Integrative Physiology

Induced Pluripotent Stem Cell (iPSC)–Derived

Extracellular Vesicles Are Safer and More Effective
for Cardiac Repair Than iPSCs
Marta Adamiak,* Guangming Cheng,* Sylwia Bobis-Wozowicz, Lin Zhao,
Sylwia Kedracka-Krok, Anweshan Samanta, Elzbieta Karnas, Yu-Ting Xuan,
Bozena Skupien-Rabian, Xing Chen, Urszula Jankowska, Magdy Girgis, Malgorzata Sekula,
Arash Davani, Slawomir Lasota, Robert J. Vincent, Michal Sarna, Kathy L. Newell,
Ou-Li Wang, Nathaniel Dudley, Zbigniew Madeja, Buddhadeb Dawn, Ewa K. Zuba-Surma

Rationale: Extracellular vesicles (EVs) are tiny membrane-enclosed droplets released by cells through membrane
budding or exocytosis. The myocardial reparative abilities of EVs derived from induced pluripotent stem cells
(iPSCs) have not been directly compared with the source iPSCs.
Objective: To examine whether iPSC-derived EVs can influence the biological functions of cardiac cells in vitro and
to compare the safety and efficacy of iPSC-derived EVs (iPSC-EVs) and iPSCs for cardiac repair in vivo.
Methods and Results: Murine iPSCs were generated, and EVs isolated from culture supernatants by sequential
centrifugation. Atomic force microscopy, high-resolution flow cytometry, real-time quantitative RT-PCR, and
mass spectrometry were used to characterize EV morphology and contents. iPSC-EVs were enriched in miRNAs
and proteins with proangiogenic and cytoprotective properties. iPSC-EVs enhanced angiogenic, migratory, and
antiapoptotic properties of murine cardiac endothelial cells in vitro. To compare the cardiac reparative capacities
in vivo, vehicle, iPSCs, and iPSC-EVs were injected intramyocardially at 48 hours after a reperfused myocardial
infarction in mice. Compared with vehicle-injected mice, both iPSC- and iPSC-EV–treated mice exhibited improved
left ventricular function at 35 d after myocardial infarction, albeit iPSC-EVs rendered greater improvement.
iPSC-EV injection also resulted in reduction in left ventricular mass and superior perfusion in the infarct zone.
Downloaded from http://ahajournals.org by on December 18, 2020

Both iPSCs and iPSC-EVs preserved viable myocardium in the infarct zone, whereas reduction in apoptosis was
significant with iPSC-EVs. iPSC injection resulted in teratoma formation, whereas iPSC-EV injection was safe.
Conclusions: iPSC-derived EVs impart cytoprotective properties to cardiac cells in vitro and induce superior cardiac
repair in vivo with regard to left ventricular function, vascularization, and amelioration of apoptosis and hypertrophy.
Because of their acellular nature, iPSC-EVs represent a safer alternative for potential therapeutic applications in
patients with ischemic myocardial damage.   (Circ Res. 2018;122:296-309. DOI: 10.1161/CIRCRESAHA.117.311769.)
Key Words: angiogenesis ■ apoptosis ■ extracellular vesicles ■ induced pluripotent stem cells
■ myocardial infarction ■ remodeling ■ stem cells

B ecause the adult mammalian heart possesses limited regen-

erative capacity after injury, cell therapy has emerged as
a potential approach for repair of the infarcted heart. Several
adult stem and progenitor cells from various sources have al-
ready been tested in patients, albeit with modest benefits.1
Although induced pluripotent stem cells (iPSCs) offer exciting
opportunities for tissue restoration, recent evidence indicates
Editorial, see p 197
In This Issue, see p 185
Meet the First Author, see p 186
that in addition to cellular replacement, paracrine factors re-
leased by injected cells are also important for cell-based heart
repair.2 These paracrine factors may be secreted directly from

Original received July 21, 2017; revision received November 3, 2017; accepted November 7, 2017. In October 2017, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 13 days.
From the Department of Cell Biology (M.A., S.B.-W., E.K., S.L., Z.M., E.K.Z.-S), Department of Physical Biochemistry (S.K.-K., B.S.-R.), and
Department of Biophysics (M. Sarna), Jagiellonian University, Krakow, Poland; Division of Cardiovascular Diseases, Cardiovascular Research Institute
(G.C., L.Z., A.S., Y.-T.X., X.C., M.G., A.D., R.J.V., O.-L.W., N.D., B.D.) and Department of Pathology and Laboratory Medicine (K.L.N.), University of
Kansas Medical Center, Kansas City; and Malopolska Centre of Biotechnology, Krakow, Poland (E.K., B.S.-R., U.J., M. Sekula, M. Sarna).
*These authors contributed equally to this article.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
117.311769/-/DC1.
Correspondence to Ewa K. Zuba-Surma, PhD, DSc, Prof JU, Department of Cell Biology, Jagiellonian University, Krakow 30-663, Poland, E-mail ewa.
zuba-surma@uj.edu.pl; or Buddhadeb Dawn, MD, Division of Cardiovascular Diseases, Cardiovascular Research Institute, University of Kansas Medical
Center, Kansas City, KS 66160, E-mail bdawn@kumc.edu
© 2017 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.117.311769

296
 Adamiak et al   iPSC-EVs for Myocardial Repair   297
Novelty and Significance
What Is Known?
• Induced pluripotent stem cells (iPSCs) differentiate into numerous cell
types and, therefore, hold great promise for heart regeneration.
• Extracellular vesicles (EVs), including the fraction of exosomes derived
from various stem cells, have been shown to ameliorate myocardial
ischemia/reperfusion injury and promote cardiac repair.
• The comparative safety and efficacy of iPSCs and iPSC-derived EVs
(iPSC-EVs) for infarct repair have not been examined.
What New Information Does This Article Contribute?
• The molecular cargo within iPSC-EVs includes numerous miRNAs that
are present in iPSCs and several additional miRNAs that are enriched
specifically in EVs. These miRNAs are known to promote cellular
proliferation and differentiation, enhance angiogenesis, and prevent
apoptosis.
• The proteomic analysis of EVs revealed molecules that stimulate car-
diomyogenesis; promote cardiac, endothelial, and smooth muscle cell
proliferation; and protect against oxidative damage.
• The iPSC-EVs induced angiogenic, migratory, and antiapoptotic prop-
erties in cardiac endothelial cells in vitro and induced superior infarct
repair in vivo compared with iPSCs. iPSC injection resulted in teratoma
formation, whereas EV injection did not produce any tumor.
Nonstandard Abbreviations and Acronyms
EVs extracellular vesicles
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iPSC induced pluripotent stem cell
iPSC-EV induced pluripotent stem cell–derived extracellular vesicles
LV left ventricle
MI myocardial infarction
the transplanted cells or released as cargo in tiny membrane-en-
closed structures termed extracellular vesicles (EVs).2,3 Given
the potential of both iPSCs and iPSC-derived EVs (iPSC-EVs)
for myocardial repair, there is a strong rationale to directly com-
pare the safety and reparative efficacy of these 2 approaches.
EVs represent small (30–1000 nm) vesicles released by
cells either by outward budding from the plasma membrane or
via exocytosis.4 Upon shedding, EVs may fuse with the recipi-
ent cells directly or after endocytosis followed by the release of
contents or interact with target cells by receptor–ligand inter-
actions.5,6 Because EVs carry proteins, mRNAs, and miRNAs,
from their parental cells, they represent an important compo-
nent of paracrine signaling and cell-to-cell communication.7
Although the regenerative abilities of EVs harvested from mes-
enchymal stem cells and endothelial and cardiac progenitors
have been reported,8–10 the biological basis of EV actions needs
further elucidation. Thus, another major rationale for this study
was to characterize the molecular contents of EVs with regard
to potential paracrine activities relevant for myocardial repair.
Finally, although iPSCs have been known to produce terato-
mas,11 the safety of iPSC-EVs remains unknown.
Accordingly, we meticulously characterized the morphol-
ogy and contents of iPSC-EVs with a multi-instrumental
approach, examined their biological function in vitro, and
iPSCs, which are able to give rise to numerous cell types, have
shown great promise for heart regeneration. EVs, minute mem-
brane-enclosed droplets released by mammalian cells, have also
been shown to ameliorate myocardial ischemia/reperfusion in-
jury. However, the myocardial reparative abilities of EVs derived
from iPSCs have not been directly compared with the source
iPSCs. The results from quantitative RT-PCR and proteomic analy-
ses revealed that EV cargo is enriched with numerous molecules
that impart cytoprotective and angiogenic benefits to cardiac
cells. After ischemia/reperfusion and transplantation in vivo, both
iPSC- and iPSC-EV–treated mice exhibited improved LV function
compared with vehicle-treated mice; however, iPSC-EVs induced
greater functional benefits and resulted in superior perfusion in
the infarct zone and prevented apoptosis. iPSC injections resulted
in teratoma formation, whereas iPSC-EV injections were safe.
These observations indicate that iPSC-EVs confer cytoprotective
properties to cardiac cells in vitro and induce superior cardiac
repair in vivo without the risk of teratoma formation seen with
parental iPSCs. Thus, iPSC-EVs offer a safer alternative for poten-
tial therapeutic applications in patients with ischemic myocardial
damage.
compared head-to-head, for the first time, the safety and effi-
cacy of iPSC-EVs with their parental iPSCs for cardiac repair
in vivo. Our results show that iPSC-EVs carry unique sets of
proteins and regulatory miRNAs and impact by angiogenic
capacity, migratory activity, and survival of cardiac endothe-
lial cells (CECs) in cytotoxic environment. In vivo, injection
of iPSC-EVs after acute myocardial infarction (MI) improves
left ventricular (LV) function and remodeling. Importantly, we
provide evidence that although injection of iPSCs leads to tu-
mor formation, the use of EVs derived from these cells is safe.
Methods
An expanded methods section is available in the Online Data
Supplement.
The authors declare that all data that support the findings of this
study are available within the article and its Online Data Supplement.
The present study was performed in accordance with the guide-
lines of the Animal Care and Use Committee of the University of
Kansas Medical Center and with the Guide for the Care and Use of
Laboratory Animals (Department of Health and Human Services, pub-
lication No. [National Institutes of Health] 86-23) and also in accor-
dance with the Polish and European legislation after approval by the
First Local Ethical Committee on Animal Testing at the Jagiellonian
University. The data that support the findings of this study are avail-
able from the corresponding authors on reasonable request.
iPSC Generation
Murine fibroblasts were transfected with lentivirus expressing Oct4,
Sox2 (sex-determining region Y-box 2), and Klf4. iPSC colonies were
picked, further expanded, and adapted to serum-free conditions. Colonies
with high contents of pluripotency-associated transcripts (Online Figure
I) were chosen for further studies. iPSCs were phenotyped by flow cy-
tometry and immunocytochemistry following standard protocols.
EV Purification
EVs were prepared from the culture supernatant of iPSCs using a pro-
tocol optimized in our laboratory (Online Figure II).12 Nanoparticle
 298  Circulation Research  January 19, 2018
tracking analysis, atomic force microscopy, high-resolution flow cytome-
try, and quantitative RT-PCR were used to characterize EVs. MicroRNA
expression profiling was performed using quantitative RT-PCR.
EV Protein Analysis by LC-MS/MS
After lysis, samples from iPSCs and iPSC-EVs for LC-MS/MS (liquid
chromatography–mass spectrometry) analysis were prepared using
the FASP (filter-aided sample preparation) method.13 Peptide fractions
were analyzed using Q Exactive high-resolution mass spectrometer
coupled with nanoHPLC. The data were analyzed using Proteome
Discoverer 1.4 and a MASCOT server against the Swissprot_201509
database. Search result validation was performed using Percolator al-
gorithm.14 Gene ontology (GO) was analyzed using FatiGO software.
Assessment of EV Function In Vitro
Murine CECs and fluorescent EVs were used for cellular uptake stud-
ies. Capillary tube formation assay was performed on Matrigel. The
movement of CECs was time-lapse recorded, and cell trajectories
were constructed and analyzed. CEC viability and apoptosis were
analyzed by flow cytometry.
Experimental Protocol for the In Vivo Studies
A total of 43 wild-type (C57BL6/J) mice were used for the in vivo
studies. The experimental protocol is summarized in Online Figure
III. All mice underwent a 30-minute coronary occlusion followed by
reperfusion. After 48 hours, chest was reopened, and mice received
intramyocardial injection of vehicle (group I), iPSCs (group II), or
iPSC-EVs (group III). Echocardiographic studies were performed 4
days before coronary occlusion/reperfusion, at 48 hours after cell in-
jection, and 35 days after MI using a Vevo 2100 Ultrasound System.15
Morphometry and Immunohistochemistry
After 35 days, mice were euthanized and hearts harvested and for-
malin fixed. All morphometric and immunohistochemical analyses
were performed on 4-μm thick sections using standard methods.15,16
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Capillary density was quantitated in isolectin-B4–stained sections.
Interstitial collagen was quantified on images taken from the nonisch-
emic myocardium of picrosirius red-stained sections using ImagePro
Plus. Myocyte apoptosis was quantitated using TUNEL (terminal de-
oxynucleotidyl transferase dUTP nick-end labeling) assay.15
Statistical Analysis
Data are expressed as mean±SEM or SD as indicated. The impact of
iPSC-EVs on the formation of CECs into capillary-like structures was
evaluated using the Mann–Whitney U test. The movement parameters
for CECs were compared using the Student t test. Morphometric and
histological data were analyzed using 1-way ANOVA, whereas serial
echocardiographic parameters were analyzed using 2-way (time and
group) ANOVA followed by Student t tests with the Bonferroni cor-
rection. Comparisons between 2 groups were performed by unpaired
Student t test. P values <0.05 were considered statistically significant.
Statistical analyses were performed using the GraphPad Prism (ver-
sion 4.03; GraphPad Software, Inc, La Jolla, CA) and SPSS software,
version 22.0 (IBM, Armonk, NY).
Results
In Vitro Studies
iPSCs Expanded in Optimized Serum- and Feeder-Free
Conditions Maintained Pluripotency as a Source of
Purified EVs
To obtain pure iPSC-EV specimens without contamination of
nanoparticles that originated from other cells or from serum
present in the culture system,17 all iPSC clones were expanded
in optimized culture conditions in serum-free media without
feeder layer cells. Using a multi-instrumental approach, we
confirmed the presence of a stable pluripotent phenotype in
the selected iPSC lines (Online Figure I). iPSCs expressed
pluripotency-related markers, including Oct3/4A, Nanog,
Sox2, and Rex1, at the mRNA and protein levels, as well as
placental alkaline phosphatase (Figure 1A through 1C; Online
Figure I). iPSCs also expressed adhesion molecules and
growth factor receptors considered important for stem cell ac-
tivities, including CD49e, CD29, CD105, and CD117, which
may be potentially transferred to iPSC-EVs (Figure 1D).
These results provide evidence that pluripotent iPSCs can be
effectively cultured in feeder- and serum-free conditions for
uncontaminated harvest of EVs.
Next, iPSC-EVs were successfully isolated from the iPSC-
conditioned media by a sequential centrifugation procedure,
including ultracentrifugation, resulting in EV specimens con-
taining both microvesicles and exosome fractions (Figure 2A).
Importantly, we confirmed the presence of iPSC-EVs accord-
ing to the current recommendations18 by examining their (1)
size distribution by nanoparticle tracking analysis platform, (2)
high-resolution morphology and membrane elasticity by atom-
ic force microscopy, and (3) expression of EV-related antigens
(eg, CD9 and CD81 tetraspanins) by Western and high-reso-
lution flow cytometry (Figure 2B through 2E). Moreover, we
confirmed the iPSC origin by detecting transcripts for pluripo-
tent transcription factors Oct3/4, Nanog, and Rex1 (Figure 2F).
These data confirm that the iPSC-EV specimens used in further
studies represent homogenous vesicular fractions that exhibit
common EV morphology and antigen expression, along with
the presence of iPSC-derived markers.
iPSC-EVs Contained iPSC-Specific Molecules and Were
Enriched in a Distinctive Set of miRNAs and Proteins
To identify the molecular contents, which may be responsible
for the biological activity and regenerative capacity of EVs, we
next performed global miRNA and proteomic profiling of iPSC-
EV specimens. Among 282 miRNAs detected in iPSCs, 199
were also present in iPSC-EVs (Figure 2G), indicating efficient
transfer of these regulatory transcripts from the parental iPSCs
to EVs. Importantly, miRNAs belonging to the miR-290-295
cluster regulating pluripotency,19 including miR-292, -293, -294,
and miR-295, were abundant not only in the parental cells but
also in the iPSC-EVs (Online Tables I and II). Moreover, among
the miRNAs present in both iPSCs and iPSC-EVs, we found
miR-19b, miR-20a, miR-126-3p, miR-130a-3p, miR-210-3p,
and the longevimir cluster miR-17-92 reportedly involved in the
promotion of angiogenesis, adaptation to hypoxic stress, regula-
tion of cell cycle, mammalian development, and aging.20
Moreover, certain miRNAs were differentially expressed
in iPSCs and iPSC-EVs (Figure 2G; Online Tables I and
II). Indeed, 33 miRNAs were detected only in iPSC-EVs by
qPCR-based method (Figure 2G; Online Table III), which
suggests their high enrichment during EV production and
release by iPSCs—a phenomenon suggested previously.21
MicroRNAs enriched in EVs, including let-7, miR-145, miR-
17-92 cluster, and ESC-specific miR-302a-5p, have been sug-
gested to play a role in late developmental timing; regulation
of cell proliferation, differentiation, apoptosis, and mainte-
nance of self-renewal and pluripotency19,20,22; and may exert
possible beneficial effects on myocardial tissue. Moreover, the
heatmap analysis indicated other signaling pathways poten-
tially regulated by miRNA contents of iPSC-EVs, including
 Adamiak et al   iPSC-EVs for Myocardial Repair   299
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Figure 1. Induced pluripotent stem cell (iPSC) characterization. A, Representative dot plots showing the expression of intracellular
markers of pluripotency in iPSCs. B–C, Representative images showing the morphology of iPSC colonies in serum- and feeder-
free culture and fluorescence staining for fetal alkaline phosphatase (PALP) activity (B), and expression of pluripotency markers
Oct3/4, Nanog, Sox2 (sex-determining region Y-box 2), and SSEA-1 (stage-specific embryonic antigen-1; C). Scale bar, 200 μm. D,
Representative histograms showing the antigenic phenotype of iPSCs by flow cytometry. The red-colored histograms represent samples
stained for specific surface antigens, whereas gray-colored ones correspond to respective unstained control samples. Data represent
mean±SD from 3 independent experiments. APC indicates allophycocyanin; BF, brightfield image; and PE, phycoerythrin.

Wnt, PI3K-Akt, and MAPK (mitogen-activated protein ki-
nase) pathways, which are involved in the regulation of actin
cytoskeleton, focal adhesion, and ECM-receptor interactions
(Online Figures IV and V). Moreover, additional examina-
tion by Ingenuity Pathway Analysis tool (Qiagen) identified
several miRNAs abundant in iPSC-EVs, including miR-294,
miR-16, miR-34, and miR-20, which may regulate VEGFA
(vascular endothelial growth factor A) signaling, thereby po-
tentially enhancing angiogenesis (Online Figure VI).
Next, we performed high-throughput global proteomic
analysis of iPSCs and iPSC-EVs using mass spectrometry.
Importantly, the number of proteins identified in challenging
iPSC-EV samples and also in iPSC samples was highly re-
producible and consistent across all replicates, confirming the
high quality of samples and proteomic procedures (Figure 3A).
Proteins within each group (iPSCs or iPSC-EVs) were consid-
ered for analysis only when proteins were reliably identified
within the samples, ie, (1) in each replicate of the group and
(2) in at least 2 of 3 replicates, on the basis of ≥2 unique pep-
tides. These strict analytic criteria were fulfilled by 4122 and
1965 proteins detected in iPSCs and iPSC-EVs, respectively
(Figure 3A). Because of the high number of common proteins
 300  Circulation Research  January 19, 2018
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Figure 2. Characterization of induced pluripotent stem cell–derived extracellular vesicles (iPSC-EVs). A, Representative iPSC-EV
size distribution histogram by nanoparticle tracking analysis. The cumulative D50 parameter indicates that 50% of the population of
vesicles is <143 nm in diameter. B (left), representative atomic force microscopy (AFM) image of iPSC-derived EVs. Scale bar, 50 nm.
Right, 3D topography of individual vesicles. Scan area: 250×250 nm. C, The Young modulus (kPa) and adhesion (pN) values by AFM,
reflecting mechanical properties of iPSC-EVs. D, High-resolution flow cytometry. Left, Histogram showing the size distribution of mixed-
size synthetic beads (PS, fluorescently labeled polystyrene calibration beads detected in FITC (fluorescein isothiocyanate) channel; Si,
unlabeled silicone calibration beads). Right, Representative dot plots show the medium-angle light scatter (MALS) related to EV size
vs fluorescence intensity indicating expression of selected EV-specific (CD81) and iPSC-specific (SSEA-1 [stage-specific embryonic
antigen-1]) antigens on iPSC-EVs. E, Representative Western immunoblot confirming the presence of typical exosomal marker CD9 in
iPSC-EV specimens. F, Presence of mRNA transcripts for pluripotency-related markers in iPSCs and iPSC-EVs by real-time RT-PCR.
Data are shown as CT mean values. G, Venn diagram showing the number of miRNAs common and specific for iPSCs and iPSC-EVs. H,
Scatter plot of miRNA expression in iPSCs (x axis) and iPSC-EVs (y axis); each dot represents 1 transcript. Data represent mean±SD from
3 independent experiments. PE indicates phycoerythrin.

detected in both groups, confirming again the effective transfer
of major molecular contents from the parental iPSCs to EVs,
we focused our analysis on comparing the unique proteins iden-
tified in all samples of a given group and none of the samples
from the other groups. A total of 1536 differential proteins were
found to be present in iPSCs and 191 in iPSC-EVs (Figure 3A).
GO analysis for molecular function, cellular component,
and biological processes related to the most apparent differ-
ences between parental iPSCs and iPSC-EVs are shown in
Figure 3B through 3E and Online Figure VII. As expected,
the proteins identified in iPSC-EVs were the most strongly
represented (by GO terms) in plasma membrane, cytosol, and
cytoskeleton but not in other organelle compartments not pres-
ent in EV structure (Figure 3C). Importantly, the GO analysis
of biological processes revealed an enrichment of differential
proteins involved in response to wound healing, regulation of
cell differentiation, as well as organ, anatomic structure, and
multicellular organism development (Figure 3D; Online Figure
 Adamiak et al   iPSC-EVs for Myocardial Repair   301

A B
Group No. of all No. of No. of Common Differential iPSCs iPSC-derived EVs
sample proteins proteins proteins proteins in proteins in
identified in identified in identified in the group* the group†
the group the sample the sample
based on 2 or
Molecular function

% of proteins in the given GO term

more unique 30
peptides
25
iPSCs
20
1 6325 5412 4156 4122 1536
15
2 5551 4315
10
3 5561 4351
iPSC-EVs 5

1 4111 3329 2253 1965 191 0

2 2923 1838
3 3427 2308
*Proteins identified in at least 2 of 3 samples on the basis of two or more unique peptides.
†Proteins identified in all samples of a given group and none of the samples from the other group.

C Cellular component
% of proteins in the given GO term

16

14

12

10

D Biological process
% of proteins in the given GO term

18
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16
14
12
10
8
6
4
2
0

E 35 Molecular function
% of proteins in the given GO term

30

25

20

15

10

Figure 3. Global proteomic contents of induced pluripotent stem cells (iPSCs) and iPSC-derived extracellular vesicles (iPSC-EVs)
by mass spectrometry. A, The number of proteins identified in iPSCs and their EVs. The absolute values are shown individually for each
replicate (n=3) and as average for each examined group. B–D, Gene ontology (GO) analysis, including common proteins detected in
both iPSCs and iPSC-EVs, with focus on molecular function, cellular components, and biological processes. E, GO enrichment analysis
for differential expression of proteins in iPSCs and iPSC-EVs, with focus on molecular function. GO terms related to the most apparent
differences between iPSCs and iPSC-EVs are shown.
 302  Circulation Research  January 19, 2018
VIIA). Importantly, several proteins involved in angiogenesis
signaling pathways (VEGF-C [vascular endothelial growth
factor C], BMP-4 [bone morphogenetic protein-4], PDGFα
[platelet-derived growth factor α], TDGF1 [teratocarcinoma-
derived growth factor 1], CTGF [connective tissue growth
factor], and thrombospondin-1) were found within GO group
guiding multicellular organism development (Online Figure
VIIIA). Moreover, proteins stimulating cardiomyogenesis
(BMP-4 and FGFs [fibroblast growth factors]); promoting
cardiac, endothelial, and smooth muscle cell proliferation
(PDGFs, IGF-2 [insulin-like growth factor-2], and FGFs); and
protecting cells against oxidative damage (hemopexin) were
identified within GO group involved in signal transduction
(Online Figure VIIIB). Thus, proteomic data indicated that the
majority of iPSC-specific proteins is transferred from the pa-
rental iPSCs to iPSC-EVs and may suggest the impact of such
protein contents on several biological processes in target cells,
including the ones associated with tissue repair.
iPSC-EVs Improved CEC Function by Enhancing
Angiogenic Capacity, Migration, and Survival In Vitro
Increasing evidence indicates that angiogenesis is a major
mechanism responsible for the improvement in LV function
with cell therapy after ischemic myocardial injury. Because
our data identified several miRNAs and proteins within the
iPSC-EVs, which may regulate target cell function, including
proliferation, differentiation, survival, and angiogenesis, we
investigated the impact of iPSC-EVs on selected functions of
murine CECs in vitro, with emphasis on features potentially
important for heart repair. First, we confirmed that iPSC-EVs
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were internalized by CECs after only 1 hour of coincubation,
as shown by optical orthogonal sectioning in xy axis by fluo-
rescent microscopy (Figure 4A). Compared with control-un-
treated cells, CECs treated with iPSC-EVs exhibited greater
ability to form endotubules on Matrigel by quantitative analy-
sis (Figure 4B; Online Figure IX; Online Movies I and II).
These data suggest that iPSC-EVs are likely to exert proan-
giogenic effects after myocardial injection in vivo.
Next, we addressed whether iPSC-EVs influence CEC mi-
gratory capacity, which has been reported as a crucial functional
property required in wound healing, tissue repair, including
myocardial regeneration, as well as during new vessel forma-
tion and maturation.23 The quantitative analysis of recorded CEC
trajectories, based on time-lapse monitoring of spontaneous un-
guided movement of single cells, showed greater speed of cell
displacement after iPSC-EV treatment compared with controls
(Figure 4C). This phenomenon was more pronounced and sta-
tistically significant after prolonged EV treatment, when migra-
tion was analyzed between 9 and 14 hours of CEC coincubation
with iPSC-EVs. These data indicate that iPSC-EVs enhance the
migratory capacity of CECs, which may augment new vessel
formation and maturation after iPSC-EV transplantation in vivo.
Finally, to examine the impact of iPSC-EVs on cell surviv-
al in cytotoxic conditions that often accompany tissue injury,
we used staurosporine (1 μM), a cytotoxic agent commonly
used to induce apoptosis, to challenge CECs before their incu-
bation with iPSC-EVs. We measured cell death processes, in-
cluding apoptosis and necrosis, after 24 hours of coincubation
of staurosporine-challenged CECs with iPSC-EVs by flow
cytometry. Exposure to iPSC-EVs significantly decreased
the incidence of staurosporine-induced apoptosis (38.1±4.7%
versus 54.4±1.7% of cells in late apoptosis among CECs in-
cubated with iPSC-EVs versus control-untreated cells, respec-
tively; P<0.05; Figure 4D). Moreover, the number of viable
cells was significantly greater in samples treated with iPSC-
EVs (29.3±5.3%) when compared with control CECs treated
with staurosporine alone (13.4±0.7%). Importantly, there was
no change in the number of cells undergoing necrosis in either
iPSC-EV–treated or untreated samples (Figure 4D). These re-
sults indicate cytoprotective and antiapoptotic effects of iPSC-
EVs on CECs in vitro, which may play an important role in
protecting ischemic myocytes in vivo.
In Vivo Studies
Exclusions
Four mice died in the perioperative period, and 3 mice died at
5, 19, and 29 days after intramyocardial injection. Eight mice
were excluded from the study because of cardiac tumor for-
mation in the iPSC-treated group, leaving a total of 9, 7, and
12 mice in vehicle, iPSC, and iPSC-EV groups, respectively
(Online Table IV).
Myocardial Infarct Size
The infarct area fraction denotes the average area of scar tis-
sue, expressed as a percentage of the LV area in 3 LV sections
0.5 to 1.0 mm apart. The average infarct area fraction did not
differ significantly among the 3 groups (Online Figure X).
Intramyocardial Injection of iPSC-EVs Induced Greater
Attenuation of LV Dysfunction
Before coronary occlusion (baseline), all parameters of LV
function were similar in groups I through III (Figure 5). At
48 hours after cell or EV transplantation (4 days after MI),
the degree of LV dysfunction was also similar among the 3
groups (Figure 5B and 5C), indicating that the injuries sus-
tained during ischemia/reperfusion and intramyocardial injec-
tion were similar in all groups. In vehicle-treated (group I)
mice, there was further LV functional deterioration between
4 and 35 days after reperfusion. In contrast, both iPSC- and
iPSC-EV–treated mice (groups II and III, respectively) exhib-
ited significantly improved global (Figure 5B through 5E) LV
function compared with group I at 35 days after MI. Mice in
groups II and III also showed enhanced regional myocardial
function in the infarct zone, as evidenced by a 10% (P<0.05)
and 19% (P<0.01) greater systolic infarct wall thickness
(Figure 5A and 5F), respectively, compared with group I. The
preservation of LV function was significantly greater in hearts
injected with iPSC-EVs compared with iPSC-injected hearts
(Figure 5B and 5C). In addition, mice in group III also exhib-
ited smaller LV end-systolic volume compared with vehicle-
treated mice (Figure 5E).
Intramyocardial Delivery of iPSCs and iPSC-EVs Halted
LV Remodeling and Hypertrophy
The echocardiographic measurements of LV remodel-
ing at 35 days mirrored the observations on LV function.
The LV end-diastolic diameter was significantly smaller
in groups II and III compared with group I (Figure 6B).
 Adamiak et al   iPSC-EVs for Myocardial Repair   303
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Figure 4. Impact of induced pluripotent stem cell–derived extracellular vesicles (iPSC-EVs) on cardiac endothelial cell (CEC)
function. A, Representative Z-stacked image showing uptake of fluorescently labeled iPSC-EVs by CECs. One set of orthogonal
slices is shown. Middle, right, and bottom represent XY, YZ, and XZ planes, respectively. YZ and XZ planes intersect according to the
crosshairs. Scale bar, 10 μm. B–D, Impact of iPSC-EVs on CEC functions: (B) angiogenic capacity on Matrigel; scale bar, 100 μm. Each
bar represents mean values±SD from 3 independent experiments (*P<0.1, **P<0.01, ***P<0.001); (C) migratory activity of untreated
CECs (control) and CECs treated with iPSC-EVs. Cell trajectories for each period are depicted as circular diagrams (axis scale in μm).
Selected migration parameters are shown on the graphs (bottom). Each bar represents mean values from 3 independent experiments
(*P<0.05); (D) representative dot plots showing the impact of treatment with iPSC-EVs on survival of CECs after exposure to cytotoxic
agent staurosporine. Data represent mean±SD from 3 independent experiments (*P<0.05). CME indicates coefficient of movement; PE,
phycoerythrin efficiency; and STS, staurosporine.

Also, compared with group I, the infarct wall thickness
in diastole was significantly greater both in groups II and
III (Figure 6C), indicating superior infarct repair. The
posterior LV wall thickness was smaller in groups II and
III compared with group I (Figure 6D), indicating less
hypertrophy of the viable myocardium. Although the im-
provements in LV end-diastolic diameter and infarct wall
thickness were numerically greater in iPSC-EV– compared
with iPSC-treated hearts, the differences were not statisti-
cally significant.
 304  Circulation Research  January 19, 2018
Intramyocardial Delivery of iPSC-EVs Attenuated
LV Hypertrophy
To assess the impact of therapy on the myocardium, interstitial
fibrosis was carefully quantitated. At 35 days after MI, inter-
stitial fibrosis was similar in vehicle- and iPSC-treated hearts
(Figure 6E and 6F). Although not statistically significant, in
Downloaded from http://ahajournals.org by on December 18, 2020
mice treated with iPSC-EVs, interstitial fibrosis was 11% less
compared with vehicle-treated hearts (Figure 6E and 6F). At
35 days after MI, the echocardiographically estimated LV mass
increased significantly in all groups compared with baseline
values. Compared with group I, the LV mass was 16% (P<0.05)
smaller in iPSC-EVs–treated mice (Figure 6G). Taken togeth-
er, these data indicate that injection of iPSC-EVs attenuates
the adverse adaptations in surviving LV myocardium after MI.
Effects of iPSC and iPSC-EV Therapy on Myocardial
Capillary Density
Because myocardial vascularity plays an important role in
cardiac repair post-MI, we assessed capillary density in the
infarct zone, borderzone, and nonischemic zone separately.
At 35 days after MI, compared with vehicle-treated hearts,
capillary numbers in nonischemic zone and infarct border-
zone were greater in both iPSC- and iPSC-EV–treated hearts
(Figure 7). In the infarct zone, compared with vehicle treat-
ment, iPSC-EV injection, but not iPSC injection, resulted in
significantly greater capillary numbers. These data indicate
that intramyocardial delivery of both iPSCs and iPSC-EVs
promotes angiogenesis in infarct border zone and nonisch-
emic zone, whereas iPSC-EV therapy extends neovasculariza-
tion in the infarct zone as well.
Effects on Viable Myocardium Within the Infarct Zone and
Myocyte Apoptosis
The effects of reparative therapies with regard to potential cel-
lular replacement and myocyte salvage were assessed quan-
titatively. Myocytes constituted 45.1±1.2%, 51.4±2.7%, and
Figure 5. Assessment of left ventricular
(LV) systolic function. A, Representative
M-mode images from vehicle-, induced
pluripotent stem cell (iPSC)-, and iPSC-
derived extracellular vesicles (iPSC-
EV)–treated mice at 35 d after coronary
occlusion/reperfusion. Compared with
the vehicle-treated heart, both iPSC- and
iPSC-EV–treated hearts exhibit improved
wall motion, smaller LV cavity (D and E),
and thicker infarct wall (F). Transplantation
of iPSC-EVs resulted in greater
improvement in LV systolic function (B
and C). Data are mean±SEM. n=7 to 12
mice per group. BSL indicates baseline.
*P<0.05 vs vehicle at 35 d, #P<0.05 vs
iPSC group at 35 d.
53.1±1.6% of the infarct zone in groups I, II, and III, respec-
tively (Figure 8A through 8D); therefore, the amount of vi-
able myocardium in the infarct zone was, on average, 14% and
18% greater in iPSC- and iPSC-EV–treated mice compared
with vehicle-treated mice, respectively (both P<0.05 versus
vehicle-treated group). However, there was no significant dif-
ference between iPSCs and iPSC-EVs groups.
In the infarct zone, the percentage of TUNEL-positive
apoptotic cardiomyocyte nuclei was similar in groups I and II,
whereas iPSC-EV treatment reduced apoptosis modestly yet
significantly (Figure 8E and 8F). In the borderzone and non-
ischemic zone, compared with vehicle injection, iPSC injec-
tion reduced the percentage of apoptotic myocytes minimally,
whereas iPSC-EV injection was associated with significant
reduction in myocyte apoptosis. These data indicate that in-
tramyocardial delivery of iPSC-EVs protects against myocyte
apoptosis during postinfarct remodeling.
Intramyocardial Delivery of iPSCs Induced Teratoma
Formation
Development of cardiac tumor was noted in a large number of
mice (8 of 15; 53%) in the iPSC-treated group. Gross morpho-
logical features, variegated tissue composition, and histologi-
cal characteristics suggesting differentiation into derivatives
of all 3 germ layers identified these tumors as teratomas
(Figure 8G; Online Figure XI). No such tumor was noted in
any mouse in iPSC-EV– or vehicle-injected groups.
Discussion
Although numerous strategies for cell-based cardiac repair
have been tested in patients with ischemic heart disease, the
benefits of such therapies are still only modest in clinical tri-
als.1,24 Growing evidence indicates that paracrine effects of
EVs released by various cells can protect the endogenous
myocardial tissue from cell death and other adverse effects,
 Adamiak et al   iPSC-EVs for Myocardial Repair   305
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Figure 6. Assessment of left ventricular (LV) remodeling and hypertrophy. A, Representative Masson trichrome-stained myocardial
sections at 35 d after myocardial infarction (MI) show improved remodeling in induced pluripotent stem cell (iPSC)- and iPSC-derived
extracellular vesicles (iPSC-EV)–treated hearts. Scar tissue and viable myocardium are identified in blue and red, respectively. Scale
bar, 500 μm. Echocardiographically estimated LV end-diastolic diameter (B) was smaller in both iPSC- and iPSC-EV–treated groups
compared with the vehicle-treated group. LV infarct wall thickness in diastole (C) was greater and posterior wall thickness (D) was smaller
in both groups. E and F, Interstitial fibrosis in the viable myocardium was quantitated in picrosirius red-stained myocardial sections (E) at
35 d after MI and quantified (F). Scale bar, 50 μm. G, Echocardiographically estimated LV mass was smaller in iPSC-EV–treated hearts
compared with vehicle-treated hearts. Data are mean±SEM. n=7 to 12 mice per group. BSL indicates baseline. *P<0.05 vs vehicle at 35 d.

leading to improved remodeling and preserved function.8,25
The current results provide novel information in this regard.
Salient Findings
We performed an extensive multidimensional evaluation of
the molecular contents, biological effects, and regenerative
capacity of iPSC-EVs, consisting of both ectosomal and exo-
somal fractions, on cardiac cell function in vitro. For the first
time, the safety and cardiac reparative efficacy of iPSC-EVs
were directly compared with those of iPSCs in a model of
reperfused MI in vivo. Our results show that (1) iPSCs release
EVs that carry significant amounts of bioactive contents of the
parent cell, along with a distinct set of miRNAs and proteins;
(2) iPSC-EVs enhance angiogenic capacity, migratory proper-
ties, and survival of heart-derived cells; (3) intramyocardial
transplantation of both iPSCs and iPSC-EVs after a reper-
fused MI improves LV function, albeit iPSC-EVs render supe-
rior benefits; (4) the in vitro effects of iPSC-EVs are translated
in vivo with reduced myocyte apoptosis and enhanced angio-
genesis; and (5) iPSC injection is associated with tumor for-
mation, whereas iPSC-EVs seem safe. Together, these results
indicate that injection of bioactive, cell-free, iPSC-EV speci-
mens represents an effective and safe approach for cardiac re-
pair after ischemic injury.
 306  Circulation Research  January 19, 2018
Paracrine activities of transplanted cells have been widely
implicated in tissue regeneration and heart repair. Indeed, our
previous work suggests that adult bone marrow-derived cells
improve heart function by increasing new vessel formation
and myocardial perfusion because of growth factors released
by injected cells.26,27 Recent evidence also indicates that tiny
vesicles termed EVs carry various bioactive molecules that
may ultimately mediate the paracrine effects of stem cells.
Although the composition of EV cargo depends on the specif-
ic cell type of origin, and influences the EV functional effects
on target cells,28 iPSC-EVs have not been characterized in suf-
ficient detail. Our current data constitute the first comprehen-
sive multi-instrumental analysis of cargo in murine iPSC-EVs
and show that iPSC-EVs are enriched in numerous mRNA,
Downloaded from http://ahajournals.org by on December 18, 2020
miRNA, and proteins that originate from parental iPSCs and
also possess a unique set of factors, which may be transferred
to cardiac cells. The miRNAs and proteins in iPSC-EVs were
related not only to pluripotency but also to activation of cell
proliferation, differentiation, and angiogenic activity.
Importantly, we cultured iPSCs in feeder- and serum-free
conditions in composition-defined medium, which are crucial
factors for the quality and purity of iPSC-EVs. It has been
shown that the presence of any serum in EV donor cell culture
significantly increased the number of serum-derived particles
coisolated with the EV specimens, which may influence the re-
sults and data interpretation.29 Moreover, several laboratories,
including ours, have reported major impacts of different expan-
sion media on the molecular composition and biological prop-
erties of EVs released by the same type of stem cell, suggesting
the critical need for appropriate EV preparation and character-
ization before their administration.30,31 Thus, we selected serum-
free, fully defined medium for iPSC culture before iPSC-EV
harvest, which simulated closely the GMP standards for cellular
expansion for future application in humans. Our results confirm
that similar to iPSCs cultured in presence of serum,32 iPSCs ex-
panded under our defined serum-free conditions preserved plu-
ripotent characteristics (Figure 1) and released iPSC-EVs with
normal morphology and antigenic phenotype (Figure 2).
By performing global miRNA analysis, we found >200
regulatory miRNAs in iPSC-EV cargo, including 33 that
were enriched in the vesicles when compared with the donor
iPSCs (Online Table I). The miRNAs enriched in iPSC-EVs
included the miR-145, let-7 family, and miR-302a-5p that are
known to regulate cell proliferation, differentiation, as well
Figure 7. Impact on myocardial
capillary density. A, Representative
images of capillary profiles in the
infarct zone (IZ), border zone (BZ), and
nonischemic zone (NZ). B, Quantitative
myocardial capillary density. Data
represent mean±SEM. Scale bar, 50
μm. EV indicates extracellular vesicle;
and iPSC, induced pluripotent stem cell.
*P<0.05 vs vehicle.
as self-renewal and pluripotency.19 Moreover, the iPSC-EV–
enriched miRNAs were accompanied with other transcripts
common for iPSCs, including miR-290-295 embryonic cluster
regulating pluripotency,19,33 as well as miR-19b, miR-20a, miR-
126-3p, miR-130a-3p, miR-210-3p, and embryonic longevimir
cluster miR-17-92, which promote angiogenesis, adaptation
to hypoxic stress, regulation of cell cycle, mammalian devel-
opment, and aging.20 These data suggest potential regulatory
effects of iPSC-EVs on survival, neovascularization, and pro-
regenerative properties after the transfer of such miRNAs into
cardiac cells.
Consistent with these findings at the miRNA level, we not
only found ≈2000 proteins that were common for iPSC-EVs
and their parental cells but also ≈200 proteins, which were en-
riched in iPSC-EVs. The GO analysis identified sets of proteins
enriched in iPSC-EVs, which are involved in response to ex-
ternal stimuli, wound healing, regulation of cell differentiation,
as well as development of organs and multicellular organisms
(Figure 3; Online Figure VIA). Importantly, several proteins in-
volved in angiogenesis and remodeling were identified as node
proteins within the network (Online Figure VII). Together, the
current proteomic data identify key novel components of the
iPSC-EV cargo that may exert reparative effects in vivo.
The above findings also indicate that the contents of iP-
SCs are efficiently transferred into EVs, thereby enabling
these vesicles as conveyors of iPSC molecular properties. The
potential role of EVs in transfer of proregenerative functions
to target cells was confirmed by increased angiogenic capac-
ity, migratory ability, and resistance to apoptosis of CECs af-
ter exposure to iPSC-EVs (Figure 4). These data also extend
our previous observations on the transfer of bioactive cargo
from human iPSC-EVs to primary cardiac mesenchymal
cells, thereby improving their proliferation, metabolic activ-
ity, as well as angiogenic and cardiomyogenic differentia-
tion.3 Furthermore, these molecular findings provide plausible
mechanisms underlying the iPSC-EV–induced benefits after
injection into the infarcted heart, which include (1) enhanced
angiogenesis, which in turn leads to superior remodeling;
(2) reduced apoptosis, which leads to greater preservation
of myocytes within the scar region; and (3) reduced fibrosis,
which leads to improved remodeling and reduced LV mass.
However, based on the size criteria, iPSC-EVs used in this
study contained both ectosomes and exosomes, described
as distinct EV subpopulations in terms of biogenesis, size,
 Adamiak et al   iPSC-EVs for Myocardial Repair   307
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Figure 8. Impact on cardiomyocyte salvage and neoplastic growth. Viable myocyte area fraction in the infarct zone (IZ). A–C,
representative examples of the infarct scar area in Masson trichrome-stained vehicle-treated (A), induced pluripotent stem cell
(iPSC)-treated (B), and iPSC-EV–treated (C) hearts. Scale bar, 50 μm. Quantitative data are presented in (D). Data are mean±SEM.
n=7 to 12 mice per group. *P<0.05 vs vehicle. Myocyte apoptosis. E, Representative images from the infarct borderzone after TUNEL
(terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining at 35 d after myocardial infarction. Apoptotic nuclei (white
arrows) are visualized by the green fluorescence. DAPI (4’,6-diamidino-2-phenylindole dihydrochloride) staining identifies nuclei
in blue. Cardiac myocytes are positive for α-sarcomeric actin (red). Scale bar, 50 μm. Quantitative data are presented in F. Data
represent mean±SEM. *P<0.05 vs vehicle, #P<0.05 vs iPSCs. BZ indicates borderzone; IZ, infarct zone; and NZ, (Continued )
 308  Circulation Research  January 19, 2018
Figure 8 Continued. nonischemic zone. G, representative gross morphologies of tumors from 2 iPSC-injected hearts (top);
photomicrographs from Masson trichrome-stained myocardial sections showing the variegated tissue composition of tumors (middle;
scale bar, 200 μm); and differentiation into ectodermal (neuroectoderm with pigment granules [arrows]; lower left), mesodermal (cartilage;
lower middle), and endodermal (respiratory epithelium with cilia [arrows]; lower right) lineages in hematoxylin and eosin–stained sections
from hearts harboring tumors. Scale bar, 50 μm.
molecular contents, and function.34 The potential differences
in bioactive molecular contents between these 2 fractions and
the mechanistic implications thereof with regard to heart re-
pair by each fraction separately remain to be explored.
Although both iPSCs and EVs have been used for car-
diac repair, each has unique advantages and disadvantages.
Therefore, we felt it was important to directly compare the
outcomes of cardiac repair with iPSCs and iPSC-EVs in vivo.
Although both improved LV function, the benefits of iPSC-
EVs were more pronounced. In addition, the improvement in
LV remodeling was superior with iPSC-EVs. This was evi-
dent in smaller LV end-systolic volume and LV mass in iPSC-
EV–treated mice. Although iPSC-EV injection also reduced
interstitial fibrosis by 11%, this did not reach statistical sig-
nificance. Furthermore, iPSC-EV injection enhanced capillary
density in the infarct zone, which may enhance the quality of
scar and further improve remodeling during longer follow-up.
This effect may directly relate to the transference of proangio-
genic properties by iPSC-EVs to host myocardial cells.
The reduced incidence of myocyte apoptosis in hearts in-
jected with iPSC-EVs constitutes another important observa-
tion. These findings are consistent with the in vitro findings
of greater survival of CECs after incubation with iPSC-EVs.
Similar cytoprotective effects of iPSC-EVs were noted in a pre-
Downloaded from http://ahajournals.org by on December 18, 2020
vious study by Wang et al,35 However, in that study, exosomes
were injected immediately after the coronary occlusion and
before reperfusion was achieved. The reduction of apoptosis
with exosome therapy was noted after 24 hours. The authors
reported reduced myocardial injury, which was explained pre-
dominantly by cytoprotective effects mediated by miR-21 and
miR-210 transferred from exosomes to cardiac cells.35 Our re-
sults show, for the first time, that iPSC-EVs confer cytoprotec-
tive benefits even when administered after reperfusion injury
has set in and these benefits persist during longer follow-up.
Although several miRNAs have been implicated in cytopro-
tection, miRNAs from the miR-17-92 cluster (eg, miR-19a,
miR-19b, and miR-20a) noted in high concentration in iPSC-
EVs may be responsible for this observation.36 Moreover, the
miR-17-92 cluster also enhances angiogenesis,37 which may
contribute toward improvement in heart function after iPSC-
EV injection. Consistently, our proteomic data also indicated
the enrichment of several proangiogenic molecules in iPSC-
EVs, including BMP-4, PDGFα, TDGF1, thrombospondin-1,
and VEGF-C. This enrichment of the molecular contents of
iPSC-EVs with proangiogenic and cytoprotective agents may
explain the beneficial effects observed in vivo.
Finally, the direct comparison of iPSCs and iPSC-EVs
also brings to light a vitally important finding with regard
to safety of regenerative therapy. Although the follow-up
duration was only 6 weeks, 8 of 15 mice injected with iP-
SCs developed teratomas within various cardiac structures.
No such neoplastic transformation was noted in the other 2
groups. Teratoma formation by iPSCs after injection in vivo
is a well-known phenomenon related to the pluripotent state
of these cells and has been observed in several prior stud-
ies.11,38,39 This phenomenon is currently one of the major
hurdles for therapeutic application of iPSCs for tissue re-
generation in humans. Our findings show that even though
the injection of parental iPSCs remains hazardous, superior
reparative benefits may be achieved safely with the injection
of EVs derived from those same cells.
Conclusions
The current findings provide comprehensive evidence that iP-
SC-EVs represent bioactive specimens that carry unique pay-
loads of molecules released by donor iPSCs, which may be
transferred to cardiac cells resulting in enhancement of biologi-
cal functions. Further, the first systematic comparison of iPSCs
and iPSC-EVs in vivo reveals superior efficacy of iPSC-EVs
for cytoprotection, vascularization, and cardiac repair. Finally,
the absence of tumor formation in the iPSC-EV group advances
the potential candidacy of the same for safe and effective heart
repair after ischemic myocardial injury in humans.
Acknowledgments
We thank Dr Grazyna Drabik, MD, from the Department of
Transplantation, Polish-American Children’s Hospital in Krakow for
assistance with pathological analysis of tumors.
Sources of Funding
This work was supported, in part, by the National Institutes of
Health grant R01 HL-117730 (B. Dawn), 2013/10/E/NZ3/00750 and
2015/16/W/NZ4/00071 grants from National Science Center (E.K.
Zuba-Surma), and TEAM-2012/9-6 grant from the Foundation for
Polish Science (E.K. Zuba-Surma). The Faculty of Biochemistry,
Biophysics, and Biotechnology at the Jagiellonian University,
Krakow, Poland, is a partner of the Leading National Research Center
(KNOW) supported by the Ministry of Science and Higher Education.
Disclosures
None.
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