Transcriptional and Functional Profiling of Human
Embryonic Stem Cell-Derived Cardiomyocytes
Feng Cao1., Roger A. Wagner2., Kitchener D. Wilson1,3., Xiaoyan Xie1, Ji-Dong Fu6, Micha Drukker4,
Andrew Lee1, Ronald A. Li6, Sanjiv S. Gambhir1,3, Irving L. Weissman4, Robert C. Robbins5, Joseph C.
1Department of Radiology, Stanford University School of Medicine, Stanford, California, United States of America, 2Department of Medicine (Division of Cardiology),
Stanford University School of Medicine, Stanford, California, United States of America, 3Department of Bioengineering, Stanford University School of Medicine, Stanford,
California, United States of America, 4Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, United States of America,
5Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California, United States of America, 6Stem Cell Program and Department of
Cell Biology and Human Anatomy, University of California Davis, Davis, California, United States of America
Human embryonicstemcells(hESCs)canserveasapotentiallylimitlesssourceof cellsthatmayenableregeneration ofdiseased
recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM
transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs
expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good
source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8
weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track
cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation
enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies
such as this will serve as the foundation for future clinical applications of stem cell therapies.
Citation: Cao F, Wagner RA, Wilson KD, Xie X, Fu J-D, et al. (2008) Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived
Cardiomyocytes. PLoS ONE 3(10): e3474. doi:10.1371/journal.pone.0003474
Editor: Marie Csete, California Institute for Regenerative Medicine, United States of America
Received May 2, 2008; Accepted September 26, 2008; Published October 22, 2008
Copyright: ? 2008 Cao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the BWF CAMS, Stanford Cardiovascular Institute, NIH HL089027, and CIRM RS1-00322 (JCW). This work was
also supported in part by NIH CA114747 (SSG), NIH HL076445 (RAW), and NIH HL72857 (RAL).
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
. These authors contributed equally to this work.
Myocardial infarction is a major cause of morbidity and
mortality worldwide. The limited ability of the surviving cardiac
cells to proliferate following an ischemic attack renders the
damaged heart susceptible to unfavorable remodeling processes
and heart failure . Currently, pharmaceutical and implantable
device management of heart failure seek only to preserve existing
viable myocardium after an ischemic attack, and thus merely slows
the progression of cardiac dysfunction. Ultimately, heart trans-
plantation is the only viable treatment option for end-stage heart
failure patients. To ‘‘regenerate’’ the heart and not only preserve
cardiac function but also recover lost or diseased muscle, stem cell
therapy has emerged as a promising therapy for heart disease
because it can provide a virtually unlimited source of cardiomy-
ocytes, endothelial cells, and other differentiated cell types. The
hope is to use these cells to replace diseased myocardium that
would otherwise progress to outright failure and regenerate the
heart to its former, healthy self.
Recently, human embryonic stem cells (hESCs) have generated
much interest because of their capacity for self-renewal and
pluripotency. In practical terms, hESCs can be cultured
indefinitely ex vivo, and can differentiate into virtually any cell
type in the adult body [2,3]. hESCs are thus an attractive source
for the derivation of large numbers of cells to be used in various
tissue repair and cell replacement therapies. However, upon
transplantation into living organisms, undifferentiated hESCs can
spontaneously differentiate into rapidly proliferating teratomas,
which are disordered amalgams of all three germs layers [2,3].
Nevertheless, under the appropriate conditions, ex vivo hESCs can
be directed to differentiate into beating cardiomyocytes via an
embryoid body (EB) intermediate . Subsequently, the cardio-
myocyte sub-population is enriched several-fold using discontin-
uous density gradient separation . Therefore, coaxing hESCs
into cardiomyocytes for therapeutic applications is an innovative
and feasible strategy that can minimize the risk of cellular
misbehavior and teratoma formation .
In order to define at a molecular level the changes occurring at
each stage of hESC differentiation into cardiomyocytes, we
performed transcriptional profiling of the cells using whole human
genome microarrays. This allowed us to examine the activation of
specific genes as well as broader developmental processes during
the progression from hESC to fetal cardiomyocyte, and to identify
novel genes that are potentially important in mediating differen-
tiation and development as well as potential novel markers of each
stage. In the future, such genes may prove vital in efforts to more
PLoS ONE | www.plosone.org1 October 2008 | Volume 3 | Issue 10 | e3474
closely direct and assess differentiation of potential therapeutic
pre-cardiomyocytes or cardiomyocytes in the repair of injured
cardiac tissues. To monitor cell survival after transplantation, we
then employ molecular imaging techniques that allow repetitive,
noninvasive assessment of transplanted ES cell engraftment,
viability, and proliferation in small animal models. Using these
genomic and imaging tools, we investigate the molecular networks
governing our differentiating cardiomyocytes, with an eye toward
transplantation and assessment of cell survival and proliferation in
vivo in a myocardial ischemia reperfusion model.
Differentiation of hESCs to cardiomyocytes
We differentiated hESCs into cardiomyocytes as shown in
Figure 1a. To understand the time course of transcriptional
changes occurring in these cells, we performed RT-PCR analysis
of hESC-derived EBs as they differentiated over the course of 42
days into beating clusters (Figure 1b). Expression of stem cell
markers (Oct4, NANOG, Rex1) decreased substantially by day 28,
while early stage cardiac transcriptional factors (Nkx2.5, MEF2C)
appeared between days 14–28. As expected, cardiac specific
markers (aMHC, ANF) appeared by day 14 and persisted through
terminal differentiation into beating EBs. Before enrichment, only
2–5% of the cells within beating EBs expressed cardiac marker
troponin-T as determined by FACS analysis. However, by
utilizing Percoll density gradient separation, we were able to
achieve cardiomyocyte-enriched populations ranging from 40–
45%, a ten-fold increase (Fig 1c & Movies S1–S3).
Major changes in gene expression between stages
highlight developmental progression
cRNA derived from four independent biological replicates at
the three stages of differentiation, and from cells isolated from four
individual human fetal hearts (19, 19, 20, and 21 weeks), was
hybridized into individual whole human genome microarrays.
Because fetal and adult hearts are composed of numerous cell
types, including cardiomyocytes, endothelial cells, smooth muscle,
fibroblasts, and many others, we isolated only primary ventricular
cardiomyocytes for microarray analysis (see Methods S1). Doing
so prevented non-cardiac cell types from contaminating our gene
expression data. The resulting data were analyzed using the SAM
algorithm  to identify genes which had changed expression
significantly between stages. A summary of our major findings is
Figure 1. Differentiation of hESCs to cardiomyocytes that express lineage-specific genes. (a) Schematic highlighting the experimental
design. hESCs are maintained in an undifferentiated state on mouse embryonic fibroblasts (MEFs), then transferred to suspension culture and allowed
to form embryoid bodies for 7 to 10 days. Upon appearance of beating clusters, the whole embryoid bodies are dissociated and separated by Percoll
density gradient enrichment to obtain cardiomyocytes (hESC-CMs). The hESC-CMs are then used for either in vitro analysis or in vivo transplantation
into the heart. (b) RT-PCR analysis of embryoid bodies over the course of 6 weeks shows expression of endodermal, (AFP), mesodermal (aMHC), and
endodermal (NeuroD) germ layer markers. GAPDH is used as loading control. (c) FACS analysis shows approximately 43.063.2% cells isolated by
Percoll density gradient separation are positive for cardiac troponin-T. Control population is cells prior to Percoll separation.
Profiling of hESC-CMs
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shown in Figure 2a. To obtain an overview of the transcriptional
landscape, we looked at the data using principal components
analysis (PCA), a dimensional reduction technique which identifies
‘‘principal components’’ or major trends in gene expression in the
overall data (Figure 2b). PCA demonstrates that each of the four
replicates from each stage has very similar transcriptional profiles
to one another, but distinctly different between stages, as expected.
‘‘Adjacent’’ stages show a progression of gene expression changes
primarily along component one, a pattern of continuously
decreasing gene expression across time, a pattern that we also
identified as prominent in clustering analyses. A hierarchical
clustering overview of the microarray experiments as a whole
(Figure 2c) likewise shows that the overall gene expressions
among replicates of each stage are very similar, with progressive
differences between more distantly separated stages.
hESCs exhibit unique biologic processes and molecular
To better understand which cellular processes are important in
the undifferentiated hESC stage, we performed statistical Gene
Ontology (GO) biological process overrepresentation analysis and
found that the most highly upregulated processes involved almost
exclusively cell cycling and mitosis, as well as nucleic acid synthesis
and metabolism (Table S2 (A7)). This was not surprising given
that hESCs’ primary mission is to self renew. hESCs are also
characterized by a network of genes important for pluripotency,
including the unique homeobox transcription factor NANOG,
which is the main downstream effector of this network. When we
compared expression patterns in hESCs to EB cells, we found that
there were 2,219 genes expressed much more highly in hESCs.
The most dramatically elevated transcript in hESCs was NANOG,
which is expressed at a level 250 times higher in hESCs than in EB
[8,9] (Table S1 (A1)). POU5F1 (also known as Oct4), upstream
of NANOG, is one of the critical regulators of pluripotency in the
mammalian embryo, and our results show that it is expressed 106–
160 fold more highly in the undifferentiated hESCs when
compared to EB. SOX2, another key pluripotency gene, is
expressed at 7.4 times the level in hESCs as in EB . Other
known markers of ES cell status are also clearly present at high
levels: TDGF1 and 3 (Crypto1 and 3) , expressed at ,100 fold
higher levels in hESCs; the SRC family kinase LCK (40 fold
higher), whose repression is associated with ES differentiation ;
the ES cell markers such as developmental pluripotency-associated
4 (Dppa4) (15.7 fold)  and homeobox expressed in ES cells 1
(Hesx1, 10 fold) . Further discussion can be found in
Supplemental Results S1.
Beating EB cells express many mesodermal and cardiac
specific gene programs
Differentiation to the beating EB stage is a very exciting and
complex time in the life of the cell population. Our microarray
results showed significant upregulation of master cardiac tran-
scriptional regulators, as well as cardiac-specific structural and
functional genes (Figure 2a, also see Supplemental Results
S1). Although this population of cells had clearly differentiated
with a significant bias toward mesodermal and cardiac lineages,
we could still see expression of genes characteristic of all three cell
layers. Analysis of the biological processes in the beating EBs
confirms these observations, with overexpression of embryonic and
organ system developmental categories including nervous system
development, kidney development, skeletal muscle development,
and heart development (Table S2 (A8)).
hESC-CMs downregulate early mesodermal genes and
upregulate cardiovascular and structural genes
Differentiation of beating EBs to cardiomyocytes is marked by a
transcriptional downregulation of 2,389 genes, including early
mesodermal genes such as TWIST1, whose expression goes down
substantially by 5 fold between the EB and CM stages, and
MEOX2, with a 15 fold reduction (Table S1 (A3)). There are
also considerable reductions in the expression of a number of
homeobox genes (HOXB3, 4, IRX, HHEX, HESX1). At the
Figure 2. Major themes in gene expression profiles at each stage of differentiation. (a) hESCs express high levels of pluripotency-
associated genes including Oct4, Sox2, NANOG, Crypto, and LCK. At the beating EB stage, the cells express high levels of mesodermal master
regulators such as Twist1, Tbx5, and Meox as well as very enriched levels of cardiogenic specific master regulators including Isl1, Hand, GATA4, 5, and
6, and MEF2C, along with high levels of cardiac specific myosins. This population also expresses genes from other cell layers, and many
developmental genes from Wnt and homeobox families. Cardiomyocytes downregulate early mesodermal genes and express more cardiac specific
and structural genes, while fetal heart cells have the highest levels of mature cardiac gene expression with very few other developmental lineages
represented. (b) Principal Components Analysis (PCA) shows that replicate experiments of each cell type are very similar while differentiation groups
separate significantly along components 1 and 2. (c) Hierarchical Clustering Analysis - Cells from each developmental stage cluster relatively close to
each other, with the most distance between hESCs and fetal heart cardiomyocytes. (d) K-means clustering analysis identifies major trends in gene
expression across the timecourse.
Profiling of hESC-CMs
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same time, we observe substantial increases in expression of 1,012
genes, including cardiac structural genes such as tropomyosin 1
and 2 (TPM1, 2, ,3 fold upregulated) , the heart and muscle
gene LMO7 (3.5 fold) , and a number of actins and actin-
regulatory genes such as beta actin (ACTNB, 3.4 fold), alpha
actinin (5 fold), coronin (2.5 fold), transgelin (4.6 fold) , and
caldesmon 1 (5 fold)  (Table S1 (A4)). Cells in this population
also exhibit evidence of maturation with the increased expression
of extracellular structural components such as vascular collagens
COL8A1 (18 fold) , COL4A3 and 4 (11 fold and 16 fold) ,
COL6A3 (4 fold) , and COL2A1 (2.4 fold) . Nevertheless,
there is still transcriptional evidence for the presence of other
mesodermal derivatives such as hematopoietic derivatives with the
increased expression of IL1A (10–20 fold), toll like receptor 3
(TLR 3, 3 fold), skeletogenic genes such as RUNX1 (4 fold),
sclerostosis (SOST, 8.7 fold), osteoprotogerin (TNFSFR11, 7 fold).
Some neuroectodermal genes are also upregulated, including
neuralized (NEURL, 2.7 fold) and neurofilament light peptide
(NEFL, 2.3 fold).
Significant changes in energy metabolism between CM
and fetal heart cardiomyocytes
One of our goals inthis study is to compareour CM population to
a cell population that would likely be optimal for cell transplantation
into the damaged heart. An optimal cell type would be committed to
the cardiomyocyte lineage but would still retain the capacity to
undergo mitosis and thereby regenerate damaged heart muscle. We
therefore chose primary fetal heart (FH) cardiomyocytes as the gold
while also maintaining a cardiac phenotype . In general, we
found that expression of cardiac structural and force generating
protein genes in the FH cells was not significantly higher than in the
CM populations. This suggests that the CM population, while still
somewhat heterogeneous, is composed of differentiated cardiomy-
ocytes that are capable of contraction but have not yet faced the
biomechanical stresses in vivo required for cardiac development. This
is further corroborated when we looked at the GO processes that are
more active in the FH cells and found a pattern suggesting increased
metabolic activity but not structural protein biogenesis (Table S2
(A11and A12)). Specifically,many ofthe increased processes inFH
cells include the TCA cycle, cellular respiration, mitochondrial
biogenesis, and lipidmetabolism. These energy-related pathways are
necessary for the mature cardiomyocyte to contract forcefully, and
their expression timing may correlate with the necessity for active
cardiac contraction in the fetus that continues up to and beyond
birth, with an interesting shift to a lipid-based metabolism in the
Electrophysiological recordings of hESC-CMs
Previous groups have studied the electrophysiology of hESC-
CMs and have reported significant heterogeneity within the
population [24,25,26]. To understand the electrophysiological
properties of our hESC-CMs, we took action potential (AP)
recordings from single cells using whole-cell patch-clamps. hESC-
CMs were categorized into pacemaker-, atrial-, or ventricular-like
phenotypes, based on such common electrophysiological charac-
teristics as the AP amplitude, upstroke velocity, as well as the
resting membrane potential. Ca2+transients that are crucial for
excitation-contraction coupling were also recorded. At 3 and 6
weeks post-differentiation, ventricular-, atrial-, and pacemaker-like
derivatives were readily observed (Figure 3). Noticeably,
ventricular-like hESC-CMs were most similar to fetal rather than
adult ventricular cells as indicated by their depolarized resting
membrane potential. Nonetheless, the AP profiles did not appear
to change significantly over the time course of our experiments.
Ideally, we would like to isolate only the ventricular-like hESC-
CMs and use those cells for transplantation studies in ischemic left
ventricles. Given the lack of specific cellular markers for
identifying ventricular/atrial/pacemaker CM types, we were
limited to using the whole population for transplantation studies.
hESC-CM transplantation improves left ventricular
function in a mouse myocardial infarction model
After analyzing the molecular changes underlying hESC
differentiation as well as their electrophysiological phenotypes,
we then assessed the effect of hESC-CM transplantation on
myocardial function. Using SCID-Beige mice, one million hESC-
CMs were transplanted by direct injection into ischemic regions of
the left ventricle after 30 minutes of temporary left anterior
descending (LAD) coronary artery occlusion. To characterize
potential functional improvements, we performed echocardiogra-
phy on post-transplant animals that received either hESC-CMs
(n=21) or PBS (n=12) as a control. Left ventricular fractional
shortening (LVFS), which is a common method for quantifying
cardiac contractility or ability of the ventricle to eject blood, was
used as the metric for comparison of the two groups’ outcomes.
Expressed as a percentage of the ventricle’s volume, diminished
LVFS is associated with a failing heart. Animals receiving hESC-
CMs showed a 12.564.2% improvement over controls at 8 weeks
as measured by LVFS (P=0.03, Figure 4). This was primarily
due to improvements in the left ventricular end systolic dimension
(LVESD), as the left ventricular end diastolic dimension (LVEDD)
remained constant between the two groups.
We also noted evidence of increased angiogenesis in the
ischemic regions of explanted mouse hearts (Figure S1), and
histology confirmed that the fibrotic scar was attenuated at 8 weeks
post-transplantationin animals that underwent
transplantation (Figure 4c and Figure S2). Using NIH Image
J software, the quantified infarct sizes (percent of LV) in hESC-
CM-treated mice and PBS controls were 21%63% (n=6) and
25%62% (n=6) (P=0.041), respectively. However, improved
LVFS was not sustained at later time point (16 weeks) (data not
shown). Although the reasons for this are unclear, we suspect that
acute donor cell death within the first month is responsible for
attenuation of the positive remodeling processes initiated by
hESC-CM paracrine effectors. To confirm this hypothesis, we
decided to use molecular imaging to track hESC-CM fate
noninvasively in living mice.
Lentiviral transduction with reporter genes does not alter
In order to follow the fate of transplanted hESC-CMs
noninvasively and longitudinally, we next employed reporter gene
imaging. Undifferentiated hESCs were stably transduced with a
firefly luciferase (Fluc) and enhanced green fluorescent protein
(eGFP) double fusion reporter gene driven by a constitutive human
ubiquitin promoter (pUB) using a lentiviral vector. The double
and hESC-CM characteristics (Figure 5a and 5b, Figure S3).
Reporter gene imaging for tracking transplanted hESC-
CM fate in vivo
To understand the fate of transplanted cells in vivo, one
regions of the myocardium.
successfully, emitting a robust and stable bioluminescent signal
for 8 weeks following transplantation (Figure 5c). Quantitative
Fluc+/eGFP+hESC-CMs were injected into peri-infarct
Profiling of hESC-CMs
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imaging analysis revealed that signal intensity declined logarith-
mically during the first 3 weeks post-injection and remained
constant thereafter (Figure 5d). This initial drop in biolumines-
cence correlates with the death of roughly 90% of the
administered cell population. Importantly,
remained localized to the heart throughout our studies. Imaging
did not reveal any cellular misbehavior, and no histological
evidence of teratoma formation was observed in any animal within
this group (n=15) (Figures 5d and 5e, Figure S4). In contrast,
injection of one million undifferentiatedFluc+/eGFP+hESCs into the
heart led to both intra-cardiac and extra-cardiac teratoma
formation in 7 out of 7 mice (Figure S5). We also performed
spike-in studies to mimic clinically relevant scenarios in which
contaminating undifferentiated hESCs are injected along with
hESC-CMs. Our results show teratoma formation with 100 k
hESCs (+400 k hESC-CMs), but not with 1 k hESCs (+499 k
hESC-CMs) or 10 k hESCs (+490 k hESC-CMs) (Figure S6).
Thus, these data suggest there is likely a threshold for the number
of contaminating hESCs within an injected hESC-CM population
that can lead to teratoma formation in the heart.
Histologic evaluation of transplanted hESC-CMs
Importantly, our histologic studies revealed minimal integration
ventricle. Though small clusters of injected cells appeared to
engraft and then persist for many weeks after transplantation
Fluc+/eGFP+hESC-CMs into infarcted areas of the left
Figure 3. Electrophysiological recordings of hESC-CMs. Action potential (AP) recordings from single cells were done using the whole-cell
patch-clamp technique. hESC-CMs were categorized into pacemaker-, atrial- or ventricular-like phenotypes, based on such common
electrophysiological characteristics as the AP amplitude (mV), upstroke velocity (mV/ms), APD50 and APD90 (ms), as well as the resting membrane
potential (RMP, mV). (a) Representative ventricular-, atrial- and pacemaker-like action potentials, demonstrating electrophysiological heterogeneity in
our hESC-CM population, and (b) Ca2+ transients recorded from hESC-derived cardiomyocytes, confirming calcium influx of these cells. See Materials
and Methods for description of experimental parameters.
Profiling of hESC-CMs
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(emiting measurable bioluminescent signal), they did not exhibit
functional organization with the surrounding host myocardium.
These findings confirm previous reports which have also found
minimal organized integration of hESC-CMs with host myocar-
dium [27,28,29]. It is therefore difficult to explain the transient
improvement in cardiac contractility at 8 weeks given the
underwhelming evidence for robust integration of transplanted
cells (Figure 5e and Figure S4). Such findings have led us and
others to hypothesize that paracrine factors may be involved by
helping to increase angiogenesis or preventing apoptosis of
ischemic host myocardial tissues [30,31,32].
In this study, we have described the hESC-CM transcriptome
within the spectrum of changes that occur between undifferenti-
ated hESCs and fetal heart cells, and used molecular imaging to
follow their survival and engraftment in the heart. Global gene
expression profiling of hESC differentiation thus enables a
systems-based analysis of the biological processes, networks, and
genes that drive hESC fate decisions. This systems biology
approach has obvious benefits over traditional PCR-based
methods, which measure only a limited number of transcripts
and so cannot define the complex regulatory networks of genes
and pathways important for hESC differentiation.
Previous studies have also analyzed the transcriptional profiles
of hESC-CMs [33,34], and we found a high degree of similarity in
the significant gene lists between our results and theirs. For
example, we also noted upregulation of cardiac markers (e.g.
MYH6, MYL4, TNNT2), cardiac transcription factors (e.g.
TBX5, MEF2C, GATA4), as well as phospholamban (PLN).
Interestingly, the two previous studies compared only spontane-
ously beating clusters of non-purified cardiomyocytes (what we
refer to as ‘‘beating embryoid bodies’’ in this study). In our
experience, only 2–5% of the cells within these clusters actually
express the cardiac-specific marker cardiac troponin-T. Because of
concern that non-cardiac and non-mesodermal cell types will
obscure the hESC-CM molecular signatures, we used Percoll
density gradient separation to achieve cardiac troponin-T-
enriched populations ranging from 40–45%. Analysis of the
expression differences between purified and non-purified CMs
revealed considerable downregulation of early mesodermal and
homeobox genes, and upregulation of cardiovascular and
structural genes such as actins and extracellular collagens. Given
Figure 4. Assessment of myocardial function after ischemic injury and hESC-CM transplantation. (a) Echocardiography demonstrates
improved cardiac contractility (left ventricular fractional shortening, LVFS) following delivery of one million hESC-CMs compared to PBS injection
alone at eight weeks post-transplant (P=0.03). This was primarily due to improvement in left ventricular end systolic dimension (LVESD, middle)
without significant changes in left ventricular end diastolic dimension (LVEDD, right). (b) Representative M-mode echocardiographic images from a
mouse receiving hESC-CM transplantation (right panels) versus a mouse receiving a control PBS injection (left panels) at 4 (top panels) and 8 weeks
(bottom panels). (c) Histological evaluation of infarct fibrosis reveals attenuation of scar in a representative animal treated with hESC-CMs (right
panels) as compared with a representative animal receiving PBS alone (left panels) at 8 weeks post-transplantation. Masson’s Trichrome stain (top
panels) produces blue connective tissue and red muscle fibers to allow easy identification of the fibrotic scar resulting from ischemia reperfusion
injury. Scale bars=10 mm.
Profiling of hESC-CMs
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this purified population of CMs, we were also able to perform
robust bioinformatics analysis of these cells and compare them
with fetal heart cardiomyocytes. This is important since we would
like to establish how closely our derived cells compare with a gold
standard. When looking at the GO processes that are more active
in the FH cells, we found a pattern suggesting increased metabolic
activity but not structural protein biogenesis. We believe these
energy-related pathways are likely necessary for the cardiomyocyte
to contract forcefully in the in vivo environment. This finding
suggests that derived hESC-CMs have not adequately matured, at
least as far as energy metabolism, and so may benefit from
exogenous mechanical or electrical stimulation in order to
upregulate energy-related pathways prior to transplantation.
Using purified hESC-CMs, our comprehensive, systems-based
approach to transcriptional analyses supports the case that each of
the stages of differentiation and selection results in a significant
enrichment in cells of the cardiomyocyte lineage, expresses
appropriate stage specific genes, and turns on appropriate biological
processes corresponding to these stages. Given the robustness of our
differentiation method, we believe the hESC-CM population would
be an ideal source of replacement cells in the in vivo setting. We also
demonstratethathESC-CMscan successfullyengraftinthe ischemic
heart for an extended duration that result in improved cardiac
function, though only transiently. This latter finding may be partly
attributed to the activation of paracrine signaling mechanisms by
Figure 5. Survival and fate ofFluc+/eGFP+hESC-CMs in vivo. (a) RT-PCR analysis of various hESC and cardiac specific markers revealed no
significant differences betweenFluc+/eGFP+hESCs and control non-transduced hESCs (see Figure 1b for comparison), other than the presence of Fluc.
(b)Fluc+/eGFP+hESC-CMs express cardiac specific markers such as a-actin, troponin-T, connexin-43, and MEF2C (all in red) and GFP (green, scale
bars=50 mm). (c) A representative animal imaged for 2 months following transplantation of 1 millionFluc+/eGFP+hESC-CMs into the heart. (d) In vivo
bioluminescence imaging (BLI) signal measured from animals in whichFluc+/eGFP+hESC-CMs were transplanted into the ischemic hearts (n=15). Signal
activity falls drastically within the first 3 weeks of transplantation and remains stable thereafter, with no evidence of tumorigenesis (left). From 21
days post-transplantation onwards, BLI signal is reduced to ,10% of the signal obtained at two days post-transplantation. (e) Histopathological
evaluation of hearts followingFluc+/eGFP+hESC-CM delivery. H&E staining (left panels) demonstrates cluster of cells within the infarcted region of the
heart (scale bars=200, 20 mm for low and high magnification images, respectively). GFP positive cells within this cluster also express cardiac
troponin-T (red, near right panel) and connexin-43 (red, far right panel). Scale bars=20 mm.
Profiling of hESC-CMs
PLoS ONE | www.plosone.org7 October 2008 | Volume 3 | Issue 10 | e3474
after acute donor cell death. Lastly, we show that cardiac
differentiation prior to transplantation can prevent teratoma
formation, which remains a major safety concern for investigators
exploring the therapeutic uses of hESCs.
In our microarray analysis, we observed high expression of
pluripotency-related genes involved in the core hESC regulatory
circuitry, including OCT4, SOX2, and NANOG, as well as
CRYPTO 1 and 3, LCK, and HESX1. Differentiation into
beating EBs was accompanied by mesodermal differentiation and
dramatic activation of TWIST1, TBX5, and MEOX transcrip-
tion, as well as the very clear induction of nearly all of the early
cardiogenic genes, including FOXC1, ISL2, HAND1, GATA4, 5,
and 6, FOXH1, and MEF2C. While it is clear that other
developmental lineages are still present in the EB population, it is
also clear from the high levels of cardiac gene expression that this
population is significantly enriched for the cardiac lineage even at
an early stage. The transcriptional analysis of the final differen-
tiation and selection of the hESC-derived CMs indicates that this
enrichment continues, with the CM population expressing
differentiated cardiomyocyte genes at levels similar to our more
advanced FH cells. Importantly, because of the cell type
heterogeneity in the fetal heart, we specifically isolated cardiomy-
ocytes from the fetal left ventricles for microarray analysis.
We now briefly discuss the four major trends in the microarray
data seen in the K-means clustering analysis (Figure 2d), which will
allow us to explore the major themes within an enormous amount
expression data. Cluster 1 is composed of 1775 genes whose
expression increases at each stage from hESC to EB to hESC-CM to
FH(Figure 2dand TableS3(B1)). Overrepresentation analysis of
this cluster of genes shows that the GO processes to which these
genes contribute include many basic differentiated cell functions
such as the establishment of cellular transport and secretory
processes, regulation of cell localization, response to cellular stresses
and hypoxia, cytoskeletal biogenesis, control of apoptosis, and
interestingly, cardioblast cell fate commitment (Table S4 (B5)).
This cluster of genes has a substantial overlap with the component
genes of principal component 1 from the PCA analysis (Figure 2b),
demonstrating how two analytic approaches can result in similar
The converse expression pattern is seen in the 2,453 genes
composing cluster 2, which are sequentially downregulated across
the groups from hESC to EB to hESC-CM to FH (Table S3
(B2)). The processes overrepresented in this cluster primarily
involve nucleic acid synthesis, DNA replication and chromatin
maintenance, cell cycle, and transcription in general (Table S4
(B6)). This theme is consistent with patterns seen in normal
embryonic development in both drosophila and mouse , and
reflects the fact that earlier undifferentiated cells are undergoing
rapid replication and production of broad ranges of transcripts,
while cell cycling slows dramatically later in development as cells
begin to express a more limited number of genes that are
appropriate for the differentiated state.
Cluster 3 is comprised of 1,009 genes whose expression
increases at each stage from hESC to EB to hESC-CM, but
which are expressed significantly less in the FH cells (Table S3
(B3)). The overrepresented processes in this cluster correspond to
non-cardiac cell differentiation pathways, particularly neuroecto-
dermal differentiation, that compose a portion of the hESC-CM
population which we have differentiated and purified it from
hESC precursors, but are not present in the harvested fetal heart
cells (Table S4 (B7)). The final interesting cluster of genes is
cluster 4 (Table S3 (B4)), representing genes which generally
increase in expression across all stages from hESC to EB to hESC-
CM to FH, but which are expressed at considerably higher levels
in the two older FH samples, 3 and 4 (at 20 and 21 weeks,
respectively), than in FH1 and 2 (19 weeks each). The processes
overrepresented in this gene group are heavily weighted toward
cardiac muscle contraction, muscle development, heart develop-
ment and other cardiac specific processes, and the genes
contributing to these processes include dozens of cardiac structural
proteins such as cardiac myosin heavy and light chains,
cardiomyocyte potassium channels such as KCNE1 , KCNQ1
 and KCNH2 , cardiomyocyte troponins including T2
and C1, as well as cardiac phospholamban and cardiac actin 1
(Table S4 (B8)). Thus the hESC-CM population’s expression of
terminal cardiac differentiation markers at a level intermediate
between younger and older FH cardiomyocytes suggests that this
population is sufficiently advanced developmentally to serve as a
potential replacement population for cells lost to ischemia.
With these very interesting and detailed gene expression studies,
we began focusing on cellular transplantation to the ischemic heart.
We observed significant improvements in echocardiographic metrics
when comparing treated and control animals. Histologic analysis
revealed reduced scar formation, but there was underwhelming
evidence of functional myocardium regeneration, confirming
previous reports [28,29,39]. To explain this disparity, the improve-
ment in cardiac function may be due to paracrine factors, as
suggested by Dzau and colleagues [30,31,32]. Our own studies
indicate some increased cytokine signaling in hypoxic hESC-CMs
(Figure S7). However, if paracrine signaling is the primary
mechanism of improvement, then long-term generation of these
factors from sufficient numbers of transplanted hESC-CMs may be
required for a sustained improvement in cardiac function. Until now
no studies have analyzed the overall survival and growth kinetics of
transplanted hESC-CMs in ischemic myocardium.
To address this lack of understanding, we employed molecular
imaging technology for understanding the fate of cells following
transplantation . Longitudinal imaging of transplanted hESC-
CMs exposes the limitations of cardiac stem cell therapy, as ,90%
of cells die within the first three weeks of delivery. Though we did
not address the specific mechanism of death in this study, poor cell
survival is likely due to widespread apoptosis and anoikis of cells
injected into an inhospitable environment. Improving cell survival
by subjecting hESC-CMs to the appropriate anti-apoptotic and
pro-survival cues may alleviate some of the survival issues, and
efforts to this end have been reported since completion of this work
. Other methods that take advantage of tissue engineering
technologies in which biopolymers and synthetic tissue constructs
are used to organize and support transplanted cells may offer
another means for increasing cell survival . Delivery
techniques other than intra-myocardial injection, such as intra-
coronary or retrograde coronary venous, may also improve cell
survival . Another confounding factor is the host immune
response, which we did not address in this study (as SCID mice
were used). With a functioning host immune system, we would
expect to see a further reduction in cell survival. Nevertheless, it is
important to note that even in our SCID mice, transplantation of
Fluc+/eGFP+hESC-CMs did not form teratomas in the post-trans-
plantation period. The lack of teratoma formation emphasizes the
robustness of our hESC-CM purification protocol in removing
undifferentiated cell contaminants.
In summary, hESC-CMs hold potential promise for treatment
of cardiovascular disease. The molecular processes that control
stem cell pluripotency, differentiation, and proliferation are
complex, justifying the need for a broad investigation that
integrates systems biological tools for transcriptome analysis with
molecular imaging tools for confirmation of survival, engraftment
and functional benefit in the in vivo setting. We found that the
Profiling of hESC-CMs
PLoS ONE | www.plosone.org8 October 2008 | Volume 3 | Issue 10 | e3474
enriched hESC-CMs expresses cardiomyocyte genes at levels
similar to 20-week fetal heart cells, making this population a good
source of potential replacement cells in the in vivo setting. Beyond a
characterization of the overall transcriptional characteristics of our
differentiated cells, we have also identified a large number of
potentially important new genes that are expressed at high levels at
distinct stages and that may play roles in the cardiogenic
developmental program. These genes may also act as specific
markers of cell differentiation in addition to being inducers of
cardiogenic differentiation, thus opening new avenues of investi-
gation into the basic biology of cardiovascular development.
However, understanding the molecular networks of differentiation
is not enough to predict the fate of differentiated cells once
transplanted in a living host. To address this lack of knowledge, we
have shown molecular imaging to be a powerful method for
assessing cellular localization, engraftment, survival, and prolifer-
ation in vivo. Taken together, gene expression and molecular
imaging studies such as this will serve as a crucial foundation for
future clinical applications of stem cell therapies.
Materials and Methods
Culture of undifferentiated hESCs
hESCs (H9 line) from passage 35–40 were initially maintained
on top of murine embryonic fibroblasts (MEF) feeder layers,
seeded onto 0.1% gelatin coated plastic dishes, and inactivated by
10 mg/ml of mitomycin C. hESCs were maintained in ES medium
containing 80% Dulbecco’s modified Eagle’s medium/F12, 1 mM
L-glutamine, 0.1 mM b-mercaptoethanol, 0.1 mN non-essential
amino acids, 20% Knockout Serum Replacement, and 8 ng/ml
hbFGF. The ES cell culture medium was changed daily and
hESCs were passaged every 4–5 days.
Lentiviral production and generation of stable hESC lines
SIN lentivirus was prepared by transient transfection of 293T
cells . hESCs were stably transduced with LV-pUB-Fluc-eGFP
at a multiplicity of infection (MOI) of 10. The infectivity was
determined by eGFP expression as analyzed on FACScan (BD
Bioscience). The eGFP positive cell populations were isolated by
fluorescence activated cell sorting (FACS) Vantage SE cell sorter
(Becton Dickinson) followed by plating on the feeder layer cells for
long-term culturing. Flow cytometry data were analyzed with
FlowJo (Treestar, San Carlos, CA) analysis software.
Embryoid body formation and cardiac specific
hESC colonies were dispersed into cell aggregates containing
approximately 500 to 1,000 cells using 1 mg/mL collagenase IV.
The cell aggregates were then suspension-cultured in ultra-low
attachment cell culture dishes with hESC differentiated medium
(without basic fibroblast growth factor) for 9 days with the media
changed every two days. To promote cardiac differentiation, 9-day
old EBs were transferred to 10 cm dishes coated with 0.1% gelatin
and grown in media consisting of DMEM supplemented with 20%
FBS and 2 mmol/L L-glutamine. During differentiation, the
media was changed every two days. Spontaneously contracting
cells appeared as clusters in outgrowths from the EBs at day 10
after differentiation. These beating EBs were maintained in long-
term cultures for up to 103 days.
Isolation of hESC-CMs
Differentiated cultures containing beating cardiomyocytes were
washed with phosphate buffered saline (PBS) and incubated with
0.56 units/ml Liberase Blendzyme IV (Roche, Indianapolis, IN) at
37uC for 25 min. After dissociation, the cell suspension was
separated by Percoll density (58.5% and 40.5%) centrifugation at
1500 g for 30 minutes at room temperature.
Cell samples collection and RNA preparation
The undifferentiated hESC, day 10 beating whole embryoid
bodies (Beating EBs), day 14 Percoll-enriched cardiomyocytes
derived from human hESCs (hESC-CM) and human fetus heart-
derived left ventricular cardiomyocytes (FH-CM) at 19, 19, 20,
and 21 weeks were collected at chosen time points. Four samples
from each group (for a total of 16 unique samples) were harvested
for RNA isolation. Total RNA was isolated as described previously
in Trizol (Invitrogen) followed by purification over a Qiagen
RNeasy column (Qiagen).
Microarray hybridization and data acquisition
A full description of RNA quality control, and labeling reaction
and hybridization is included in Methods S1. Using Agilent Low
RNA Input Fluorescent Linear Amplification Kits, cDNA was
reverse transcribed from each of 16 RNA samples representing
four biological quadruplicates, as well as the pooled reference
control, and cRNA was then transcribed and fluorescently labeled
with Cy5/Cy3. cRNA was purified using an RNeasy kit (Qiagen,
Valencia, CA, USA). 825 ng of Cy3- and Cy5- labeled and
amplified cRNA was hybridized to Agilent 4644 K whole human
genome microarrays (G4112F) and processed according to the
manufacturer’s instructions. The array was scanned using Agilent
G2505B DNA microarray scanner. The image files were extracted
using Agilent Feature Extraction software version 9.5.1 applying
LOWESS background subtraction and dye-normalization.
Microarray data analysis
The data were analyzed using the SAM algorithm  with
multiple testing correction to identify genes which had statistically
significantly changed expression between each stage, and K-means
clustering to identify clusters of genes having unique temporal
expression profiles. For hierarchical clustering, we used positive
correlation for distance determination and required complete
linkage. Gene Ontology overrepresentation analysis was performed
using Fisher’s Exact test and High Throughput GOMiner software.
Action potential (AP) recordings from single cells were done
using the whole-cell patch-clamp technique. Patch pipettes were
prepared from 1.5 mm thin-walled borosilicate glass tubes using a
Sutter Micropipette Puller (P-97) and typically had resistances of
4–6 M when filled with an internal solution containing (mM): 110
K+aspartate, 20 KCl, 1 MgCl2, 0.1 Na-GTP, 5 Mg-ATP, 5 Na2-
phospocreatine, 1 EGTA, 10 HEPES, pH adjusted to 7.3 with
KOH. The external Tyrode’s bath solution consisted of (mM): 140
NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES, pH
adjusted to 7.4 with NaOH. Upon seal formation and following
membrane rupture, APs were recorded using the current-clamp
mode. Data were filtered at 10 KHz. Axopatch 200B, Digitize
1322 and pClamp8 (Axon Burlingame, CA, USA) were used for
data amplification and acquisition. hESC-CMs were categorized
into pacemaker-, atrial- or ventricular-like phenotypes, based on
such common electrophysiological characteristics as the AP
amplitude (mV), upstroke velocity (mV/ms), APD50 and APD90
(ms), as well as the resting membrane potential (RMP, mV). We
primarily used the AP profiles as signatures of different chamber-
specific CM types. Nodal-like AP phenotype was defined as those
that exhibited: a) prominent phase-4 depolarization, b) slow
Profiling of hESC-CMs
PLoS ONE | www.plosone.org9 October 2008 | Volume 3 | Issue 10 | e3474
upstroke (dV/dt), c) small action potential amplitude (APA), d)
relatively depolarized MDP, and e) spontaneous firing. By
contrast, like others, we defined the ventricular-like phenotype as
those that displayed: i) a significant plateau phase, ii) longer APD
(vs. those of atrial and nodal), iii) rapid upstroke, and iv) a flat
phase 4. Atrial APs were those that displayed a triangular shape.
Of note, in comparison to neonatal and adult human ventricular
and atrial CMs, the AP parameters of hESC-CMs exhibit MDP
and upstroke velocities that were positive (,240 vs. ,280 mV)
and slow (,10 V/s vs. 100–300 V/s), respectively. (See Methods
S1 for further Electrophysiology Methods).
Measurements of cytosolic Ca2+
A spectrofluorometric method with Fura-2/AM as the Ca2+
indicator was used for measuring [Ca2+]i. FLV- or hESC-CMs
were incubated with 5 mM Fura-2/AM and 0.2% pluronic F-127
for 30 min at 37uC. Fluorescent signals obtained upon excitation
at 340 nm (F340) and 380 nm (F380) were recorded from cells
perfused with Tyrode solution containing (mM): 140 NaCl, 5.0
KCl, 1.0 CaCl2, 1.0 MaCl2, 10.0 glucose and 10 HEPES (pH 7.4)
unless otherwise indicated. Data were analyzed using the
Ionwizard software (Version 5, IonOptix) to generate the Ca2+
transient parameters. The F340/F380 ratio was used to represent
cytosolic [Ca2+]. To induce cytoplasmic Ca2+transients, hESC-
CMs were electrically stimulated. Ca2+transients were recorded
and analyzed after a series of depolarizations that enabled each
transient to fully decay so as to establish steady-state SR content.
Effect of reporter genes on hESC proliferation and
Reverse transcription polymerase chain reaction (RT-PCR) was
used to compare the expression of embryonic markers (Oct4,
NANOG, Rex1), cardiac transcription factors (Nkx2.5, MEF2C),
ventricular specific proteins (aMHC, ANF), and Fluc reporter
gene between control non-transduced hES andFluc+/eGFP+hESCs.
RNA was isolated from hES andFluc+/eGFP+hESCs using Trizol
reagent. Two mg of total RNA extracted from EBs was reverse-
transcribed using ThermoScript RT-PCR system (Invitrogen,
Carlsbad, CA). One ml of cDNA sample was PCR amplified with
gene-specific primers (see Methods S1) using optimized PCR
cycles to obtain amplified reactions in a linear range. PCR
products were separated on 1% agarose gel by electrophoresis and
quantified by using Labworks 4.6 Image Acquisition and analysis
software (UVP Bio-imaging systems, Upland, CA).
Transplantation of hESC-CMs into ischemic myocardium
A total of 50 adult female SCID Beige mice (Charles River
Laboratories) weighing 20–25 gm (8 weeks old) were used. All
procedures were performed in accordance with protocols approved
by the Stanford Animal Research Committee guidelines. Following
induction of anesthesia with isoflurane (3–4%), animals were
orotracheally intubated and ventilated with a mixture of oxygen
and 2–3% isoflurane with a volume-cycled rodent ventilator as
described . A lateral thoracotomy was performed followed by
ligation of the mid left anterior descending (LAD) artery for
30 minutes. Myocardial infarction was confirmed by blanching and
EKG changes. Subsequently, 3 groups received direct myocardial
injection of: (1) 16106 Fluc+/eGFP+hESC-derived cardiomyocytes in
40 ml of PBS (n=16), (2) 16106non-transduced hESC-derived
cardiomyocytes (n=6), and (3) 40 ml of PBS as control (n=12).
Another set of 16 animals were used to evaluate the potential for
teratoma formation following intramyocardial injection of undiffer-
Fluc+/eGFP+hESCs. Animals were injected with 16106
tiatedFluc+/eGFP+hESCs spiked with 46105non-transduced hESC-
CMs (n=3), 16104undifferentiatedFluc+/eGFP+hESCs spiked with
4.96105non-transduced hESC-CMs (n=3), and 16103undiffer-
hESC-CMs (n=3). Post-operative analgesia was provided by a one-
time, subcutaneous injection of buprenorphine (0.1 mg/kg body
weight). Animals were recovered in a warmed, humidified chamber.
Fluc+/eGFP+hESCs spiked with 4.996105non-transduced
Bioluminescence imaging (BLI) of hESC and hESC-CM
Cardiac BLI was performed by an independent, blinded operator
using the Xenogen In Vivo Imaging System. Mice were anesthetized
with 2% isoflurane and D-Luciferin was administered intraperitone-
ally at a dose of 375 mg/kg body weight. At the time of imaging,
animals were placed in a light-tight chamber, and photons emitted
from luciferase expressing hESCs transplanted into the animals were
collected with integration times of 1–10 min, depending on the
intensity of the bioluminescence emission. Ventral images were
mice were scanned repetitively over 12 months as per the study
design. Bioluminescence was quantified in units of maximum
photons persecond percentimetersquare persteridian (p/s/cm2/sr).
Assessment of left ventricular contractility
Echocardiography was performed by an independent, blinded
operator using the Siemens-Acuson Sequioa C512 system
equipped with a multi-frequency (8–14 MHz) 15L8 transducer.
Mice were assessed pre-operatively, and 2, 4, 8, and 16 weeks post-
transplant. Animals received continuous inhaled isoflurane (1.5–
2%) for the duration of the imaging session (10–15 minutes).
Animals were imaged in the supine position resting on a
specialized platform allowing for continual inhaled anesthesia
while maintaining optimal exposure of the left chest. M-mode
short axis views of the LV were obtained and archived. Analysis of
the M-Mode images was performed using the Siemens built-in
analysis software. Left ventricular end diastolic diameter (LVEDD)
and end-systolic diameter (LVESD) were measured and used to
calculate fractional shortening (FS) by the following formula:
Cell and tissue immunohistochemical analysis
To confirm their undifferentiated state, cultured hESCs were
plated onto 8 chamber slides and fixed with acetone on ice for
20 minutes, then stained for immunofluorescence with the
appropriate antibodies. Microscopy was performed using a ZEISS
Axiovert microscropy (Sutter Instrument). Hearts were excised two
months after transplantation and prepared in 10-micron thick
frozen sections. Immunofluorescent labeling was analyzed using a
Zeiss LSM 510 Confocal Laser Scanning Microscope equipped
with a Coherent Mira 900 tunable Ti:Sapphire laser for two-
photon excitation (Zeiss, Minneapolis, MN).
Unless otherwise noted, non-microarray data are presented as
mean6S.D. Data were compared using standard or repeated
measures, using ANOVA where appropriate. Pairwise compari-
sons were performed using a two-tailed Student’s t–test. For
electrophysiology data, data are expressed as mean6SEM. One-
way ANOVA followed by Newman-Keuls multiple comparison
tests or paired t test was carried out to test for differences between
the mean values within the same study. For all data, differences
were considered significant for P-values,0.05.
Profiling of hESC-CMs
PLoS ONE | www.plosone.org10October 2008 | Volume 3 | Issue 10 | e3474
analysis of microarray data.
Found at: doi:10.1371/journal.pone.0003474.s001 (0.07 MB PDF)
Document contains additional
Found at: doi:10.1371/journal.pone.0003474.s002 (0.11 MB PDF)
Document contains supplemental methods.
CD31 (mouse) shows upregulation of capillary density in ischemic
hearts at week 8. The hESC-CM-treated group showed significant
augmentation of CD31 positive capillary density (P,0.05).
Capillary densities were examined by counting the number of
capillaries stained with anti-CD31 in five random fields on two
different sections (approximately 3 mm apart) from each mouse.
Images were analyzed using Image J software.
Found at: doi:10.1371/journal.pone.0003474.s003 (2.08 MB TIF)
Quantitative analysis of the endothelial cell marker
plantation. (a) Histological evaluation of infarct fibrosis reveals
attenuation of scar in a representative animal treated with hESC-
CMs (right panels) as compared with a representative animal
receiving PBS alone (left panels) at 8 weeks post-transplantation.
Masson’s Trichrome stain (bottom panels) produces blue connec-
tive tissue and red muscle fibers to allow easy identification of the
fibrotic scar resulting from ischemia reperfusion injury. (b) The
quantified infarct sizes (percent of LV) in hESC-CM-treated mice
and PBS controls were 21%63% (n=6) and 25%62% (n=6)
(P=0.041), respectively. Scale bars=10 mm.
Found at: doi:10.1371/journal.pone.0003474.s004 (40.89 MB
Ventricular scar formation after hESC-CM trans-
fusion (DF) reporter gene. (a) Schema of the DF reporter gene
containing Fluc and eGFP with brightfield (left) and fluorescent
(right) images ofFluc+/eGFP+hESCs (scale bars=200 mm). (b) Stably
correlation between cell number and reporter gene activity. Raw
bioluminescence images of increasing numbers of
hESCs in vitro are shown below graph. (c)
maintain firefly luciferase activity over successive passages. (d)
Fluc+/eGFP+hESCs maintain pluripotent stem cell markers such as
SSEA-4, Oct-4, and AKP, but remain negative for differentiation
marker SSEA-1. Scale bars=50 mm. (e) RT-PCR analysis of
embryoid bodies over the course of 7 weeks shows expression of
endodermal (AFP), mesodermal (aMHC), and ectodermal (Neu-
roD) germ layer markers for both control non-transduced hESCs
andFluc+/eGFP+hESCs. GAPDH is used as loading control.
Found at: doi:10.1371/journal.pone.0003474.s005 (3.70 MB TIF)
Stable lentiviral transduction of hESCs with double
Fluc+/eGFP+hESCs (collected by FACS) show robust
CMs do not integrate into host myocardium but continue to express
cardiac markers. Representative histopathological images of ex-
planted hearts taken two months after
delivery. GFP positive cells (transplanted
express cardiac troponin-T and connexin-43, but do not appear to
be well integrated with the surrounding host myocardium.
Found at: doi:10.1371/journal.pone.0003474.s006 (5.26 MB TIF)
Histopathological evaluation demonstrates that hESC-
undifferentiatedFluc+/eGFP+hES cells transplanted into the heart.
(a) Representative images from a single animal receiving one
million undifferentiatedFluc+/eGFP+hES cells. Undifferentiated hES
cells rapidly form teratomas with extra-cardiac spread within 3 to
4 weeks of transplantation. (b) Quantification of imaging signals
from animals receiving undifferentiatedFluc+/eGFP+hESCs (n=6)
orFluc+/eGFP+hESC-CMs (n=15) shows logarithmic increases in
Bioluminescence imaging and histological fate of
BLI signals in the undifferentiated group (*P,0.001) vs. the hESC-
CM group due to teratoma formation. (c) Histology demonstrating
typical teratoma formation in the heart following transplantation
low-power field of teratoma (I), respiratory epithelium (II), and
cartilage formation (III) can be identified (scale bars=50 mm). The
border of the graft area shows that only host myocardium stains
positive for cardiac markers such as cardiac troponin-T (cTnT),
while cardiac markers are absent from the eGFP+ region (IV).
Found at: doi:10.1371/journal.pone.0003474.s007 (3.84 MB TIF)
Fluc+/eGFP+hES cells. Histological features of
Fluc+/eGFP+hESCs mixed with non-transduced hESC-derived
cardiomyocytes after transplantation to SCID mouse heart. This
study represents a clinically relevant scenario in which undiffer-
entiated hESC contaminants are mixed in with the hESC-CM
population. We observed teratoma formation in the 100 k hESC
contaminant group, but not in the 10 k or 1 k hESC groups. Data
presented as mean6SEM.
Found at: doi:10.1371/journal.pone.0003474.s008 (2.41 MB TIF)
Fluc+/eGFP+hESC-CMs upregulate secretion of angio-
genic growth factors under hypoxic conditions. (a) Culture media
from hESC-CMs under hypoxia (1% O2/5% CO2/94% N2) or
normoxia (20% O2/5% CO2) was washed over an antibody array
to assess angiogenic protein secretion levels. (b) Hypoxia induces
significant up-regulation of multiple cytokines byFluc+/eGFP+hESC-
CMs. Following 12 hours of hypoxia in vitro, media from
Fluc+/eGFP+hESC-CMs had increased levels of FGF, IL-6, IL-8
and VEGF as compared to cells maintained in normoxic
Found at: doi:10.1371/journal.pone.0003474.s009 (0.81 MB TIF)
Found at: doi:10.1371/journal.pone.0003474.s010 (13.12 MB
Microarray data tables of differentially-regulated
Found at: doi:10.1371/journal.pone.0003474.s011 (0.30 MB
Gene Ontology analysis of differentially-regulated
Found at: doi:10.1371/journal.pone.0003474.s012 (3.37 MB
K-means clustering analysis of microarray data.
Found at: doi:10.1371/journal.pone.0003474.s013 (0.20 MB
Gene Ontology analysis of K-means clustering data.
Found at: doi:10.1371/journal.pone.0003474.s014 (1.78 MB
Beating embryoid body.
Found at: doi:10.1371/journal.pone.0003474.s015 (0.93 MB
Beating left ventricular fetal cardiomyocytes.
Found at: doi:10.1371/journal.pone.0003474.s016 (0.39 MB
Beating hESC-CMs after Percoll purification.
Conceived and designed the experiments: FC RW KDW RAL ILW RR
JCW. Performed the experiments: FC RW KDW XX. Analyzed the data:
FC RW KDW JDF MD AL RAL SSG ILW RR JCW. Contributed
reagents/materials/analysis tools: XX JDF MD AL RAL SSG ILW RR.
Wrote the paper: RW KDW.
Profiling of hESC-CMs
PLoS ONE | www.plosone.org11 October 2008 | Volume 3 | Issue 10 | e3474
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PLoS ONE | www.plosone.org 12October 2008 | Volume 3 | Issue 10 | e3474