Sca-1+ cardiosphere-derived cells are enriched for Isl1-expressing cardiac precursors and improve cardiac function after myocardial injury.
ABSTRACT Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.
Using "middle aged" mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1(+)CD45(-) cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1(+)CD45(-) cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1(+)CD45(-) cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.
These studies demonstrate that cloned Sca-1(+)CD45(-) cells derived from CSs from infarcted "middle aged" hearts are enriched for second heart field (i.e., Isl-1(+)) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.
- SourceAvailable from: Perpétua Pinto-do-Ó[Show abstract] [Hide abstract]
ABSTRACT: The identification, in the adult, of cardiomyocyte (CM) turnover events and of cardiac progenitor cells (CPCs) has revolutionized the field of cardiovascular medicine. However, the low rate of CPCs differentiation events reported both in vitro and in vivo, even after injury, raised concerns on the biological significance of these subsets. In this Comprehensive Review, we discuss the current understanding of cardiac Lin-Sca-1+ cells in light of what is also known for similar phenotype cell-compartments in other organs.Stem Cells and Development 06/2014; · 4.20 Impact Factor
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ABSTRACT: Stem cell therapy has the potential to regenerate heart tissue after myocardial infarction (MI). The regeneration is dependent upon cardiac differentiation of the delivered stem cells. We hypothesized that timing of the stem cell delivery determines the extent of cardiac differentiation as cell differentiation is dependent on matrix properties such as biomechanics, structure and morphology, and these properties in cardiac extracellular matrix continuously vary with time after MI. Towards elucidating the relationship between ECM properties and cardiac differentiation, we created an in vitro model based on ECM-mimicking fibers and a type of cardiac progenitor cell, cardiosphere-derived cells (CDCs). A simultaneous fiber electrospinning and cell electrospraying technique was utilized to fabricate constructs. By blending a highly soft hydrogel with a relatively stiff polyurethane and modulating fabrication parameters, tissue constructs with similar cell adhesion property but different global modulus, single fiber modulus, fiber density, and fiber alignment were achieved. The CDCs remained alive within the constructs during a 1-week culture period. CDC cardiac differentiation was dependent on the scaffold modulus, fiber volume fraction, and fiber alignment. Two constructs with relatively low scaffold modulus ∼50-60 kPa most significantly directed the CDC differentiation into mature cardiomyocytes as evidenced by gene expressions of cardiac troponin T (cTnT), calcium channel (CACNA1c) and cardiac myosin heavy chain (MYH6), and protein expressions of cardiac troponin I (cTnI) and connexin 43 (CX43). Of these two low modulus constructs, the extent of differentiation was greater for lower fiber alignment and higher fiber volume fraction. These results suggest that cardiac ECM properties may have an effect on cardiac differentiation of delivered stem cells.Acta biomaterialia 04/2014; · 5.68 Impact Factor
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ABSTRACT: The adult mammalian heart was once believed to be a post-mitotic organ without any capacity for regeneration, but recent findings have challenged this dogma. A modified view assigns the mammalian heart a measurable capacity for regeneration throughout its lifetime, with the implication that endogenous regenerative capacity can be therapeutically stimulated in the injury setting. Although extremely limited in adult mammals, the natural capacity for organ regeneration is a conserved trait in certain vertebrates. Urodele amphibians and teleosts are well known examples of such animals that can efficiently regenerate various organs including the heart as adults. By understanding how these animals regenerate a damaged heart, one might obtain valuable insights into how regeneration can be augmented in injured human hearts. Among the regenerative vertebrate models, the teleost zebrafish, Danio rerio, is arguably the best characterized with respect to cardiac regenerative responses. Knowledge is still limited, but a decade of research in this model has led to results that may help to understand how cardiac regeneration is naturally stimulated and maintained. This review surveys recent advances in the field and discusses current understanding of the endogenous mechanisms of cardiac regeneration in zebrafish.Stem Cell Research 07/2014; · 3.91 Impact Factor
Sca-1+Cardiosphere-Derived Cells Are Enriched for Isl1-
Expressing Cardiac Precursors and Improve Cardiac
Function after Myocardial Injury
Jianqin Ye1, Andrew Boyle1,3, Henry Shih1, Richard E. Sievers1, Yan Zhang1, Megha Prasad1, Hua Su5,
Yan Zhou6, William Grossman1,2, Harold S. Bernstein2,3,4, Yerem Yeghiazarians1,3*
1Division of Cardiology, Department of Medicine, University of California San Francisco, San Francisco, California, United States of America, 2Cardiovascular Research
Institute, University of California San Francisco, San Francisco, California, United States of America, 3Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell
Research, University of California San Francisco, San Francisco, California, United States of America, 4Department of Pediatrics, University of California San Francisco, San
Francisco, California, United States of America, 5Department of Anesthesia and Perioperative Care, University of California San Francisco, San Francisco, California, United
States of America, 6Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California, United States of America
Background: Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI).
However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been
proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by
the tissue milieu remains unclear.
Methodology/Principal Finding: Using ‘‘middle aged’’ mice as CSs donors, we found that acute MI induced a dramatic
increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also
observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac
function. To determine the role of Sca-1+CD45-cells within CSs, we cloned these from single cell isolates. Expression of Islet-
1 (Isl1) in Sca-1+CD45-cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1+CD45-cells had the ability to
differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells
engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and
improved cardiac function in post-MI hearts.
Conclusions/Significance: These studies demonstrate that cloned Sca-1+CD45-cells derived from CSs from infarcted
‘‘middle aged’’ hearts are enriched for second heart field (i.e., Isl-1+) precursors that give rise to both myocardial and vascular
tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.
Citation: Ye J, Boyle A, Shih H, Sievers RE, Zhang Y, et al. (2012) Sca-1+Cardiosphere-Derived Cells Are Enriched for Isl1-Expressing Cardiac Precursors and
Improve Cardiac Function after Myocardial Injury. PLoS ONE 7(1): e30329. doi:10.1371/journal.pone.0030329
Editor: Marcello Rota, Brigham & Women’s Hospital - Harvard Medical School, United States of America
Received April 14, 2011; Accepted December 13, 2011; Published January 17, 2012
Copyright: ? 2012 Ye et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Wayne and Gladys Valley Foundation, the UCSF Cardiac Stem Cell Fund and the Harold Castle Foundation (all to Y.Y.
and W.G.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Growing evidence demonstrates the existence of endogenous
cardiac progenitor cells in the adult mammalian heart, which can
divide and differentiate into cardiomyocytes, endothelial cells and
smooth muscle cells, and potentially play an important role in
maintaining normal cardiac homeostasis  as well as myocardial
response to injury [2–6]. Various methods have been used to
isolate cardiac progenitor cells, including immune selection of cells
using various cell surface markers [2–4] or in vitro culture of
cardiospheres (CSs) [7–9]. Endogenous cardiac progenitor cells
could be collected from the hearts of patients by myocardial
biopsy, expanded in vitro , and then potentially be transplanted
back to the same patient to repair damaged myocardium. This
approach would avoid immune rejection and may therefore
represent an ideal model for cell therapy to achieve long term
reconstitution of lost myocardium and preservation of cardiac
function [10–12]. However, the myocardiogenic potential of CSs
and adult cardiac progenitor cells has recently been questioned
[13,14]. In fact, a recent report by Andersen et al suggested that
CSs are merely fibroblasts and, therefore, not a potential source of
therapeutic cardiac progenitor cells . In addition, whether CSs
obtained from age-appropriate tissues have the ability to function
in myocardial rehabilitation has not been studied.
During fetal development, the LIM homeodomain transcription
factor Islet-1 (Isl1) is expressed in a cell population that gives rise to
second heart field structures and the myocardial vasculature, and
is accepted as a marker of endogenous cardiac progenitors
[5,6,15]. The existence of these cells in the adult heart is not
clear . Since Isl1 is expressed in the nucleus, it has been difficult
to isolate and purify genetically unmodified endogenous Isl1+cells
for therapeutic evaluation. Whereas cells bearing the surface
PLoS ONE | www.plosone.org1January 2012 | Volume 7 | Issue 1 | e30329
markers c-kit and Sca-1 have been isolated from the adult heart
and recognized as adult resident cardiac progenitor cells [2–4].
Questions remain regarding the behavior and cellular compo-
sition of CSs and their response to signals from the myocardial
tissue environment, including: 1) whether acute myocardial
infarction (MI) effects the generation of CSs; 2) whether CSs
derived from injured myocardium have therapeutic potential to
repair ischemically damaged hearts in vivo; and 3) whether specific
subpopulations of CS cells bear the therapeutic potential of CSs in
vivo. We now demonstrate that the Sca-1+CD452CS subpopu-
lation from post-MI ‘‘middle-aged’’ hearts are enriched in Isl1+
cells, have the potential to differentiate into both cardiomyocytes
and vascular cells, and can be used to improve cardiac function in
the injured heart.
CSs enrichment with injury
To determine whether myocardial injury influences the
generation of CS-forming cells, whole hearts including the infarct
area were removed from mice following experimental MI, as well
as from sham-operated and non-operated mice, and were cut to
small pieces as ‘‘explants’’. A monolayer of fibroblast-like cells
migrated out from the cardiac explants over several weeks in
culture. From this monolayer, small, round, phase-bright cells (CS-
forming cells) were seen to emerge (Figure S1A, B). CS-forming
cells from non-operated hearts contained several populations of
cells, based on their expression of Sca-1, c-kit, CD45, CD133,
CD34, Flk1 and CD31 (Figure S2A, B). We observed that explants
took less time to form confluent monolayer in culture when
isolated from injured hearts (1461, 1361 and 1862 days from 1-,
2- and 4-weeks post-MI hearts, respectively), compared to explants
derived from sham-operated and non-operated hearts (2161 and
3262 days, respectively). Thus, cells derived from 1- and 2-week
post-MI cardiac explants expanded more rapidly than both
control groups (P,0.004), however, the growth rate of these cells
was attenuated by 4-weeks post-MI and was not significantly
different from the sham-operated hearts (P.0.05).
In addition to faster growth rates, the number of putative CS-
forming cells harvested from hearts at 1-week (5.12610560.456105/
heart) and 2-weeks (3.75610560.526105/heart) post-MI was
significantly higher than those from sham-operated (2.20610560.70
6105/heart) and non-operated hearts (1.67610560.266105cells/
as many CS-forming cells in approximately half the culture time,
equivalent to an approximate four-fold increase in proliferative rate.
However,thenumberofputative CS-formingcells harvested 4-weeks
post-MI (2.88610560.466105cells/heart) hearts was not signifi-
cantly different from sham-operated and non-operated hearts
(P.0.05), suggesting that the increase in the number of CS-forming
cells was also attenuated by 4-weeks post-MI (Figure 1A).
Consistent with our observation of an increase in CS-forming
cell number with injury, we observed that the number of CSs
derived from hearts harvested 1-week (354650/heart) and 2-
weeks (213638/heart) post-MI were significantly higher than
from sham-operated hearts (80633/heart) (p,0.001) and non-
operated hearts (18614/heart) (P,0.01) (Figure 1B). Our results
also showed that the number of CSs from hearts 4-weeks post-MI
(141642/heart) was not significantly different compared to sham
operated hearts (P.0.05), but still higher than from non-operated
hearts (P=0.02) (Figure 1B). Thus, the effect of injury on CS
formation was attenuated by 4-weeks post-MI.
We have demonstrated that at each of the three stages of CS
formation (explant outgrowth, CS-forming cell generation and
number of CSs), the proliferative was greater at 1–2 weeks post-
MI, and this then returned toward baseline by 4 weeks post-MI.
No regional differences in CS-yield from infarcted hearts
To determine the CS generating potential of different regions of
the heart, we separated the hearts 1-week post-MI into five
regions: left ventricle (LV) excluding scar, right ventricle (RV),
septum, left atrium (LA) and right atrium (RA), and cultured them
separately. The number of CSs from each region of the heart was
counted and adjusted for tissue weight. We observed no
statistically significant regional differences in CS production (LV:
2.660.6 CSs/mg; RV: 3.861.0; septum: 2.360.5; LA: 2.260.2;
RA: 2.960.7; P.0.05) (Figure S3).
CSs contain Isl1+cells
We used fluorescence-activated cell sorting (FACS) to determine
the cellular composition of CSs derived from control and infarcted
hearts after 14 days in culture. CSs from non-operated hearts
contained several populations of cells, based on their expression of
Sca-1, c-kit, CD45, CD133, CD34, Flk1 and CD31 (Figure 2A, B
and C). Immunocytochemical staining showed that the cardiac
transcription factors, Isl1, Nkx2-5 and GATA4, were expressed in
5.061.0%, 13.661.4%, and 60.165.6% of CS cells, respectively
(Figure 2D and E). These were confirmed by real-time RT-PCR
(Figure 3A) and semi-quantitative RT-PCR (Figure 4C). To
determine whether Isl1 expression occurred only with culture ex
Figure 1. Myocardial injury increases the production of CSs. (A)
Numbers of CS-forming cells harvested from hearts at 1-week and 2-
weeks post-MI was significantly higher than those from sham-operated
and non-operated hearts (N=6). (B) Hearts at 1- and 2-weeks post-MI
generated more CSs than sham-operated and non-operated hearts. #
P,0.03. Hearts at 4-weeks post-MI produced similar number of CSs to
sham-operated hearts (N=6). Data in Figure shown as mean6SEM.
Second Heart Field Cardiac Precursors
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vivo, we checked Isl1 expression in adult whole heart by real-time
RT-PCR, and found that Isl1 was indeed expressed in whole
heart. This finding is consistent with a recent report by Khattar et
al.  that showed cells expressing Isl1 protein in adult non-
operated murine hearts by immunohistochemical staining. Nota-
bly however, the expression level of Isl1 mRNA in CSs was 17-fold
higher than in the adult heart (Figure 3A).
To determine the source of Isl1 enrichment in CSs, we analyzed
RNA from Sca-1+CD452and CD45+cells sorted from CSs, and
found that Isl1 expression in Sca-1+CD452cells was 3-fold higher
than in whole CS cells, and that the CD45+fraction did not express
Isl1 (Figure 3B). These results suggested that Sca-1+CD452cells
derived from CSs are enriched for Isl1+cells, and, as expected, no
Isl1+cells were detected among hematopoietic cells of bone marrow
origin. In addition, with the exception of CD45+cells, which likely
represent inflammatory cells that migrate into the heart post-MI, the
proportions of other cell populations in CSs from infarcted heart
werenotaltered compared to non-operated hearts(Figure 4A, B,C).
We investigated whether Isl1 protein expression was detected in
the post-MI hearts of ‘‘middle aged’’ mice using immunohisto-
chemical staining and found Isl1+cells in the epicardium at the
border of the infarct region at 1 week post-MI (Figure S4).
Consistent with our findings, Smart et al  also have found
significant number of Isl1+cells in the epicardium and subepicar-
Figure 2. Cellular composition of CSs from mouse hearts. (A) Flow cytometric analysis of Sca-1, CD45 and c-kit expression in disaggregated CS
cells. Typical results are shown (N=6). (B, C) Bar graph showing the profile of progenitor cell markers in CSs by FACS (N=6). (D) Immunocytochemical
staining demonstrates that CS cells express Isl1, Nkx2-5 and GATA4. Arrows point to positive staining cells (red). Nuclei stained with DAPI. Scale
bar=35 mm and 15 mm (NKx2-5). (E) Bar graph shows the profile of Isl1, Nkx2-5, GATA4 positive cells in CSs by immunocytochemical staining. Data in
Figure shown as mean6SEM.
Second Heart Field Cardiac Precursors
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dial regions at the border of the infarct scar at 7 days post-MI by
genetic tracing and immunohistochemical staining. Together,
these indicate that Isl1-expressing cells are present in the post-MI
heart of middle aged mice.
CS cells derived from infarcted myocardium engraft in
ischemic myocardium in vivo
To determine whether CSs from infarcted myocardium
differentiate into cardiac cells in vivo, we harvested CSs derived
from 1-week post-MI hearts of GFP transgenic mice, injected 105
GFP+cells into the peri-infarct zone (PZ) of syngeneic wild type
mice at 3 days post-MI, and analyzed hearts by immunohisto-
chemistry at 25 days post-injection. Numerous GFP+cells were
found in infarct and peri-infarct zones (Figure 5A, B and C).
Approximately 10% of engrafted GFP+cells expressed cardiac
Troponin I, and ,10% of engrafted GFP+cells expressed either
the endothelial cell marker, CD31, or the smooth muscle cell
marker, a smooth muscle actin (a-SMA) (Figure 5A, B and C).
However, Troponin I+GFP+cells lacked the sarcomeric structure
seen in typical mature cardiomyocytes, and CD31+or SMA+cells
were not incorporated into vascular structures. Nevertheless, our
findings demonstrated that the injected CSs cells derived from
infarcted heart survived in the ischemic, inflammatory microen-
vironment for at least 25 days in vivo, and expressed markers of
nascent cardiac muscle, endothelium, and vascular smooth
CSs from infarcted myocardium promote angiogenesis in
To determine whether transplantation of CSs promoted
angiogenesis in ischemic myocardium, we quantified the capillary
and arteriole density in hearts at day 25 post-injection The results
showed a greater density of CD31+vessels in the infarct zone (IZ)
(10.061.9% vs. 5.362.8%, P=0.002) and PZ (10.362.4% vs.
6.062.4%, P=0.0004) in CS-injected versus control hearts
(Figure 6A). Moreover, the CS-injected group had a significantly
higher number of arterioles (SMA+) in the IZ and PZ vs. control
(3.861.6 and 3.261.1/high power field (HPF) vs. 0.860.2 and
1.260.5/HPF, P,0.0005) respectively (Figure 6B). Vessel counts
did not differ between the CS-injected and control groups in the
remote zone (RZ) (CD31+vessels: 4.061.9% vs. 3.462.3%,
P.0.05; SMA+arterioles 0.660.3 vs. 0.860.3/HPF, P.0.05)
(Figure 6A, B). Because engrafted CD31+and SMA+cells did not
contribute to vessel-like structures at this time point, these results
suggest that injection of CSs promoted endogenous angiogenesis.
Injected CSs from infarcted myocardium reduced infarct
size and improved cardiac function
To determine whether injection of CSs from infarcted
myocardium improved cardiac function in a MI mouse model,
we evaluated left ventricular ejection fraction (LVEF) by
echocardiography and measured infarct size by histochemical
staining at 25 days post-injection. LVEF was significantly reduced
from an average of 51.261.7% before MI to 35.162.9% at 2 days
post-MI in both groups, with no significant difference between two
groups (P.0.05). At 28 days post-MI (25 days post-injection),
LVEF was significantly higher in the CS-injected group
(Figure 6C). Furthermore, we found that the CS-injected group
had significantly smaller relative infarct size compared to control
(24.8616.5% vs. 48.4619.8%, P=0.02) (Figure 6D and E). These
findings suggest that CSs from injured myocardium have a
beneficial effect in the MI mouse model.
Figure 3. Isl1+cells are present in adult heart and CSs. Real-time
RT-PCR was used to compare Isl1 expression in heart, indicated cells,
and liver (negative control). Results show as Isl1 mRNA expression
relative to Hypoxanthine phosphoribosyltransferase (HPRT). (A) Isl1
expression in CSs (N=10) was 17-fold higher than in the hearts (n=4).
(B) Isl1 expression in Sca-1+CD452cells (N=6) derived from CSs was 3-
fold higher compared to that in whole CSs (N=10). (C) Isl1 expression in
cloned Sca-1+CD452cells (n=5) was similar to primary Sca-1+CD452
cells isolated from CSs (N=6). Isl1 expression dropped significantly with
differentiation (N=3). CM: cardiomyocytes.
Second Heart Field Cardiac Precursors
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Sca-1+CD45-cells in CSs have the characteristics of
cardiac progenitor cells in vitro
In our studies, the Sca-1+CD45-subpopulation comprised the
largest fraction of CS cells, and contained more Isl1+cells.
To further investigate the origin of Sca-1+CD45-in CSs,
chimeric mice were generated by transplantation of bone marrow
cells isolated from GFP transgenic mice to lethally irradiated
C57BL/6 mice. Flow cytometric analysis demonstrated that
8860.5% of peripheral blood mononuclear cells were GFP+in
the chimeric mice 5 months post-transplantation (Figure S5A),
indicating stable bone marrow engraftment. About 18.464.5% of
cells in CSs derived from the no-surgery or 2-week post-MI hearts
of chimeric mice co-expressed GFP and CD45 (Figure S5B),
indicating they originated from bone marrow. All of the
Sca-1+CD452cells were GFP negative (Figure S5C), suggesting
that they did not originate from the bone marrow.
To further investigate whether these cells play a key role in
restoring cardiac function and reducing infarct size, we sorted Sca-
1+CD452cells from CSs from 1-week post-MI hearts of adult
GFP transgenic mice, and clonally expanded these in culture from
single cells. After culturing for 14 days, 5.6% (16/288) of the single
cells grew to colonies. About 30% (3/10) of clones grew to .106
cells after 30 days in culture. To determine the cellular
composition of cloned cells derived from a single Sca-1+CD452
cell, we analyzed the cell-types of cloned cells by FACS after 30
days at passage 4, 15 and 22. FACS analysis showed that
61.8612.4% of the clonally derived cells was still Sca-1+CD452
Figure 4. Cellular composition of CSs from injured hearts. (A) The percentages of Sca-1+CD452in CSs were not altered by MI (N=6). (B) The
percentages of total CD45+cells in CSs was increased at 1-week post-MI and attenuated at 2-week post-MI (N=6). (C) Semi-quantitative RT-PCR
analysis showed that CSs from all groups expressed similar level of Nkx2-5, GATA4, Flk-1 and SMA. HPRT was used as control. N: Non-surgery. S: Sham-
operated. 1W: 1 week post-MI. 2W: 2 weeks post-MI. 4W: 4 weeks post-MI. H: mouse heart (positive control). SM: skeletal muscle (negative control).
Data in Figure shown as mean6SEM.
Second Heart Field Cardiac Precursors
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0.0360.02% CD133+, 1.160.6% CD34+, 0.660.3% CD31+and
0.360.2% Flk1+. In addition, GATA4, Nkx2-5, and Isl1 were
expressed in 60%, 20% and 10% of cloned cells, respectively
(Figure 7B, C and D).
After treatment with 5-azacytidine, transforming growth factor
b1 (TGF-b1), and vitamin C , cloned Sca-1+CD452cells at 4
and 20 passages differentiated into cardiomyocytes (,25% of cells
expressed sarcomeric a actinin (SA) (Figure 8B), and ,60% of
cells expressed connexin-43 (Figure 8C), the first connexin to be
expressed in developing cardiomyocytes ). Alternatively,
treatment with by vascular endothelial growth factor (VEGF)
resulted in differentiation into endothelial cells (,20% of cells
expressed CD31, Von Willebrand Factor (VWF), and FLK-1, and
accumulated acetylated low density lipoprotein labeled with 1,19-
(Dil-ac-LDL)) and smooth muscle cells (,34% of cells expressed
SMA) (Figure 8D, E, F, G and H). Thus, the Sca-1+CD452cells
within CSs were clonogenic, multipotent (i.e., formed cardiomy-
ocytes, endothelial cells, and smooth muscle cells), and were
capable of long-term self-renewal in vitro, all characteristics of
cardiac progenitor cells.
To determine whether Isl1 expression persisted in clonally
expanded Sca-1+CD452cells, we compared Isl1 transcript levels
between primary Sca-1+CD452isolates, clonally expanded Sca-
1+CD452cells, and cardiomyocytes differentiated from Sca-
1+CD452clones by real-time RT-PCR. We found that expression
of Isl1 was not significantly different between primary Sca-
1+CD452cells and cloned cells. However, Isl1 expression
decreased in cardiomyocytes, consistent with differentiation
beyond the progenitor stage (Figure 3C).
Transplanted Sca-1+CD452cells differentiate into
endothelial and smooth muscle cells in vivo
To determine whether cloned Sca-1+CD452cells can differen-
tiate after implantation in vivo, we injected 106dissociated cloned
GFP+Sca-1+CD452cells into the PZ of syngeneic wild-type mice
3 days post-MI. Twenty five days after injection, the hearts showed
numerous implanted cells present in the PZ, but none of the
injected cells had differentiated into cardiomyocytes (Figure 9A),
endothelial cells and smooth muscle cells at this early time-point
after implantation. In contrast, hearts harvested 75 days after cell
injection showed that ,10% of retained transplanted GFP+cells
differentiated into CD31+endothelial cells (Figure 9C) or SMA+
smooth muscle cells (Figure 9D), but not troponin I+cardiomy-
ocytes (Figure 9B). These findings demonstrate that the cloned
Sca-1+CD452cells not only survived in the ischemic microenvi-
ronment at 75 days post-injection, but also differentiated into two
vascular lineages in vivo.
To quantify the level of engraftment and persistence of injected
cells in infarcted hearts, cloned Sca-1+CD452(GFP+) cells (106)
were injected into infarcted hearts of wild type mice. The injected
hearts were harvested at 1 hour and 1, 3, 7, 14 and 25 days post-
injection. RNA was isolated from whole heart and mRNA
expression of GFP was quantified by real-time RT-PCR as a
surrogate for the number of engrafted cells. The expression level of
GFP in the heart collected 1 hour post-injection was used to
represent 100% of injected cells. Approximately 15% and 4% of
injected cells were detected in injected heart 3 and 7 days post-
injection respectively. Approximately 3% of injected cells were
detected 14 to 25 days post-injection (Figure S6).
Cloned Sca-1+CD452cells promote angiogenesis in
To determine whether transplantation of cloned Sca-1+CD452
cells promote angiogenesis in ischemic myocardium, we quantified
the capillary and arteriole density in hearts 25 days after cell-
injection. The results showed that there were more CD31+vessels
at the IZ (13.262.9% vs. 6.262.4%, P=0.001) and PZ
(11.763.6% vs. 7.26 1.9%, P=0.02) in the cell-injected group
vs. control (Figure 10A). Moreover, the cell-injected group had a
significantly higher number of SMA+arterioles in the IZ (3.961.1
vs. 1.960.3/HPF, P=0.001) and PZ (5.062.0 vs. 2.160.9/HPF,
Figure 5. CS cells engraft in ischemic myocardium. CS cells from 1-week post-MI GFP transgenic mice were injected into the peri-infarct zone
(PZ) of syngeneic wild type mice 3 days post-MI. Injected cells were detected in the PZ at 25 days after delivery. The surviving cells expressed cardiac
Troponin I (A), CD31 (B), and SMA (C). Nuclei were stained with DAPI. Typical results are shown (N=7). Scale bars=15 mm in A, B and C.
Second Heart Field Cardiac Precursors
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P=0.009) vs. control (Figure 10B). Cell-injection had no effect on
vascular density in the RZ (CD31+vessels: 6.461.7% vs.
5.761.1%, P.0.05; SMA+arterioles 0.860.3 vs. 0.760.3/HPF,
P.0.05) (Figure 10A, B). However, none of the vessels contained
GFP+cells 25 days after cell-injection, suggesting that cloned Sca-
1+CD452cells promoted endogenous angiogenesis by paracrine
mechanisms, rather than by direct participation in new blood
vessel formation at this time point. Together, it is likely that
transplanted cloned Sca-1+CD452cells induce angiogenesis
through both paracrine effects and transdifferentiation. However,
the small number of vessels with GFP+cells detected 75 days post-
transplantation suggests that angiogenesis is induced mainly
through paracrine effects of the engrafted cells.
Cloned Sca-1+CD452cells reduce apoptosis of
To evaluate the effects of Sca-1+CD452cells injection on
cardiomyocyte apoptosis, we used terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) and co-staining
troponin I in the hearts 25 days after cell-injection. Cell injection
resulted in significant reduction in the number of TUNEL+/
troponin I+cells in PZ compared to control (0.2560.17 vs.
1.0860.35/low power field (LPF), P,0.05) (Figure S7). The
number of TUNEL+/troponin I+cells did not differ between cell-
injected and control groups in IZ and RZ (Figure S7).
Cloned Sca-1+CD452cells reduce infarct size and
improve cardiac function
To determine whether the cloned Sca-1+CD452cells alone
improve cardiac function in the MI mouse model, we evaluated
LVEF by echocardiography and measured infarct size by
histochemistry 25 days after cell injection. LVEF was significantly
reduced from an average of 51.261.5% before MI to 36.362.0%
at 2 days post-MI in both groups, with no significant difference
between the two groups (P=0.9). At 28 days post-MI (25 days
post-injection), LVEF was significantly higher in the cell-injected
group compared to control (39.763.2% versus 30.966.6%,
P=0.02) (Figure 10C). In addition, the myocardium was
significantly thicker in the peri-infarct wall in the cell injected
group compared to control (0.5760.05 vs. 0.4460.06 mm,
P=0.002), but there was no statistically significant difference in
the thickness of the posterior wall (0.7060.05 vs. 0.6960.06 mm,
Figure 6. Injected CS cells promote angiogenesis, limited infarct size and improve cardiac function. At 25 days post-injection, engrafted
cells resulted in both increased vessel density (A) and numbers of arterioles (B) in the infarct zone and peri-infarct zone, but not remote zone (N=7).
*P=0.002, #P=0.0004 (C) LVEF improved with CS cell injection compared to control. Each line represents the mean of one group (N=6). (D) Mice
treated with CS cells had smaller infarct sizes (circumferential extent of scar) compared to control (N=7).
Second Heart Field Cardiac Precursors
PLoS ONE | www.plosone.org7 January 2012 | Volume 7 | Issue 1 | e30329
P=N.S.), septum (1.060.08 vs. 0.9560.08 mm, P=N.S.) or scar
(0.2960.03 vs. 0.2560.03 mm, P=N.S.) between the groups
(Figure 10D). The cell-injected group had significantly smaller
infarcts compared to control (24.4613.8% vs. 49.4621.5%,
P=0.037) (Figure 10E, D). These studies suggest that the cloned
Sca-1+CD452cells confer the therapeutic benefits of CSs in the
mouse MI model.
In this study, we have shown that: 1) there is a significant
increase in the proliferative capacity of CS-forming cells isolated
from the ‘‘middle aged’’ heart following acute MI resulting in a
significant rise in the number of CSs in vitro; 2) this increase is time-
dependent and is most pronounced within the first week post-MI
in the animal model studied; 3) transplanted CSs from infarcted
myocardium engraft in ischemic myocardium, improve cardiac
function, and promote endogenous angiogenesis; 4) adult heart
contains Isl1+cells and Isl1 expression in CSs is 17-fold higher
than in total adult cardiac tissue; 5) Isl1 expression in the Sca-
1+CD452subpopulation within CSs is 3-fold higher than in total
CSs; 6) Sca-1+CD452cells in CSs can be cloned, expanded, and
have the characteristics of multipotent cardiac progenitor cells in
vitro; and 7) after injection into ischemic myocardium, cloned Sca-
1+CD452cells not only survive long-term, but also differentiate
into endothelial and smooth muscle cells, promote endogenous
angiogenesis, reduce cardiomyocyte apoptosis, reduce infarct size,
and improve cardiac function. Of special note, these experiments
have been performed in middle-aged mice, rather than in young
adults, to simulate a more clinically relevant disease model.
Previous studies  have shown that the number of Sca-1+
cardiac progenitor cells increases post-MI. Our data agrees with
these reports and further shows that the potential for CSs
generation in vitro is highly time dependent post-MI. The number
of CSs from 1- and 2-week post-MI hearts greatly increases
compared to uninjured hearts, and this increase is attenuated by 4
weeks post-MI. This suggests that acute MI induces the
proliferation of cardiac progenitor cells, and this increase in
proliferation gradually dissipates over a 4-week period post-MI.
Therefore early acquisition of tissue from post-MI hearts would
facilitate higher yields of CSs in vitro. However, the optimal timing
in humans may be different than the ones reported in our study
and future research is necessary to better define the ideal timing of
tissue acquisition in patients post-MI.
A previous report  showed that stem cell niches distribute
preferentially to the apex and atria of the heart, where the wall
stress is relatively low. MI may affect their distribution. Our results
show that various regions of 1-week post-MI hearts have similar
abilities to produce CSs. This suggests that although MI mainly
affects the LV, the CS-forming cells throughout the heart are
Figure 7. Analysis of cloned Sca-1+CD45-cells. (A) Flow cytometry showed that ,60% of cloned cells were Sca-1+CD452. Typical results are
shown (N=6). (B–D) Immunocytochemical staining showed that cloned Sca-1+CD452cells expressed Isl-1 (B), Nkx2-5 (C) and GATA4 (D). Nuclei were
stained with DAPI. Scale bar=35 mm in B3; Scale bar=15 mm in C3 and D3. Typical results are shown (N=3).
Second Heart Field Cardiac Precursors
PLoS ONE | www.plosone.org8 January 2012 | Volume 7 | Issue 1 | e30329
activated post-MI. As such, taking tissue from any region of the
heart appears to yield similar numbers of CSs per unit of tissue.
The mechanism by which MI increases CSs production is worthy
of further investigation. Importantly, the septum and right
ventricle yield the same numbers of CSs. Thus, percutaneous
right ventricular endomyocardial biopsy, as is performed routinely
for other clinical indications, could potentially be used to generate
CSs in the post-MI setting.
It has been previously reported that CSs from non-infarcted
hearts could differentiate into cardiac cells and preserve cardiac
function. Whether CSs derived from early stage post-MI hearts
have the same abilities has not been demonstrated until now. Our
results show for the first time that the CS cells obtained from 1-
week post-MI hearts engraft in ischemic myocardium and restore
cardiac function at 25 days post-injection in vivo. However, we did
not find evidence for differentiation of these cells into mature
cardiomyocytes or new vessels. Our data demonstrate that the
injected CSs promoted angiogenesis in vivo, suggesting that
engrafted CSs cells likely have a paracrine, pro-angiogenic effect
in the ischemic myocardium. Secreted VEGF from engrafted CSs
may contribute to angiogenesis in the infarcted hearts . This
paracrine effect may play an important role in attenuating adverse
LV remodeling and preserving cardiac function.
Recent reports have found Isl1+cells in adult mouse, rat and
human hearts [16,17,23,24]. Our results not only confirm the
presence of Isl1+cells in adult murine hearts, but we show that
these can be efficiently isolated and expanded by culturing
Sca-1+CD452cells from CSs. Since Isl1 is not expressed on
the cell surface, it has been difficult to isolate and purify these
cells by immune selection. However, we now demonstrate that
isolating Sca-1+CD452cells from CSs results in an enriched
population of Isl1+cardiac progenitors for autologous cardiac
While cloned Sca-1+CD452cells improved cardiac function
post-MI in transplanted mice, we did not find evidence for
differentiation of these cells into cardiomyocytes in vivo. One
possible explanation is that the cloned Sca-1+CD452cells may
need a longer time to differentiate into cardiomyocytes in situ. This
is supported by the observation that there was no differentiation
seen from cloned cells in the first 25 days, while endothelial and
smooth muscle cell differentiation occurred only after 75 days.
Another possible explanation is that there may be subpopulations
of Sca-1+CD452CS cells with distinct differentiation capacities.
Whether resident cardiac progenitor cells in adult hearts are
capable of cardiomyogenic differentiation in vivo remains contro-
versial [14,25–27]. The therapeutic effects of CS-derived Sca-
1+CD452cells in vivo do suggest, however, that these cells might be
responsible for the overall effects of CSs. Since the Sca-1+CD452
cells can be clonally expanded in vitro, they provide a feasible
approach to rapidly generating therapeutic quantities of cardiac
A recent report has suggested that CSs are composed of
fibroblasts, and not cardiac progenitor cells . Although many
fibroblasts grow out from the cardiac explant during the first stage
of culture, we demonstrate here that our CSs contain cardiac
progenitor cells that are capable of clonal expansion and multi-
lineage cardiac differentiation. Furthermore, our demonstration
that cloned Sca-1+CD452cells have a beneficial therapeutic
effect, similar to heterogeneous CSs, argues strongly against the
hypothesis that fibroblasts are the major contributors to cardiac
repair in CSs.
There are several limitations to this study. First, under the
experimental conditions used, we did not find evidence for
differentiation of cloned Sca-1+CD452cells into cardiomyocytes in
vivo. It is possible that a longer follow-up period might be required
for this differentiation to be observed in vivo. Despite this, we have
Figure 8. Cloned Sca-1+CD452cells differentiate into cardiac cells in vitro. (A–C) After treatment with 5-azacytidine, TGF-b and vitamin C,
cloned Sca-1+CD452cells differentiated in vitro into cardiomyocytes expressing cardiac Troponin T (A), sarcomeric a-actinin+(SA; B), and connexin-43
(C). (D–H) After treatment VEGF, the cloned cells differentiated into endothelial cells expressing CD31 (D), Von Willebrand Factor (VWF; F), and Flk1
(G), and smooth muscle cells expressing SMA (H). The endothelial cells also demonstrated acetylated LDL uptake (Dil-ac-LDL; E). Nuclei were stained
with DAPI. Scale bar=15 mm in A, D, E, Scale bar=35 mm in B, C, F, G and H. Typical results are shown (N=3).
Second Heart Field Cardiac Precursors
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demonstrated that these cells do indeed have ‘‘progenitor’’
characteristics given their ability to differentiate into other cell
types both in vivo and in vitro. Second, we show increased
angiogenesis and reduced cardiomyocyte apoptosis after injection
of cloned Sca-1+CD452cells. However, given our experimental
conditions, we are unable to address whether the injected cells
recruit and influence endogenous cardiac progenitors in vivo.
Recently, using a genetic lineage mapping approach, Loffredo et
al  have reported new cardiomyocyte formation in infarcted
hearts derived from endogenous cardiac stem cells 8 weeks after
injecting with murine bone marrow c-kit+cells. There are several
possible explanations for the consistent improvements in ventric-
ular function including: reduction in cardiomyocyte apoptosis
[22,29,30], prevention of infarct scar expansion by mechanically
stiffening the infarct zone, facilitation of hypertrophy of the border
zone cardiomyocytes by enhanced angiogenesis [22,29–31]. Other
groups have reported stimulation of the endogenous adult
cardiomyocytes to re-enter cell cycle and divide  and
recruitment and/or activation of resident cardiac progenitors
[22,28,32] as possible additional mechanisms. Each of these
mechanisms has been implicated in improved cardiac function
seen with cell therapy. However, the exact contribution of each of
these mechanisms to the overall benefit of therapy remains the
focus of intense research.
In summary, our data suggest that the cloned Sca-1+CD452cells
derived from CSs from post-MI hearts are enriched in Isl1+
progenitors, have the characteristics of progenitor cells, and are an
attractive source ofautologous cellsformyocardialtherapypost-MI.
Materials and Methods
a-actin promoter driving EGFP expression were purchased from the
Jackson Laboratory (Bar Harbor, Maine). Allanimals werehoused in
theanimalcarefacilityat The UniversityofCalifornia,SanFrancisco
(UCSF), and all experiments were approved by, and conducted in
accordance with the guidelines of, the Institutional Animal Care and
Use Committee of UCSF (Approval number: AN078431).
Myocardial infarction model
Nine month-old, male C57BL/6J mice were used for all
experiments to simulate ‘‘middle-aged’’ subjects. Mice underwent
total permanent ligation of the left anterior descending coronary
artery (LAD) to induce MI, and hearts were collected 1, 2, and 4
weeks post-MI (n=6/group). Hearts were also harvested from
animals that had undergone sham operation or no surgery (n=6/
group). The surgical procedure for MI has been previously
described [29,33,34]. Briefly, mice were anesthetized using
isoflurane, intubated and ventilated. A midline thoracotomy
incision was made to expose the heart for surgery. LAD was
ligated permanently. Sham operation was performed by passing a
suture under the LAD and removing it without ligation.
Figure 9. Cloned Sca-1+CD452cells differentiate into endothelial and smooth muscle cells in vivo. Cloned Sca-1+CD452GFP+cells were
injected into the peri-infarct zone (PZ) of infarcted myocardium of syngeneic wild type mice 3 days post-MI. Injected cells were detected by GFP
expression 25 days after transplantation, but there was no evidence of cardiac differentiation at this time point (A) (N=7). At 75 days post-injection
(C–D), transplanted cells were detected in the PZ, and expressed CD31 (C) and SMA (D), but not Troponin I (B). Nuclei were stained with DAPI. Scale
bar=35 mm in A, B, C and D. Typical results are shown (N=2).
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Generating chimeric mouse
Bone marrow cells were harvested from 8–10 week old GFP
transgenic mice and transplanted into lethally irradiated (9.5 Gy) 2
month -old C57BL/6 mice through tail vein injection (2x106
nucleated unfractionated cells per mouse). The expression of GFP
by peripheral blood mononuclear cells was analyzed by FACS 5–6
months later. CSs were isolated from no-surgery or 2-week post-
MI hearts of chimeric mice.
Cell transplant studies
Nine month-old, male C57BL/6J GFP transgenic mice (n=12)
were used as CSs donors. One week post-MI, hearts were
Figure 10. Injected Sca-1+CD452cells promote angiogenesis, limit infarct size and improve cardiac function. At 25 days post-injection
of cloned Sca-1+CD452cells, engrafted cells resulted in both increased vessel density (A) and number of arterioles (B) in the infarct zone (IZ) and peri-
infarct zone (PZ) but not the remote zone (RZ). *P,0.02, #P,0.009. HPF, high power field at 40X (N=6). (C) Echocardiography showed that LVEF
improved in mice treated with cloned cells compared to control (PBS). Each line represents the mean of one group (N=6). (D) Left ventricular wall
thickness evaluated by the echocardiography showed a significant increase in PZ wall thickness in mice treated with cloned cells versus control
(N=6). (E) Infarct size was determined morphometrically with picosirius red staining (N=6). (F) Mice treated with cloned cells exhibited smaller infarct
sizes than control, as measured by circumferential extent of the scar (N=6).
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harvested and used to generate CSs for injection. CSs were
dissociated into single cell suspension by Blendzyme 4 and
resuspended with phosphate buffered saline (PBS). Alternatively,
cloned Sca-1+CD452cells were dissociated into single cell
suspension by trypsin and also resuspended with PBS. These cells
in 10 ml PBS were injected into the hearts of 3-day post-MI mice
by ultrasound-guided injection, a technique developed and
reported by our laboratory [29,34]. PBS only was injected into
infarcted hearts as control. Two month-old, wild-type C57BL/6J
mice were used as recipients. The recipient mice received either
CS cells (n=7), Cloned Sca-1+CD452cells (n=8) or PBS (n=12),
and hearts were harvested 25 days post-injection or 75 days post-
injection (Sca-1+CD452cell injections only). For studying the
retention of injected cloned Sca-1+CD452cells, eighteen wild-type
mice were used as recipients and whole hearts were harvested 1
hour and 1, 3, 7, 14 and 25 days post-injection (n=3/each time
point) for RNA isolation.
CSs were generated using the method described by Messina et
al.  with minor modification. The whole heart was removed
from the mice and cut into 1–2 mm3pieces. After being washed
with Ca++Mg++free PBS and digested three times, 5 minutes each
at 370C with 0.25% trypsin (Invitrogen, Carlsbad, CA) and 0.1%
collagenase D (Roche Diagnostics, Indianapolis, IN), the tissue
pieces were cultured as ‘‘explants’’ on fibronectin (Sigma, St Louis,
MO) coated 6-well plates, 2 wells for each heart in IMDM
medium with 10% FBS and 0.1 mM b-mercaptoethanol at 370C
with 5% CO2. A layer of fibroblast-like cells grew from explants,
over which small, round phase-bright cells (CS-forming cells)
appeared 2 to 4 weeks after initiating the culture. Once the
fibroblast-like cells grew to 90% confluence determined visually,
the cells surrounding the explants were harvested by two washes
with PBS, one wash with 0.53 mmol/L EDTA and one wash with
0.05% trypsin (Invitrogen) at room temperature. The harvested
cells were filtered by 70 mm cell strainer (BD Biosciences, San
Jose, CA), and then cultured at a density of 1x105cells/ml in each
well of 24-well plates coated with Poly-D-Lysine (BD Biosciences)
in cardiosphere growth medium (CGM), which included 35%
IMDM, 65% DMEM-F12, 3.5% FBS, 0.1 mM b-mercaptoeth-
anol, 2% B27 (Invitrogen), 10 ng/ml EGF (R&D systems), 20 ng/
ml bFGF (R&D systems), 40 nmol/L thrombin (R&D systems) and
4 nmol/L cardiotrophin (R&D systems). The number of CSs in
each well was counted on the 7thdays after the CS-forming cells
Flow cytometry and cells sorting
CSs were dissociated into single cell suspension by Blendzyme 4
(5.6u/ml) (Roche). The following phycoerythrin (PE) or allophy-
cocyanin (APC) conjugated rat anti-mouse antibodies and
conjugated isotype-matched control antibodies were used: Sca-1-
PE, c-kit-PE, CD133-PE, CD34-PE, CD45-APC, Flk-1-APC and
CD31-APC (eBioscience). The cells were incubated with antibod-
ies for 25 min on ice, washed with PBS containing 0.2% BSA, and
analyzed by FACSCabilur with CellQuest software (BD Biosci-
For cell sorting, the dissociated CS cells from hearts of 1-week
post-MI GFP transgenic mice were stained by the following
antibodies: Sca-1-PE and CD45-APC. The Sca-1+CD452cells
were sorted by FACSAria with FACSdiva software (BD Biosci-
ences) and were dropped into a 96-well plate, one cell/well, on top
of mitomycin-C treated murine embryonic fibroblast cells
(Millipore, New Jersey). The cells were then cultured with CGM
at 370C with 5% CO2. For isolating RNA, sorted Sca-1+CD452
cells or CD45+cells from CSs were collected into a tube with 1 ml
of CGM respectively.
Directed differentiation in vitro
Cloned Sca-1+CD452cells were loaded into chamberslides
coated with gelatin at 15,000 cells/cm2in differentiation medium,
treated by 5-Azacytidine (5 mM) for 3 days, and then we added
TGF-b1 (1 ng/ml) and vitamin C (0.1mM) for three weeks to
induce cardiomyocyte differentiation. The cells were treated
with VEGF (20 ng/ml) in IMDM medium with 10% FBS for 2
weeks for endothelial and smooth muscle cells differentiation .
The cells cultured in chamberslides were washed with PBS,
fixed with cold methanol for 5 min or 4% paraformaldehyde/PBS
for 15 min, and blocked with Dako antibody diluent (DakoCyto-
mation, Carpinteria, CA) for 1 hour. When using mouse derived
monoclonal antibody, we also used Rodent Block M (Biocare
Medical, Concord, CA) blocking for 30 min. The cells were
incubated with the following primary antibodies diluted in Dako
antibody diluent at 40C overnight: rabbit anti-Nkx2-5, GATA4
(Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-Isl-1
(39.4D5) (Developmental Studies Hybridoma Bank, Iowa City,
IA), mouse anti-a SMA (Sigma), mouse anti-Troponin T (Thermo
Fisher Scientific, Fremont, CA), mouse anti-SA (Abcam, Cam-
bridge, MA), rabbit anti-connexin-43 (Sigma), mouse anti-CD31
(Abcam) and rabbit anti-VWF (Abcam). The cells were then
incubated with the Alexa Fluor 546 labeled goat anti-rabbit
antibody or goat anti-mouse antibody (Invitrogen) at room
temperature for 1 hour. The slides were mounted with ProLong
Gold antifade reagent with DAPI (Invitrogen) and viewed with a
Nikon E800 fluorescence microscope using Openlab software
(Improvision, Lexington, MA).
Acetylated-LDL uptake assay
Acetylated low density lipoprotein labeled with Dil-ac-LDL
(Invitrogen) was added into the medium with cells at 2ug/ml as
final concentration and incubated at 370C with 5% CO2for 1
hour. The medium was removed. The cells were washed with PBS
and fixed with 4% paraformaldehyde/PBS for 15 min. The slides
were mounted and viewed same as immunocytochemical staining.
RT-PCR and Real-time RT-PCR
The total RNA from CSs and tissues were isolated by TRIzol
reagent (Invitrogen). cDNA was generated from 0.3 mg of total
RNA by using SuperScript III First-Strand Synthesis kit (Invitro-
gen). RT-PCR was performed using 1 ml of cDNA and Advantage
2 PCR kit (Clontech, Mountain View, CA) with the following
program: 95uC 3 min, (95uC 30 s– 68uC– 3 min)630 cycles, 68uC
10 min. PCR products were separated on 2% agarose gel. Every
pair of PCR primers was designed to span one or several introns to
distinguish the signals amplified from genomic DNA contamina-
tion. The primers sequence of Nkx2-5, GATA4, Flk-1, SMA and
internal control hypoxanthine phosphoribosyltransferase (HPRT)
are from previous publications [4,35,36].
The total RNA from sorted cells was isolated and the cDNA was
generated by Taqman Gene Expression Cells-to-Ct kit (Applied
Biosystems, Foster City, CA, USA). The primers and probe for
murine Isl1 and HPRT were purchased from Applied Biosystems.
The real-time PCR were performed by ABI PISM 7300 (Applied
Biosystems) using Taqman Master Mix (Applied Biosystems) in
duplicates and the average threshold cycles (CT) of duplicate were
used to calculate the relative value of Isl-1 in different cells and
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tissues. The CT for HPRT was used to normalize the samples.
Expression of Isl1 mRNA relative to HPRT mRNA was calculated
based on the CT, DCTIsl1=CTIsl1-CTHPRT. The relative values
of Isl1 were calculated as 22DCTIsl1.
For studying the retention of injected cloned GFP+Sca-
1+CD452cells, total RNA from whole heart was isolated by
TRIzol, genomic DNA was removed from total RNA by RNeasy
Mini Kit with RNase-free DNase (Qiagen) and 25-ng cDNA was
used for real-time PCR. The sequences of primers and probes for
GFP and histone 3.3A were as previously published .
Expression of GFP mRNA relative to histone 3.3A mRNA was
calculated based on the CT, DCTGFP=CTGFP-CThistone. The
relative values of GFP were calculated as 22DCTGFP.
Tissue was analyzed by two blinded reviewers. Mice were
sacrificed 25 days post-injection of cells (28 days post-MI) or 75 day
post-injection. The hearts were arrested in diastole with KCl,
perfusion and fixed with 10% formalin, embedded in paraffin, cut
into 5 mm sections and blocked with Dako antibody diluent for 1
hour. When using mouse or rat derived monoclonal antibody, we
also incubated the sections with Rodent Block M or R blocking for
30 min. To detectGFP and troponinI double positive cells, sections
were stained with anti-troponin I (Abcam) and rabbit anti-GFP
(Invitrogen) overnight at 4uC. Alexa Fluor 546 goat anti mouse IgG
and Alexa Fluor 660 goat anti rabbit were used as secondary
antibodies (Invitrogen). Detection of GFP and CD31 double
positive cells were stained with rat anti-CD31 (Biocare Medical)
and Alexa Fluor goat anti rat 546 were used secondary antibodies.
GFP and a-SMA double positive cells were stained with mouse anti
a-SMA and without prior antigen retrieval but otherwise followed
the steps described above. The slides were mounted with ProLong
Gold antifade reagent with DAPI and viewed with a Nikon E800
fluorescence microscope using Openlab software.
In order to assess vascular density in the hearts, the sections
from mid-ventricular level were stained by antibodies of rat anti-
CD31 and mouse anti- a-SMA at room temperature for 1–
2 hours. A CD31 signal was detected using a Rat on Mouse HRP-
Polymer kit (Biocare) and 3,39Diaminobenzidine (DAB) (Biocare).
a-SMA signal was detected by a MM AP-Polymer kit (Biocare)
and a Vulcan Fast Red Chromogen kit (Biocare) for color
development. The slides were mounted and observed as described
above. ImagePro Plus 6.0 software (MediaCybernetics, Bethesda,
MD) was used to analyze the percentage area occupied by CD31
positive vessels. The number of arterioles, defined as vessels with
CD31+endothelial cells surrounded by a-SMA+smooth muscle
cells, per HPF in each region was counted .
For Isl1 staining, the hearts were perfused and fixed with 4%
paraformaldehyde overnight, equilibrated with 20–30% sucrose
and frozen in OCT for tissue sectioning using a cryostat. The
sections were blocked with Rodent Block M and Dako antibody
diluent for 30 min respectively, stained with mouse anti-Isl-1
(39.4D5) overnight at 4uC and Alexa Fluor 546 goat anti mouse
IgG used as secondary antibody.
TUNEL staining was performed with ApopTagH Plus Perox-
idase In Situ Apoptosis Detection Kit (Chemicon, Temecula, CA)
according to manufacture’s protocol and DAB was used for color
development. For co-staining troponin I, the sections from mid-
ventricular level were treated with denature solution (Biocare),
blocked with Rodent Block M and then incubated with mouse
anti-troponin I. The mouse-on-mouse alkaline phosphatase
polymer (Biocare) was used as secondary antibody. Vulcan Fast
Red Chromogen kit was used for color development. Finally the
sections were counterstained with hematoxylin. TUNEL-positive
cardiomyocytes were defined by the presence of both DAB nuclear
staining and completely surrounded by troponin I staining.
In order to assess the size of infarct scar, the sections from mid-
ventricular level (mid-papillary) were stained by picosirius red. The
scar was stained as dark red. The slides were mounted and viewed
same as above. All histological sections were examined with a
Nikon Eclipse E800 microscope using a 1x objective with the use
of Openlab software (Improvision, Lexington, MA). To assess the
circumferential extent of the infarct, the epicardial and endocar-
dial infarct lengths, epicardial and endocardial circumferences of
LV were traced manually using the ImagePro Plus 6.0 software.
Epicardial infarct ratio was obtained by dividing the epicardial
infarct length by the epicardial circumference of LV. Endocardial
infarct ratio was calculated by dividing the endocardial infarct
length by the endocardial circumference of LV. The circumfer-
ential extent of the infarct scar was calculated as [(epicardial
infarct ratio + endocardial infarct ratio)/2]6100.
Echocardiography was accomplished under isoflurane anesthe-
sia with the use of a Vevo-660 (VisualSonic, Toronto) equipped
with a 30 MHz transducer. Echocardiograms were obtained at
baseline, 2 days post-MI (before injection), and day 28 post-MI.
We measured LVEF and wall thickness. Wall thickness was
measured at the apical anterior wall (infarct wall thickness) and at
the mid-anterior segment (peri-infarct wall thickness) separately on
the parasternal long-axis view; posterior wall thickness was
obtained at the papillary muscle level. Three cycles were measured
for each assessment and average values were obtained [29,33].
Echocardiograms were analyzed by a blinded reviewer.
difference among multiple groups. Student’s t test was used to analyze
differences between two groups. Values were expressed as mean6SD
unless otherwise specified, with P,0.05 considered significant. SPSS
15.0 software was used to conduct all statistical analysis.
one day after placing into culture. (B) CS-forming cells are seen as
small, round, phase-bright cells arising from the fibroblast-like
monolayer around the attached explant after 14 days. (C) CSs
appear 3 days after the CS-forming cells are re-plated in separate
wells. Typical results are shown (N=12). Scale bar=200 mm.
CS culture. (A) Typical explants of cardiac tissue,
from mouse hearts. (A) Flow cytometric analysis of Sca-1,
CD45 and c-kit expression in disaggregated CS cells (N=5). (B)
Bar graph showing the profile of progenitor cell markers in CSs by
FACS (N=5). Data are shown as mean6SEM.
Cellular composition of CS-forming cells
Different cardiac regions generated similar number of CSs per
milligram of tissue at 1 week post-MI. LV, left ventricle excluding
scar; RV, right ventricle; LA, left atrium and RA, right atrium.
Data are shown as mean6SEM (N=4).
Isl1+cells in post-MI heart. After 7 days post-MI,
Isl1+cells were detected in epicardium at the border of infarct
region by immunohistochemical staining in 9 month old mice at
CSs generated from different cardiac regions.
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PLoS ONE | www.plosone.org13January 2012 | Volume 7 | Issue 1 | e30329
lower power (scale bar=35 mm) (A) and higher power (scale
bar=100 mm) (B) (N=3).
Sca-1+CD45-cells in CSs from chimeric
mouse are GFP negative. Flow cytometric analysis GFP+cells
in peripheral blood mononuclear cells in wild type mouse (A1) and in
chimeric mouse 5 months post-transplantation (A2). FACS showed
CD45+cells in CSs from wild type mouse (B1) and CD45+GFP+cells
in CSs from chimeric mouse (B2). FACS showed Sca-1+CD45-cells in
CSs from chimeric mouse were GFP negative, whereas Sca-1+CD45+
cells were GFP positive (C). Typical results are shown (N=4).
injected cells in infarcted hearts. Real-time RT-PCR
(Taqman) was used to compare GFP expression in hearts injected
with cloned Sca-1+CD45-GFP+cells. The injected hearts were
harvested at 1 hour and 1, 3, 7, 14 and 25 days post-injection.
Results show as GFP mRNA expression relative to histone 3.3A.
The expression level of GFP in the heart collected 1 hour post-
injection was used to represent 100% of injected cells. H: hour; D:
day. Typical results are shown (N=3).
The level of engraftment and persistence of
ocyte apoptosis. Typical image showed TUNEL+/Troponin I+
cells (black arrow) (A). Sca-1+cell injection resulted in a
significant reduction of TUNEL+/Troponin I+cells in the peri-
infarct zone (PZ), but not in the infarct zone (IZ) and remote zone
(RZ) compared to the control group 25 days post-injection (B).
TnI, troponin I; TUNEL, terminal deoxynucleotidyl transferase
dUTP nick end labeling; CM, cardiomyocyte; LPF, low power
field (20 x magnification); Data are shown as mean6SEM
Injected Sca-1+CD45-cells reduce cardiomy-
We thank Drs. Zhien Wang, Junya Takagawa, Muhammad Khan, Yagai
Yang, Meenakshi Gaur and Mr. Brian Lee for technical input and
Conceived and designed the experiments: JY AB WG HSB YY. Performed
the experiments: Jy H. Shih RES Y. Zhang Y. Zhou. Analyzed the data: JY
AB HSB YY. Wrote the paper: JY AB HSB YY.
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