNA polymorphisms. Such informa-
tion is shared through large publicly available (e.g.,
PharmGKB) or private (e.g., PGMD) databases, with the aim
of contributing towards more effective, and safer medications
and doses that will be tailored to a person’s genetic make-up.
A. Examples of Known Polymorphisms
Implicated in Drug-Induced Toxicity

It is clear that there are roles for both patient recruitment
and genome editing, and it is becoming the general consen-
sus that both approaches should complement each other to
establish large biorepository banks that can serve as imme-
diate resources for the academic and pharmaceutical com-
munities (299).
IX. PHARMACOGENOMICS
When a patient is administered a new drug, three outcomes
are possible: 1) the patient experiences a beneficial effect in
relation to the condition they are being treated for, 2) the
patient’s condition does not improve, or 3) the patient ex-
periences adverse cytotoxic effects or even death. In many
cases, the same medication can elicit any of these three
responses in different patients, and our understanding of
the parameters that influence this outcome is greatly lack-
ing. The field of pharmacogenomics aims to bridge this gap,
by combining pharmacology and genomics to determine
how individuals’ genomic variation can affect responses to
One example of a pharmacogenomic study focused on the
therapeutic efficacy of simvastatin, a HMG CoA reductase
inhibitor with cholesterol-lowering abilities that is used to
lower the risk of stroke, heart attack, and other heart com-
plications in people with diabetes or coronary heart disease.
This study involved a cohort of 156 human subjects with
low-density lipoprotein (LDL) cholesterol levels of 160 mg/
dl, who were treated with simvastatin at varying doses (40,
80, and 160 mg/day) over the period of 6 weeks. Simvasta-
tin treatment led to reduction of LDL cholesterol by 41, 47,
and 53%, respectively, therefore providing strong evidence
that this drug has a high therapeutic value in reducing LDL
cholesterol (49). However, a small subset of the cohort (⬃5%)
showed little to no reduction in LDL cholesterol levels, even at
160 mg/day. Furthermore, 0.7 and 2.1% of patients devel-
oped liver toxicity, as measured by elevated activity of alanine
aminotransferase in the plasma, at the 80 and 160 mg/day
doses. Importantly, one subject developed myopathy (0.7%)
at the 160 mg/day dose. This variation in clinical outcome was
later linked with genetic polymorphisms in the HMG CoA
reductase gene HMGCR (e.g., p.A12T and p.T29G), as well

1112 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 HUMAN INDUCED PLURIPOTENT STEM CELLS
as in the solute carrier drug transporter SLCO1B1 (e.g.,
p.V174A), and the ATP-binding cassette transporter ABCG2
(e.g., c.421C⬎A) genes that have varying frequencies among
different ethnic groups. The latter genes are known to regulate
efflux of statins and statin metabolites and, therefore, contrib-
ute to the variability in efficacy and side effects of cholesterol-
lowering drugs such as simvastatin, pravastatin, or rosuvasta-
tin (35, 200, 273).
Another example is concerned with drug-induced LQT syn-
drome. In a cohort of 98 patients with drug-induced LQTS,
the KCNE2 variant c.T8A, which is found in ⬃1.6% of the
general population, was linked to adverse QT interval prolon-
gation following administration of trimethoprim/sulfame-
thoxazole for the treatment of urinary tract infections (237).
Screening for KCNE2-blocking activity is now routinely per-
formed during the development process of any new drug to
predict any risk for drug-induced QT prolongation that could
lead to sudden death in humans. However, testing is typically
performed in transgenic cell lines overexpressing KCNE2,
which lack the functional complexity of human CMs such as
the interaction between opposing ion currents, as well as the
ability to reflect deleterious effects associated with SNPs and
other types of individualized gDNA variation.
Patient-specific gDNA variation has also been shown to
affect cardiotoxicity induced by the drug doxorubicin, an
anthracycline antibiotic with broad antineoplastic activity
which is currently one of the most common and most effec-
tive chemotherapy agents (265). Despite its anti-cancer ef-
ficacy, doxorubicin treatment is associated with fatal heart
failure in 5–50% of patients (depending on administered
dose) that was not predicted in preclinical drug-testing
stages (113). Increased susceptibility to cardiotoxicity has
been linked to polymorphisms in genes related to mitochon-
drial function (e.g., NADPH oxidase subunits) (283, 300).
Specific examples include the 212A⬎G variant of the gene
encoding the NCF4 subunit of NADPH oxidase (230), and
the rs17863783 variant in the UDP glucuronosyltransferase
UGT1A6 (284). Interestingly, some SNPs found in efflux
transporters (e.g., ABCB1/MDR1, ABCC2, ABCC3,
ABCG2, and RALBP1) improve doxorubicin pharmacoki-
netics/clearance and have been shown to be cardioprotec-
tive against this drug (271). Further study regarding the
cardiotoxic or cardioprotective effects of such variants in
patient-specific or engineered hiPSC models could lead to a
deeper understanding of the genetic determinants of chemo-
therapeutic cardiotoxicity.
B. Use of hiPSCs in Pharmacogenomic
Investigations
Preclinical drug testing in hiPSC-CMs can circumvent the
limitations of drug testing in transgenic lines (196). Al-
ready, hiPSC platforms have been utilized to highlight
differences in drug response underlined by genomic vari-
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
ation. In a study that generated a library of hiPSC-CMs
from healthy patients as well as patients carrying muta-
tions related to LQT syndrome, DCM, or HCM, it was
found that LQT and HCM hiPSC-CMs were more sus-
ceptible to development of cardiotoxicity (e.g., EADs and
DADs) compared with DCM and control hiPSC-CMs
when treated with cisapride (161), a gastroprokinetic
agent that was voluntarily withdrawn from the United
States market in 2000 due to arrhythmic and sudden
cardiac death episodes in patients with preexisting heart
conditions such as LQT syndrome and heart failure
(223). In a separate study, Braam et al. (17) observed that
two potent blockers of the IKs current (HMR1556 and
JNJ303) were cardiotoxic in LQT hiPSC-CMs, but not in
controls. These studies have important clinical implica-
tions showing that genetic predisposition to toxicity is
reflected in disease-specific hiPSC-CMs and may predict
adverse drug responses more accurately than tests in
transgenic lines overexpressing a single ion channel or
even tests using a single control hiPSC line.
X. POTENTIAL APPLICATIONS OF hiPSCs
IN PRECISION MEDICINE
A. Safety and Efficacy Assessment of Novel
Compounds in Preclinical Trials
Despite strict guidelines by regulatory agencies, several
drugs continue to present adverse side effects subsequent
to their release to the clinic, with deleterious cardiovas-
cular events (e.g., torsades de pointes, ventricular tachy-
cardia, ventricular fibrillation, and sudden cardiac ar-
rest) being implicated in 28% of drug withdrawals (16,
86). For example, milrinone, a phosphodiesterase-4
(PDE4) inhibitor, is an inotropic drug thought to have
great clinical promise against heart failure, but was
found during clinical trials to be associated with increase
in mortality and lack of efficacy due to calcium overload
and DADs in some patients (69). Cardiotoxicity has been
observed not only with drugs targeted to act on the heart,
but also with drugs aimed at other organs. For example,
anthracycline chemotherapy agents (e.g., doxorubicin,
daunorubicin, idarubicin, and epirubicin) have been fre-
quently linked to cardiac complications such as arrhyth-
mias, or dilated cardiomyopathy which develop either
acutely or chronically (216, 285).
It is possible that unanticipated drug-induced cardiotoxic-
ity may be in part attributed to the lack of appropriate
preclinical screening platforms. Current drug testing is reli-
ant on preclinical evaluation using human or animal trans-
genic cell lines [e.g., Chinese hamster ovary (CHO) cells and
human embryonic kidney (HEK) cells overexpressing the
human ether-à-go-go-related gene (hERG)], followed by in
vivo evaluation in small and large animal models (99, 232)
1113
 MATSA ET AL.

(FIGURE 3). Despite having several advantages, including
ease of maintenance and simplicity in developing assay for
transgenic lines and the ability to study systemic effects for
animal models, these platforms also have several draw-
backs. Transgenic lines are only engineered to overexpress
certain human proteins (e.g., the IKr protein hERG) and
lack the structural and functional complexity of relevant
human cell types (218). For example, transgenic lines over-
expressing a single ion channel failed to predict the false-
positive outcome related to the drug verapamil, because
while it blocks outward ion flow through the rapid delayed
rectifier current, IKr, it also blocks inward ion flow through
the L-type calcium channel ICa-L, therefore producing a neu-
tral outcome in functional CMs (191). Notably, Navarette
et al. (203) showed no cardiotoxicity in response to vera-
pamil in hiPSC-CMs. In addition, these transgenic immor-
talized lines accumulate several karyotype aneuploidies that
may alter their response to drugs (253, 256). As even SNP
changes are known to contribute to variability in drug re-
sponse (253), aneuploidies causing whole gene duplications
or deletions as seen in transgenic lines may significantly
alter recorded phenotypes.
In vivo testing in small and large animal models is also
limited by significant interspecies variation between ani-
mals and humans. For example, mice have a heart beat rate
that is eight times faster than that of humans (500 vs. 60
beats/min), and cardiac repolarization in murine CMs relies
primarily on Ito, IK,slow1, IK,slow2, and ISS ion currents,
whereas in humans, repolarization is mostly dependent on
the potassium channels IKs and IKr (226). Calcium handling
(e.g., phospholamban; PLN), myofilament (e.g., MYH6/
MYH7), and surface (e.g., SIRPA) proteins also have differ-
ent expression patterns and pathophysiological roles in
mouse versus human hearts. As an example, while trunca-
tion mutations have been shown to result in considerably
reduced myocardial PLN protein content and loss of PLN
inhibition of SERCA2a, leading to development of heart
failure and early mortality in homozygous humans, in mice
PLN deficiency enhances myocardial inotropy and lusi-
tropy without adverse effects (87). Large animals such as
rats, pigs, sheep, or dogs have hearts with closer cardiac
electromechanical properties to those of humans, but are
more difficult to handle, and more expensive to maintain in
animal facilities. In addition, they are known to have differ-
ent drug dose sensitivity compared with humans. For exam-
ple, relative to humans, canines can tolerate up to 100-fold
higher concentrations of chemotherapy agents such as thio-
TEPA, myleran, actinomycin D, mitomycin C, mithramy-
cin, and fludarabine (220). It is estimated that animal mod-

Predicted Paradigm Shift in Clinical Trial Model

Transgenic lines
Traditional model

Costly
Slow
Small animals
Low predictive
value
Large animals

Selection of therapeutic
Pre-clinical testing Clinical testing
molecule
New model

Fast

Increased
confidence prior to
hiPSC-CMs from patients with diverse costly clinical trials
ethnicity, gender, cardiovascular history

FIGURE 3. Use of hiPSCs to transform the current clinical trial model. Of great importance is the application
of hiPSCs in drug discovery and toxicology testing as a part of clinical trials. In the current drug development
model, novel compounds are initially tested on human or animal transgenic lines (e.g., HEK and CHO) which not
only lack the complexity of functionally relevant human cell types, but also carry several aneuploidies that may
affect their response to drugs. This often results in false-positive or false-negative data. In vivo testing in small,
and later on, large animal models is also an FDA requirement prior to approval of novel compounds for human
clinical testing. Although this allows for systemic characterization of drug function, the vast species differences
between animal models and humans often lead to the release of cytotoxic compounds to the clinic. Introduction
of hiPSCs to the current drug development model will allow testing of novel compounds on functionally relevant
human cells derived from individuals with diverse ethnicities, gender, and disease history (e.g., cardiovascular
disease). Preclinical drug testing against a diverse human genetic background may prevent the costly release
of cytotoxic drugs to the clinic, and will enable informed decisions about drug prescriptions based on gender,
ethnic group, disease status, and other relevant categories.

1114 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 HUMAN INDUCED PLURIPOTENT STEM CELLS

els have a combined predictability of human behavior of
only 10% (185, 238).
In contrast to transgenic and animal platforms, in vitro
derived hiPSC-CMs display many of the complex charac-
teristics associated with in vivo development of the heart,
including gene and protein expression, ion channel forma-
tion, electrophysiological responsiveness, calcium han-
dling, and excitation-contraction coupling (320). Although
their current relatively early maturation state warrants ad-
ditional attempts to drive further maturation, hiPSC-CMs
provide an excellent platform to further improve preclinical
drug testing (FIGURE 3). Beyond their properties as a func-
tionally relevant human platform for preclinical drug test-
ing, the introduction of hiPSCs to the current drug develop-
ment model may allow testing of novel compounds on hu-
man cells which will be derived from individuals with
diverse ethnicities, gender, and disease history (e.g., cardio-
vascular disease). This will take into account the expanding
role of pharmacogenomics in the management of cardiovas-
cular disorders (313). Preclinical drug testing against a di-
verse human genetic background has the potential to refine
drug response-to-genotype correlations, prevent the release
of cytotoxic chemicals to the clinic, and enable informed
decisions about drug prescriptions based on gender, ethnic
group, disease status, etc. A growing body of evidence in-
dicates that hiPSC-CMs can offer the pharmaceutical indus-
try a powerful, human, and physiologically relevant tool for
validating new drug candidates in early drug development
stages (65).
B. Clinical Management
Another goal of precision medicine is to enable physicians
to prescribe appropriate medications at the right dose and
schedule for individual patients to achieve maximal thera-
peutic benefit with minimal, tolerable adverse effects (133).
In current clinical protocols, clinicians typically rely on the
patient’s history and test indications to estimate the best
pharmacotherapy, which can be conditional upon careful
subsequent monitoring of patients to assess the effective-
ness and toxicity of the drug. Often, the clinician may pre-
scribe several different drugs to identify the optimum treat-
ment for each patient (FIGURE 4). During this lengthy pro-
cess, patients may experience unwanted side effects, and
their condition may deteriorate. Furthermore, side effects
may be induced by polypharmacy (i.e., the use of multiple
medications by a patient). Avoidable adverse drug events
are among the most serious consequences of inappropriate
drug prescribing (219). However, effective protocols for
predicting such events are lacking. A hiPSC-based model for

Predicted Paradigm Shift in Drug Treatment

Patient history
Traditional model

Slow
Clinical examinations
Ineffective

Serial testing of multiple drugs

Diseased patient Investigation of possible therapies Selection of best drug

Patient-specific
New model

hiPSC-CMs Fast

Selection of
optimum drug
treatment
Drug screen

FIGURE 4. hiPSC-based personalized medicine as a novel model for clinical pharmacotherapy. The hiPSC field
is growing at an unparalleled pace, with the promise that these cells will become major tools in the advance-
ment of personalized medicine. When treating a patient, clinicians nowadays typically rely on the patient’s
history and clinical test indications to choose the presumed best pharmacotherapy. Since there is limited
predictive confidence in these indications, the clinician may need to prescribe several different drugs before
they can identify one that best suits each patient. During this sometimes lengthy process, the patient may
experience unwanted and potentially serious side effects, and their condition may deteriorate. In contrast, a
hiPSC-based model for personalized medicine may allow safe and patient-specific testing of several drugs on
hiPSC-differentiated derivatives (e.g., cardiomyocytes), and a pharmacogenomics analysis using hiPSC-derived
cells may enable precise prediction of the patient’s response to each drug. This paradigm-shifting model of
clinical treatment could vastly increase our confidence in the prescription of appropriate medication, saving
patients from dangerous side effects or toxicities, and optimally addressing their disease symptoms.

Physiol Rev • VOL 96 • JULY 2016 • www.prv.org 1115

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 MATSA ET AL.

precision medicine has the potential to provide patient-spe-
cific information about drug response by taking into ac-
count individualized information such as genomic, tran-
scriptomic, metabolomic, pharmacokinetic, and pharmaco-
dynamic profiles that are reflected in hiPSCs. This may
allow safe and patient-specific laboratory-based testing of
several drugs on hiPSC differentiated derivatives (e.g.,
CMs) to enable precise prediction of the patient’s response
to a drug or to polypharmacy, as well as to guide the dosing
and frequency of drug administration. Overall, this model
of clinical treatment could provide increased confidence in
the prescription of appropriate medication in the future,
which could protect patients from side effects or toxicities,
and optimally address their disease symptoms.
In a hiPSC-based study in which the proband had a de novo
SCN5A LQT3-associated mutation (F1473C) and a poly-
morphism (K897T) in KCNH2, the gene associated with
LQT2 syndrome, biophysical and molecular analysis of ion
channels expressed in hiPSC-CMs demonstrated that phe-
notypic abnormalities (e.g., arrhythmias) observed in vitro
were attributed to the SCN5A mutation, whereas the
KCNH2 polymorphism was not a contributing factor to
disease severity (264). The abnormally augmented INaL cur-
rent was corrected either by faster pacing of hiPSC-CMs or
treatment with the Na⫹ channel blocker mexiletine. There-
fore, in vitro testing in hiPSCs was able to provide func-
tional information as to the mechanisms of disease, indicat-
ing that Na⫹ channel blockers, rather than K⫹ channel
activators, would serve as a better pharmacological treat-
ment for this specific patient.
Similar principles can be applied to disease prognosis by
utilizing patient-specific hiPSC derivatives to enable exten-
sive phenotypic screening and early prediction of disease
outcome and severity (FIGURE 5). Whereas current clinical
protocols for prognosis rely on family history and long-
term monitoring, hiPSCs may provide a fast track to diag-
nosis in certain clinical scenarios, thus enabling implemen-
tation of prevention or early treatment plans that lead to the
best outcome.
One limitation towards clinical applications of hiPSCs is the
time required (⬃3 months) to reprogram somatic cells to
hiPSCs and subsequently differentiate them to functional
cell types, such as CMs. Optimization of differentiation
protocols has already shortened the time needed for hiPSC
differentiation to CMs from ⬃18 –25 to ⬃9 –11 days (24).
As further improvements in this field are under develop-
ment, large-scale bio-repositories of both hiPSCs and their
differentiated derivatives provide an optimal way to reduce
the time required to obtain patient-specific cells for func-
tional assays. It is also possible that hiPSC derivation and
bio-banking could become routine, to enable population-
wide individualized medicine in the future.
XI. CONCLUSIONS AND FUTURE
PERSPECTIVES
Precision medicine approaches in which the combination of
any given patient’s unique clinical, genomic, and in vitro
cellular characteristics can fine-tune decisions regarding the

Predicted Paradigm Shift in Disease Diagnosis

Family history
Traditional model

? Slow

? Ineffective

Health monitoring

Familial disease Investigation of possible diagnosis Disease outcome

Fast
Patient-specific
New model

hiPSC-CMs

Predict disease
development &
Phenotypic screen progression

FIGURE 5. hiPSC-based personalized diagnostics for disease prediction. Familial disease often has a large
social impact on family life. In the absence of a known disease-associated mutation, clinicians adopt a disease
prediction strategy for young offspring that involves long-term monitoring of biomarkers and heart function.
However, this strategy is time-consuming and delays implementation of prevention or early treatment plans.
Generation of hiPSC-CMs from family members may enable extensive phenotypic screening and early prediction
of disease severity and outcome, to improve patient care.

1116 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org

Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
 HUMAN INDUCED PLURIPOTENT STEM CELLS
diagnosis, treatment, and prevention of human disease are
starting to take shape thanks to in vitro hiPSC-CM models.
To facilitate this field and to promote more efficient devel-
opment of novel therapies, biobanking of hiPSCs has
bloomed over the past 5 years, with participation of several
academic groups (e.g., Stanford Cardiovascular Institute)
and commercial institutions (e.g., Coriell Institute, Cellular
Dynamics International, Stem Cell Theranostics, Pfizer, and
Roslin Cells) which are typically funded in partnership with
government entities (e.g., NIH, CIRM, etc.). The Interna-
tional Stem Cell Banking Initiative (ISCBI) alongside the
International Stem Cell Initiative (ISCI) have also coordi-
nated teams of stem cell biologists, bio-banking centers,
stem cell society representatives (e.g., ISSCR, ISCT), eth-
ics committees (e.g., ISCF Ethics Working Party), and
regulators from over 20 countries to standardize the der-
ivation, culture, and characterization methods of hiPSCs
(7). These large multinational efforts have one goal in
common: to expedite the clinical uses promised by the
hiPSC technology.
While hiPSCs avoid the main controversies associated with
hESCs (i.e., the destruction of human embryos during hESC
derivation), new ethical concerns have not only clouded the
use of hiPSCs, but also the generation and privatization of
patient-specific “omic” data. For example, when the com-
pany 23andMe initiated sales of a saliva-based Personal
Genome Service (PGS) that provides genotyping reports on
254 diseases and conditions without prior marketing clear-
ance or approval from the Food and Drug Administration,
this was considered a risk. The concerns were not only that
this kit should have been regulated and marketed as a med-
ical device, but also that patients were receiving complex
information about their possible predisposition to diseases
without appropriate genetic counseling, and without con-
sideration for false-positive or false-negative outcomes
(11). This and other examples illustrate the need to set
ethical boundaries and rules for generation and distribution
of patient-specific NGS data related to hiPSC generation
and analysis (43). Careful regulation of genome editing
technologies will also be necessary to prevent eugenic mis-
use (140).
In addition to ethical issues, technical challenges also need
to be addressed for each technology. For the hiPSC technol-
ogy, one major concern is the immature state of differenti-
ated derivatives. This topic has been extensively discussed
elsewhere (128, 309), and several physical, chemical, and
electrical signals are under investigation to enable these cells
to mature to a state more closely resembling the adult hu-
man heart (124, 208, 266, 276, 309). Subtype specification
of CMs (i.e., into pacemaker, atrial, or ventricular cells of
either the right or left side of the heart) is also under inves-
tigation. Although a few protocols claim to enable enhance-
ment of the specific generation of ventricular (129, 296),
atrial (53, 119, 263), and pacemaker (127, 326) CM sub-
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
types, thus far none has become standard practice. Improv-
ing our ability to generate specific CM subtypes from hiP-
SCs will also enable more precise modeling of diseases that
preferentially affect either the ventricles (e.g., HCM, DCM,
ARVD), or atria (e.g., atrial arrhythmias), in addition to
opening new prospects for creating patient-specific bioen-
gineered pacemakers.
Significant limitations for the genome editing technology
include not only the length and cost of the process, but more
importantly, its efficiency, and off-target effects, as dis-
cussed extensively in previous sections. In addition, the
NGS field needs to address the issue of storage and manage-
ment of the vast datasets generated, as well as to develop
more effective methods of data analysis (288). Sequencing
throughputs have rapidly outpaced even Moore’s Law for
computing power, which states that the number of transis-
tors in a dense integrated circuit doubles approximately
every two years (195). Improvement of data compression
algorithms would help to address the growing data-to-cen-
tral processing unit (CPU) ratio (287). To date, thousands
of WG and hundreds of thousands of exomes have been
sequenced. However, meaningful biomedical contribution
of these data has been greatly outpaced by the rate at which
new samples are sequenced (156). Therefore, an additional
challenge for the NGS technology is to accelerate the rate of
data analysis. Efficient data analysis would lead to the iden-
tification of new disease-associated mutations, epigenetic
modifications, or gene expression pathways which could in
turn generate innovative therapeutic targets, improve the
predictive abilities of genetic testing, and ultimately trans-
late into public health benefits. In the future, clinical and
hiPSC-based sequencing of patients suffering from disease
is expected to guide diagnosis and treatment decisions. The
precision medicine initiative is here to ensure that outstand-
ing issues associated with the technologies involved are suf-
ficiently addressed so that these tools can serve to benefit
patients. Precision health represents a revolution in patient
care, by moving beyond incremental improvements in diag-
nosis and treatments for acute or chronic disease to achieve
fundamental understanding of underlying disease causes
and treating individuals based on their unique genetic com-
position (45). By combining the strengths of basic scientific
research, biomedical data science, genome editing, and
transformative biomedical platforms such as hiPSCs (299),
we can truly begin to understand individual variability in
disease development, progression, and response to therapy.
Although precision health requires big thinking and bold
action, its promise of a revolution in healthcare away from
after-the-fact diagnosis, towards prediction and prevention,
is approaching fast.
ACKNOWLEDGMENTS
We thank Dr. Joseph Gold and Blake Wu for critical read-
ing of the manuscript. Due to space limitations, we are
unable to include all of the important papers relevant to the
1117
 MATSA ET AL.
use of hiPSCs in cardiovascular research; we apologize to
the investigators who have made significant contributions
to this field.
Address for reprint requests and other correspondence: J. C.
Wu, 265 Campus Dr., Rm. G1120B, Stanford, CA 94305-
5454 (e-mail: joewu@stanford.edu) or E. Matsa (e-mail:
ematsa@stanford.edu).
GRANTS
This work was supported by the American Heart Associa-
tion; Burroughs Wellcome Foundation; National Institutes
of Health (NIH) Grants R01 HL123968, R01 HL126527,
R01 HL128170, and R01 HL130020 (to J. C. Wu); NIH
Progenitor Cell Biology Jump Start Award, Stanford Car-
diovascular Institute Seed Grant, and American Heart As-
sociation Beginning Grant in Aid (to E. Matsa).
DISCLOSURES
J. Wu is a co-founder and Scientific Advisory Board member
for Stem Cell Theranostics.
REFERENCES
1. Abi Khalil C. The emerging role of epigenetics in cardiovascular disease. Ther Adv
Chronic Dis 5: 178 –187, 2014.
2. Aggarwal P, Turner A, Matter A, Kattman SJ, Stoddard A, Lorier R, Swanson BJ, Arnett
DK, Broeckel U. RNA expression profiling of human ipsc-derived cardiomyocytes in
a cardiac hypertrophy model. PLoS One 9: e108051, 2014.
3. Alex Buerkle C, Gompert Z. Population genomics based on low coverage sequencing:
How low should we go? Mol Ecol 22: 3028 –3035, 2013.
4. Amit M, Winkler ME, Menke S, Bruning E, Buscher K, Denner J, Haverich A, Itskovitz-
Eldor J, Martin U. No evidence for infection of human embryonic stem cells by feeder
cell-derived murine leukemia viruses. Stem Cells 23: 761–771, 2005.
5. Andreoletti L, Leveque N, Boulagnon C, Brasselet C, Fornes P. Viral causes of human
myocarditis. Arch Cardiovasc Dis 102: 559 –568, 2009.
6. Ang SL, Constam DB. A gene network establishing polarity in the early mouse em-
bryo. Semin Cell Dev Biol 15: 555–561, 2004.
7. Aoi T, Stacey G. Impact of national and international stem cell banking initiatives on
progress in the field of cell therapy: Iabs-jst joint workshop: summary for session 5.
Biologicals 43: 399 – 401, 2015.
8. Applegarth DA, Toone JR, Lowry RB. Incidence of inborn errors of metabolism in
British Columbia, 1969 –1996. Pediatrics 105: e10, 2000.
9. Baghbaderani BA, Tian X, Neo BH, Burkall A, Dimezzo T, Sierra G, Zeng X, Warren
K, Kovarcik DP, Fellner T, Rao MS. cGMP-manufactured human induced pluripotent
stem cells are available for pre-clinical and clinical applications. Stem Cell Reports 5:
647– 659, 2015.
10. Ban H, Nishishita N, Fusaki N, Tabata T, Saeki K, Shikamura M, Takada N, Inoue M,
Hasegawa M, Kawamata S, Nishikawa S. Efficient generation of transgene-free human
induced pluripotent stem cells (ipscs) by temperature-sensitive sendai virus vectors.
Proc Natl Acad Sci USA 108: 14234 –14239, 2011.
11. Barlas S. Precision medicine initiative aims for a new generation of diagnostics and
treatments: but is the promise of genetic targeting overinflated? PT 40: 340 –352,
2015.
12. Bayart E, Cohen-Haguenauer O. Technological overview of ips induction from human
adult somatic cells. Curr Gene Ther 13: 73–92, 2013.
1118 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
13. Bellin M, Casini S, Davis RP, D’Aniello C, Haas J, Ward-van Oostwaard D, Tertoolen
LG, Jung CB, Elliott DA, Welling A, Laugwitz KL, Moretti A, Mummery CL. Isogenic
human pluripotent stem cell pairs reveal the role of a kcnh2 mutation in long-qt
syndrome. EMBO J 32: 3161–3175, 2013.
14. Bilic J, Izpisua Belmonte JC. Concise review: induced pluripotent stem cells versus
embryonic stem cells: Close enough or yet too far apart? Stem Cells 30: 33– 41, 2012.
15. Bondue A, Lapouge G, Paulissen C, Semeraro C, Iacovino M, Kyba M, Blanpain C.
Mesp1 acts as a master regulator of multipotent cardiovascular progenitor specifica-
tion. Cell Stem Cell 3: 69 – 84, 2008.
16. Braam SR, Passier R, Mummery CL. Cardiomyocytes from human pluripotent stem
cells in regenerative medicine and drug discovery. Trends Pharmacol Sci 30: 536 –545,
2009.
17. Braam SR, Tertoolen L, Casini S, Matsa E, Lu HR, Teisman A, Passier R, Denning C,
Gallacher DJ, Towart R, Mummery CL. Repolarization reserve determines drug re-
sponses in human pluripotent stem cell derived cardiomyocytes. Stem Cell Res 10:
48 –56, 2013.
18. Bruneau BG, Nemer G, Schmitt JP, Charron F, Robitaille L, Caron S, Conner DA,
Gessler M, Nemer M, Seidman CE, Seidman JG. A murine model of holt-oram syn-
drome defines roles of the t-box transcription factor tbx5 in cardiogenesis and dis-
ease. Cell 106: 709 –721, 2001.
19. Brunel-Guitton C, Levtova A, Sasarman F. Mitochondrial diseases and cardiomyopa-
thies. Can J Cardiol 31: 1360 –1376, 2015.
20. Burridge PW, Diecke S, Matsa E, Sharma A, Wu H, Wu JC. Modeling cardiovascular
diseases with patient-specific human pluripotent stem cell-derived cardiomyocytes.
Methods Mol Biol. 1353: 119 –130, 2016.
21. Burridge PW, Fuga Y, Matsa E, Wu H, Ong G, Sharma A, Holmström A, Chang AC,
Coronado MJ, Ebert AD, Knowles JW, Telli ML, Witteles RM, Blau HM, Bernstein D,
Altman RB, Wu JC. Human induced pluripotent stem cells recapitulate breast cancer
patients’ predilection to doxorubicin-induced cardiotoxicity. Nature Med. 22: 547–
556, 2016.
22. Burridge PW, Holmstrom A, Wu JC. Chemically defined culture and cardiomyocyte
differentiation of human pluripotent stem cells. Curr Protocol Hum Genet 87: 21 3 1 3
15547–21, 2015.
23. Burridge PW, Keller G, Gold JD, Wu JC. Production of de novo cardiomyocytes:
Human pluripotent stem cell differentiation and direct reprogramming. Cell Stem Cell
10: 16 –28, 2012.
24. Burridge PW, Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD, Lan F, Diecke S,
Huber B, Mordwinkin NM, Plews JR, Abilez OJ, Cui B, Gold JD, Wu JC. Chemically
defined generation of human cardiomyocytes. Nat Methods 11: 855– 860, 2014.
25. Burridge PW, Sharma A, Wu JC. Genetic and epigenetic regulation of human cardiac
reprogramming and differentiation in regenerative medicine. Annu Rev Genet 49:
20.1.24855–20, 2015.
26. Burridge PW, Thompson S, Millrod MA, Weinberg S, Yuan X, Peters A, Mahairaki V,
Koliatsos VE, Tung L, Zambidis ET. A universal system for highly efficient cardiac
differentiation of human induced pluripotent stem cells that eliminates interline vari-
ability. PLoS One 6: e18293, 2011.
27. Burrows CK, Banovich NE, Pavlovic BJ, Patterson K, Gallego Romero I, Pritchard JK,
Gilad Y. Genetic variation, not cell type of origin, underlies the majority of identifiable
regulatory differences in ipscs. PLoS Genet 12: e1005793, 2016.
28. Callis TE, Pandya K, Seok HY, Tang RH, Tatsuguchi M, Huang ZP, Chen JF, Deng Z,
Gunn B, Shumate J, Willis MS, Selzman CH, Wang DZ. MicroRNA-208a is a regulator
of cardiac hypertrophy and conduction in mice. J Clin Invest 119: 2772–2786, 2009.
29. Cao X, Deng W, Qu R, Yu Q, Li J, Yang Y, Cao Y, Gao X, Xu X, Yu J. Non-viral
co-delivery of the four yamanaka factors for generation of human induced pluripotent
stem cells via calcium phosphate nanocomposite particles. Adv Functional Materials 23:
5403–5411, 2013.
30. Carlson C, Koonce C, Aoyama N, Einhorn S, Fiene S, Thompson A, Swanson B, Anson
B, Kattman S. Phenotypic screening with human ips cell-derived cardiomyocytes:
Hts-compatible assays for interrogating cardiac hypertrophy. J Biomol Screen 18:
1203–1211, 2013.
 HUMAN INDUCED PLURIPOTENT STEM CELLS
31. Carvajal-Vergara X, Sevilla A, D’Souza SL, Ang YS, Schaniel C, Lee DF, Yang L, Kaplan
AD, Adler ED, Rozov R, Ge Y, Cohen N, Edelmann LJ, Chang B, Waghray A, Su J,
Pardo S, Lichtenbelt KD, Tartaglia M, Gelb BD, Lemischka IR. Patient-specific induced
pluripotent stem-cell-derived models of leopard syndrome. Nature 465: 808 – 812,
2010.
32. Caspi O, Huber I, Gepstein A, Arbel G, Maizels L, Boulos M, Gepstein L. Modeling of
arrhythmogenic right ventricular cardiomyopathy with human induced pluripotent
stem cells. Circ Cardiovasc Genet 6: 557–568, 2013.
33. Chamberlain AA, Lin M, Lister RL, Maslov AA, Wang Y, Suzuki M, Wu B, Greally JM,
Zheng D, Zhou B. DNA methylation is developmentally regulated for genes essential
for cardiogenesis. J Am Heart Assoc 3: e000976, 2014.
34. Chandrasekera PC, Pippin JJ. The human subject: an integrative animal model for
21(st) century heart failure research. Am J Transl Res 7: 1636 –1647, 2015.
35. Chasman DI, Posada D, Subrahmanyan L, Cook NR, Stanton VP Jr, Ridker PM.
Pharmacogenetic study of statin therapy and cholesterol reduction. JAMA 291: 2821–
2827, 2004.
36. Chatterjee K, Zhang J, Honbo N, Karliner JS. Doxorubicin cardiomyopathy. Cardiology
115: 155–162, 2010.
36a.Chen IY, Matsa E, Wu JC. Induced pluripotent stem cells: at the heart of cardiovascular
precision medicine. Nat Rev Cardiol 13: 333–349, 2016.
37. Chen VC, Ye J, Shukla P, Hua G, Chen D, Lin Z, Liu JC, Chai J, Gold J, Wu J, Hsu D,
Couture LA. Development of a scalable suspension culture for cardiac differentiation
from human pluripotent stem cells. Stem Cell Res 15: 365–375, 2015.
38. Chen ZY, He CY, Ehrhardt A, Kay MA. Minicircle DNA vectors devoid of bacterial
DNA result in persistent and high-level transgene expression in vivo. Mol Ther 8:
495–500, 2003.
39. Chester AH, Yacoub MH. The role of endothelin-1 in pulmonary arterial hyperten-
sion. Glob Cardiol Sci Pract 2014: 62–78, 2014.
40. Choi J, Lee S, Mallard W, Clement K, Tagliazucchi GM, Lim H, Choi IY, Ferrari F,
Tsankov AM, Pop R, Lee G, Rinn JL, Meissner A, Park PJ, Hochedlinger K. A compar-
ison of genetically matched cell lines reveals the equivalence of human ipscs and escs.
Nat Biotechnol 33: 1173–1181, 2015.
41. Churko JM, Burridge PW, Wu JC. Generation of human ipscs from human peripheral
blood mononuclear cells using non-integrative sendai virus in chemically defined con-
ditions. Methods Mol Biol 1036: 81– 88, 2013.
42. Churko JM, Mantalas GL, Snyder MP, Wu JC. Overview of high throughput sequenc-
ing technologies to elucidate molecular pathways in cardiovascular diseases. Circ Res
112: 1613–1623, 2013.
43. Clarke AJ. Managing the ethical challenges of next-generation sequencing in genomic
medicine. Br Med Bull 111: 17–30, 2014.
44. Colamonici OR, Domanski P, Sweitzer SM, Larner A, Buller RM. Vaccinia virus b18r
gene encodes a type i interferon-binding protein that blocks interferon alpha trans-
membrane signaling. J Biol Chem 270: 15974 –15978, 1995.
45. Collins FS, Varmus H. A new initiative on precision medicine. N Engl J Med 372:
793–795, 2015.
46. Corrigan-Curay J, O’Reilly M, Kohn DB, Cannon PM, Bao G, Bushman FD, Carroll D,
Cathomen T, Joung JK, Roth D, Sadelain M, Scharenberg AM, von Kalle C, Zhang F,
Jambou R, Rosenthal E, Hassani M, Singh A, Porteus MH. Genome editing technolo-
gies: Defining a path to clinic. Mol Ther 23: 796 – 806, 2015.
47. Corwin EJ. The concept of epigenetics and its role in the development of cardiovas-
cular disease: Commentary on “new and emerging theories of cardiovascular dis-
ease.” Biol Res Nurs 6: 11–16, 2004.
48. David L, Polo JM. Phases of reprogramming. Stem Cell Res 12: 754 –761, 2014.
49. Davidson MH, Stein EA, Dujovne CA, Hunninghake DB, Weiss SR, Knopp RH, Illing-
worth DR, Mitchel YB, Melino MR, Zupkis RV, Dobrinska MR, Amin RD, Tobert JA.
The efficacy and six-week tolerability of simvastatin 80 and 160 mg/day. Am J Cardiol
79: 38 – 42, 1997.
50. Davis RP, Nemes C, Varga E, Freund C, Kosmidis G, Gkatzis K, de Jong D, Szuhai K,
Dinnyes A, Mummery CL. Generation of induced pluripotent stem cells from human
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
foetal fibroblasts using the sleeping beauty transposon gene delivery system. Differ-
entiation 86: 30 –37, 2013.
51. Denning C, Allegrucci C, Priddle H, Barbadillo-Munoz MD, Anderson D, Self T, Smith
NM, Parkin CT, Young LE. Common culture conditions for maintenance and cardio-
myocyte differentiation of the human embryonic stem cell lines, bg01 and hues-7. Int
J Dev Biol 50: 27–37, 2006.
52. Denning C, Borgdorff V, Crutchley J, Firth KSA, George V, Kalra S, Kondrashov A,
Hoang MD, Mosqueira D, Patel A, Prodanov L, Rajamohan D, Skarnes WC, Smith
JGW, Young LE. Cardiomyocytes from human pluripotent stem cells: from labo-
ratory curiosity to industrial biomedical platform. Biochim Biophys Acta 4889:
367–375, 2015.
53. Devalla HD, Schwach V, Ford JW, Milnes JT, El-Haou S, Jackson C, Gkatzis K, Elliott
DA, Chuva de Sousa Lopes SM, Mummery CL, Verkerk AO, Passier R. Atrial-like
cardiomyocytes from human pluripotent stem cells are a robust preclinical model for
assessing atrial-selective pharmacology. EMBO Mol Med 7: 394 – 410, 2015.
54. Di Pasquale E, Lodola F, Miragoli M, Denegri M, Avelino-Cruz JE, Buonocore M,
Nakahama H, Portararo P, Bloise R, Napolitano C, Condorelli G, Priori SG. CaMKII
inhibition rectifies arrhythmic phenotype in a patient-specific model of catecholamin-
ergic polymorphic ventricular tachycardia. Cell Death Dis 4: e843, 2014.
55. Dick E, Matsa E, Bispham J, Reza M, Guglieri M, Staniforth A, Watson S, Kumari R,
Lochmuller H, Young L, Darling D, Denning C. Two new protocols to enhance the
production and isolation of human induced pluripotent stem cell lines. Stem Cell Res 6:
158 –167, 2011.
56. Diecke S, Lisowski L, Kooreman NG, Wu JC. Second generation codon optimized
minicircle (comic) for nonviral reprogramming of human adult fibroblasts. Methods
Mol Biol 1181: 1–13, 2014.
57. Diecke S, Lu J, Lee J, Termglinchan V, Kooreman NG, Burridge PW, Ebert AD,
Churko JM, Sharma A, Kay MA, Wu JC. Novel codon-optimized mini-intronic plasmid
for efficient, inexpensive, and xeno-free induction of pluripotency. Sci Rep 5: 8081,
2015.
58. Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF,
Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. Induced pluri-
potent stem cells generated from patients with als can be differentiated into motor
neurons. Science 321: 1218 –1221, 2008.
59. Ding Q, Lee YK, Schaefer EA, Peters DT, Veres A, Kim K, Kuperwasser N, Motola
DL, Meissner TB, Hendriks WT, Trevisan M, Gupta RM, Moisan A, Banks E, Friesen
M, Schinzel RT, Xia F, Tang A, Xia Y, Figueroa E, Wann A, Ahfeldt T, Daheron L,
Zhang F, Rubin LL, Peng LF, Chung RT, Musunuru K, Cowan CA. A talen genome-
editing system for generating human stem cell-based disease models. Cell Stem Cell
12: 238 –251, 2013.
60. Dowey SN, Huang X, Chou BK, Ye Z, Cheng L. Generation of integration-free human
induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid
vector expression. Nature Protocols 7: 2013–2021, 2012.
61. Drawnel FM, Boccardo S, Prummer M, Delobel F, Graff A, Weber M, Gerard R, Badi
L, Kam-Thong T, Bu L, Jiang X, Hoflack JC, Kiialainen A, Jeworutzki E, Aoyama N,
Carlson C, Burcin M, Gromo G, Boehringer M, Stahlberg H, Hall BJ, Magnone MC,
Kolaja K, Chien KR, Bailly J, Iacone R. Disease modeling and phenotypic drug screening
for diabetic cardiomyopathy using human induced pluripotent stem cells. Cell Rep 9:
810 – 821, 2014.
62. Dubois NC, Craft AM, Sharma P, Elliott DA, Stanley EG, Elefanty AG, Gramolini A,
Keller G. Sirpa is a specific cell-surface marker for isolating cardiomyocytes derived
from human pluripotent stem cells. Nat Biotechnol 29: 1011–1018, 2011.
63. Ebert AD, Kodo K, Liang P, Wu H, Huber BC, Riegler J, Churko J, Lee J, de Almeida
P, Lan F, Diecke S, Burridge PW, Gold JD, Mochly-Rosen D, Wu JC. Characterization
of the molecular mechanisms underlying increased ischemic damage in the aldehyde
dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell
model system. Sci Transl Med 6: 255ra130, 2014.
64. Ebrahimi B. Reprogramming barriers and enhancers: Strategies to enhance the effi-
ciency and kinetics of induced pluripotency. Cell Regen 4: 10, 2015.
65. Edwards AM, Arrowsmith CH, Bountra C, Bunnage ME, Feldmann M, Knight JC,
Patel DD, Prinos P, Taylor MD, Sundstrom M, Barker P, Barsyte D, Bengtson MH,
Bell C, Bowness P, Boycott KM, Buser-Doepner C, Carpenter CL, Carr AJ, Clark K,
Das AM, Dhanak D, Dirks P, Ellis J, Fantin VR, Flores C, Fon EA, Frail DE, Gileadi O,
O’Hagan RC, Howe T, Isaac JT, Jabado N, Jakobsson PJ, Klareskog L, Knapp S, Lee
1119
 MATSA ET AL.
WH, Lima-Fernandes E, Lundberg IE, Marshall J, Massirer KB, MacKenzie AE, Maruy-
ama T, Mueller-Fahrnow A, Muthuswamy S, Nanchahal J, O’Brien C, Oppermann U,
Ostermann N, Petrecca K, Pollock BG, Poupon V, Prinjha RK, Rosenberg SH, Rouleau
G, Skingle M, Slutsky AS, Smith GA, Verhelle D, Widmer H, Young LT. Preclinical
target validation using patient-derived cells. Nat Rev Drug Discov 14: 149 –150, 2015.
66. Elanzew A, Sommer A, Pusch-Klein A, Brustle O, Haupt S. A reproducible and ver-
satile system for the dynamic expansion of human pluripotent stem cells in suspen-
sion. Biotechnol J 10: 1589 –1599, 2015.
67. Fatima A, Kaifeng S, Dittmann S, Xu G, Gupta MK, Linke M, Zechner U, Nguemo F,
Milting H, Farr M, Hescheler J, Saric T. The disease-specific phenotype in cardiomy-
ocytes derived from induced pluripotent stem cells of two long qt syndrome type 3
patients. PLoS One 8: e83005, 2013.
68. Fatima A, Xu G, Shao K, Papadopoulos S, Lehmann M, Arnaiz-Cot JJ, Rosa AO,
Nguemo F, Matzkies M, Dittmann S, Stone SL, Linke M, Zechner U, Beyer V, Hennies
HC, Rosenkranz S, Klauke B, Parwani AS, Haverkamp W, Pfitzer G, Farr M, Cleemann
L, Morad M, Milting H, Hescheler J, Saric T. In vitro modeling of ryanodine receptor
2 dysfunction using human induced pluripotent stem cells. Cell Physiol Biochem 28:
579 –592, 2011.
69. Feldman MD, Copelas L, Gwathmey JK, Phillips P, Warren SE, Schoen FJ, Grossman
W, Morgan JP. Deficient production of cyclic amp: Pharmacologic evidence of an
important cause of contractile dysfunction in patients with end-stage heart failure.
Circulation 75: 331–339, 1987.
70. Foley AC, Mercola M. Heart induction by wnt antagonists depends on the homeodo-
main transcription factor hex. Genes Dev 19: 387–396, 2005.
71. Frishman WH. 〉-adrenergic blockade in cardiovascular disease. J Cardiovasc Pharma-
col Ther 18: 310 –319, 2013.
72. Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of transgene-
free human pluripotent stem cells using a vector based on sendai virus, an rna virus
that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85:
348 –362, 2009.
73. Gaber N, Gagliardi M, Patel P, Kinnear C, Zhang C, Chitayat D, Shannon P, Jaeggi E,
Tabori U, Keller G, Mital S. Fetal reprogramming and senescence in hypoplastic left
heart syndrome and in human pluripotent stem cells during cardiac differentiation. Am
J Pathol 183: 720 –734, 2013.
74. Gaj T, Gersbach CA, Barbas CF 3rd. Zfn, talen, and crispr/cas-based methods for
genome engineering. Trends Biotechnol 31: 397– 405, 2013.
75. Gaj T, Mercer AC, Gersbach CA, Gordley RM, Barbas CF 3rd. Structure-guided
reprogramming of serine recombinase DNA sequence specificity. Proc Natl Acad Sci
USA 108: 498 –503, 2011.
76. Gaj T, Mercer AC, Sirk SJ, Smith HL, Barbas CF 3rd. A comprehensive approach to
zinc-finger recombinase customization enables genomic targeting in human cells.
Nucleic Acids Res 41: 3937–3946, 2013.
77. Gersbach CA, Perez-Pinera P. Activating human genes with zinc finger proteins,
transcription activator-like effectors and crispr/cas9 for gene therapy and regenera-
tive medicine. Expert Opin Ther Targets 18: 835– 839, 2014.
78. Gersh BJ, Maron BJ, Bonow RO, Dearani JA, Fifer MA, Link MS, Naidu SS, Nishimura
RA, Ommen SR, Rakowski H, Seidman CE, Towbin JA, Udelson JE, Yancy CW. 2011
ACCF/AHA guideline for the diagnosis and treatment of hypertrophic cardiomyopa-
thy: a report of the american college of cardiology foundation/american heart associ-
ation task force on practice guidelines. Circulation 124: e783– 831, 2011.
79. Gilchrist KH, Lewis GF, Gay EA, Sellgren KL, Grego S. High-throughput cardiac safety
evaluation and multi-parameter arrhythmia profiling of cardiomyocytes using micro-
electrode arrays. Toxicol Appl Pharmacol 288: 249 –257, 2015.
80. Gonzalez F, Boue S, Izpisua Belmonte JC. Methods for making induced pluripotent
stem cells: reprogramming a la carte. Nat Rev Genet 12: 231–242, 2011.
81. Gonzalez R, Lee JW, Schultz PG. Stepwise chemically induced cardiomyocyte speci-
fication of human embryonic stem cells. Angew Chem Int Ed Engl 50: 11181–11185,
2011.
82. Grobarczyk B, Franco B, Hanon K, Malgrange B. Generation of isogenic human ips cell
line precisely corrected by genome editing using the crispr/cas9 system. Stem Cell Rev
11: 774 –787, 2015.
1120 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
83. Guilinger JP, Pattanayak V, Reyon D, Tsai SQ, Sander JD, Joung JK, Liu DR. Broad
specificity profiling of talens results in engineered nucleases with improved DNA-
cleavage specificity. Nat Methods 11: 429 – 435, 2014.
84. Gurdon JB. Adult frogs derived from the nuclei of single somatic cells. Dev Biol 4:
256 –273, 1962.
85. Guzun R, Kaambre T, Bagur R, Grichine A, Usson Y, Varikmaa M, Anmann T, Tepp K,
Timohhina N, Shevchuk I, Chekulayev V, Boucher F, Dos Santos P, Schlattner U,
Wallimann T, Kuznetsov AV, Dzeja P, Aliev M, Saks V. Modular organization of cardiac
energy metabolism: Energy conversion, transfer and feedback regulation. Acta Physiol
213: 84 –106, 2015.
86. Gwathmey JK, Tsaioun K, Hajjar RJ. Cardionomics: a new integrative approach for
screening cardiotoxicity of drug candidates. Expert Opin Drug Metab Toxicol 5: 647–
660, 2009.
87. Haghighi K, Kolokathis F, Pater L, Lynch RA, Asahi M, Gramolini AO, Fan GC, Tsiapras
D, Hahn HS, Adamopoulos S, Liggett SB, Dorn GW 2nd, MacLennan DH, Kremasti-
nos DT, Kranias EG. Human phospholamban null results in lethal dilated cardiomy-
opathy revealing a critical difference between mouse and human. J Clin Invest 111:
869 – 876, 2003.
88. Han D, Pal S, Yang Y, Jiang S, Nangreave J, Liu Y, Yan H. DNA gridiron nanostructures
based on four-arm junctions. Science 339: 1412–1415, 2013.
89. Han L, Li Y, Tchao J, Kaplan AD, Lin B, Li Y, Mich-Basso J, Lis A, Hassan N, London B,
Bett GC, Tobita K, Rasmusson RL, Yang L. Study familial hypertrophic cardiomyopa-
thy using patient-specific induced pluripotent stem cells. Cardiovasc Res 104: 258 –269,
2014.
90. Hankowski KE, Hamazaki T, Umezawa A, Terada N. Induced pluripotent stem cells
as a next-generation biomedical interface. Lab Invest 91: 972–977, 2011.
91. Hannah-Shmouni F, Seidelmann SB, Sirrs S, Mani A, Jacoby D. The genetic challenges
and opportunities in advanced heart failure. Can J Cardiol 31: 1338 –1350, 2015.
92. Hattori F, Chen H, Yamashita H, Tohyama S, Satoh YS, Yuasa S, Li W, Yamakawa H,
Tanaka T, Onitsuka T, Shimoji K, Ohno Y, Egashira T, Kaneda R, Murata M, Hidaka K,
Morisaki T, Sasaki E, Suzuki T, Sano M, Makino S, Oikawa S, Fukuda K. Nongenetic
method for purifying stem cell-derived cardiomyocytes. Nat Methods 7: 61– 66, 2010.
93. Hinson JT, Chopra A, Nafissi N, Polacheck WJ, Benson CC, Swist S, Gorham J, Yang
L, Schafer S, Sheng CC, Haghighi A, Homsy J, Hubner N, Church G, Cook SA, Linke
WA, Chen CS, Seidman JG, Seidman CE. Heart disease. Titin mutations in ips cells
define sarcomere insufficiency as a cause of dilated cardiomyopathy. Science 349:
982–986, 2015.
94. Hiratsuka M, Uno N, Ueda K, Kurosaki H, Imaoka N, Kazuki K, Ueno E, Akakura Y,
Katoh M, Osaki M, Kazuki Y, Nakagawa M, Yamanaka S, Oshimura M. Integration-
free ips cells engineered using human artificial chromosome vectors. PLoS One 6:
e25961, 2011.
95. Hiroi Y, Kudoh S, Monzen K, Ikeda Y, Yazaki Y, Nagai R, Komuro I. Tbx5 associates
with nkx2-5 and synergistically promotes cardiomyocyte differentiation. Nat Genet
28: 276 –280, 2001.
96. Holmgren G, Synnergren J, Bogestal Y, Ameen C, Akesson K, Holmgren S, Lindahl A,
Sartipy P. Identification of novel biomarkers for doxorubicin-induced toxicity in hu-
man cardiomyocytes derived from pluripotent stem cells. Toxicology 328: 102–111,
2015.
97. Hotta A, Yamanaka S. From genomics to gene therapy: Induced pluripotent stem cells
meet genome editing. Annu Rev Genet. 49: 47–70, 2015.
98. Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, Zhao T, Ye J, Yang W, Liu K, Ge J, Xu J,
Zhang Q, Zhao Y, Deng H. Pluripotent stem cells induced from mouse somatic cells
by small-molecule compounds. Science 341: 651– 654, 2013.
99. Houser SR, Margulies KB, Murphy AM, Spinale FG, Francis GS, Prabhu SD, Rockman
HA, Kass DA, Molkentin JD, Sussman MA, Koch WJ, American Heart Association
Council on Basic Cardiovascular Sciences. Animal models of heart failure: a scientific
statement from the american heart association. Circ Res 111: 131–150, 2012.
100. Hsu PD, Lander ES, Zhang F. Development and applications of crispr-cas9 for ge-
nome engineering. Cell 157: 1262–1278, 2014.
101. Huang HP, Chen PH, Hwu WL, Chuang CY, Chien YH, Stone L, Chien CL, Li LT,
Chiang SC, Chen HF, Ho HN, Chen CH, Kuo HC. Human pompe disease-induced
 HUMAN INDUCED PLURIPOTENT STEM CELLS
pluripotent stem cells for pathogenesis modeling, drug testing and disease marker
identification. Hum Mol Genet 20: 4851– 4864, 2011.
102. Huang ZP, Chen J, Seok HY, Zhang Z, Kataoka M, Hu X, Wang DZ. Microrna-22
regulates cardiac hypertrophy and remodeling in response to stress. Circ Res 112:
1234 –1243, 2013.
103. Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S, Muhlestein W,
Melton DA. Induction of pluripotent stem cells from primary human fibroblasts with
only oct4 and sox2. Nat Biotech 26: 1269 –1275, 2008.
104. Hung SC, Kang MS, Kieff E. Maintenance of epstein-barr virus (ebv) orip-based epi-
somes requires ebv-encoded nuclear antigen-1 chromosome-binding domains, which
can be replaced by high-mobility group-i or histone h1. Proc Natl Acad Sci USA 98:
1865–1870, 2001.
105. Hussain W, Moens N, Veraitch FS, Hernandez D, Mason C, Lye GJ. Reproducible
culture and differentiation of mouse embryonic stem cells using an automated micro-
well platform. Biochem Eng J 77: 246 –257, 2013.
106. Hussein SM, Batada NN, Vuoristo S, Ching RW, Autio R, Narva E, Ng S, Sourour M,
Hamalainen R, Olsson C, Lundin K, Mikkola M, Trokovic R, Peitz M, Brustle O,
Bazett-Jones DP, Alitalo K, Lahesmaa R, Nagy A, Otonkoski T. Copy number variation
and selection during reprogramming to pluripotency. Nature 471: 58 – 62, 2011.
107. Illingworth R, Kerr A, Desousa D, Jorgensen H, Ellis P, Stalker J, Jackson D, Clee C,
Plumb R, Rogers J, Humphray S, Cox T, Langford C, Bird A. A novel cpg island set
identifies tissue-specific methylation at developmental gene loci. PLoS Biol 6: e22,
2008.
108. Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS. The ribosome profiling
strategy for monitoring translation in vivo by deep sequencing of ribosome-protected
mrna fragments. Nat Protoc 7: 1534 –1550, 2012.
109. Itzhaki I, Maizels L, Huber I, Gepstein A, Arbel G, Caspi O, Miller L, Belhassen B, Nof
E, Glikson M, Gepstein L. Modeling of catecholaminergic polymorphic ventricular
tachycardia with patient-specific human-induced pluripotent stem cells. J Am Coll
Cardiol 60: 990 –1000, 2012.
110. Itzhaki I, Maizels L, Huber I, Zwi-Dantsis L, Caspi O, Winterstern A, Feldman O,
Gepstein A, Arbel G, Hammerman H, Boulos M, Gepstein L. Modelling the long qt
syndrome with induced pluripotent stem cells. Nature 471: 225–229, 2011.
111. Jacoby D, McKenna WJ. Genetics of inherited cardiomyopathy. Eur Heart J 33: 296 –
304, 2012.
112. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome inte-
grates intrinsic and environmental signals. Nat Genet 33 Suppl: 245–254, 2003.
113. Jensen BC, McLeod HL. Pharmacogenomics as a risk mitigation strategy for chemo-
therapeutic cardiotoxicity. Pharmacogenomics 14: 205–213, 2013.
114. Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z, Panetta NJ, Chen ZY, Robbins RC,
Kay MA, Longaker MT, Wu JC. A nonviral minicircle vector for deriving human ips
cells. Nat Methods 7: 197–199, 2010.
115. Jiang Y, Habibollah S, Tilgner K, Collin J, Barta T, Al-Aama JY, Tesarov L, Hussain R,
Trafford AW, Kirkwood G, Sernagor E, Eleftheriou CG, Przyborski S, Stojkovic M,
Lako M, Keavney B,Armstrong L. An induced pluripotent stem cell model of hypoplas-
tic left heart syndrome (hlhs) reveals multiple expression and functional differences in
hlhs-derived cardiac myocytes. Stem Cells Transl Med 3: 416 – 423, 2014.
116. Jiang Y, Habibollah S, Tilgner K, Collin J, Barta T, Al-Aama JY, Tesarov L, Hussain R,
Trafford AW, Kirkwood G, Sernagor E, Eleftheriou CG, Przyborski S, Stojkovic M,
Lako M, Keavney B, Armstrong L. An induced pluripotent stem cell model of hyp-
oplastic left heart syndrome (hlhs) reveals multiple expression and functional differ-
ences in hlhs-derived cardiac myocytes. Stem Cells Transl Med, 2014.
10.5966/sctm.2013-0105.
117. Jinek M. A programmable dual-rna-guided DNA endonuclease in adaptive bacterial
immunity. Science 337: 816 – 821, 2012.
118. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable
dual-rna-guided DNA endonuclease in adaptive bacterial immunity. Science 337: 816 –
821, 2012.
119. Josowitz R, Lu J, Falce C, D’Souza SL, Wu M, Cohen N, Dubois NC, Zhao Y, Sobie EA,
Fishman GI, Gelb BD. Identification and purification of human induced pluripotent
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression. PLoS One
9: e101316, 2014.
120. Jouni M, Si-Tayeb K, Es-Salah-Lamoureux Z, Latypova X, Champon B, Caillaud A,
Rungoat A, Charpentier F, Loussouarn G, Baro I, Zibara K, Lemarchand P, Gaborit N.
Toward personalized medicine: Using cardiomyocytes differentiated from urine-de-
rived pluripotent stem cells to recapitulate electrophysiological characteristics of type
2 long qt syndrome. JAMA 4: 9, 2015.
121. Jung CB, Moretti A, Schnitzler MM, Iop L, Storch U, Bellin M, Dorn T, Ruppenthal S,
Pfeiffer S, Goedel A, Dirschinger RJ, Seyfarth M, Lam JT, Sinnecker D, Gudermann T,
Lipp P, Laugwitz KL. Dantrolene rescues arrhythmogenic ryr2 defect in a patient-
specific stem cell model of catecholaminergic polymorphic ventricular tachycardia.
EMBO Mol Med 4: 180 –191, 2012.
122. Kadari A, Mekala S, Wagner N, Malan D, Koth J, Doll K, Stappert L, Eckert D, Peitz M,
Matthes J, Sasse P, Herzig S, Brustle O, Ergun S, Edenhofer F. Robust generation of
cardiomyocytes from human ips cells requires precise modulation of bmp and wnt
signaling. Stem Cell Rev 11: 560 –569, 2014.
123. Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K. Virus-free induction
of pluripotency and subsequent excision of reprogramming factors. Nature 458: 771–
775, 2009.
124. Kamakura T, Makiyama T, Sasaki K, Yoshida Y, Wuriyanghai Y, Chen J, Hattori T,
Ohno S, Kita T, Horie M, Yamanaka S, Kimura T. Ultrastructural maturation of
human-induced pluripotent stem cell-derived cardiomyocytes in a long-term culture.
Circ J 77: 1307–1314, 2013.
125. Kang HB, Kim JS, Kwon HJ, Nam KH, Youn HS, Sok DE, Lee Y. Basic fibroblast growth
factor activates erk and induces c-fos in human embryonic stem cell line mizhes1.
Stem Cells Dev 14: 395– 401, 2005.
126. Kang X, Yu Q, Huang Y, Song B, Chen Y, Gao X, He W, Sun X, Fan Y. Effects of
integrating and non-integrating reprogramming methods on copy number variation
and genomic stability of human induced pluripotent stem cells. PLoS One 10:
e0131128, 2015.
127. Kapoor N, Liang W, Marban E, Cho HC. Direct conversion of quiescent cardiomyo-
cytes to pacemaker cells by expression of tbx18. Nat Biotech 31: 54 – 62, 2013.
128. Karakikes I, Ameen M, Termglinchan V, Wu JC. Human induced pluripotent stem
cell-derived cardiomyocytes: insights into molecular, cellular, and functional pheno-
types. Circ Res 117: 80 – 88, 2015.
129. Karakikes I, Senyei GD, Hansen J, Kong CW, Azeloglu EU, Stillitano F, Lieu DK, Wang
J, Ren L, Hulot JS, Iyengar R, Li RA, Hajjar RJ. Small molecule-mediated directed
differentiation of human embryonic stem cells toward ventricular cardiomyocytes.
Stem Cells Transl Med 3: 18 –31, 2014.
130. Karakikes I, Stillitano F, Nonnenmacher M, Tzimas C, Sanoudou D, Termglinchan V,
Kong CW, Rushing S, Hansen J, Ceholski D, Kolokathis F, Kremastinos D, Katoulis A,
Ren L, Cohen N, Gho JM, Tsiapras D, Vink A, Wu JC, Asselbergs FW, Li RA, Hulot JS,
Kranias EG, Hajjar RJ. Correction of human phospholamban r14del mutation associ-
ated with cardiomyopathy using targeted nucleases and combination therapy. Nat
Commun 6: 6955, 2015.
131. Kattman SJ, Witty AD, Gagliardi M, Dubois NC, Niapour M, Hotta A, Ellis J, Keller G.
Stage-specific optimization of activin/nodal and bmp signaling promotes cardiac dif-
ferentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8: 228 –
240, 2011.
132. Kempf H, Olmer R, Kropp C, Ruckert M, Jara-Avaca M, Robles-Diaz D, Franke A,
Elliott DA, Wojciechowski D, Fischer M, Roa Lara A, Kensah G, Gruh I, Haverich A,
Martin U, Zweigerdt R. Controlling expansion and cardiomyogenic differentiation of
human pluripotent stem cells in scalable suspension culture. Stem Cell Reports 3:
1132–1146, 2014.
133. Khoury MJ, Iademarco MF, Riley WT. Precision public health for the era of precision
medicine. Am J Prevent Med 50: 398 – 401, 2016.
134. Kim C, Wong J, Wen J, Wang S, Wang C, Spiering S, Kan NG, Forcales S, Puri PL,
Leone TC, Marine JE, Calkins H, Kelly DP, Judge DP, Chen HS. Studying arrhythmo-
genic right ventricular dysplasia with patient-specific ipscs. Nature 494: 105–110,
2013.
135. Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza
R, Kim KS. Generation of human induced pluripotent stem cells by direct delivery of
reprogramming proteins. Cell Stem Cell 4: 472– 476, 2009.
1121
 MATSA ET AL.
136. Kim JS, Lee HJ, Carroll D. Genome editing with modularly assembled zinc-finger
nucleases. Nat Methods 7: 91–92, 2010.
137. Kim WH, Jung DW, Williams DR. Making cardiomyocytes with your chemistry set:
Small molecule-induced cardiogenesis in somatic cells. World J Cardiol 7: 125–133,
2015.
138. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD,
Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. Genome-scale transcrip-
tional activation by an engineered crispr-cas9 complex. Nature 517: 583–588, 2015.
139. Kowalski MP, Yoder A, Liu L, Pajak L. Controlling embryonic stem cell growth and
differentiation by automation: Enhanced and more reliable differentiation for drug
discovery. J Biomol Screen 17: 1171–1179, 2012.
140. Krishan K, Kanchan T, Singh B. Human genome editing and ethical considerations. Sci
Eng Ethics 22: 597–599, 2016.
141. Krueger F, Kreck B, Franke A, Andrews SR. DNA methylome analysis using short
bisulfite sequencing data. Nature Methods 9: 145–151, 2012.
142. Kujala K, Paavola J, Lahti A, Larsson K, Pekkanen-Mattila M, Viitasalo M, Lahtinen AM,
Toivonen L, Kontula K, Swan H, Laine M, Silvennoinen O, Aalto-Setala K. Cell model
of catecholaminergic polymorphic ventricular tachycardia reveals early and delayed
afterdepolarizations. PLoS One 7: e44660, 2012.
143. Kushwaha SS, Fallon JT, Fuster V. Restrictive cardiomyopathy. N Engl J Med 336:
267–276, 1997.
144. Labaj PP, Leparc GG, Linggi BE, Markillie LM, Wiley HS, Kreil DP. Characterization
and improvement of rna-seq precision in quantitative transcript expression profiling.
Bioinformatics 27: i383–391, 2011.
145. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, Dupras SK, Reinecke
H, Xu C, Hassanipour M, Police S, O’Sullivan C, Collins L, Chen Y, Minami E, Gill EA,
Ueno S, Yuan C, Gold J, Murry CE. Cardiomyocytes derived from human embryonic
stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotech-
nol 25: 1015–1024, 2007.
146. Lam HY, Clark MJ, Chen R, Chen R, Natsoulis G, O’Huallachain M, Dewey FE,
Habegger L, Ashley EA, Gerstein MB, Butte AJ, Ji HP, Snyder M. Performance com-
parison of whole-genome sequencing platforms. Nat Biotechnol 30: 78 – 82, 2012.
147. Lan F, Lee AS, Liang P, Sanchez-Freire V, Nguyen PK, Wang L, Han L, Yen M, Wang
Y, Sun N, Abilez OJ, Hu S, Ebert AD, Navarrete EG, Simmons CS, Wheeler M, Pruitt
B, Lewis R, Yamaguchi Y, Ashley EA, Bers DM, Robbins RC, Longaker MT, Wu JC.
Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy
pathology in patient-specific induced pluripotent stem cells. Cell Stem Cell 12: 101–
113, 2013.
149. Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. Crispr interference
(crispri) for sequence-specific control of gene expression. Nat Protocols 8: 2180 –
2196, 2013.
150. Laslett LJ, Alagona P, Clark BA, Drozda JP, Saldivar F, Wilson SR, Poe C, Hart M. The
worldwide environment of cardiovascular disease: prevalence, diagnosis, therapy,
and policy issues a report from the American College of Cardiology. J Am Coll Cardiol
60: S1–S49, 2012.
151. Lee CH, Kim JH, Lee HJ, Jeon K, Lim H, Choi H, Lee ER, Park SH, Park JY, Hong S, Kim
S, Cho SG. The generation of ips cells using non-viral magnetic nanoparticle based
transfection. Biomaterials 32: 6683– 6691, 2011.
152. Lee J, Sayed N, Hunter A, Au KF, Wong WH, Mocarski ES, Pera RR, Yakubov E,
Cooke JP. Activation of innate immunity is required for efficient nuclear reprogram-
ming. Cell 151: 547–558, 2012.
153. Lee YK, Lau YM, Ng KM, Lai WH, Ho SL, Tse HF, Siu CW, Ho PW. Efficient attenu-
ation of friedreich’s ataxia (frda) cardiomyopathy by modulation of iron homeostasis-
human induced pluripotent stem cell (hipsc) as a drug screening platform for frda. Int
J Cardiol 203: 964 –971, 2016.
154. Lescroart F, Chabab S, Lin X, Rulands S, Paulissen C, Rodolosse A, Auer H, Achouri Y,
Dubois C, Bondue A, Simons BD, Blanpain C. Early lineage restriction in temporally
distinct populations of mesp1 progenitors during mammalian heart development. Nat
Cell Biol 16: 829 – 840, 2014.
155. Li-Pook-Than J, Snyder M. Ipop goes the world: Integrated personalized omics pro-
filing and the road toward improved health care. Chem Biol 20: 660 – 666, 2013.
1122 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
156. Li C, Liakata M, Rebholz-Schuhmann D. Biological network extraction from scientific
literature: state of the art and challenges. Brief Bioinform 15: 856 – 877, 2014.
157. Li X, Heyer WD. Homologous recombination in DNA repair and DNA damage
tolerance. Cell Res 18: 99 –113, 2008.
158. Li Y, Zhang Q, Yin X, Yang W, Du Y, Hou P, Ge J, Liu C, Zhang W, Zhang X, Wu Y,
Li H, Liu K, Wu C, Song Z, Zhao Y, Shi Y, Deng H. Generation of ipscs from mouse
fibroblasts with a single gene, oct4, and small molecules. Cell Res 21: 196 –204, 2011.
159. Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM, Raval KK, Zhang J, Kamp
TJ, Palecek SP. Robust cardiomyocyte differentiation from human pluripotent stem
cells via temporal modulation of canonical wnt signaling. Proc Natl Acad Sci USA 109:
E1848 –1857, 2012.
160. Liang G, Zhang Y. Embryonic stem cell and induced pluripotent stem cell: an epige-
netic perspective. Cell Res 23: 49 – 69, 2013.
161. Liang P, Lan F, Lee AS, Gong T, Sanchez-Freire V, Wang Y, Diecke S, Sallam K,
Knowles JW, Nguyen PK, Wang PJ, Bers DM, Robbins RC, Wu JC. Drug screening
using a library of human induced pluripotent stem cell-derived cardiomyocytes re-
veals disease specific patterns of cardiotoxicity. Circulation 127: 1677–1691, 2013.
162. Limongelli G, Tome-Esteban M, Dejthevaporn C, Rahman S, Hanna MG, Elliott PM.
Prevalence and natural history of heart disease in adults with primary mitochondrial
respiratory chain disease. Eur J Heart Fail 12: 114 –121, 2010.
163. Lin B, Kim J, Li YX, Pan HY, Carvajal-Vergara X, Salama G, Cheng T, Li Y, Lo CW,
Yang L. High-purity enrichment of functional cardiovascular cells from human ips cells.
Cardiovasc Res 95: 327–335, 2012.
164. Lin B, Li Y, Han L, Kaplan AD, Ao Y, Kalra S, Bett GC, Rasmusson RL, Denning C, Yang
L. Modeling and studying mechanism of dilated cardiomyopathy using induced pluri-
potent stem cells derived from duchenne muscular dystrophy (dmd) patients. Dis
Model Mech 8: 457– 466, 2015.
165. Liu L, Li Y, Li S, Hu N, He Y, Pong R, Lin D, Lu L, Law M. Comparison of next-
generation sequencing systems. J Biomed Biotechnol 2012: 251364, 2012.
166. Lu J, Zhang F, Kay MA. A mini-intronic plasmid (mip): a novel robust transgene
expression vector in vivo and in vitro. Mol Ther 21: 954 –963, 2013.
167. Lu J, Zhang F, Xu S, Fire AZ, Kay MA. The extragenic spacer length between the 5’ and
3’ ends of the transgene expression cassette affects transgene silencing from plasmid-
based vectors. Mol Ther 20: 2111–2119, 2012.
168. Lujan E, Zunder ER, Ng YH, Goronzy IN, Nolan GP, Wernig M. Early reprogramming
regulators identified by prospective isolation and mass cytometry. Nature 521: 352–
356, 2015.
169. Luna-Zurita L, Stirnimann CU, Glatt S, Kaynak BL, Thomas S, Baudin F, Samee AH, He
D, Small EM, Mileikovsky M, Nagy A, Holloway AK, Pollard KS, CMüller CW, Bruneau
BG. Complex interdependence regulates heterotypic transcription factor distribution
and coordinates cardiogenesis. Cell 164: 999 –1014, 2016.
170. Ma D, Wei H, Lu J, Ho S, Zhang G, Sun X, Oh Y, Tan SH, Ng ML, Shim W, Wong P,
Liew R. Generation of patient-specific induced pluripotent stem cell-derived cardio-
myocytes as a cellular model of arrhythmogenic right ventricular cardiomyopathy. Eur
Heart J 34: 1122–1133, 2013.
171. Ma D, Wei H, Lu J, Huang D, Liu Z, Loh LJ, Islam O, Liew R, Shim W, Cook SA.
Characterization of a novel kcnq1 mutation for type 1 long qt syndrome and assess-
ment of the therapeutic potential of a novel iks activator using patient-specific induced
pluripotent stem cell-derived cardiomyocytes. Stem Cell Res Ther 6: 39, 2015.
172. Ma D, Wei H, Zhao Y, Lu J, Li G, Sahib NB, Tan TH, Wong KY, Shim W, Wong P, Cook
SA, Liew R. Modeling type 3 long qt syndrome with cardiomyocytes derived from
patient-specific induced pluripotent stem cells. Int J Cardiol 168: 5277–5286, 2013.
173. Ma Q, Lu AY. Pharmacogenetics, pharmacogenomics, and individualized medicine.
Pharmacol Rev 63: 437– 459, 2011.
174. Maddah M, Heidmann JD, Mandegar MA, Walker CD, Bolouki S, Conklin BR, Loewke
KE. A non-invasive platform for functional characterization of stem-cell-derived car-
diomyocytes with applications in cardiotoxicity testing. Stem Cell Reports 4: 621– 631,
2015.
175. Malan D, Zhang M, Stallmeyer B, Muller J, Fleischmann BK, Schulze-Bahr E, Sasse P,
Greber B. Human ips cell model of type 3 long qt syndrome recapitulates drug-based
phenotype correction. Basic Res Cardiol 111: 14, 2016.
 HUMAN INDUCED PLURIPOTENT STEM CELLS
176. Malik LH, Singh GD, Amsterdam EA. The epidemiology, clinical manifestations, and
management of chagas heart disease. Clin Cardiol 38: 565–569, 2014.
177. Mandal PK, Rossi DJ. Reprogramming human fibroblasts to pluripotency using modi-
fied mRNA. Nat Protoc 8: 568 –582, 2013.
178. Mardis ER. A decade’s perspective on DNA sequencing technology. Nature 470:
198 –203, 2011.
179. Maron BJ, Towbin JA, Thiene G, Antzelevitch C, Corrado D, Arnett D, Moss AJ,
Seidman CE, Young JB. Contemporary definitions and classification of the cardiomy-
opathies: An american heart association scientific statement from the council on
clinical cardiology, heart failure and transplantation committee; quality of care and
outcomes research and functional genomics and translational biology interdisciplinary
working groups; and council on epidemiology and prevention. Circulation 113: 1807–
1816, 2006.
180. Masaki H, Ishikawa T, Takahashi S, Okumura M, Sakai N, Haga M, Kominami K, Migita
H, McDonald F, Shimada F, Sakurada K. Heterogeneity of pluripotent marker gene
expression in colonies generated in human ips cell induction culture. Stem Cell Res 1:
105–115, 2007.
181. Matsa E, Burridge PW, Wu JC. Human stem cells for modeling heart disease and for
drug discovery. Sci Transl Med 6: ps6, 2014.
182. Matsa E, Denning C. In vitro uses of human pluripotent stem cell-derived cardiomy-
ocytes. J Cardiovasc Transl Res 5: 581–592, 2012.
183. Matsa E, Dixon JE, Medway C, Georgiou O, Patel MJ, Morgan K, Kemp PJ, Staniforth
A, Mellor I, Denning C. Allele-specific rna interference rescues the long-qt syndrome
phenotype in human-induced pluripotency stem cell cardiomyocytes. Eur Heart J 5:
1078 –1087, 2014.
184. Matsa E, Rajamohan D, Dick E, Young L, Mellor I, Staniforth A, Denning C. Drug
evaluation in cardiomyocytes derived from human induced pluripotent stem cells
carrying a long qt syndrome type 2 mutation. Eur Heart J 32: 952–962, 2011.
185. May JE, Xu J, Morse HR, Avent ND, Donaldson C. Toxicity testing: the search for an
in vitro alternative to animal testing. Br J Biomed Sci 66: 160 –165, 2009.
186. Mehta A, Chung YY, Ng A, Iskandar F, Atan S, Wei H, Dusting G, Sun W, Wong P,
Shim W. Pharmacological response of human cardiomyocytes derived from virus-free
induced pluripotent stem cells. Cardiovasc Res 91: 577–586, 2011.
187. Mehta A, Sequiera GL, Ramachandra CJ, Sudibyo Y, Chung Y, Sheng J, Wong KY, Tan
TH, Wong P, Liew R, Shim W. Re-trafficking of herg reverses long qt syndrome 2
phenotype in human ips-derived cardiomyocytes. Cardiovasc Res 102: 497–506, 2014.
188. Mendjan S, Mascetti VL, Ortmann D, Ortiz M, Karjosukarso DW, Ng Y, Moreau T,
Pedersen RA. Nanog and cdx2 pattern distinct subtypes of human mesoderm during
exit from pluripotency. Cell Stem Cell 15: 310 –325, 2014.
189. Mercer AC, Gaj T, Fuller RP, Barbas CF. Chimeric tale recombinases with program-
mable DNA sequence specificity. Nucleic Acids Res 40: 11163–11172, 2012.
190. Merriman B, Ion Torrent R, Team D, Rothberg JM. Progress in ion torrent semicon-
ductor chip based sequencing. Electrophoresis 33: 3397–3417, 2012.
191. Meyer T, Leisgen C, Gonser B, Gunther E. Qt-screen: high-throughput cardiac safety
pharmacology by extracellular electrophysiology on primary cardiac myocytes. Assay
Drug Dev Technol 2: 507–514, 2004.
192. Milan DJ, MacRae CA. Animal models for arrhythmias. Cardiovasc Res 67: 426 – 437,
2005.
193. Mioulane M, Foldes G, Ali NN, Schneider MD, Harding SE. Development of high
content imaging methods for cell death detection in human pluripotent stem cell-
derived cardiomyocytes. J Cardiovasc Transl Res 5: 593– 604, 2012.
194. Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, Nishikawa S, Tane-
mura M, Mimori K, Tanaka F, Saito T, Nishimura J, Takemasa I, Mizushima T, Ikeda M,
Yamamoto H, Sekimoto M, Doki Y, Mori M. Reprogramming of mouse and human
cells to pluripotency using mature microRNAs. Cell Stem Cell 8: 633– 638, 2011.
195. Moore GE. Cramming more components onto integrated circuits. Electronics 38:
114 –117, 1965.
196. Mordwinkin NM, Lee AS, Wu JC. Patient-specific stem cells and cardiovascular drug
discovery. JAMA 310: 2039 –2040, 2013.
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
197. Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flugel L, Dorn T, Goedel A,
Hohnke C, Hofmann F, Seyfarth M, Sinnecker D, Schomig A, Laugwitz KL. Patient-
specific induced pluripotent stem-cell models for long-qt syndrome. N Engl J Med 363:
1397–1409, 2010.
198. Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, de Ferranti S,
Despres JP, Fullerton HJ, Howard VJ, Huffman MD, Judd SE, Kissela BM, Lackland DT,
Lichtman JH, Lisabeth LD, Liu S, Mackey RH, Matchar DB, McGuire DK, Mohler ER,
3rd Moy CS, Muntner P, Mussolino ME, Nasir K, Neumar RW, Nichol G, Palaniappan
L, Pandey DK, Reeves MJ, Rodriguez CJ, Sorlie PD, Stein J, Towfighi A, Turan TN,
Virani SS, Willey JZ, Woo D, Yeh RW, Turner MB, American Heart Association. Heart
disease and stroke statistics–2015 update: a report from the American Heart Asso-
ciation. Circulation 131: e29 –322, 2015.
199. Mussolino C, Mlambo T, Cathomen T. Proven and novel strategies for efficient editing
of the human genome. Curr Opin Pharmacol 24: 105–112, 2015.
200. Mwinyi J, Johne A, Bauer S, Roots I, Gerloff T. Evidence for inverse effects of oatp-c
(slc21a6) 5 and 1b haplotypes on pravastatin kinetics. Clin Pharmacol Ther 75: 415–
421, 2004.
201. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K,
Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells
without myc from mouse and human fibroblasts. Nat Biotechnol 26: 101–106, 2008.
202. Narsinh KH, Jia F, Robbins RC, Kay MA, Longaker MT, Wu JC. Generation of adult
human induced pluripotent stem cells using nonviral minicircle DNA vectors. Nat
Protoc 6: 78 – 88, 2011.
203. Navarrete EG, Liang P, Lan F, Sanchez-Freire V, Simmons C, Gong T, Sharma A,
Burridge PW, Patlolla B, Lee AS, Wu H, Beygui RE, Wu SM, Robbins RC, Bers DM, Wu
JC. Screening drug-induced arrhythmia [corrected] using human induced pluripotent
stem cell-derived cardiomyocytes and low-impedance microelectrode arrays. Circu-
lation 128 Suppl 1: S3–13, 2013.
204. Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M. Crispr-cas9-based photo-
activatable transcription system. Chem Biol 22: 169 –174, 2014.
205. Nimura K, Ura K, Shiratori H, Ikawa M, Okabe M, Schwartz RJ, Kaneda Y. A histone
h3 lysine 36 trimethyltransferase links nkx2-5 to wolf-hirschhorn syndrome. Nature
460: 287–291, 2009.
206. Novak A, Barad L, Lorber A, Gherghiceanu M, Reiter I, Eisen B, Eldor L, Itskovitz-
Eldor J, Eldar M, Arad M, Binah O. Functional abnormalities in ipsc-derived cardio-
myocytes generated from cpvt1 and cpvt2 patients carrying ryanodine or calseques-
trin mutations. J Cell Mol Med 19: 2006 –2018, 2014.
207. Novak A, Barad L, Zeevi-Levin N, Shick R, Shtrichman R, Lorber A, Itskovitz-Eldor J,
Binah O. Cardiomyocytes generated from cpvtd307h patients are arrhythmogenic in
response to beta-adrenergic stimulation. J Cell Mol Med 16: 468 – 482, 2012.
208. Nunes SS, Miklas JW, Liu J, Aschar-Sobbi R, Xiao Y, Zhang B, Jiang J, Masse S, Gagliardi
M, Hsieh A, Thavandiran N, Laflamme MA, Nanthakumar K, Gross GJ, Backx PH,
Keller G, Radisic M. Biowire: a platform for maturation of human pluripotent stem
cell-derived cardiomyocytes. Nat Methods 10: 781–787, 2013.
209. Okita K, Yamakawa T, Matsumura Y, Sato Y, Amano N, Watanabe A, Goshima N,
Yamanaka S. An efficient nonviral method to generate integration-free human-in-
duced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells
31: 458 – 466, 2013.
210. Olivotto I, d’Amati G, Basso C, Van Rossum A, Patten M, Emdin M, Pinto Y, Tomberli
B, Camici PG, Michels M. Defining phenotypes and disease progression in sarcomeric
cardiomyopathies: Contemporary role of clinical investigations. Cardiovasc Res 105:
409 – 423, 2015.
212. Paige SL, Osugi T, Afanasiev OK, Pabon L, Reinecke H, Murry CE. Endogenous
wnt/beta-catenin signaling is required for cardiac differentiation in human embryonic
stem cells. PLoS One 5: e11134, 2010.
213. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan
C, Hochedlinger K, Daley GQ. Disease-specific induced pluripotent stem cells. Cell
134: 877– 886, 2008.
214. Pattanayak V, Guilinger JP, Liu DR. Determining the specificities of talens, cas9, and
other genome-editing enzymes. Methods Enzymol 546: 47–78, 2014.
215. Paull D, Sevilla A, Zhou H, Hahn AK, Kim H, Napolitano C, Tsankov A, Shang L,
Krumholz K, Jagadeesan P, Woodard CM, Sun B, Vilboux T, Zimmer M, Forero E,
1123
 MATSA ET AL.
Moroziewicz DN, Martinez H, Malicdan MC, Weiss KA, Vensand LB, Dusenberry CR,
Polus H, Sy KT, Kahler DJ, Gahl WA, Solomon SL, Chang S, Meissner A, Eggan K,
Noggle SA. Automated, high-throughput derivation, characterization and differentia-
tion of induced pluripotent stem cells. Nat Methods 12: 885– 892, 2015.
216. Pereira GC, Silva AM, Diogo CV, Carvalho FS, Monteiro P, Oliveira PJ. Drug-induced
cardiac mitochondrial toxicity and protection: from doxorubicin to carvedilol. Curr
Pharm Des 17: 2113–2129, 2011.
217. Phan D, Rasmussen TL, Nakagawa O, McAnally J, Gottlieb PD, Tucker PW, Richard-
son JA, Bassel-Duby R, Olson EN. Bop, a regulator of right ventricular heart develop-
ment, is a direct transcriptional target of mef2c in the developing heart. Development
132: 2669 –2678, 2005.
218. Pouton CW, Haynes JM. Embryonic stem cells as a source of models for drug discov-
ery. Nat Rev Drug Discov 6: 605– 616, 2007.
219. Pretorius RW, Gataric G, Swedlund SK, Miller JR. Reducing the risk of adverse drug
events in older adults. American Family Physician 87: 329 –334, 2013.
220. Price PS, Keenan RE, Swartout JC. Characterizing interspecies uncertainty using data
from studies of anti-neoplastic agents in animals and humans. Toxicol Appl Pharmacol
233: 64 –70, 2008.
221. Puri MC, Nagy A. Concise review: embryonic stem cells versus induced pluripotent
stem cells: the game is on. Stem Cells 30: 10 – 4, 2012.
222. Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR, Bertoni A, Swerd-
low HP, Gu Y. A tale of three next generation sequencing platforms: Comparison of
ion torrent, pacific biosciences and illumina miseq sequencers. BMC Genomics 13: 341,
2012.
223. Quigley EM. Cisapride: what can we learn from the rise and fall of a prokinetic? J Dig
Dis 12: 147–156, 2011.
224. Raha D, Hong M, Snyder M. Chip-seq: a method for global identification of regulatory
elements in the genome. Curr Protoc Mol Biol 21: 1–14, 2010.
225. Rais Y, Zviran A, Geula S, Gafni O, Chomsky E, Viukov S, Mansour AA, Caspi I,
Krupalnik V, Zerbib M, Maza I, Mor N, Baran D, Weinberger L, Jaitin DA, Lara-Astiaso
D, Blecher-Gonen R, Shipony Z, Mukamel Z, Hagai T, Gilad S, Amann-Zalcenstein D,
Tanay A, Amit I, Novershtern N, Hanna JH. Deterministic direct reprogramming of
somatic cells to pluripotency. Nature 502: 65–70, 2013.
226. Rajamohan D, Matsa E, Kalra S, Crutchley J, Patel A, George V, Denning C. Current
status of drug screening and disease modelling in human pluripotent stem cells. Bioes-
says 35: 281–298, 2013.
227. Raval KK, Tao R, White BE, De Lange WJ, Koonce CH, Yu J, Kishnani PS, Thomson JA,
Mosher DF, Ralphe JC, Kamp TJ. Pompe disease results in a golgi-based glycosylation
deficit in human induced pluripotent stem cell-derived cardiomyocytes. J Biol Chem
290: 3121–3136, 2015.
228. Reuter Jason A, Spacek DV, Snyder MP. High-throughput sequencing technologies.
Mol Cell 58: 586 –597, 2015.
229. Rhoads A, Au KF. Pacbio sequencing and its applications. Genomics Proteomics Bioin-
formatics 13: 278 –289, 2015.
230. Rossi D, Rasi S, Franceschetti S, Capello D, Castelli A, De Paoli L, Ramponi A, Chiap-
pella A, Pogliani EM, Vitolo U, Kwee I, Bertoni F, Conconi A, Gaidano G. Analysis of
the host pharmacogenetic background for prediction of outcome and toxicity in
diffuse large b-cell lymphoma treated with r-chop21. Leukemia 23: 1118 –1126, 2009.
231. Rouhani F, Kumasaka N, de Brito MC, Bradley A, Vallier L, Gaffney D. Genetic
background drives transcriptional variation in human induced pluripotent stem cells.
PLoS Genet 10: e1004432, 2014.
232. Sallam K, Li Y, Sager PT, Houser SR, Wu JC. Finding the rhythm of sudden cardiac
death: New opportunities using induced pluripotent stem cell-derived cardiomyo-
cytes. Circ Res 116: 1989 –2004, 2015.
232a.Sanchez-Freire V, Lee AS, Hu S, Abilez OJ, Liang P, Lan F, Huber BC, Ong SG, Hong
WX, Huang M, Wu JC. Effect of human donor cell source on differentiation and
function of cardiac induced pluripotent stem cells. Am J Cell Cardiol 64: 436 – 448,
2014.
233. Sato Y, Kobayashi H, Higuchi T, Shimada Y, Era T, Kimura S, Eto Y, Ida H, Ohashi T.
Disease modeling and lentiviral gene transfer in patient-specific induced pluripotent
stem cells from late-onset pompe disease patient. Mol Ther 2: 15023, 2015.
1124 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
233a.Sayed N, Liu C, Wu JC. Translation of human-induced pluripotent stem cells: from
clinical trial in a dish to precision medicine. J Am Coll Cardiol 67: 2161–2176, 2016.
234. Schmid-Burgk JL, Schmidt T, Kaiser V, Honing K, Hornung V. A ligation-independent
cloning technique for high-throughput assembly of transcription activator-like effec-
tor genes. Nat Biotechnol 31: 76 – 81, 2013.
235. Schroeder M, Niebruegge S, Werner A, Willbold E, Burg M, Ruediger M, Field LJ,
Lehmann J, Zweigerdt R. Differentiation and lineage selection of mouse embryonic
stem cells in a stirred bench scale bioreactor with automated process control. Bio-
technol Bioeng 92: 920 –933, 2005.
237. Sesti F, Abbott GW, Wei J, Murray KT, Saksena S, Schwartz PJ, Priori SG, Roden DM,
George AL Jr, Goldstein SA. A common polymorphism associated with antibiotic-
induced cardiac arrhythmia. Proc Natl Acad Sci USA 97: 10613–10618, 2000.
238. Shanks N, Greek R, Greek J. Are animal models predictive for humans? PEHM 4: 2,
2009.
239. Sharma A, Marceau C, Hamaguchi R, Burridge PW, Rajarajan K, Churko JM, Wu H,
Sallam KI, Matsa E, Sturzu AC, Che Y, Ebert A, Diecke S, Liang P, Red-Horse K,
Carette JE, Wu SM, Wu JC. Human induced pluripotent stem cell-derived cardiomy-
ocytes as an in vitro model for coxsackievirus b3-induced myocarditis and antiviral
drug screening platform. Circ Res 115: 556 –566, 2014.
240. Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang
X, Skarnes WC. Efficient genome modification by crispr-cas9 nickase with minimal
off-target effects. Nat Methods 11: 399 – 402, 2014.
241. Shirai M, Osugi T, Koga H, Kaji Y, Takimoto E, Komuro I, Hara J, Miwa T, Yamauchi-
Takihara K, Takihara Y. The polycomb-group gene rae28 sustains nkx2.5/csx expres-
sion and is essential for cardiac morphogenesis. J Clin Invest 110: 177–184, 2002.
242. Sirenko O, Crittenden C, Callamaras N, Hesley J, Chen YW, Funes C, Rusyn I, Anson
B, Cromwell EF. Multiparameter in vitro assessment of compound effects on cardi-
omyocyte physiology using ipsc cells. J Biomol Screen 18: 39 –53, 2013.
243. Siu CW, Lee YK, Ho JC, Lai WH, Chan YC, Ng KM, Wong LY, Au KW, Lau YM, Zhang
J, Lay KW, Colman A, Tse HF. Modeling of lamin a/c mutation premature cardiac aging
using patient-specific induced pluripotent stem cells. Aging 4: 803– 822, 2012.
244. Smith C, Gore A, Yan W, Abalde-Atristain L, Li Z, He C, Wang Y, Brodsky RA, Zhang
K, Cheng L, Ye Z. Whole-genome sequencing analysis reveals high specificity of
crispr/cas9 and talen-based genome editing in human ipscs. Cell Stem Cell 15: 12–13,
2014.
245. Smith JG, Celiz AD, Patel AK, Short RD, Alexander MR, Denning C. Scaling human
pluripotent stem cell expansion and differentiation: Are cell factories becoming a
reality? Regen Med 10: 925–930, 2015.
246. Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, Hargus G, Blak A,
Cooper O, Mitalipova M, Isacson O, Jaenisch R. Parkinson’s disease patient-derived
induced pluripotent stem cells free of viral reprogramming factors. Cell 136: 964 –977,
2009.
247. Solloway MJ, Harvey RP. Molecular pathways in myocardial development: A stem cell
perspective. Cardiovasc Res 58: 264 –277, 2003.
248. Spears DA, Gollob MH. Genetics of inherited primary arrhythmia disorders. Appl Clin
Genet 8: 215–233, 2015.
249. Spies D, Ciaudo C. Dynamics in transcriptomics: advancements in rna-seq time
course and downstream analysis. Comput Struct Biotechnol J 13: 469 – 477, 2015.
250. Stadtfeld M, Hochedlinger K. Induced pluripotency: History, mechanisms, applica-
tions. Genes Dev 24: 2239 –2263, 2010.
251. Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. Induced pluripotent stem
cells generated without viral integration. Science 322: 945–949, 2008.
252. Steiner D, Khaner H, Cohen M, Even-Ram S, Gil Y, Itsykson P, Turetsky T, Idelson M,
Aizenman E, Ram R, Berman-Zaken Y, Reubinoff B. Derivation, propagation and
controlled differentiation of human embryonic stem cells in suspension. Nat Biotech-
nol 28: 361–364, 2010.
253. Storchova Z, Kuffer C. The consequences of tetraploidy and aneuploidy. J Cell Sci 121:
3859 –3866, 2008.
254. Sun N, Yazawa M, Liu J, Han L, Sanchez-Freire V, Abilez OJ, Navarrete EG, Hu S,
Wang L, Lee A, Pavlovic A, Lin S, Chen R, Hajjar RJ, Snyder MP, Dolmetsch RE, Butte
 HUMAN INDUCED PLURIPOTENT STEM CELLS
MJ, Ashley EA, Longaker MT, Robbins RC, Wu JC. Patient-specific induced pluripo-
tent stem cells as a model for familial dilated cardiomyopathy. Sci Transl Med 4:
130ra47, 2012.
255. Suzuki K, Yu C, Qu J, Li M, Yao X, Yuan T, Goebl A, Tang S, Ren R, Aizawa E, Zhang
F, Xu X, Rupa Soligalla D, Chen F, Kim J, Na Kim Y, Liao HK, Benner C, Concepcion
Esteban R, Jin Y, Liu GH, Li Y, Izpisua Belmonte JC. Targeted gene correction mini-
mally impacts whole-genome mutational load in human-disease-specific induced plu-
ripotent stem cell clones. Cell Stem Cell 15: 31–36, 2014.
256. Swanton C, Nicke B, Schuett M, Eklund AC, Ng C, Li Q, Hardcastle T, Lee A, Roy R,
East P, Kschischo M, Endesfelder D, Wylie P, Kim SN, Chen JG, Howell M, Ried T,
Habermann JK, Auer G, Brenton JD, Szallasi Z, Downward J. Chromosomal instability
determines taxane response. Proc Natl Acad Sci USA 106: 8671– 8676, 2009.
257. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S.
Induction of pluripotent stem cells from adult human fibroblasts by defined factors.
Cell 131: 861– 872, 2007.
259. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic
and adult fibroblast cultures by defined factors. Cell 126: 663– 676, 2006.
260. Takaya T, Kawamura T, Morimoto T, Ono K, Kita T, Shimatsu A, Hasegawa K.
Identification of p300-targeted acetylated residues in gata4 during hypertrophic re-
sponses in cardiac myocytes. J Biol Chem 283: 9828 –9835, 2008.
261. Tam PP, Loebel DA. Gene function in mouse embryogenesis: get set for gastrulation.
Nat Rev Genet 8: 368 –381, 2007.
262. Tanaka A, Yuasa S, Mearini G, Egashira T, Seki T, Kodaira M, Kusumoto D, Kuroda Y,
Okata S, Suzuki T, Inohara T, Arimura T, Makino S, Kimura K, Kimura A, Furukawa T,
Carrier L, Node K, Fukuda K. Endothelin-1 induces myofibrillar disarray and contrac-
tile vector variability in hypertrophic cardiomyopathy-induced pluripotent stem cell-
derived cardiomyocytes. J Am Heart Assoc 3: e001263, 2014.
263. Tanwar V, Bylund JB, Hu J, Yan J, Walthall JM, Mukherjee A, Heaton WH, Wang WD,
Potet F, Rai M, Kupershmidt S, Knapik EW, Hatzopoulos AK. Gremlin 2 promotes
differentiation of embryonic stem cells to atrial fate by activation of the jnk signaling
pathway. Stem Cells 32: 1774 –1788, 2014.
264. Terrenoire C, Wang K, Tung KW, Chung WK, Pass RH, Lu JT, Jean JC, Omari A,
Sampson KJ, Kotton DN, Keller G, Kass RS. Induced pluripotent stem cells used to
reveal drug actions in a long qt syndrome family with complex genetics. J Gen Physiol
141: 61–72, 2013.
265. Tewey KM, Chen GL, Nelson EM, Liu LF. Intercalative antitumor drugs interfere with
the breakage-reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259:
9182–9187, 1984.
266. Thavandiran N, Dubois N, Mikryukov A, Masse S, Beca B, Simmons CA, Deshpande
VS, McGarry JP, Chen CS, Nanthakumar K, Keller GM, Radisic M, Zandstra PW.
Design and formulation of functional pluripotent stem cell-derived cardiac microtis-
sues. Proc Natl Acad Sci USA 110: E4698 – 4707, 2013.
267. Thomas RJ, Anderson D, Chandra A, Smith NM, Young LE, Williams D, Denning C.
Automated, scalable culture of human embryonic stem cells in feeder-free conditions.
Biotechnol Bioeng 102: 1636 –1644, 2009.
268. Thompson JF, Steinmann KE. Single molecule sequencing with a heliscope genetic
analysis system. Curr Protoc Mol Biol 7: 10, 2010.
269. Thompson R, Drew CJ, Thomas RH. Next generation sequencing in the clinical
domain: clinical advantages, practical, and ethical challenges. Adv Protein Chem Struct
Biol 89: 27– 63, 2012.
270. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS,
Jones JM. Embryonic stem cell lines derived from human blastocysts. Science 282:
1145–1147, 1998.
271. Thorn CF, Oshiro C, Marsh S, Hernandez-Boussard T, McLeod H, Klein TE, Altman
RB. Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenet
Genomics 21: 440 – 446, 2011.
272. Tohyama S, Hattori F, Sano M, Hishiki T, Nagahata Y, Matsuura T, Hashimoto H,
Suzuki T, Yamashita H, Satoh Y, Egashira T, Seki T, Muraoka N, Yamakawa H, Ohgino
Y, Tanaka T, Yoichi M, Yuasa S, Murata M, Suematsu M, Fukuda K. Distinct metabolic
flow enables large-scale purification of mouse and human pluripotent stem cell-de-
rived cardiomyocytes. Cell Stem Cell 12: 127–137, 2013.
Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
273. Tomlinson B, Hu M, Lee VW, Lui SS, Chu TT, Poon EW, Ko GT, Baum L, Tam LS, Li
EK. Abcg2 polymorphism is associated with the low-density lipoprotein cholesterol
response to rosuvastatin. Clin Pharmacol Ther 87: 558 –562, 2010.
274. Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee
MJ, Joung JK. Dimeric crispr rna-guided foki nucleases for highly specific genome
editing. Nat Biotech 32: 569 –576, 2014.
275. Tse HF, Ho JC, Choi SW, Lee YK, Butler AW, Ng KM, Siu CW, Simpson MA, Lai WH,
Chan YC, Au KW, Zhang J, Lay KW, Esteban MA, Nicholls JM, Colman A, Sham PC.
Patient-specific induced-pluripotent stem cells-derived cardiomyocytes recapitulate
the pathogenic phenotypes of dilated cardiomyopathy due to a novel des mutation
identified by whole exome sequencing. Hum Mol Genet 22: 1395–1403, 2013.
276. Tzatzalos E, Abilez OJ, Shukla P, Wu JC. Engineered heart tissues and induced pluri-
potent stem cells: macro- and microstructures for disease modeling, drug screening,
and translational studies. Adv Drug Deliv Rev 15: 213–216, 2015.
277. Uosaki H, Fukushima H, Takeuchi A, Matsuoka S, Nakatsuji N, Yamanaka S, Ya-
mashita JK. Efficient and scalable purification of cardiomyocytes from human embry-
onic and induced pluripotent stem cells by vcam1 surface expression. PLoS One 6:
e23657, 2011.
278. Vaissiere T, Sawan C, Herceg Z. Epigenetic interplay between histone modifications
and DNA methylation in gene silencing. Mutat Res 659: 40 – 48, 2008.
279. Van Dijk EL, Auger H, Jaszczyszyn Y, Thermes C. Ten years of next-generation
sequencing technology. Trends Genet 30: 418 – 426, 2014.
280. Van Hoof D, Dormeyer W, Braam SR, Passier R, Monshouwer-Kloots J, Ward-van
Oostwaard D, Heck AJ, Krijgsveld J, Mummery CL. Identification of cell surface
proteins for antibody-based selection of human embryonic stem cell-derived cardio-
myocytes. J Proteome Res 9: 1610 –1618, 2010.
281. Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Talkowski ME,
Musunuru K. Low incidence of off-target mutations in individual crispr-cas9 and talen
targeted human stem cell clones detected by whole-genome sequencing. Cell Stem
Cell 15: 27–30, 2014.
282. Vicini P, Fields O, Lai E, Litwack ED, Martin AM, Morgan TM, Pacanowski MA,
Papaluca M, Perez OD, Ringel MS, Robson M, Sakul H, Vockley J, Zaks T, Dolsten M,
Sogaard M. Precision medicine in the age of big data: The present and future role of
large scale unbiased sequencing in drug discovery and development. Clin Pharmacol
Ther 99: 198 –207, 2016.
283. Visscher H, Ross CJ, Rassekh SR, Barhdadi A, Dube MP, Al-Saloos H, Sandor GS,
Caron HN, van Dalen EC, Kremer LC, van der Pal HJ, Brown AM, Rogers PC, Phillips
MS, Rieder MJ, Carleton BC, Hayden MR, Canadian Pharmacogenomics Network for
Drug Safety. Pharmacogenomic prediction of anthracycline-induced cardiotoxicity in
children. J Clin Oncol 30: 1422–1428, 2012.
284. Visscher H, Ross CJD, Rassekh SR, Sandor GSS, Caron HN, van Dalen EC, Kremer
LC, van der Pal HJ, Rogers PC, Rieder MJ, Carleton BC, Hayden MR. Validation of
variants in slc28a3 and ugt1a6 as genetic markers predictive of anthracycline-induced
cardiotoxicity in children. Pediatr Blood Cancer 60: 1375–1381, 2013.
285. Volkova M, Russell R 3rd. Anthracycline cardiotoxicity: Prevalence, pathogenesis and
treatment. Curr Cardiol Rev 7: 214 –220, 2011.
286. Wall JD, Tang LF, Zerbe B, Kvale MN, Kwok PY, Schaefer C, Risch N. Estimating
genotype error rates from high-coverage next-generation sequence data. Genome Res
24: 1734 –1739, 2014.
287. Wandelt S, Bux M, Leser U. Trends in genome compression. Curr Bioinformatics 9:
315–326, 2014.
288. Wandelt S, Rheinländer A, Bux M, Thalheim L, Haldemann B, Leser U. Data manage-
ment challenges in next generation sequencing. Datenbank-Spektrum 12: 161–171,
2012.
289. Wang G, McCain ML, Yang L, He A, Pasqualini FS, Agarwal A, Yuan H, Jiang D, Zhang
D, Zangi L, Geva J, Roberts AE, Ma Q, Ding J, Chen J, Wang DZ, Li K, Wang J, Wanders
RJ, Kulik W, Vaz FM, Laflamme MA, Murry CE, Chien KR, Kelley RI, Church GM,
Parker KK, Pu WT. Modeling the mitochondrial cardiomyopathy of barth syndrome
with induced pluripotent stem cell and heart-on-chip technologies. Nat Med 20:
616 – 623, 2014.
290. Wang Y, Liang P, Lan F, Wu H, Lisowski L, Gu M, Hu S, Kay MA, Urnov FD, Shinnawi
R, Gold JD, Gepstein L, Wu JC. Genome editing of isogenic human induced pluripo-
1125
 MATSA ET AL.
tent stem cells recapitulates long qt phenotype for drug testing. J Am Coll Cardiol 64:
451– 459, 2014.
291. Wang Z. An integrated chip for the high-throughput synthesis of transcription activa-
tor-like effectors. Angew Chem Int Ed Engl 51: 8505– 8508, 2012.
292. Wang Z, Oron E, Nelson B, Razis S, Ivanova N. Distinct lineage specification roles for
nanog, oct4, and sox2 in human embryonic stem cells. Cell Stem Cell 10: 440 –554,
2012.
293. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD,
Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ. Highly
efficient reprogramming to pluripotency and directed differentiation of human cells
with synthetic modified mRNA. Cell Stem Cell 7: 618 – 630, 2010.
294. Warren L, Ni Y, Wang J, Guo X. Feeder-free derivation of human induced pluripotent
stem cells with messenger RNA. Sci Rep 2: 657, 2012.
295. Watanabe Y, Zaffran S, Kuroiwa A, Higuchi H, Ogura T, Harvey RP, Kelly RG, Buck-
ingham M. Fibroblast growth factor 10 gene regulation in the second heart field by
tbx1, nkx2-5, and islet1 reveals a genetic switch for down-regulation in the myocar-
dium. Proc Natl Acad Sci USA 109: 18273–18280, 2012.
296. Weng Z, Kong CW, Ren L, Karakikes I, Geng L, He J, Chow MZ, Mok CF, Keung W,
Chow H, Leung AY, Hajjar RJ, Li RA, Chan CW. A simple, cost-effective but highly
efficient system for deriving ventricular cardiomyocytes from human pluripotent stem
cells. Stem Cells Dev 23: 1704 –1716, 2014.
297. Wicks EC, Elliott PM. Genetics and metabolic cardiomyopathies. Herz 37: 598 – 610,
2012.
298. Willmann CA, Hemeda H, Pieper LA, Lenz M, Qin J, Joussen S, Sontag S, Wanek P,
Denecke B, Schuler HM, Zenke M, Wagner W. To clone or not to clone? Induced
pluripotent stem cells can be generated in bulk culture. PLoS One 8: e65324, 2014.
298a.Wilson KD, Shen P, Fung E, Karakikes I, Zhang A, InanlooRahatloo K, Odegaard J,
Sallam K, Davis RW, Lui GK, Ashley EA, Scharfe C, Wu JC. A rapid, high-quality,
cost-effective, comprehensive and expandable targeted next generation sequencing
assay for inherited heart diseases. Circ Res 117: 603– 611, 2015.
299. Wilson KD, Wu JC. Induced pluripotent stem cells. JAMA 313: 1613–1614, 2015.
300. Wojnowski L, Kulle B, Schirmer M, Schluter G, Schmidt A, Rosenberger A, Vonhof S,
Bickeboller H, Toliat MR, Suk EK, Tzvetkov M, Kruger A, Seifert S, Kloess M, Hahn H,
Loeffler M, Nurnberg P, Pfreundschuh M, Trumper L, Brockmoller J, Hasenfuss G.
Nad(p)h oxidase and multidrug resistance protein genetic polymorphisms are associ-
ated with doxorubicin-induced cardiotoxicity. Circulation 112: 3754 –3762, 2005.
300a.World Health Organization. World Health Statistics Report 2008. Geneva, Switzerland:
WHO, 2008.
301. Wu H, Lee J, Vincent LG, Wang Q, Gu M, Lan F, Churko JM, Sallam KI, Matsa E,
Sharma A, Gold JD, Engler AJ, Xiang YK, Bers DM, Wu JC. Epigenetic regulation of
phosphodiesterases 2a and 3a underlies compromised beta-adrenergic signaling in an
ipsc model of dilated cardiomyopathy. Cell Stem Cell 17: 89 –100, 2015.
302. Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD, Dadon DB, Cheng AW, Trevino AE,
Konermann S, Chen S, Jaenisch R, Zhang F, Sharp PA. Genome-wide binding of the
crispr endonuclease cas9 in mammalian cells. Nat Biotechnol 32: 670 – 676, 2014.
303. Wu Y, Gao T, Wang X, Hu Y, Hu X, Hu Z, Pang J, Li Z, Xue J, Feng M, Wu L, Liang D.
Tale nickase mediates high efficient targeted transgene integration at the human
multi-copy ribosomal DNA locus. Biochem Biophys Res Commun 446: 261–266, 2014.
304. Wyles SP, Li X, Hrstka SC, Reyes S, Oommen S, Beraldi R, Edwards J, Terzic A, Olson
TM, Nelson TJ. Modeling structural and functional deficiencies of rbm20 familial
dilated cardiomyopathy using human induced pluripotent stem cells. Hum Mol Genet
25: 254 –265, 2016.
305. Xu C, Police S, Rao N, Carpenter MK. Characterization and enrichment of cardio-
myocytes derived from human embryonic stem cells. Circ Res 91: 501–508, 2002.
306. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step generation of
mice carrying reporter and conditional alleles by crispr/cas-mediated genome engi-
neering. Cell 154: 1370 –1379, 2013.
307. Yang L, Soonpaa MH, Adler ED, Roepke TK, Kattman SJ, Kennedy M, Henckaerts E,
Bonham K, Abbott GW, Linden RM, Field LJ, Keller GM. Human cardiovascular
1126 Physiol Rev • VOL 96 • JULY 2016 • www.prv.org
Downloaded from www.physiology.org/journal/physrev (031.223.142.132) on November 16, 2019.
progenitor cells develop from a kdr⫹ embryonic-stem-cell-derived population. Na-
ture 453: 524 –528, 2008.
308. Yang L, Yang JL, Byrne S, Pan J, Church GM. Crispr/cas9-directed genome editing of
cultured cells. Curr Protoc Mol Biol 107: 31 1 1 1 17524 –31, 2014.
309. Yang X, Pabon L, Murry CE. Engineering adolescence: maturation of human pluripo-
tent stem cell-derived cardiomyocytes. Circ Res 114: 511–523, 2014.
310. Yasuno T, Osafune K, Sakurai H, Asaka I, Tanaka A, Yamaguchi S, Yamada K, Hitomi
H, Arai S, Kurose Y, Higaki Y, Sudo M, Ando S, Nakashima H, Saito T, Kaneoka H.
Functional analysis of ipsc-derived myocytes from a patient with carnitine palmitoyl-
transferase II deficiency. Biochem Biophys Res Commun 448: 175–181, 2014.
311. Yazawa M, Hsueh B, Jia X, Pasca AM, Bernstein JA, Hallmayer J, Dolmetsch RE. Using
induced pluripotent stem cells to investigate cardiac phenotypes in timothy syn-
drome. Nature 471: 230 –234, 2011.
312. Yazawa M, Hsueh B, Jia X, Pasca AM, Bernstein JA, Hallmayer J, Dolmetsch RE. Using
induced pluripotent stem cells to investigate cardiac phenotypes in timothy syn-
drome. Nature 471: 230 –234, 2011.
313. Yip VL, Pirmohamed M. Expanding role of pharmacogenomics in the management of
cardiovascular disorders. Am J Cardiovasc Drugs 13: 151–162, 2013.
314. Yokoo N, Baba S, Kaichi S, Niwa A, Mima T, Doi H, Yamanaka S, Nakahata T, Heike
T. The effects of cardioactive drugs on cardiomyocytes derived from human induced
pluripotent stem cells. Biochem Biophys Res Commun 387: 482– 488, 2009.
315. Yoshioka N, Gros E, Li HR, Kumar S, Deacon DC, Maron C, Muotri AR, Chi NC, Fu
XD, Yu BD, Dowdy SF. Efficient generation of human ipscs by a synthetic self-
replicative RNA. Cell Stem Cell 13: -2462–54, 2013.
316. Yu J, Chau KF, Vodyanik MA, Jiang J, Jiang Y. Efficient feeder-free episomal repro-
gramming with small molecules. PLoS One 6: e17557, 2011.
317. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin, II, Thomson JA. Human induced pluri-
potent stem cells free of vector and transgene sequences. Science 324: 797–801, 2009.
318. Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA. Human induced pluri-
potent stem cells free of vector and transgene sequences. Science 324: 797–801, 2009.
319. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J,
Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Induced pluripotent stem
cell lines derived from human somatic cells. Science 318: 1917–1920,
2007.
320. Zeevi-Levin N, Itskovitz-Eldor J, Binah O. Cardiomyocytes derived from human plu-
ripotent stem cells for drug screening. Pharmacol Ther 134: 180 –188, 2012.
321. Zhang J, Klos M, Wilson GF, Herman AM, Lian X, Raval KK, Barron MR, Hou L,
Soerens AG, Yu J, Palecek SP, Lyons GE, Thomson JA, Herron TJ, Jalife J, Kamp TJ.
Extracellular matrix promotes highly efficient cardiac differentiation of human
pluripotent stem cells: the matrix sandwich method. Circ Res 111: 1125–1136,
2012.
322. Zhang XH, Haviland S, Wei H, Saric T, Fatima A, Hescheler J, Cleemann L, Morad M.
Ca2⫹ signaling in human induced pluripotent stem cell-derived cardiomyocytes (ips-
cm) from normal and catecholaminergic polymorphic ventricular tachycardia (cpvt)-
afflicted subjects. Cell Calcium 54: 57–70, 2013.
323. Zhi D, Irvin MR, Gu CC, Stoddard AJ, Lorier R, Matter A, Rao DC, Srinivasasai-
nagendra V, Tiwari HK, Turner A, Broeckel U, Arnett DK. Whole-exome sequencing
and an ipsc-derived cardiomyocyte model provides a powerful platform for gene
discovery in left ventricular hypertrophy. Front Genet 3: 92, 2012.
324. Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuzdak
G, Scholer HR, Duan L, Ding S. Generation of induced pluripotent stem cells using
recombinant proteins. Cell Stem Cell 4: 381–384, 2009.
325. Zhou W, Freed CR. Adenoviral gene delivery can reprogram human fibroblasts to
induced pluripotent stem cells. Stem Cells 27: 2667–2674, 2009.
326. Zhu WZ, Xie Y, Moyes KW, Gold JD, Askari B, Laflamme MA. Neuregulin/erbb
signaling regulates cardiac subtype specification in differentiating human embryonic
stem cells. Circ Res 107: 776 –786, 2010.
327. Zunder ER, Lujan E, Goltsev Y, Wernig M, Nolan GP. A continuous molecular road-
map to ipsc reprogramming through progression analysis of single-cell mass cytom-
etry. Cell Stem Cell 16: 323–337, 2015.