Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction

Abstract

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.

Full text

© 2013 Nature America, Inc. All rights reserved.
nature biotechnology ADVANCE ONLINE PUBLICATION 1
A R T I C L E S
Myocardial infarction in a human causes the death of billions of
cardiomyocytes. The heart’s limited capacity to regenerate these lost
cardiomyocytes leads to compromised cardiac function and high
morbidity and mortality. As a result, there has been intense interest
in developing treatments to reduce or reverse myocardial injury.
A number of strategies have been proposed for regenerative cardio-
vascular therapeutics, including transplantation of cells expanded
ex vivo, delivery of therapeutic genes on naked DNA plasmids or
viral vectors, and administration of recombinant proteins. Thus
far, these approaches have had mixed results. Cell-based therapies
have shown limited long-term engraftment and low efficacy. Gene-
based methods have been hampered by poor control of dosage and
duration, low gene-transfer efficiency, risk of genomic integration
and associated tumorigenesis, and antiviral immune responses.
Recombinant-protein approaches have been characterized by fleet-
ing protein half-lives, poor targeting to the heart and complications
due to systemic release. modRNA, in which one or more nucleotides
is replaced by modified nucleotides, represents a potential alter-
native. Previous work has shown that modRNA mediates highly
efficient, transient protein expression in vitro and in vivo without
eliciting an innate immune response
1–6
. We therefore hypothesized
that modRNA might provide an effective means to control the spatial
and temporal delivery of gene products to enhance tissue repair or
regeneration after injury.
The fetal and post-natal mammalian heart contains a diverse set
of endogenous cardiovascular progenitors
7–17
, but native expansion,
mobilization and differentiation of progenitors in vivo are inadequate
to restore myocardial function after injury
18
, and inducing these proc-
esses for therapeutic benefit has proved difficult. Paracrine factors
play key roles in regulating progenitor cell activity in heart develop-
ment, and recent studies have likewise implicated paracrine factors in
promoting cardiac repair and regeneration after myocardial infarction
in experimental model systems
14,16,19
. In part, paracrine factors pro-
mote heart regeneration by stimulating the cardiomyogenic activity
of poorly defined endogenous heart progenitors
14,19,20
. Given that
native paracrine signals are often transient and precisely regulated in
time and space, we further hypothesized that the pulse-like expression
profile of modRNA might be well suited to delivering paracrine-factor
signals that modulate heart progenitor activity and thereby promote
heart repair or regeneration.
Modified mRNA directs the fate of heart progenitor
cells and induces vascular regeneration after
myocardial infarction
Lior Zangi
1–5,14
, Kathy O Lui
1,2,6,14
, Alexander von Gise
3,5
, Qing Ma
3,5
, Wataru Ebina
1,4
, Leon M Ptaszek
1,2,7
,
Daniela Später
1,2
, Huansheng Xu
1,2,6
, Mohammadsharif Tabebordbar
1,8
, Rostic Gorbatov
9
, Brena Sena
9
,
Matthias Nahrendorf
9
, David M Briscoe
10
, Ronald A Li
6,11
, Amy J Wagers
1,12
, Derrick J Rossi
1,4
, William T Pu
3,5
& Kenneth R Chien
1,2,13
In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter
the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of
synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and
directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly
improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of
epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with
DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A.
Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control
progenitor cell fate and thereby enhance long-term organ repair.
1
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.
2
Cardiovascular Research Center, Massachusetts General
Hospital, Boston, Massachusetts, USA.
3
Department of Cardiology, Children’s Hospital Boston, Boston, Massachusetts, USA.
4
Immune Disease Institute and Program
in Cellular and Molecular Medicine, Children’s Hospital Boston, Boston, Massachusetts, USA.
5
Boston and Harvard Stem Cell Institute, Cambridge, Massachusetts,
USA.
6
Stem Cell & Regenerative Medicine Consortium, LKS Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong.
7
Cardiac Arrhythmia Service,
Massachusetts General Hospital, Boston, Massachusetts, USA.
8
Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts, USA.
9
Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.
10
Division of Nephrology, Children’s Hospital
Boston, Boston, Massachusetts, USA.
11
Cardiovascular Research Center, Mount Sinai School of Medicine, New York, New York, USA.
12
Howard Hughes Medical
Institute, Cambridge, Massachusetts, USA.
13
Department of Cell and Molecular Biology and Medicine, Karolinska Institutet, Stockholm, Sweden.
14
These authors
contributed equally to this work. Correspondence should be addressed to K.R.C. (kenneth.chien@ki.se).
Received 10 May; accepted 6 August; published online 8 September 2013; doi:10.1038/nbt.2682

© 2013 Nature America, Inc. All rights reserved.
2 ADVANCE ONLINE PUBLICATION nature biotechnology
A R T I C L E S
To test these hypotheses, we studied the kinetics and efficacy of
modRNA-mediated gene transfer in a mouse myocardial infarction
model. Previous work has shown that epicardial heart progenitors are
activated within 48 h after mouse myocardial infarction and are ampli-
fied in a thickened epicardial layer at the surface of the heart. However,
these cells are not mobilized to enter the myocardium and differentiate
toward cardiovascular lineages as they do in the fetal heart
13,15,21
, but
rather remain on the heart surface and differentiate largely into fibrob-
lasts and myofibroblasts. For initial proof-of-concept experiments, we
studied modRNA encoding the VEGF-A protein, a potent angiogenic
factor. Based on its angiogenic activity, VEGF-A has been proposed
as a therapeutic agent to improve myocardial outcome after ischemic
injury (Supplementary Appendix and Supplementary Tables 1 and 2).
Previous human and animal studies of delivery of VEGF-A after injury
using naked DNA plasmids, recombinant proteins and engineered
viruses showed limited efficacy after myocardial infarction, but these
negative results may have been due to suboptimal delivery and/or anti-
viral immune responses
22–25
. Temporal and spatial control of VEGF-A
expression is likely to be critical for its therapeutic efficacy, as two inde-
pendent groups showed that prolonged exposure of normal muscle to
VEGF-A caused excessive vascular permeability
26–28
. Furthermore, our
recent work identified a novel role for VEGF-A as a cell fate switch for
multipotent ISL1
+
human heart progenitors, driving their differentiation
away from cardiac muscle and toward the endothelial lineage
29
. The
potential activity of VEGF-A on endogenous cardiac progenitors has not
been studied in vivo or in the context of myocardial injury.
Here we asked whether in vivo delivery of VEGF-A modRNA
given at the time of epicardial progenitor activation after myocardial
infarction would stimulate their mobilization or modulate their dif-
ferentiation. We found that modRNA medi-
ates ‘pulse-like’ expression of VEGF-A and
is superior to plasmid DNA in reducing inf-
arct size, enhancing myocardial perfusion
and improving survival. In part, this effect was due to a previously
unknown effect of VEGF-A on epicardial progenitors. VEGF-A
modRNA amplified these progenitors, mobilized their migration into
the myocardium and redirected their differentiation toward cardio-
vascular lineages. These results indicate that modRNA gene transfer
drives in vivo heart progenitor cell fate to enhance cardiac repair.
RESULTS
Pulse-like kinetics of modRNA gene delivery to heart and
skeletal muscle
We evaluated the suitability of modRNA for gene transfer to heart and
skeletal muscle—tissues that have been historically difficult to transfect.
Notably, modRNA transfected primary fetal human, neonatal mouse,
and adult rat cardiomyocytes or adult mouse skeletal myotubes with
high efficiency (89%, 72%, 68% and 100%, respectively; Supplementary
Fig. 1) and minimal toxicity (~80% cell survival, comparable to trans-
fection vehicle control). This high efficiency represents a 10- to 40-fold
increase compared to typical transfection efficiencies attained using
nonviral DNA–mediated transfection
22,30
. modRNA likewise medi-
ated efficient protein production in cardiac cells in vivo. Direct, single,
intramyocardial injection of luciferase (Luc) modRNA yielded a robust
bioluminescent signal indicative of dose- and time-dependent luciferase
protein expression localized to the injection site (Fig. 1a–c). Luc was
immediately expressed and reached high levels after only 3 h, peaked
at 18 h and returned to baseline at 144–150 h (Fig. 1c). These kinetics
differed considerably from luciferase DNA, which peaked at 72 h and
retained a high signal for more than 10 d after injection.
To examine cardiac cell types transduced by modRNA, we
injected Cre recombinase DNA plasmid or modRNA into
e
DNA
0 50 100255
h
modRNA
0 50 100255
i
ii
iii
i
ii
iii
VE-cadherin
TNNI3
smMHC
i
VE-cadherin
TNNI3
smMHC
β-gal/DAPI
X-gal
f g
β-gal/DAPI
X-gal
i j
3 h
18 h
24 h
48 h
72 h
240 h
DNA
0 50 100
b
modRNA
0 50 100
Luc dose (µg) Cre dose (µg)
Intramyocardial (IM) injection of different doses
of luciferase (Luc) DNA or modRNA
0 5
Days
BALB/c Bioluminescence
10
a d
0
Days
R26
fsLacZ
X-gal staining
7
IM injection of different doses
of Cre DNA or modRNA
k
Cardiac transfection
(% left ventriclular area)
40
30
20
10
0
Gene delivery
method
DNA modRNA
P = 0.001
12
10
8
6
4
0
240 48 72 96 120 144
Luciferase signal
Time (h)
c
Control modRNA DNA
2
4
3
2
1
0
240 48 72 96 120 144
VEGF-A protein (ng/ml)
Time (h)
l
Control modRNA DNA
Figure 1 Highly efficient, transient gene
transfer in vivo using modRNA. (a,b) Luciferase
DNA or modRNA was delivered by myocardial
injection (a). Protein expression was assayed
by bioluminescence (b). (c) Time course of
luciferase activity after injection of luciferase
DNA or modRNA (100 µg) or vehicle only
(control) per heart. (d) Cre DNA or modRNA,
delivered by myocardial injection, catalyzed
cardiac recombination, detected by X-gal
staining (blue) for Cre-activated expression of
LacZ from R26
fsLacZ
. (e–g) Indicated doses of
Cre DNA were injected intramyocardially into
R26
fsLacZ
hearts. DNA plasmid inefficiently
transfected cells expressing endothelial,
cardiomyocyte and smooth muscle lineage
markers. Lines labeled i, ii and iii indicate
planes of section in f. Scale bar, 400 µm (e)
or 50 µm (f,g). (h–j) Indicated doses of Cre
modRNA were injected intramyocardially into
R26
fsLacZ
hearts. modRNA efficiently transfected
cells expressing endothelial, cardiomyocyte and
smooth muscle lineage markers. Lines labeled
i, ii and iii indicate planes of section in i. Scale
bar, 400 µm (h) or 50 µm (i,j). (k) Summary
of transfection efficiency of single 100 µg
DNA or modRNA injection into cardiac muscle
(left ventricle). (l) Kinetics of VEGFA modRNA
expression in cardiac cells cultured in vitro. For
a–l, n = 3, representative of two independent
experiments. Error bars (c,l), s.d.

© 2013 Nature America, Inc. All rights reserved.
nature biotechnology ADVANCE ONLINE PUBLICATION 3
A R T I C L E S
Rosa26-lox-stop-lox-LacZ (R26
fsLacZ
) mouse hearts, followed by
5-bromo-4-chloro-3-indolyl-β--galactoside (X-gal) staining for
Cre-activated β-galactosidase (blue stain, Fig. 1d–j). Whereas using
DNA plasmids resulted in infrequent cell transfection (Fig. 1e–g),
the vast majority of cells in the injection region were transfected by
modRNA, including endothelial cells (~90%), cardiomyocytes (~80%)
and smooth muscle cells (~90%) (Fig. 1h–j). A single injection of
modRNA into the cardiac apex efficiently transfected a substantial area
of myocardium (~25% of the left ventricle), a level of transfection that
was at least ten times greater than that achieved by injection of naked
plasmid DNA (Fig. 1k). Similarly efficient in vivo modRNA transfec-
tion was observed in skeletal muscle (Supplementary Fig. 2).
VEGF-A modRNA improves myocardial outcome after
myocardial infarction
The pulse-like kinetics of modRNA gene delivery led us to hypothesize
that modRNA might be an effective means to deliver paracrine factors
that could alter progenitor cell fate and thereby durably modify organ
injury responses. We therefore set out to determine whether VEGF-A
modRNA has beneficial activity in myocardial infarction, and we
compared its performance to traditional, nonviral, VEGF-A delivery
by plasmid DNA. First, we examined the kinetic profile of VEGF-A
expression after VEGF-A modRNA administration in vivo and in vitro.
Previous work established that cardiac VEGF-A ectopic expression does
not elevate VEGF-A protein levels in serum
31
. In our in vitro experi-
ments, however, cardiac cells transfected with VEGF-A modRNA
translated and secreted VEGF-A protein with pulse-like kinetics that
peaked rapidly and declined to basal levels after 2–3 d (Fig. 1l). In
contrast, transfection with VEGF-A DNA plasmid induced a much
broader secretion profile that peaked at a lower level at 3 d and gradu-
ally declined, with protein levels remaining above baseline at day 10.
Next, we evaluated the toxicity and immunogenicity of VEGF-A
modRNA in vitro and in vivo. VEGF-A modRNA induced greater
VEGF-A protein secretion than VEGF-A mRNA (Supplementary
Fig. 3a). Unlike mRNA, modRNA did not cause apoptosis or upregu-
lation of RIG-1, INF-α or INF-β, hallmarks of the innate immune
response (Supplementary Fig. 3b–c). VEGF-A modRNA likewise
exhibited minimal immunogenicity when delivered to skeletal
e
Control
Luc
modRNA
VEGF-A
modRNA
VEGF-A
DNA
DAPI Isolectin B4 PECAM-1
FITC-
dextran
Merge
VEGF-A
DNA +
bevacizumab
VEGF-A
modRNA +
bevacizumab
g
Sham Luc modRNA VEGF-A modRNA VEGF-A DNA
Ventral Dorsal Ventral Dorsal Ventral Dorsal Ventral Dorsal
VEGF-A DNA
+ bevacizumab
Ventral Dorsal
VEGF-A modRNA
+ bevacizumab
Ventral Dorsal
d
Days
0 7
MI + intramyocardial
injection of
modRNA/DNA
± Avastin
Tail injection of
isolectin B4 and 70 kd
FITC-dextran beads
c
P = 0.03
Scar area (%)
0
Vehicle
SU5614
PTK787
10
20
30
40
50
P = 0.04
P = 0.05
Vehicle
SU5614
PTK787
0
50
100
150
P = 0.05
Luminal density
(no./300 µm
2
)
P = 0.03
Vehicle
SU5614
PTK787
0
2
4
6
8
10
P = 0.04
TUNEL
+
cells (%)
Luminal density
(no./300 µm
2
)
TUNEL
+
(%)
Transfect
Modality
Veh
–
Luc
mod
mod
DNA
Veh
–
Luc
mod
mod
DNA
0
50
100
150
0
2
4
6
8
0
50
100
150
200
0
1
2
3
4
5
P = 0.05
P = 0.02
P = 0.04
P = 0.05
b
1 Week 4 Weeks
Scar area (%)
0
20
40
60
0
20
40
60
P = 0.02 P = 0.01
VEGF-A VEGF-A
a
MI + vehicle (control)
MI + Luc modRNA (100 µg) MI + VEGF-A modRNA (100 µg) MI + VEGF-A DNA (100 µg)
f
0
25
50
75
100
125
150
175
Luminal structure density
(no./300 µm
2
)
No. of vessels No. of leaky vessels
P = 0.002
P = 0.01
Transfect
Modality
Veh
–
Luc
mod
mod
DNA
VEGF-A
Avastin – – – – + +
DNA
mod
Veh
–
Luc
mod
mod
– – – – + +
VEGF-A
mod
DNA
DNA
Figure 2 VEGF-A modRNA enhanced formation of functional, nonleaky vessels. (a) VEGF-A modRNA, injected into the infarct region at the time of
experimental myocardial infarction (MI), increased vascular density in the peri-infarct region. Seven days after myocardial infarction, the vascular plexus
was highlighted by Microfil followed by imaging of cleared hearts. The indicated treatments were injected within the region demarcated by the dashed
lines. (b) VEGF-A modRNA reduced scar area and TUNEL
+
cells and increased capillary density at 1 week and 4 weeks after myocardial infarction.
Capillary density and TUNEL
+
fraction were measured in infarct border zone (left ventricle). Masson’s trichrome was used to evaluate scar area
(Supplementary Fig. 4). n = 3. Error bars, s.d. (c) Beneficial activity of VEGF-A modRNA required KDR signaling. Mice were treated with myocardial
infarction and VEGF-A modRNA. KDR inhibitors SU5614 or PTK787, administered from one day before myocardial infarction to tissue collection at 7 d
after myocardial infarction, blocked beneficial effect of VEGF-A modRNA. (d) Experimental design to assess functional angiogenesis. VEGF-A modRNA
was injected into the myocardium at the time of LAD ligation. After 1 week, isolectin B4 and FITC-dextran beads (70 kDa) were injected into the tail
vein to assess connection to the systemic vasculature and vascular permeability, respectively. (e) Vessels formed under the influence of VEGF-A DNA,
but not VEGF-A modRNA, or VEGF-A DNA with bevacizumab, neutralizing VEGF-A antibodies (given intraperitoneally twice a week) were permeable
to FITC-dextran (yellow arrows). Scale bar, 50 µm. (f) Density of luminal structures and leaky vessels quantification for different treatments.
(g) Macroscopic myocardial edema in hearts treated with VEGF-A DNA (yellow frame), but not with VEGF-A modRNA. Scale bar, 5 mm. *For a–g, n = 3,
representative of two independent experiments.

© 2013 Nature America, Inc. All rights reserved.
4 ADVANCE ONLINE PUBLICATION nature biotechnology
A R T I C L E S
muscle in vivo (Supplementary Fig. 3c). In addition, DNA plasmid
delivery in vivo into cardiac muscle upregulated INF-β and RIG-1.
These results are consistent with prior studies that demonstrated low
modRNA toxicity and immunogenicity
1–5,32
and immunogenicity
of DNA plasmid
33,34
. Together, these data show that modRNA is an
efficient and nontoxic approach for transient, highly efficient and
localized gene delivery to heart and skeletal muscle.
To assess the efficacy of VEGF-A modRNA in the mouse myo-
cardial infarction model, we administered it at the time of coronary
artery ligation by direct intramyocardial injection into the ischemic
region. VEGF-A modRNA stimulated formation of systemically
perfused vessels in the area of injection (Fig. 2a). Both VEGF-A
modRNA and VEGF-A DNA reduced infarct size and apoptotic cell
frequency, and increased capillary density at 1 and 4 weeks after myo-
cardial infarction (Fig. 2b, P ≤ 0.05; Supplementary Fig. 4). These
beneficial effects required VEGF-A signaling through its canonical
receptor KDR (also known as VEGFR2), because they were blocked
by either SU5614 or PTK787, specific small-molecule inhibitors of
KDR (Fig. 2c; P ≤ 0.05).
Although both VEGF-A modRNA and DNA increased myocardial
capillary density and reduced infarct size and cell death, the vessels
formed by these treatments were functionally different. In VEGF-A
DNA–treated hearts, vessels showed excessive permeability as dem-
onstrated by extravasation of 70-kd dextran beads (Fig. 2d–f). In con-
trast, vessels in VEGF-A modRNA–treated hearts did not show this
abnormal vascular permeability (Fig. 2d–f). The difference in vascular
permeability was readily apparent on inspection of hearts: VEGF-
A modRNA–treated hearts were similar in shape to control hearts,
whereas VEGF-A DNA–treated hearts displayed obvious edema
(Fig. 2g). This difference in vessel function was likely due to prolonged
exposure to VEGF-A with DNA-mediated gene transfer, as increased
vascular permeability is a known consequence of lengthy VEGF-A
expression
35–37
. To further evaluate the importance of expression
kinetics on outcome, we administered bevacizumab (Avastin), a neu-
tralizing antibody specific for transfected human VEGF-A starting at
3 d after myocardial infarction. Bevacizumab did not detectably affect
VEGF-A modRNA activity in increasing vessel density (Fig. 2d–g)
but it blocked vessel induction by VEGF-A DNA, consistent with the
differing expression kinetics of each modality. On the other hand,
bevacizumab reduced the abnormal vascular permeability and edema
induced by VEGF-A DNA. These data indicate that the rapid, brief
pulse of VEGF-A delivered by modRNA stimulated growth of func-
tional vessels, whereas the more prolonged VEGF-A expression deliv-
ered by DNA stimulated formation of leaky vessels.
We next evaluated the effect of VEGF-A modRNA and DNA on
short-term survival after myocardial infarction (Fig. 3a). Although
VEGF-A DNA augmented vessel number, it caused increased mortality
compared to vehicle controls (P = 0.02 versus vehicle control), con-
sistent with earlier reports
28
. This detrimental effect was likely due to
increased vascular permeability and cardiac edema from prolonged
VEGF-A exposure, because bevacizumab blockade of human VEGF-A
beginning on post-myocardial infarction day 3 restored mortality to
control levels (P = 0.04 in presence versus absence of bevacizumab).
Unlike VEGF-A DNA, VEGF-A modRNA did not have an adverse
effect on short-term survival, and, consistent with VEGF-A
modRNA expression kinetics, this was not significantly changed by
bevacizumab given 3 d after myocardial infarction (Fig. 3a). Together,
these data point to the importance of VEGF-A expression kinetics
mediated by different gene expression systems in determining their
biological effect (Supplementary Table 3).
To assess the long-term effect of VEGF-A modRNA on outcome
after myocardial infarction, we monitored the survival of control mice
and mice treated with VEGF-A modRNA for a year after myocardial
infarction. VEGF-A DNA–treated mice were not included in this
long-term study because of their poor survival in the first month
following MI. In the control group, the 1-month survival was ~60%,
similar to that in other reported studies
38–40
. Survival was significantly
higher in the VEGF-A modRNA group than in controls at this late
end point (Fig. 3b; P = 0.04; n = 14). The beneficial effect of VEGF-
A modRNA on survival was reflected in its effect on cardiac func-
tion, as determined by cardiac magnetic resonance imaging (MRI)
measurement of cardiac ejection fraction (EF; Fig. 3c,d). At day 1
following myocardial infarction, EF was reduced to a similar extent in
control and VEGF-A modRNA groups, indicating equivalent severity
of myocardial injury (Fig. 3d). At 21 d after myocardial infarction,
EF was better preserved in the VEGF-A modRNA group (Fig. 3d;
P = 0.001, n = 4; Supplementary Movies 1–3). Consistent with
declining heart function in controls compared to VEGF-A modRNA
treatment, heart rate increased between days 1 and 21 after myocar-
dial infarction in controls, and this effect was blocked by VEGF-A
Day 1
Day 21
P = 0.001
70
60
50
40
Ejection fraction (%)
Sham Control VEGF-A
modRNA
P = 0.04
Survival (%)
0
20
40
60
80
100
0 60 120 180 240 300 365
0 5 10 15 20 25 30
0
20
40
60
80
100
Survival (%)
Days post MI
Days post MI
MI + vehicle (n = 14)
MI + VEGF-A modRNA (n = 14)
MI + vehicle (n = 14)
MI + VEGF-A modRNA (n = 14)
MI + VEGF-A modRNA + bevacizumab (n = 5)
MI + VEGF-A DNA + bevacizumab (n = 5)
MI + VEGF-A DNA (n = 8)
Sham
Control
VEGF-
A
modRNA
P = 0.02 vs. vehicle
P = 0.04 vs. bevacizumab ( )
Diastole Systole
d
a
b
c
Figure 3 VEGF-A modRNA improved outcome in
a mouse myocardial infarction model. (a) Short-
term survival curve after myocardial infarction
and the indicated treatments. VEGF-A modRNA,
DNA or vehicle were injected into the infarct
region at the time of LAD ligation. Bevacizumab
was injected twice weekly starting on post-
myocardial infarction day 3. P-values were
calculated using the Mantel-Cox log-rank test.
(b) Long-term survival curve after myocardial
infarction and VEGF-A modRNA or control
treatments. VEGF-A modRNA improved survival
at 1 year compared to control treatment. P-value
was calculated as in a. (c) MRI assessment of left
ventricular systolic function. Images show left
ventricular chamber (outlined in red) in diastole
and systole. (d) Left ventricular systolic function
(ejection fraction) was better preserved 21 d
after LAD ligation in the VEGF-A modRNA group
compared to control. P-value was calculated
using paired t-test. Sham control, n = 3, control
or VEGF-A modRNA group, n = 5.

© 2013 Nature America, Inc. All rights reserved.
nature biotechnology ADVANCE ONLINE PUBLICATION 5
A R T I C L E S
modRNA (Supplementary Fig. 5). Other MRI indices were not sig-
nificantly different between groups (Supplementary Fig. 5). MRI
assessment at later time points was not done because excess mortal-
ity in the control group would have introduced strong survivor bias.
Collectively these data indicate that VEGF-A modRNA has sustained
beneficial effects on myocardial outcome and on long-term survival
after myocardial infarction.
VEGF-A modRNA activates epicardial cardiac progenitor cells
through KDR
To further investigate mechanisms that underlie the beneficial activ-
ity of VEGF-A modRNA, we measured the expression of known
cardiovascular progenitor and differentiated cell lineage markers in
peri-infarct tissue of control and VEGF-A modRNA–treated hearts.
qRT-PCR analysis showed upregulation of the cardiomyocyte marker
Tnnt2 and the endothelial cell markers Pecam1 and Kdr (Fig. 4a and
Supplementary Fig. 6). Among cardiac progenitor markers, Wilms’
tumor gene 1 (Wt1) was highly upregulated by VEGF-A modRNA
compared to control treatment, whereas other heart progenitor mark-
ers, such as Isl1 (refs. 7–11) and Nkx2-5, were not substantially changed
(Fig. 4a and Supplementary Fig. 6). In the heart, Wt1 is a marker
of an epicardial progenitor population that has an important role in
heart injury responses
16
. Upregulation of Wt1 was confined to the
heart and was not observed in other compartments including those
that might be sources of blood-borne cells (Supplementary Fig. 7).
Wt1 upregulation by VEGF-A modRNA suggested that the VEGF-A
pulse affected the epicardial progenitor population. We confirmed a
marked increase in WT1
+
cells in the peri-infarct region by immuno-
histochemistry (Fig. 4b). Notably, this amplification of WT1
+
cells by
VEGF-A modRNA required activation of epicardial cells by injury,
as it was not observed in VEGF-A modRNA–treated, sham-operated
hearts (Fig. 4b). KDR inhibition by either SU5614 or PTK787 blocked
the effect of VEGF-A modRNA (Fig. 4c), indicating that KDR action
requires canonical VEGF-A signaling through the KDR receptor. These
results were confirmed in an unbiased and independent fluorescence-
activated cell sorting (FACS)-based approach that took advantage of the
Wt1
GFPCre
mouse line, in which a GFP-Cre fusion protein is knocked
into the endogenous Wt1 locus
15
. Consistent with quantification of
WT1
+
cells by immunohistochemistry, FACS-based quantification of
the number of WT1
+
cells by GFP signal showed that myocardial inf-
arction alone substantially expanded this cell population (Fig. 4d), as
we reported previously
16
. VEGF-A modRNA, but not Luc modRNA,
further strongly amplified this population by about fourfold. Again,
this effect was blocked by KDR inhibitors SU5614 and PTK787.
To determine whether VEGF-A acts directly on WT1
+
epicardial
progenitors to drive their amplification, we measured the abundance
Control
Luc
modRNA
VEGF-A
modRNA
Control
Luc
modRNA
VEGF-A
modRNA
Control
Luc
modRNA
VEGF-A
modRNA
WT1
+
cells (%)
b
0
10
20
30
40
50
Sham, 1 MI, 4
P = 0.01
MI, 1
P = 0.02
WT1
+
cells (%)
c
0
5
10
15
20
25
Vehicle
SU5614
PTK787
MI + VEGF-A
modRNA
0
3
6
9
Relative expression
12
15
18
a
Wt1
Nkx2-5
Isl1
Vimentin
Tcf21
Tnnt2
smMHC
Pecam1
Kdr
Injected area with
VEGF-A modRNA ( )
vs. control (dashed line)
f
1
2
3
4
5
6
7
0
8
No. of cells ×10
5
n = 3
P = 0.005
0 4 8 12 16
Days
PBS
VEGF-A
VEGF-A + DMSO
VEGF-A + PTK787
VEGF-A + SU5614
FL4 - KDR (log
10
)
No. of cells
Wt1
+
cells isolated from
MFI = 4.9
MFI = 30
MFI = 2.1
e
Sham heart,
isotype control
Sham heart
MI heart
MI + VEGF-A modRNA–
treated heart
MFI = 165
CFW control Sham
MI +
vehicle treated
MI +
VEGF-A modRNA
MI +
Luc modRNA
MI +
VEGF-A modRNA
+ DMSO
MI +
VEGF-A modRNA
+ PTK787
MI +
VEGF-A modRNA
+ SU5614
0.3% 1.1% 3.3% 3.8%
12.7% 10.4% 3.6% 1.9%
FL1 Wt1
FL2
VEGF-A or
Luc modRNA
injection + MI
Wt1
GFPCre
FACS
analysis
Wt1
+
d
Weeks
0 1
Figure 4 VEGF-A modRNA reduced scar area
and apoptosis and increased capillary density
and WT1
+
cells proliferation after myocardial
infarction in a KDR-dependent manner. (a) Marker
gene analysis showed that VEGF-A modRNA
dramatically upregulated Wt1 expression. qRT-
PCR was performed on peri-infarct tissue 3
d after myocardial infarction. Expression was
calculated relative to vehicle-treated heart
(dashed line). (b) Quantification of Wt1
+
cells
in the infarct border zone (left ventricle) of
immunostained heart sections. VEGF-A modRNA
increased frequency of WT1
+
cells at 1 week and
4 weeks after myocardial infarction but not after
sham treatment. (c) Increase in Wt1
+
cells in
myocardial infarction + VEGF-A modRNA–treated
hearts required signaling through KDR. Samples
were analyzed as in b, 1 week after myocardial
infarction. (d) FACS-based quantitation of Wt1
+
cells after myocardial infarction and control or
VEGF-A modRNA treatment. WT1
+
epicardial
progenitors were isolated from dissociated
Wt1
GFPCre
heart by GFP FACS sorting. VEGF-A
modRNA treatment increased the frequency
of GFP
+
(Wt1-expressing) cells 1 week after
myocardial infarction. Red numbers within the
region of interest indicate the fraction of cells
that were GFP
+
(WT1
+
). (e) KDR expression
on WT1
+
epicardial progenitors was measured
by FACS. Dissociated Wt1
GFPCre/+
hearts were
stained for KDR, then analyzed by FACS.
The histogram shows KDR immunostaining
intensity on WT1
+
epicardial progenitors (GFP
+
).
Myocardial infarction and VEGF-A modRNA
treatment increased KDR mean fluorescence
intensity (MFI) on these progenitors. (f) VEGF-A
protein increased, and KDR antagonists reduced
proliferation of FACS-purified WT1
+
epicardial
progenitors. Cell number was measured using an
automated cell counter at days 4, 8 and 14 of
cell culture. *For a–f, n = 3, representative of two
independent experiments. Error bars, s.d.

© 2013 Nature America, Inc. All rights reserved.
6 ADVANCE ONLINE PUBLICATION nature biotechnology
A R T I C L E S
of KDR receptors on these cells by FACS (Fig. 4e). In sham-operated
hearts, WT1
+
cells expressed low levels of the KDR receptor (mean
KDR fluorescence intensity was about twofold above isotype control
background). WT1
+
cell expression of the KDR receptor increased
dramatically (about sixfold) after myocardial infarction, and VEGF-
A modRNA treatment further upregulated KDR expression by
5.5-fold, indicating a positive-feedback response in which VEGF-A
reinforces KDR expression, as previously noted in endothelial cells
41
.
Upregulation of KDR after myocardial infarction coincides with the
general activation of numerous epicardial genes after injury
16
and
likely accounts for the requirement of myocardial infarction to enable
WT1
+
epicardial cells to respond to VEGF-A modRNA (Fig. 4b). To
further determine whether VEGF-A acts directly on post-myocardial
infarction WT1
+
epicardial cells, we purified these cells by FACS and
measured their proliferation in response to recombinant VEGF-A
(Fig. 4f). VEGF-A increased proliferation of cultured WT1
+
epicar-
dial cells, and KDR inhibition powerfully blocked their proliferation.
These data indicate that VEGF-A acts directly through KDR on acti-
vated, post-myocardial infarction WT1
+
epicardial progenitors.
VEGF-A modRNA induced WT1
+
epicardial progenitor
differentiation into endothelial cells in vitro
During heart development, WT1
+
epicardial progenitors undergo
an epithelial-to-mesenchymal transition. The resulting epicardium-
derived cells (EPDCs) migrate into the myocardium and pre-
dominantly differentiate into fibroblasts and smooth muscle cells.
Infrequently, EPDCs contribute to the endothelial lineage
15,42
, and
they have also been found to differentiate into cardiomyocytes
13,17,42
.
VEGF-A modRNA increased capillary density and upregulated
endothelial markers in peri-infarct tissue (Fig. 2b). We hypothesized
that VEGF-A might alter the fate of the WT1
+
epicardial progeni-
tors and enhance their endothelial differentiation. qRT-PCR analysis
of FACS-purified, post-myocardial infarction, WT1
+
epicardial
progenitors indicated that VEGF-A modRNA strongly upregulated
expression of endothelial markers Pecam1 and Kdr (Fig. 5a). VEGF-A
stimulation of endothelial differentiation of epicardial progenitors was
further demonstrated by culturing FACS-purified, post-myocardial
infarction, Wt1
+
cells for 7 d in the presence or absence of recom-
binant VEGF-A. Analysis of marker genes by qRT-PCR revealed
that VEGF-A stimulation strongly upregulated endothelial markers
VE-Cadherin, Pecam1 and Kdr (Supplementary Fig. 8a). This result
was corroborated by FACS analysis, which showed that VEGF-A
treatment markedly increased the frequency of KDR
+
/VE-cadherin
+
endothelial cells (68% versus 14%; Supplementary Fig. 8b), and by
immunofluorescence imaging, which demonstrated co-expression of
VE-cadherin and GFP (expressed from Wt1
GFPCre
) in cultures stimu-
lated by VEGF-A (Supplementary Fig. 8c).
We further tested the hypothesis that VEGF-A influences WT1
+
epicardial progenitor cell lineage decisions using an in vitro clonal
assay. FACS-purified, post-myocardial infarction, WT1
+
epicardial
cells were individually plated in 96-well dishes, clonally expanded and
assessed for differentiation into the major cardiac lineages in the pres-
ence or absence of recombinant VEGF-A by qRT-PCR (Fig. 5b). This
assay demonstrated the multipotency of WT1
+
epicardial progeni-
tors at the clonal level (Fig. 5c). VEGF-A treatment for 7 d increased
the fraction of clones that were positive for the endothelial marker
VE-Cadherin from 12% to 52% (Fig. 5c). To further demonstrate
that this was a clonal event rather than polyclonal contamination of
endothelial cells, we subcloned a clone that was initially VE-Cadherin
negative in the absence of VEGF-A and repeated the clonal differ-
entiation assay. VEGF-A again strongly stimulated differentiation
toward an endothelial fate (24% versus 0%, Fig. 5d). These clonal
assays confirm the multipotency of WT1
+
epicardial progenitor cells,
and demonstrate at the clonal level that VEGF-A biases epicardial
progenitor fate decisions toward the endothelial lineage.
In summary, these studies identify epicardial progenitors as a target
for VEGF acting as a vasculogenic cell fate switch, in addition to its
already well-known effect on promoting the proliferation of already
differentiated endothelial cells.
VEGF-A modRNA induced WT1
+
epicardial cell differentiation
into endovascular cell types in vivo
To directly track the fate of EPDCs to different lineages after myo-
cardial infarction, we performed genetic lineage tracing using
Figure 5 VEGF-A modRNA induced WT1
+
epicardial progenitor proliferation and shifted
differentiation toward the endothelial lineage.
(a) VEGF-A modRNA increased endothelial
marker gene expression in FACS-purified
GFP
+
cells after myocardial infarction. Gene
expression, determined by qRT-PCR, was
calculated relative to GFP
+
cells isolated from
control-treated, post-myocardial infarction
hearts. n = 3, representative of two independent
experiments. (b) Experimental design of clonal
assays to assess VEGF-A modulation of WT1
+
epicardial cell fate decisions. Individual FACS-
purified cardiac WT1
+
cells were deposited
in 96-well dishes, clonally expanded and
assessed for differentiation to the indicated
lineages by qRT-PCR. One VEGF-A–naive,
VE-cadherin–negative clone was subcloned and
the individual subclones were further tested
in the clonal differentiation assay. (c) VEGF-A
modRNA promoted Wt1
+
epicardial progenitor
differentiation toward the endothelial lineage.
Each row represents an individual clone, and
each column indicates the relative qRT-PCR–measured expression of the indicated lineage marker. The percentage of clones with detectable expression
of each lineage marker is indicated at the bottom of each column. (d) VEGF-A modRNA effect on Wt1
+
epicardial progenitor differentiation was
recapitulated in the subclonal assay. This confirmed multipotency of WT1
+
epicardial cells and made polyclonal contamination highly unlikely.
c
Clonal assay
Clones (%) Clones (%)
smMHC
VE-cadherin
Tnni3
Vimentin
smMHC
Tnni3
Vimentin
VE-cadherin
Control VEGF-A
100
92
12
0
100
88
52
0
Subclonal assay
Subclonal assay
Control VEGF-A
100
64
0
0
100
72
24
0
smMHC
VE-cadherin
TNNI3
Vimentin
smMHC
VE-cadherin
TNNI3
Vimentin
d
Wt1
Isl1
Nkx2-5
Vim
Tcf21
Tnnt2
smMHC
Pecam1
Kdr
0
1
2
3
4
5
Relative expression
Wt1 epicardial progenitors from VEGF-A modRNA
( ) vs. vehicle-treated hearts (dashed line)
a
Explant of
Wt1
GFPCre
heart
after MI
FACS purify
Wt1
+
cells
Clonal assay Subclonal assay
+VEGF-A -VEGF-A +VEGF-A -VEGF-A
b
WT1
+

© 2013 Nature America, Inc. All rights reserved.
nature biotechnology ADVANCE ONLINE PUBLICATION 7
A R T I C L E S
WT1
CreERT2/+
mice, in which tamoxifen-activated CreERT2 was
expressed from the endogenous Wt1 locus
16
. Treatment of adult
mice with tamoxifen triggered recombination of the R26
mTmG
reporter
43
in Wt1-expressing cells, irreversibly labeling them with
membrane-localized GFP and simultaneously inactivating expres-
sion of membrane-localized tomato fluorescent protein. We defined
these cardiac GFP
+
cells, consisting of Wt1-expressing cells and their
descendants, as EPDCs
16
. After allowing clearance of tamoxifen for 1
week, myocardial infarction was induced by injection of VEGF-A or
Luc (control gene) modRNA into the peri-infarct zone. After 7 d, the
fate of Wt1
CreERT2
-labeled EPDCs was evaluated by FACS, qRT-PCR
and immunofluorescent imaging (Fig. 6a–e and Supplementary
Fig. 9). VEGF-A modRNA increased the number of EPDCs four-
fold (Fig. 6b). The fraction of PECAM1
+
cells was 26% greater in
VEGF-A modRNA versus Luc modRNA hearts (44% versus 18%).
Therefore, we estimate that ~23% (6% of Wt1
CreERT2
-labeled EPDCs /
26% total endothelial cells) of the VEGF-A–stimulated increase in
PECAM1
+
cells arose from EPDCs. This result was supported by
qRT-PCR of FACS-purified EPDCs, which showed that VEGF-A
modRNA increased expression of Pecam1 and Kdr (Fig. 6c). Moreover,
EPDCs in the VEGF-A modRNA group upregulated the cardiomyo-
cyte marker Tnnt2, suggesting that VEGF-A enhanced EPDC dif-
ferentiation toward the cardiomyocyte lineage.
Confocal analysis of immunostained sections further substantiated
these conclusions. In controls without injury or with myocardial inf-
arction and vehicle treatment, EPDCs stayed on the epicardial surface
of the heart
16
(Supplementary Fig. 9). In contrast, myocardial infarc-
tion and VEGF-A modRNA mobilized EPDCs so that they migrated
into the myocardium (Supplementary Fig. 9) and increased differen-
tiation toward the endothelial lineage (58% with VEGF-A modRNA
versus 16% with Luc modRNA; Fig. 6d,e). We also detected EPDCs
that co-expressed the cardiomyocyte marker TNNI3 in VEGF-A
Figure 6 VEGF-A modRNA promoted
differentiation of EPDCs toward the
cardiovascular lineage in vivo. (a) Genetic
lineage tracing was used to follow the fate
of EPDCs after myocardial infarction with
VEGF-A or Luc modRNA treatment. Tamoxifen
treatment of Wt1
CreERT2/+
øR26
mTmG
mice
before myocardial infarction irreversibly labeled
epicardial cells and their descendants with GFP.
(b) FACS analysis of dissociated hearts 1 week
after myocardial infarction indicated that VEGF-
A modRNA increased the frequency of EPDCs
expressing the endothelial marker PECAM1
(% indicated in red). (c) VEGF-A modRNA
increased endothelial and cardiomyocyte
marker gene expression in FACS-sorted EPDCs
1 week after myocardial infarction. Expression
of each marker was measured by qRT-PCR and
displayed relative to expression in control-
treated EPDCs. (d) Immunofluorescent analysis
of EPDC fate by Wt1
CreERT2
genetic lineage
tracing. Expression of smooth muscle (smMHC),
endothelial (PECAM1) and cardiomyocyte
(TNNI3) markers by GFP
+
EPDCs was assessed
by immunostaining and confocal microscopy.
Scale bar, 30 µm. (e) Quantification of d.
A minimum of 2,000 EPDCs were analyzed in
each post-myocardial infarction heart treated
with Luc modRNA (n = 2) or VEGF-A modRNA
(n = 5) from two independent experiments. The
graph shows the percentage of GFP
+
EPDCs that
co-expressed the indicated lineage marker.
(f) Cre modRNA gel-mediated tracing of
epicardial cell fate was used to follow the fate
of EPDCs after myocardial infarction with VEGF-
A or Luc modRNA treatment. Cre modRNA
gel, applied to R26
mTmG
mice 2 weeks before
myocardial infarction, irreversibly labeled
epicardial cells and their descendants with
GFP. (g) Cre modRNA gel selectively labeled
epicardial cells with GFP in R26
mTmG
mice.
Note that labeled cells were restricted to the
epicardium in controls (sham or myocardial
infarction and Luc modRNA treatment).
However in myocardial infarction hearts injected
with VEGF-A modRNA, labeled cells were found
both in the epicardial layer and within the myocardium and differentiated into myocytes (yellow asterisks) and nonmyocytes (white arrowheads). Scale
bar, 50 µm. (h) Immunofluorescent analysis of EPDC fate with Cre modRNA gel lineage tracing. Scale bar, 30 µm. (i) Quantification of h. A minimum of
2,000 Cre-gel–labeled cells were analyzed in each post-myocardial infarction heart treated with Luc modRNA (n = 3) or VEGF-A modRNA (n = 4) from
two independent experiments. Error bars, s.d. The graph shows the percentage of GFP
+
EPDCs that co-expressed the indicated lineage marker.
Isotype control
MI +
Vehicle treated
MI +
Luc modRNA
MI +
VEGF-A modRNA
FL-1 Wt1
CreERT2
FL-4 PECAM1
1% 24% 17% 38%0% 0.5% 0.5% 6.3%
7.7%3.2%3%0%
b
d
i
e
a
g
Sham
+ Cre modRNA gel
MI + Cre modRNA gel
+ Luc modRNA
MI + Cre modRNA gel + VEGF-A modRNA
EPDCs (Cre modRNA gel lineage)/DAPI/TNNI3
*
*
EPDC (Cre modRNA gel lineage) fate
TNNI3
+
PECAM1
+
smMHC
+
Marker
+
EPDCs (%)
0 20 40 60
EPDC (Wt1
CreERT2
lineage) fate
TNNI3
+
PECAM1
+
smMHC
+
Marker
+
EPDCs (%)
0 20 40 60
h
smMHC
PECAM1
TNNI3
EPDCs
(Cre modRNA
gel lineage)/
DAPI
Lineage marker/
DAPI
Merge
EPDCs
(Wt1
CreERT2
lineage)/
DAPI
Lineage marker/
DAPI
Merge
smMHC
PECAM1
TNNI3
Wt1
CreERT2
genetic lineage tracing
0 1 2 3
Tam
Weeks
Wt1
CreERT2/+
::R26
mTmG
FACS, qRT-PCR
and immunostaining
Week
IM injection of VEGF-A
or Luc modRNA
+ MI
0
14
f
IM injection of VEGF-A
or Luc modRNA
+ MI
Apply Cre
modRNA gel
Immunostaining
Weeks Week
Cre modRNA gel lineage tracing
10
0 21
R26
mTmG
VEGF-A modRNALuc modRNA
c
Wt1
Isl1
Nkx2-5
Vim
Tcf21
Tnnt2
smMHC
Pecam1
Kdr
0
3
6
9
12
15
18
Relative expression
FACS-purified EPDCs from VEGF-A modRNA treated ( ) vs.
vehicle treated (dashed line)

© 2013 Nature America, Inc. All rights reserved.
8 ADVANCE ONLINE PUBLICATION nature biotechnology
A R T I C L E S
modRNA hearts, but not in controls (5% versus 0%, respectively;
Fig. 6d,e). It is unlikely that this result was due to VEGF-A–induced
upregulation of Wt1 (and therefore the CreERT2 lineage tracer) in
cardiomyocytes, as qRT-PCR indicated that VEGF-A had no effect
on adult cardiomyocyte Wt1 expression (Supplementary Fig. 10).
Furthermore, tamoxifen labeling was done before either myocar-
dial infarction or VEGF-A modRNA treatment, and the level of
tamoxifen-independent Cre activity in myocardial infarction and
VEGF-A modRNA–treated hearts was trivial (0.005% of cardiomyo-
cytes and 0.003% of endothelial cells were GFP
+
).
To confirm our results using an independent system that did not criti-
cally depend on Wt1-driven marker alleles, we used a Cre modRNA–
containing biocompatible gel to selectively label and trace the fate of
epicardium-derived cells in the adult heart (Fig. 6f). When applied to
R26
mTmG
hearts, Cre modRNA gel selectively labeled cells in the epi-
cardial layer (Fig. 6g). We tested the kinetics of gene transfer by means
of modRNA gel by applying Luc modRNA gel onto the hearts of CFW
strain mice (Supplementary Fig. 11a,b). Luciferase bioluminescence was
detected at near-peak levels by 3 h and peak levels at 24 h, and it was no
longer detectable at 72 h. Similarly, Cre modRNA gel expressed Cre pro-
tein in cells confined to the epicardial layer 2 d after gel application, but
Cre protein was no longer detectable at 14 d (Supplementary Fig. 11c).
Based on these data, we developed a Cre modRNA gel-based prela-
beling strategy (Fig. 6f) that minimized the possibility of nonepicar-
dial labeling in the complex environment induced by the myocardial
infarction. We applied Cre modRNA gel to the heart to label epicardial
cells, waited 2 weeks for decay of Cre activity, and then performed
LAD ligation and concurrent myocardial injection of VEGF-A or Luc
modRNA into the infarct region. One week later, we assessed the fate
of Cre modRNA–labeled EPDCs by confocal analysis of immunos-
tained cryosections. Consistent with the Wt1
CreERT2
labeling result,
EPDCs remained in the epicardial layer in control hearts with sham
operations or hearts with myocardial infarction plus Luc modRNA.
In contrast, myocardial infarction plus VEGF-A modRNA mobi-
lized EPDCs from the epicardial layer and allowed their migration
into the myocardium (Fig. 6g and Supplementary Fig. 12). EPDCs
co-expressed smooth muscle, endothelial and cardiomyocyte line-
age markers (Fig. 6h and Supplementary Fig. 12). Quantification
of the percentage of EPDCs expressing each lineage marker showed
that VEGF-A modRNA directed EPDCs toward an endothelial fate
(48% versus 9%), and led to a small but reproducible subset of EPDCs
co-expressing cardiomyocyte markers (Fig. 6g–i). Collectively, the
Wt1
CreERT2
and Cre modRNA gel fate mapping experiments demon-
strate through two independent approaches that VEGF-A modRNA
alters EPDC fate in the postnatal heart, driving EPDC differentiation
into endothelial cells and potentially to cardiomyocytes.
DISCUSSION
Our study advances an approach to solid-organ repair and regenera-
tion in which delivery of appropriate signal(s) at the right time and
place modifies endogenous progenitor cell activity and thereby pro-
motes longstanding therapeutic benefits. We show that modRNA is
an effective, robust approach to implement this approach. modRNA
avoids several of the apparent problems that have arisen with
conventional cardiac gene therapy vectors
30,44
, including lack of
genomic integration, persistence of expression, immunogenicity,
difficulty in scalability and production, need for life-long monitor-
ing for tumorigenesis and other adverse clinical outcomes, and the
potential for vector escape into the systemic circulation and long-term
expression elsewhere in the body. For these reasons, modRNA has
considerable translational potential.
One of the keys to paracrine signal therapeutics is to deliver a
transient, strong signal at a time and place that coincides with initial
activation of an endogenous progenitor pool. As shown in this study,
a transient pulse delivered in this manner can achieve long-term benefit
through modification of progenitor cell activity and fate. Specifically,
we demonstrated that a single intramyocardial injection of VEGF-A
modRNA improved myocardial outcome and survival after myocardial
infarction. This salutary response was due to improved formation of
functional vessels in the peri-infarct region, which is associated with
altered activity of epicardial progenitors. Pulse-like VEGF-A expres-
sion after myocardial infarction amplified the WT1
+
epicardial pro-
genitor pool and enhanced their differentiation toward the endothelial
lineage (Fig. 7), forming a substantial subset of the additional endothe-
lial cells generated under VEGF-A stimulation. This VEGF-A effect
on epicardial progenitors is reminiscent of the effect of VEGF-A on
multipotent Isl1 heart progenitors that we recently reported
29
, and
indeed VEGF-A may similarly affect other cardiac progenitor popu-
lations. The unique kinetics of modRNA delivery were required to
obtain benefit, as it permitted pulse-like VEGF-A delivery at precisely
the time that myocardial injury activates epicardial cells from their
quiescent state in the normal heart. The transient nature of VEGF-A
modRNA delivery was also crucial, as sustained VEGF-A delivery by
DNA injection led to adverse effects on vascular function.
EDPC lineage tracing using several different genetic labels has
indicated that a subset of EPDCs differentiate into cardiomyocytes
under certain conditions in the developing and adult heart
6,12,13,45,46
.
Consistent with prior studies
15,16
, we found that adult EPDCs have
little native potential to differentiate toward the cardiomyocyte line-
age. However, VEGF-A stimulation appeared to increase cardiomyo-
cyte differentiation to consistently detectable, albeit low, levels. We
confirmed this result using an independent lineage tracing system,
bolstering the evidence that EPDCs differentiate into cardiomyocytes.
However, given the pitfalls and limitations of genetic lineage tracing
approaches
47,48
and the lack of cardiomyocyte differentiation in the
in vitro clonal assay (which might be attributable to inadequacies of
the in vitro culture system), additional studies are needed to further
support this conclusion. The number of cardiomyocytes formed by
EPDCs was reproducible but low and likely not sufficient to account
for the therapeutic benefit of VEGF-A modRNA. Nevertheless, this
finding suggests that additional paracrine signals might be identi-
fied that will achieve differentiation of EPDCs to cardiomyocytes at
therapeutically meaningful levels.
Native state VEGF-A modRNA
EPDCs EPDCs
Endothelial cells Endothelial cells
Smooth muscle cells Smooth muscle cells
Cardiomyocytes Cardiomyocytes
WT1
+
WT1
+
Figure 7 Suggested model for the role of VEGF-A modRNA on EPDCs
differentiation in vivo. Schematic summary of results. In the native
state, myocardial infarction stimulates amplification of WT1
+
EPDCs,
which remain confined to the epicardial layer. VEGF-A modRNA, in the
context of myocardial infarction, augments amplification of WT1
+
EPDCs,
increases their mobilization into the myocardial layer and enhances their
differentiation toward the endothelial lineage.

© 2013 Nature America, Inc. All rights reserved.
nature biotechnology ADVANCE ONLINE PUBLICATION 9
A R T I C L E S
METHODS
Methods and any associated references are available in the online
version of the paper.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
ACKNOWLEDGMENTS
This work was funded by US National Institutes of Health U01H100408 (K.R.C.),
U01HL098166 (K.R.C.), U01JL100401 (W.T.P.), R01HL094683 (W.T.P.),
RC1HL099618 (K.R.C., W.T.P.) and UO1HL100402 (A.J.W.). K.O.L. held a Croucher
Foundation Fellowship and A.J.W. is an Early Career Scientist of the Howard
Hughes Medical Institute. We thank R. Liao, J. Guan, J. Truelove, L. Bu, M. Stachel,
K. Buac, V. Priestly, R. Gazit, K. Ketman, N. Barteneva, A. He, S. Stevens, B. Zhou
and L.Warren for all their help in this project. Adult cardiomyocytes were a kind
gift from R. Liao (Biological and Biomaterial Science, Department of Medicine,
Brigham and Women’s Hospital, Harvard Medical School).
AUTHOR CONTRIBUTIONS
L.Z. (lzangi@enders.tch.harvard.edu) worked in the Rossi, Chien and Pu
laboratories, and designed and carried out most of the experiments, analyzed
most of the data, and wrote the manuscript. K.O.L. in the Chien lab designed
and performed experiments and analyzed the qRT-PCR and immunostaining
data, and wrote the manuscript. Her contribution is similar in significance to the
contributions of L.Z. A.v.G. performed and analyzed the Wt1-related experiments.
Q.M. and R.G. carried out myocardial infarction experiments. W.E. carried out
plasmid preparation. L.M.P. performed blinded analysis of imaging data and wrote
the manuscript. D.S. performed and analyzed skeletal muscle in vivo transfection.
H.X. performed isolation of neonatal mouse cardiomyocytes. M.T. performed
and analyzed in vitro transfection of mouse adult myotubes. B.S. carried out
and analyzed the MRI experiment. M.N., D.M.B., R.A.L. and A.J.W. designed
experiments, analyzed data, and revised the manuscript. D.J.R. (Derrick.Rossi@
childrens.harvard.edu) designed in vitro cardiomyocyte experiments and revised
the manuscript. K.R.C. (kchien@harvard.edu; kenneth.chien@ki.se) conceived the
initial project and experimental studies, and with W.T.P. (wpu@enders.tch.harvard.
edu) designed further experiments, analyzed data, and wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details are available in the online
version of the paper.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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© 2013 Nature America, Inc. All rights reserved.
nature biotechnology
doi:10.1038/nbt.2682
ONLINE METHODS
Construction of IVT templates and synthesis of modRNA. Production of
in vitro transcription (IVT) template constructs and subsequent RNA synthesis
have been described previously
32
. All oligonucleotide reagents were synthe-
sized by Integrated DNA Technologies (Coralville). ORFs were amplified by
PCR from plasmids encoding GFP, mCherry, firefly luciferase, Cre recombi-
nase and human VEGF-A (165) (Addgene, see Supplementary Table 4 for
ORF sequences). PCR reactions were performed with HiFi Hotstart (KAPA
Biosystems) according to the manufacturer’s instructions. Splint-medi-
ated ligations were carried out with Ampligase Thermostable DNA Ligase
(Epicenter Biotechnologies). UTR ligations were conducted in the presence
of 200 nM UTR oligos and 100 nM splint oligos. All intermediate PCR and
ligation products were purified with QIAquick spin columns (Qiagen) before
further processing. Template PCR amplicons were subcloned with the pcDNA
3.3-TOPO TA cloning kit (Invitrogen). Plasmid inserts were excised by restric-
tion digest and recovered with SizeSelect gels (Invitrogen) before being used
to template Poly A tail PCRs. RNA was synthesized with the MEGAscript
T7 kit (Ambion), with 1.6 µg of purified tail PCR product to template each
40 µl reaction. A custom ribonucleoside blend was used comprising 3′-O-
Me-m7G(5′)ppp(5′)G cap analog (New England Biolabs), ATP and guano-
sine triphosphate (USB), 5-methylcytidine triphosphate and pseudouridine
triphosphate (TriLink Biotechnologies). Final nucleotide concentrations in the
reaction mixture were 6 mM for the cap analog, 1.5 mM for guanosine triphos-
phate and 7.5 mM for the other nucleotides. RNA was purified with Ambion
MEGAclear spin columns and then treated with Antarctic Phosphatase (New
England Biolabs) for 30 min at 37 °C to remove residual 5′-phosphates. Treated
RNA was repurified, quantified by Nanodrop (Thermo Scientific) and precipi-
tated with 5 M ammonium acetate according to the manufacturer’s instruc-
tions. modRNA was resuspended in 10 mM Tris HCl, 1 mM EDTA at 100 ng/µl
for in vitro use or 20–30 µg/µl for in vivo use.
modRNA Transfection. modRNA and RNAiMAX (Invitrogen) transfection
agent were each dissolved separately in Opti-MEM (Invitrogen), combined
and then incubated for 15 min at room temperature to generate the transfec-
tion mixture. 5 or 0.5 µl of RNAiMAX reagent was used for every microgram
of modRNA for in vitro or in vivo transfection. In vitro transfection was per-
formed by adding the transfection mixture to cells plated in DMEM with 2%
FBS and 200 ng/ml B18R (eBioscience, San Diego, CA). For in vivo transfec-
tion the transfection mixture was injected directly into the cardiac or skeletal
muscle of animals.
Epicardial lineage tracing using modRNA gel. The Cre modRNA gel, with
a mixture of Cre modRNA (10 µl of modRNA at 20 µg/µl)), lipofectamine
(30 µl), and 0.05% polyacrylic acid (10 µl; Sigma), was painted on the surface
of the Rosa26 (R26)
mTmG
hearts 2 weeks before LAD ligation and injection of
VEGF-A or Luc modRNA. VEGF-A– or Luc modRNA–treated hearts were
assessed for expression of GFP and different myocardial markers to examine
the cell fate of the EPDC derivatives 7 d after myocardial infarction.
Mice. Wt1
GFPCre/+
, Wt1
CreERT2
, R26
fsLacZ
and R26
mTmG
alleles have been
described previously
15–17,43,49
. Genetically engineered mice were in a mixed
C57BL6/CFW background and both male and female mice were used.
Tamoxifen-free base (Tam) was dissolved in sunflower seed oil at 12 mg/ml by
sonication. 0.12 mg/g body weight Tam was administered to adult mice twice
weekly for 3 weeks to induce CreERT2-mediated recombination. One week
after completion of Tam dosing (to allow Tam clearance), myocardial infarc-
tion was induced by ligation of the left anterior descending coronary artery
as described below. Hearts were subsequently assessed using a combination of
FACS, immunofluorescence and real-time-qPCR (RT-qPCR) analyses for GFP
expression and myocardial markers after 7 d. Wt1
GFPCre
was used for isolating
Wt1
+
progenitor cells by the Wt1-driven GFP marker. The fate of Wt1
+
epicar-
dial progenitors was determined using adult-stage irreversible labeling in the
Wt1
CreERT2/+
øR26
mTmG
model. Mice that were Luc modRNA–treated after
Tam induction in the presence of myocardial infarction were used as controls.
To examine “leaky” CreERT2 activity that might occur under stress in the
absence of Tam, mice were treated with sunflower seed oil without Tam before
myocardial infarction and subsequently underwent myocardial infarction
and VEGF-A modRNA or control treatments in parallel with Tam-treated
mice. Wild-type were CFW strain (only males). Animals were not randomized,
but procedures were done with researchers blinded to genotype and treatment
group. All animals that started an experimental protocol and that survived to
the measurement point were included. All mice housing and handling were
performed in accordance with protocols approved by Institutional Animal
Care and Use Committees at Massachusetts General Hospital or Children’s
Hospital Boston or Harvard University.
Cell culture. Adult CFW or WT1
GFPCre/+
hearts were digested using the
Neomyt Cardiomyocyte Isolation kit (Cellutron) to achieve a single-cell sus-
pension of adult mouse cardiac cells, according to the manufacturer’s instruc-
tions. Adult mouse cardiac cells were cultured in Mesenchymal Stem Cell
Growth Medium (Lonza) containing 10% FBS. Primary cultures of human fetal
cardiomyocytes (obtained from Advanced Bioscience Resources, Inc. at gesta-
tional age 20 weeks) were prepared from human fetal ventricles as described
before
9
. Briefly, cardiomyocytes were dissociated by means of repeated (6 ×)
enzymatic digestion with collagenase II solution (Life Technologies) at 37 °C.
Dissociated cells were pelleted (30 g × 2 min) and plated at a density of 1 × 10
5
cells/cm
2
on 35-mm culture dishes with 2 ml of culture medium (3:1 DMEM:
M-199 medium with 5% FCS and 10% neonatal calf serum). Pre-plating of
seeded cells onto 100-mm culture dishes to remove noncardiomyocytes (for
three consecutive days) yielded cultures containing ~80% cTnT heavy chain-
positive cardiac myocytes. Mouse neonatal hearts were dissociated to single
cells by collagenase II (Sigma) as described previously
16
. Mouse neonatal car-
diomyocytes were cultured in DMEM containing 5% FBS, 10% horse serum
and 1 µg/ml insulin. Rat adult cardiomyocytes were a kind gift from R. Liao,
and isolation and culture methods have been described previously
50
. All cell
lines were found negative for mycoplasma contamination.
The secretion of VEGF-A protein was measured using supernatant of mouse
adult cardiac cells after transfection with VEGF-A modRNA or DNA or RNA
using ELISA (R&D systems). Cell transfection efficiency and survival after
modRNA transfection was determined as follows: 4 wells were transfected
with each different concentration of modRNA GFP (with 0, 0.3, 1 or 3 µg per
10
5
cells in a well of a 6-well plate). 16 h after transfection of cardiac cells,
2 wells from each treatment were trypsinized and stained for trypan blue. The
percentage of intact cells was calculated as the number of trypan blue–negative
cells per treatment well/number of trypan blue–negative cells per well without
any treatment *100. To determine transfection efficiency, the two remaining
wells were stained for TNNT2 (red) and GFP (green), and double-positive
cells were measured using ImageJ software.
Wt1-GFP
+
cell isolation and in vitro clonal assays. WT1
+
epicardial pro-
genitors were isolated from the heart explants of WT1
GFPCre/+
mice 7 d after
myocardial infarction. Cardiac cells (nonmyocytes) were allowed to expand
from heart explant cultures. As a control, hearts from uninjured Wt1
GFPCre/+
or Wt1
CreERT2/+
øR26
mTmG
mice treated either with vehicle or hVEGF-A
modRNA were also analyzed. After 1–2 weeks, cells were FACS sorted (FACS
Aria III) for GFP
+
cells (WT1
+
). Single cells or pooled cells of WT1
+
epicar-
dial progenitors were plated in a fibronectin-coated (5 ng/ml for 2 h at 37 °C)
96-well plate or 1 well of a 12-well plate, respectively. Cell proliferation of
the WT1
+
epicardial progenitors was assessed in the presence or absence of
VEGF-A (50 ng/ml) or different KDR inhibitors, including SU5614 or PTK787
(10 nM/l) or DMSO control at different time points (4, 8 and 14 d). Media were
changed every 3 d. Calibration curve of DMSO and KDR inhibitors indicate
that the optimal range (ratio of cell death of vehicle treatment (DMSO control)
to KDR inhibitors) of using these inhibitors in vitro is 4–10 nmol/l. Cells were
counted using an automated cell counter (Invitrogen).
For clonal assays, epicardial explants from Wt1
GFPCre/+
myocardial infarc-
tion mice were cultured as described
17
. Explant outgrowths were then disso-
ciated and WT1
+
epicardial cells were FACS sorted. Single sorted cells were
deposited into fibronectin-coated 96-well plates and clonally expanded in the
presence or absence of VEGF-A (100 µg/ml) for 7–14 d before examination for
their cell fate change in vitro. For FACS analyses, sorted cells were incubated
with fluorochrome-conjugated primary antibodies at 4 °C for 30 min fol-
lowed by three washes with PBS/2% FBS and resuspended in Hank’s balanced
salt solution. Flow cytometric analyses were done using a BD FACSCanto

© 2013 Nature America, Inc. All rights reserved.
nature biotechnology
doi:10.1038/nbt.2682
analyzer. GFP
+
fibroblasts, cardiomyocytes, smooth muscle and endothelial
cells were assessed by immunostaining and treatment-blind cell counts were
done through serial sections using ImageJ software.
Immunodetection methods. Immunostaining was performed on cryosec-
tions using standard protocols with the antibodies listed in Supplementary
Table 5. Isolectin B4 (Vector Lab) was used to stain endothelial cells in
cryosections to determine capillary density. TUNEL (Roche) or Annexin V
staining (eBiosciences) was done to detect apoptosis, according to the manu-
facturer’s instructions. To examine blood vessel leakiness, a mixture of 250 µl
isolectin B4 (0.5 mg/ml, Vector Lab) and 250 µl 70 kD FITC-dextran beads
(50 mg/ml, Sigma) was injected into the tail vein 7 d after myocardial infarc-
tion. Hearts were removed for histological analysis 30 min after tail vein injec-
tion. Quantification of immunostaining in cardiac sections was done using the
ImageJ Software. For each image, color channels (red, blue and green) were
first separated into different images. After separation, the intensity of single-
color signals within each image was quantified by the software. Specific struc-
tures in the images (e.g., blue ovals corresponding to DAPI-stained nuclei)
were defined by intensity threshold analysis. Definition of discrete structures
by the software was further refined by contour and area analysis.
Statistical analyses. Statistical significance was determined by paired t-test for
the MRI results, Log-rank (Mantel-Cox) test for survival curves or Student’s
t-test for other experiments, with P < 0.05 taken as significant. Values were
reported as mean ± s.e.m. Two-sided Student’s t-test based on assumed normal
distributions. Sample sizes were selected for 80% power to detect a biologically
meaningful effect given our past experience with intragroup variance.
Experimental MI model. All surgical and experimental procedures with
mice were done in accordance with protocols approved by Institutional
Animal Care and Use Committees at Massachusetts General Hospital or
Children’s Hospital Boston. MI was induced in CFW, C57Bl/6 and R26fsLacZ,
R26mTmG, Wt1ERT2::R26mTmG by permanent ligation of the LAD, as pre-
viously described
51
. Briefly, the left thoracic region was shaved and sterilized.
After intubation, the heart was exposed through a left thoracotomy. A suture
was placed to ligate the LAD. The thoracotomy and skin were sutured closed
in layers. Excess air was removed from the thoracic cavity, and the mouse
was removed from ventilation when normal breathing was established. In
order to determine the effect of hVEGF-A modRNA in cardiovascular out-
come after MI, lipofectamine vehicle, hVEGF-A modRNA (100 µg/heart) or
hVEGF-A DNA (100 µg/heart) were injected into the infarct zone immedi-
ately after LAD ligation. Sham controls were the same as the MI operation but
without LAD ligation. Where indicated, DMSO or VEGF receptor inhibitors
(0.5 mg/mouse intraperitoneally) were administered daily from 1 d before to
7 d after MI. Inhibitors used were SU5614 (Sigma) or PTK787 (Selleckchem).
In short-term survival experiments mice that were injected with VEGF-A
modRNA or DNA after MI were treated twice a week with humanized anti-
VEGF monoclonal antibody (Avastin, a kind gift from D.M.B.) for 4 weeks.
For analysis, the peri-infarct zone near the apex was either snap-frozen for
RNA isolation and subsequent real-time qPCR studies or was fixed in 4% PFA
for cryosectioning and immunostaining. For Cre gel experiments, the gel was
delivered through a lower intercostal space than that used for LAD ligation
2 weeks later. In all experiments, the surgeon was blinded to treatment group.
To obtain a three-dimensional cast of the vasculature, a ligature was placed
on the aorta and yellow MicroFil (Flow Tech, Inc.) was injected proximally
to fill and opacify the coronary vasculature. Hearts were cleared by wash-
ing through an ethanol-methyl salicylate series (25% for 1 d, 50% for 1 d,
75% for 1 d, 92% for 2 d, 100% for 1 d). To examine the localization of X-
gal
+
cells, X-gal (Fermentas) staining was done according to manufacturer’s
instruction. To examine the degree of fibrosis, short-axis slices of the heart
were created at defined intervals from the apex to the base of the left ventri-
cle. These slices were stained with Masson’s trichrome (Leica) according to
the manufacturer’s instructions. In each slice, areas of fibrosis (revealed by
blue staining) were measured with the UTHSCSA ImageTool software pack-
age
52
. Detection of luciferase+ cells in vivo using the IVIS system. Vehicle
(a mixture of 75 µl RNAiMAX and 5 µl opti-MEM basal medium) or Luc
modRNA (100 µg/heart) was administered intramuscularly into the left
ventricle of hearts of BALB/c mice or skeletal muscle (biceps femoris) of
CFW mice. Bioluminescence imaging of the injected mice was taken at dif-
ferent time points (3–240 h) which each unit represent p/sec/cm2/srX106
(Luc signal). To visualize Luc
+
cells, luciferin (150 µg/g body weight; Sigma)
was injected intraperitoneally. After 10 min, mice were anesthetized with iso-
flurane (Abbott Laboratories), and imaged using an IVIS100 charge-coupled
device imaging system for 2 min. Imaging data were analyzed and quantified
with Living Image Software. The strength of the signal was indicated by the
spectrum of 12 different colors. Hearts or skeletal muscles that were injected
with the vehicle only served as baseline for Luc expression.
MRI. C57Bl/6 (6–8 weeks old) treated with vehicle or hVEGF-A modRN