J. of Cardiovasc. Trans. Res.

(2011) 4:154160
DOI 10.1007/s12265-010-9245-z

Adult Stem Cells: From New Cell Sources to Changes

in Methodology
Beatriz Pelacho & Manuel Mazo & Juan Jose Gavira &
Felipe Prsper

Received: 18 October 2010 / Accepted: 15 November 2010 / Published online: 2 December 2010
# Springer Science+Business Media, LLC 2010

Abstract Cardiovascular diseases constitute the first cause

of mortality and morbidity worldwide. Alternative treatments like transplantation of (stem) cell populations derived
from several adult tissue sources, like the bone marrow,
skeletal muscle, or even adipose tissue, have been already
employed in diverse clinical trials. Results from these
studies and previous animal studies have reached to the
conclusion that stem cells induce a benefit in the treated
hearts, which is exerted mainly through paracrine mechanisms and not through direct differentiation as it was
initially expected. However, a strong technical limitation
for the stem cell therapy, which is the low level of cell
survival and engraftment, diminishes their potential. Thus,
new strategies like combination of the cells with bioengineering techniques have been developed and are being
subject of intense research, suggesting that new strategies
may improve the efficacy of these therapies. In this review,
we will discuss the different therapeutic approaches, drawbacks, and future expectations of new regenerative therapies for cardiovascular diseases.

Introduction
Cardiovascular diseases constitute the first cause of mortality
and morbidity worldwide [1], leading the group is the
myocardial infarction, which causes more than 10% of the
deaths [2]. Fortunately, medical advances at the interventional, pharmacological, and surgical levels have significantly
decreased the rate of mortality at the acute stage of the
infarct. However, irreversible loss of the cardiac tissue
predisposes the heart to arrhythmias, which can derive in
ventricular fibrillation and cardiac failure. In view of the
palliative rather than curative effect of the traditional treatments, new alternative therapies have been assayed for more
than a decade already, including gene (reviewed in [3, 4]),
protein (reviewed in [5, 6]), and stem cell (SC; reviewed in
[7, 8]) therapies. An overview of the results obtained from all
the intensive work performed until now, conclusions from it,
and future studies direction will be discussed in this review.

Stem Cell Therapy: What Have we Learned?

Keywords Cardiovascular diseases . Stem cell therapy .
Trophic mechanisms . Cardiac patches . Bioartificial hearts
B. Pelacho : M. Mazo : F. Prsper (*)
Hematology and Cell Therapy and Foundation for Applied
Medical Research, Division of Cancer,
Clnica Universidad de Navarra, University of Navarra,
Av. Po XII 36,
Pamplona 31008 Navarra, Spain
e-mail: fprosper@unav.es
J. J. Gavira
Department of Cardiology and Cardiovascular Surgery,
Clnica Universidad de Navarra, University of Navarra,
Av. Po XII 36,
Pamplona 31008 Navarra, Spain

Probably, the most intensive work among the new therapies

assayed for the treatment of the cardiac diseases has been
performed with stem cells, which, once shown their pluri/
multipotential capacity and putative cardiovascular differentiation capacity, were considered as the optimal option
for regenerating the damaged myocardium. Thus, different
cell types derived from a wide variety of tissue sources
have been tested, going from the most undifferentiated ones
like the embryonic stem cells (and most recently discovered,
the induced pluripotent stem cells (iPS)) to more differentiated
ones derived from different adult tissues like the bone
marrow, the cord blood, the skeletal and cardiac muscle, or
the adipose tissue.

J. of Cardiovasc. Trans. Res. (2011) 4:154160

An impressive number of animal studies and clinical

trials have already been performed to determine the
therapeutic potential of stem cells [7, 8]. However, although
initial expectancies are relayed in the regeneration of the
myocardium through cardiovascular differentiation of the
transplanted cells, it is now accepted that despite the
reported ability of some adult stem cell populations to in
vivo differentiate and directly contribute to the cardiovascular lineages of the heart (mainly to the vascular lineages),
this contribution is rather low and is not responsible for the
functional improvement observed in the treated hearts. In
view of this aspect and the fact that a relatively similar
positive effect was induced independently of the transplanted cell type, a trophic effect was proposed as the main
mechanism responsible for cardiac repair. This hypothesis
has been reinforced by the fact that injection of conditioned
media recovered from cultured SC can also provoke a
benefit in the injected hearts [9, 10].
The Paracrine Effect of Stem Cell Therapy
Therefore, transplanted cells secrete cytokines and factors that
intervene at different levels in the protection and regeneration
of the heart, like the induction of tissue revascularization, cell
proliferation and survival, positive induction of extracellular
matrix composition changes, and cardiac contractility and
metabolism effects (reviewed in [11]).
Starting with the probably most widely tested cell
source, the bone marrow, several studies have demonstrated
that these cells (specially the mesenchymal cells (MSC))
secrete a broad spectrum of cytoprotective molecules like
VEGF, bFGF, IGF-1, SDF1, PDGF, IL1b, IGF-1, HGF, or
TB4, among others [12, 13], contributing to diminish the
apoptosis degree and infarct size of the damaged hearts.
Moreover, secretion of some of these molecules occurs to a
greater extent when cells are exposed to hypoxia, potentiating in that way their effect when cells are transplanted
into the ischemic heart.
Together with this protective effect, it has been also
widely analyzed the vasculogenic potential of the transplanted cells mediated by differentiation-independent
mechanisms, significantly contributing to tissue revascularization. Secretion of molecules involved in the induction of
endothelial and smooth muscle cells migration, proliferation, vessel enlargement, and maturation like the VEGF,
bFGF, HGF, or angiopoietins have been demonstrated [9].
Thus, an increase in tissue reperfusion not only at the
capillary but most importantly at the collateral level, has
been determined [14]. Also, together with the BM-derived
stem cells, the adipose tissue and skeletal muscle derived
cells can induce strong angiogenic/arteriogenic effects [15].
On the other hand, cell therapy has been associated with
a positive remodeling of the heart that affects the

155

extracellular matrix composition. An anti-fibrotic effect

has been detected in the scar tissue, which presented lower
levels of the collagen types 1 and 3, the metalloproteinase
inhibitor TIMP1, or fibrotic inducer molecules like TGF. As
a consequence, treated hearts presented a lower LV dilatation
and greater myocardial thickness. Also, some studies have
determined some of the molecules involved in these processes, secreted for example by the mesenchymal cells, which
includes metalloproteinases, serine-proteases, or serineprotease inhibitors [16]. Finally, although a clear in vivo
demonstration has not been shown, in vitro data suggests the
activation of cardiac progenitor cells through stem cell trophic
effect. Thus, release of HGF and IGF-1 by MSC could induce
migration, proliferation, and differentiation of cardiac stem
cells [17], helping to regenerate the myocardium.
In summary, it is clear that despite the relative low
differentiation capacity of the adult stem cells in vivo (at
least with the actual techniques of implantation), they
present an important therapeutic potential through the
secretion of active molecules responsible for many positive
actions in the damaged heart. The identification of the
factors and the molecular pathways involved would greatly
help to understand the mechanisms of action of the adult
stem cells in the ischemic heart allowing potentiation of the
actual therapeutic treatments for the damaged heart.
Stem Cell Tissue Contribution: Looking for the Ideal
Cardiac Progenitor
As we have just discussed, the in vivo differentiation
potential of the previously described adult stem cells is quite
limited, able to contribute mostly to vascular tissue but not to
provide cardiac tissue. Few population posses a robust
cardiomyogenic potential, namely embryonic stem cells
(ES), iPS, and the cardiac progenitors present in the heart.
Discovered over 20 years ago [18], embryonic stem cells
have demonstrated a strong differentiation capacity towards
any cell type derived from each of the three germ layers,
including cardiomyocytes. Their myogenic potential has
been demonstrated both in vitro by embryoid body
formation, and in vivo, after injection into the myocardium
[1922]. However, it has been shown that when undifferentiated human ES are injected into the hind limb, they give
rise to the same proportion of cardiomyocytes than when
they are injected in the heart, indicating that the myocardium does not guide the ES cardiac differentiation [22].
This fact, together with the tumorigenic potential of the ES,
has directed the studies towards new approaches where
injected cells were previously differentiated and selected in
vitro. Selection of genetic modified cells expressing
fluorescent reporters or antibiotic resistance genes controlled under a cardiac-specific promoter [23, 24], allowed
to obtain almost pure populations of cardiomyocytes that

156

did not induce tumor formation when injected in vivo.

However, despite the efficiency of the techniques, these
approaches are not really feasible in the clinic. Recently, the
groups of Dr. Terzic and Dr. Murry have demonstrated the
isolation and characterization of a pure cardiac population,
obtained by guiding ES cardiac specification with a specific
cytokine treatment (TNF- or Activin-A plus BMP4
combination, respectively). Once transplanted in the infarcted heart, these cardiac ES-derived cells were able to
partially regenerate the muscle with no tumor formation
[25, 26]. However, despite the positive results, it would be
necessary to perform exhaustive and longer-term studies in
order to assure the safety and also benefit of the transplanted cells. Other major issues like the ethical concerns of
their use and the immunological rejection are still subject of
debate and study.
On the other hand, the recent described capacity of
genetically reprogrammed somatic cells towards pluripotent
ones (termed induced pluripotent stem cells) could bypass
these last issues by allowing the generation of autologous cells
from the diseased patient. This new pluripotent cell source
initially obtained by overexpression of the pluripotent genes
Klf-4, Oct4, Sox2, and cMyc [2729] has opened great
expectations not only for cardiac regeneration but also for
many other diseases. Importantly, iPS derived from different
species, including human, have demonstrated cardiac potential [30, 31] with similar efficiency than the ES cells to
differentiate functional cardiomyocytes with nodal-, atrial-,
and ventricular-like electrophysiological phenotypes [32].
Still, similarly with the ES cells, the tumorigenic aspect has
to be strictly controlled before any clinical use can be
foreseen. Other aspects like the removal of genomeintegrating viruses from the iPS generation protocol need to
be achieved. In general, many open questions still remain to
be answered for this newly discovered cell population,
allowing a better understanding of the epigenetic mechanisms involved in the reprogramming of the cells.
An alternative approach is the direct generation of
lineage-committed cells by transdifferentiation of mesodermal/adult cells. Transdifferentiation of murine mesoderm
towards beating cardiomyocytes has been already demonstrated by transfection with two cardiac transcription
factors: Gata4 and Tbx5 and the cardiac-specific subunit
of the BAF-chromatin-remodeling complexes, Baf60c [33].
Also, very recently, the group of Dr. Srivastava has shown
that the transdifferentiation of postnatal cardiac and dermal
fibroblasts into cardiomyocytes could be achieved by
overexpressing the transcription factors Gata4, Mef2c, and
Tbx5 [34]. These cardiomyocyte-like cells contracted
spontaneously, expressed specific cardiac markers, and
presented a cardiac gene profile. Strikingly, when 1-day
transfected fibroblasts were transplanted into the heart, they
also rapidly differentiated towards cardiac cells. These new

J. of Cardiovasc. Trans. Res. (2011) 4:154160

data suggests new strategies for the generation of cardiomyocytes, avoiding the previous generation of iPS, and in
that way, their tumor capacity.
Finally, it has been shown that despite the traditional
dogma regarding the lack of endogenous renewal capacity of
the heart, this organ possess an intrinsic regenerative potential,
which depends on the presence of cardiac progenitors
(reviewed in [35]). Importantly, these progenitors that are
localized in small clusters at the interstitium of the heart can
be isolated, grown, and in vitro differentiated towards mature
cardiomyocytes and also, vascular cells. Moreover, an
increase of the CPC pool of the heart has been demonstrated
after acute myocardial infarction [36] and also, their
cardiovascular contribution and improvement of the cardiac
function when transplanted into the ischemic heart [37].
This adult and autologous population without tumorigenic risk could represent an ideal source; however, aspects like
the origin or phenotypical characterization of this stem cell
population still need to be unraveled as recent controversial
results suggest that the capacity to generate new cardiomyocytes is not due to true cardiac progenitor cells but rather to
the potential of cardiomyocytes to de-differentiate, proliferate, and differentiate into new cardiac cells [38]. It is also
somehow confusing the present description of the cardiac
cell populations, which have been characterized by the lack
of expression of some markers whose expression just
defines other CPC population. Thus, for example, the first
reported cardiac progenitors were defined as a Sca-1c-Kit+
population [37], whereas almost simultaneously, another
study showed the existence of a Sca-1+c-Kit cell population in the heart [39]. Other groups have shown also the
existence of Sca-1+ CPC populations in the heart [40, 41]
being some of them also defined by the expression of the
transporter protein Abcg-2 [42].
The ability of some murine and human heart-derived
cells to form clusters in vitro when cultured in suspension
(named cardiospheres) has also been demonstrated [43].
These clusters contain clonally derived cells, which
organize in a core composed by proliferating c-Kit positive
cells and a surrounding layer of spontaneously differentiated cells that express markers characteristic of cardiac,
endothelial, and mesenchymal cells. Their transplantation
into immunosuppressed infarcted mice improved cardiac
function [44]. Finally, a new population isolated from the
embryo and adult atrium of the heart characterized by the
expression of the transcription factor Islet-1, but negative
for the c-Kit or the Sca1 markers, has been described [45].
As well as the other cell populations, these cells possess
self-renewing, clonogenic, and multipotent abilities, including cardiac differentiation potential.
In view of all this, it is of great relevance to better
characterize these progenitors in order to be able to isolate
them in a reproducible and consistent manner. It is

J. of Cardiovasc. Trans. Res. (2011) 4:154160

intriguing if all these populations corresponds to a unique

one at different differentiation stages or if there are several
populations from different origin.

Main Limitations of SC Therapy and New Approaches

Despite the initial hope, the clinical results using stem cells
in patients with myocardial infarction have not achieved the
expected impact. However, common alternative mechanisms of action based on their paracrine effects has been
demonstrated, which has allowed to give a step further
towards therapeutically using them. On the other hand,
some important technical limitations have been evidenced,
like the low degree of cell engraftment and survival into the
heart. As an average, more than 70% of the cells die during
the first 48 h after injection, being progressively lost during
the following days [46] due to the hypoxic, inflammatory,
and/or fibrotic environment. It is however remarkable that
despite the low numbers of engrafted cells and their short
time presence into the tissue, a functional improvement has
been generally demonstrated after cell treatment. Therefore,
it has been hypothesized that an increase in the survival rate
of the cells would improve their positive effects by
reinforcing their trophic effect or even through their
differentiation. Different approaches have been tested in
order to favor cell retention that are discussed next.
Initial experiments were performed by combining the
cells with injectable biomaterials like collagen, fibrin, or
gelatin. Also, matrigel or other factors that provided a
favorable environment rich in cytokines and growth factors
were tested. In general, these early studies showed an
increased survival rate of the transplanted cells and
consequently, a greater improvement of the cardiac function
of the treated hearts [47, 48]. These approaches, however,
were still limited and did not assure complete cell retention
or an adequate distribution of the cells. Techniques, like the
creation of cell sheets and patches and microtissues, are
being now developed in order to allow, together with a
greater cell survival, a more homogeneous and organized
distribution of the cells (reviewed in [49]).
Cell Patches and Sheets
The creation of cellular patches has been developed by using
different materials characterized by their biocompatibility
and biodegradability. These biomaterials act as a delivery
platform for the cells, assuring their engraftment and
interaction with the tissue. Two types of materials have been
mainly tested: hydrogel/extracellular matrix (ECM)-based
matrices or porous biomaterials.
Regarding the first ones, the cells are usually embedded
in soluble hydrogels matrices that can condensate after

157

temperature changes, forming in that way a cellularized

patch that can be applied to the heart pericardium. It has
been shown that creation of a patch with MSC entrapped in
a collagen-I matrix and applied to rat infarcted hearts
induces an increase of cell engraftment and consequently, a
functional improvement [50]. Thanks to this relatively
simple approach, the trophic effect exerted by the MSC is
potentiated by increasing their survival and engraftment in the
tissue. However, although this method significantly improves
the previous ones, it is obvious that the degree of cell
distribution has to be improved. More sophisticated
approaches have been performed by the group of Dr.
Zimmermann that created 3D contractile loops of mixed
collagen and neonatal cardiomyocytes that when applied to
the damaged heart, could support its contractile function [51].
On the other hand, porous biomaterials have also been
tested as cell scaffolds. Fabricated from different materials,
such as alginate or polymers like the poly-glycolide-colactide, can be easily seeded with cells and transplanted into
the pericardium. Application of adult stem cells or
cardiomyocytes of fetal origin or derived from ES cells
has been reported, observing an improvement in heart
remodeling and function after transplantation [52, 53].
Interestingly, it has been shown that combination of these
scaffolds with ECM components increases cell retention.
Despite these advances, their use still presents some
drawbacks like the lack of control for a homogenous
seeding and distribution of the cells. In order to solve this
problem, new strategies like microtemplating or electrospinning have been incorporated in order to create scaffolds
that mimic the natural heart extracellular matrix. A recent
study has reported the creation of a poly(2-hydroxyethyl
methacrylate-co-methacrylic acid) hydrogel construct organized as parallel channels that can direct an aligned
cardiomyocyte distribution. Moreover, the presence of
micrometer-sized, spherical, interconnected pores also
enhances angiogenesis while reducing scarring after transplantation into the heart [54].
Another approach, originally developed by the group of
Dr. Okano, is the creation of cell sheets without scaffold
support. In that way, putative immunogenic and inflammatory reactions against the biomaterials that constitute the
scaffolds would be avoided. This technique has been
possible by using thermo-responsive dishes that allow the
detachment of the cells once they have grown until
confluence. Also, creation of several cell layers constructs
is possible just by adhesion of one cell sheet to another. The
beneficial potential of this technique has been demonstrated
by the transplant of a monolayer of ADSC cells in the
infarcted heart [55] and later on, a three-layer-sheet
composed of cardiac and endothelial cells [56]. In both
cases, an increase in tissue revascularization together with a
positive heart remodeling responsible for the improvement

158

in cardiac function has been demonstrated. Furthermore, it

has been recently shown that transplantation of in vitro
created 3D cardiospheres improves engraftment of the
cardiac progenitors and the in vivo differentiation towards
cardiac and vascular cells [57].
Overall, an important aspect to take into account
regarding the creation of cell patches is their vascularization, which will determine the thickness of the
constructs without the limitation of cell apoptosis by
hypoxia. Interestingly, the group of Dr. Murry has
created a scaffold-free cardiac tissue patch by growing
hES-derived cardiomyocyte mixed with umbilical cord
endothelial cells and embryonic fibroblasts. It has been
shown that these cardiac patches can actively contract
and be electrically paced, but also importantly, engraft
and form vascularized human myocardium when
implanted into the mice hearts [58].
Finally, the use of decellularized tissues as scaffold for
cell transplantation has also been explored. Different tissues
like the porcine intestine submucose [59], bovine pericardium [60], and omental wrapping [61] have been used as a
support for different cell types like the mesenchymal cells,
with the aim to improve their paracrine effect. Moreover,
folding of these sheets allows reinforcing their effect in the
myocardium. An increase in cardiac revascularization and
an improvement in heart function have been shown after
transplantation.
Bioartificial Hearts
The idea of replacing a sick area of the heart by using a
cardiac patch could be useful when relatively small areas of
the heart are affected; however, when the heart has become
nonfunctional and organ transplantation is required, this
option would not be sufficient. In these cases, availability
of bioartificial hearts would be ideal, avoiding patient
waiting lists for organ transplantation or immune suppression. Also, problems associated with mechanical hearts
like, e.g., thromboembolism formation would be avoided.
The idea of creating a new heart is not new, being more
than a decade since this was considered as an option [62].
Recently, the possibility of an in vitro heart reconstruction by using decellularized cadaveric hearts has been
demonstrated. In that way, keeping the heart skeleton as a
mold where the extracellular matrix has been preserved, it
is possible to re-vascularize and re-muscularize the new
heart. For that, endothelial, cardiac, and fibroblastic cells
are perfused by using a bioreactor, which provides a pulsate
flow and pacing, mimicking the physiological conditions.
Strikingly, spontaneous contraction of the newly formed
heart has been shown [63]. Of course, many parameters still
need to be controlled, being for example mandatory to
possess a cardiac cell source either by optimizing the

J. of Cardiovasc. Trans. Res. (2011) 4:154160

isolation and expansion of cardiac progenitors, through

differentiation of pluripotent cells followed by the isolation
of cardiomyocytes,or by trans-differentiating other cells to a
myogenic phenotype. Issues like prevention of fatal
arrythmias would need to be carefully analyzed in order
to advance in the development of these bioartificial organs
before their clinical application can be realistically contemplated. In any case, demonstration of the possibility to
create these organs represents a great step forward for the
treatment of cardiac diseases and in fact, for many other
diseases, for which the technique can also be applied [64].

Conclusions
In summary, great advances have been accomplished by
combining the stem cells with the bioengeneering, which
on one hand, has improved the paracrine effect exerted by
the cells and on the other hand, found new ways for the
creation of physiological cardiac patches and even new
bioartificial organs. Still, hurdles like a real availability of
cardiac cell sources with no tumoral or immunological risks
and the ability to create patches/organs that can mimic the
structure and function of the heart without fatal arrythmias
development, are issues that will need a thorough and long
way before they can be readily applied in the clinic
Acknowledgments This work was supported in part by ISCIII
PI050168, PI070474, and CP09/00333 and ISCIII-RETIC RD06/
0014, MICCIN PLE2009-0116, and PSE SINBAD, Gobierno de
Navarra (Departamento de Educacin), Comunidad de Trabajo de los
Pirineos (CTP), European Union Framework Project VII (INELPY),
Caja de Ahorros de Navarra (Programa Tu Eliges: Tu Decides), and
the UTE project CIMA.

References
1. Rosamond, W., Flegal, K., Furie, K., Go, A., Greenlund, K.,
Haase, N., et al. (2008). Heart disease and stroke statistics2008
update: a report from the American Heart Association Statistics
Committee and Stroke Statistics Subcommittee. Circulation, 117
(4), e25146. doi:10.1161/CIRCULATIONAHA.107.187998.
2. Organization WH (2004) The world health report 2004.
3. Gaffney, M. M., Hynes, S. O., Barry, F., & OBrien, T. (2007).
Cardiovascular gene therapy: current status and therapeutic
potential. British Journal of Pharmacology, 152(2), 175188.
doi:10.1038/sj.bjp.0707315.
4. Rissanen, T. T., & Yla-Herttuala, S. (2007). Current status of
cardiovascular gene therapy. Molecular Therapy, 15(7), 1233
1247. doi:10.1038/sj.mt.6300175.
5. Shah, P. B., & Losordo, D. W. (2005). Non-viral vectors for gene
therapy: clinical trials in cardiovascular disease. Advances in
Genetics, 54, 339361. doi:10.1016/S0065-2660(05)54014-8.
6. Maulik, N., & Thirunavukkarasu, M. (2008). Growth factors and cell
therapy in myocardial regeneration. Journal of Molecular and
Cellular Cardiology, 44(2), 219227. doi:10.1016/j.yjmcc.
2007.11.012.

J. of Cardiovasc. Trans. Res. (2011) 4:154160

7. Passier, R., van Laake, L. W., & Mummery, C. L. (2008). Stemcell-based therapy and lessons from the heart. Nature, 453(7193),
322329. doi:10.1038/nature07040.
8. Segers, V. F., & Lee, R. T. (2008). Stem-cell therapy for cardiac
disease. Nature, 451(7181), 937942. doi:10.1038/nature06800.
9. Kinnaird, T., Stabile, E., Burnett, M. S., Lee, C. W., Barr, S.,
Fuchs, S., et al. (2004). Marrow-derived stromal cells express
genes encoding a broad spectrum of arteriogenic cytokines and
promote in vitro and in vivo arteriogenesis through paracrine
mechanisms. Circulation Research, 94(5), 678685.
10. Gnecchi, M., He, H., Noiseux, N., Liang, O. D., Zhang, L.,
Morello, F., et al. (2006). Evidence supporting paracrine hypothesis
for Akt-modified mesenchymal stem cell-mediated cardiac
protection and functional improvement. The FASEB Journal, 20
(6), 661669.
11. Mirotsou M, Jayawardena TM, Schmeckpeper J, Gnecchi M, Dzau
VJ Paracrine mechanisms of stem cell reparative and regenerative
actions in the heart. J Mol Cell Cardiol. doi:10.1016/j.yjmcc.
2010.08.005
12. Uemura, R., Xu, M., Ahmad, N., & Ashraf, M. (2006). Bone
marrow stem cells prevent left ventricular remodeling of ischemic
heart through paracrine signaling. Circulation Research, 98(11),
14141421.
13. Xu, M., Uemura, R., Dai, Y., Wang, Y., Pasha, Z., & Ashraf, M.
(2007). In vitro and in vivo effects of bone marrow stem cells on
cardiac structure and function. Journal of Molecular and Cellular
Cardiology, 42(2), 441448. doi:10.1016/j.yjmcc.2006.10.009.
14. Kinnaird, T., Stabile, E., Burnett, M. S., Shou, M., Lee, C. W., Barr, S.,
et al. (2004). Local delivery of marrow-derived stromal cells augments
collateral perfusion through paracrine mechanisms. Circulation, 109
(12), 15431549. doi:10.1161/01.CIR.0000124062.31102.57.
15. Perez-Ilzarbe, M., Agbulut, O., Pelacho, B., Ciorba, C., San JoseEneriz, E., Desnos, M., et al. (2008). Characterization of the
paracrine effects of human skeletal myoblasts transplanted in
infarcted myocardium. European Journal of Heart Failure, 10
(11), 10651072. doi:10.1016/j.ejheart.2008.08.002.
16. Ohnishi, S., Yasuda, T., Kitamura, S., & Nagaya, N. (2007). Effect
of hypoxia on gene expression of bone marrow-derived mesenchymal stem cells and mononuclear cells. Stem Cells, 25(5),
11661177. doi:10.1634/stemcells.2006-0347.
17. Linke, A., Muller, P., Nurzynska, D., Casarsa, C., Torella, D.,
Nascimbene, A., et al. (2005). Stem cells in the dog heart are selfrenewing, clonogenic, and multipotent and regenerate infarcted
myocardium, improving cardiac function. Proceedings of the
National Academy of Sciences of the United States of America,
102(25), 89668971.
18. Evans, M. J., & Kaufman, M. H. (1981). Establishment in culture
of pluripotential cells from mouse embryos. Nature, 292(5819),
154156.
19. Kehat, I., Kenyagin-Karsenti, D., Snir, M., Segev, H., Amit, M.,
Gepstein, A., et al. (2001). Human embryonic stem cells can
differentiate into myocytes with structural and functional properties of cardiomyocytes. Journal of Clinical Investigation, 108(3),
407414.
20. Behfar, A., Faustino, R. S., Arrell, D. K., Dzeja, P. P., PerezTerzic, C., & Terzic, A. (2008). Guided stem cell cardiopoiesis:
discovery and translation. Journal of Molecular and Cellular
Cardiology, 45(4), 523529. doi:10.1016/j.yjmcc.2008.09.122.
21. Beqqali, A., van Eldik, W., Mummery, C., & Passier, R. (2009).
Human stem cells as a model for cardiac differentiation and
disease. Cellular and Molecular Life Sciences, 66(5), 800813.
doi:10.1007/s00018-009-8476-0.
22. Nussbaum, J., Minami, E., Laflamme, M. A., Virag, J. A., Ware,
C. B., Masino, A., et al. (2007). Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation
and immune response. The FASEB Journal, 21(7), 13451357.

159
23. Huber, I., Itzhaki, I., Caspi, O., Arbel, G., Tzukerman, M.,
Gepstein, A., et al. (2007). Identification and selection of
cardiomyocytes during human embryonic stem cell differentiation. The FASEB Journal, 21(10), 25512563.
24. Schroeder, M., Niebruegge, S., Werner, A., Willbold, E., Burg,
M., Ruediger, M., et al. (2005). Differentiation and lineage
selection of mouse embryonic stem cells in a stirred bench scale
bioreactor with automated process control. Biotechnology and
Bioengineering, 92(7), 920933.
25. Behfar, A., Perez-Terzic, C., Faustino, R. S., Arrell, D. K.,
Hodgson, D. M., Yamada, S., et al. (2007). Cardiopoietic
programming of embryonic stem cells for tumor-free heart repair.
The Journal of Experimental Medicine, 204(2), 405420.
26. Laflamme, M. A., Chen, K. Y., Naumova, A. V., Muskheli, V.,
Fugate, J. A., Dupras, S. K., et al. (2007). Cardiomyocytes derived
from human embryonic stem cells in pro-survival factors enhance
function of infarcted rat hearts. Nature Biotechnology, 25(9),
10151024.
27. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T.,
Tomoda, K., et al. (2007). Induction of pluripotent stem cells from
adult human fibroblasts by defined factors. Cell, 131(5), 861872.
doi:10.1016/j.cell.2007.11.019.
28. Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget,
J., Frane, J. L., Tian, S., et al. (2007). Induced pluripotent stem
cell lines derived from human somatic cells. Science, 318(5858),
19171920. doi:10.1126/science.1151526.
29. Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem
cells from mouse embryonic and adult fibroblast cultures by defined
factors. Cell, 126(4), 663676. doi:10.1016/j.cell.2006.07.024.
30. Martinez-Fernandez, A., Nelson, T. J., Yamada, S., Reyes, S.,
Alekseev, A. E., Perez-Terzic, C., et al. (2009). iPS programmed
without c-MYC yield proficient cardiogenesis for functional heart
chimerism. Circulation Research, 105(7), 648656. doi:10.1161/
CIRCRESAHA.109.203109.
31. Mauritz, C., Schwanke, K., Reppel, M., Neef, S., Katsirntaki, K.,
Maier, L. S., et al. (2008). Generation of functional murine cardiac
myocytes from induced pluripotent stem cells. Circulation, 118
(5), 507517. doi:10.1161/CIRCULATIONAHA.108.778795.
32. Zhang, J., Wilson, G. F., Soerens, A. G., Koonce, C. H., Yu, J.,
Palecek, S. P., et al. (2009). Functional cardiomyocytes derived
from human induced pluripotent stem cells. Circulation Research,
104(4), e3041. doi:10.1161/CIRCRESAHA.108.192237.
33. Takeuchi, J. K., & Bruneau, B. G. (2009). Directed transdifferentiation of mouse mesoderm to heart tissue by defined
factors. Nature, 459(7247), 708711. doi:10.1038/nature08039.
34. Ieda, M., Fu, J. D., Delgado-Olguin, P., Vedantham, V., Hayashi,
Y., Bruneau, B. G., et al. (2010). Direct reprogramming of
fibroblasts into functional cardiomyocytes by defined factors.
Cell, 142(3), 375386. doi:10.1016/j.cell.2010.07.002.
35. Martin-Puig, S., Wang, Z., & Chien, K. R. (2008). Lives of a heart
cell: tracing the origins of cardiac progenitors. Cell Stem Cell, 2
(4), 320331. doi:10.1016/j.stem.2008.03.010.
36. Urbanek, K., Torella, D., Sheikh, F., De Angelis, A., Nurzynska,
D., Silvestri, F., et al. (2005). Myocardial regeneration by
activation of multipotent cardiac stem cells in ischemic heart
failure. Proceedings of the National Academy of Sciences of the
United States of America, 102(24), 86928697.
37. Beltrami, A. P., Barlucchi, L., Torella, D., Baker, M., Limana, F.,
Chimenti, S., et al. (2003). Adult cardiac stem cells are multipotent
and support myocardial regeneration. Cell, 114(6), 763776.
38. Jopling, C., Sleep, E., Raya, M., Marti, M., Raya, A., &
Belmonte, J. C. (2010). Zebrafish heart regeneration occurs by
cardiomyocyte dedifferentiation and proliferation. Nature, 464
(7288), 606609. doi:10.1038/nature08899.
39. Oh, H., Bradfute, S. B., Gallardo, T. D., Nakamura, T., Gaussin,
V., Mishina, Y., et al. (2003). Cardiac progenitor cells from adult

160

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

J. of Cardiovasc. Trans. Res. (2011) 4:154160

myocardium: homing, differentiation, and fusion after infarction.
Proceedings of the National Academy of Sciences of the United
States of America, 100(21), 1231312318.
Oyama, T., Nagai, T., Wada, H., Naito, A. T., Matsuura, K.,
Iwanaga, K., et al. (2007). Cardiac side population cells have a
potential to migrate and differentiate into cardiomyocytes in vitro
and in vivo. The Journal of Cell Biology, 176(3), 329341.
Matsuura, K., Nagai, T., Nishigaki, N., Oyama, T., Nishi, J.,
Wada, H., et al. (2004). Adult cardiac Sca-1-positive cells
differentiate into beating cardiomyocytes. The Journal of Biological
Chemistry, 279(12), 1138411391.
Martin, C. M., Meeson, A. P., Robertson, S. M., Hawke, T. J.,
Richardson, J. A., Bates, S., et al. (2004). Persistent expression of
the ATP-binding cassette transporter, Abcg2, identifies cardiac SP
cells in the developing and adult heart. Developmental Biology,
265(1), 262275.
Messina, E., De Angelis, L., Frati, G., Morrone, S., Chimenti, S.,
Fiordaliso, F., et al. (2004). Isolation and expansion of adult
cardiac stem cells from human and murine heart. Circulation
Research, 95(9), 911921.
Smith, R. R., Barile, L., Cho, H. C., Leppo, M. K., Hare, J. M.,
Messina, E., et al. (2007). Regenerative potential of cardiospherederived cells expanded from percutaneous endomyocardial biopsy
specimens. Circulation, 115(7), 896908.
Laugwitz, K. L., Moretti, A., Lam, J., Gruber, P., Chen, Y., Woodard,
S., et al. (2005). Postnatal isl1+ cardioblasts enter fully differentiated
cardiomyocyte lineages. Nature, 433(7026), 647653.
Muller-Ehmsen, J., Whittaker, P., Kloner, R. A., Dow, J. S.,
Sakoda, T., Long, T. I., et al. (2002). Survival and development of
neonatal rat cardiomyocytes transplanted into adult myocardium.
Journal of Molecular and Cellular Cardiology, 34(2), 107116.
Cortes-Morichetti, M., Frati, G., Schussler, O., Duong Van Huyen,
J. P., Lauret, E., Genovese, J. A., et al. (2007). Association
between a cell-seeded collagen matrix and cellular cardiomyoplasty for myocardial support and regeneration. Tissue Engineering,
13(11), 26812687. doi:10.1089/ten.2006.0447.
Ryu, J. H., Kim, I. K., Cho, S. W., Cho, M. C., Hwang, K. K., Piao,
H., et al. (2005). Implantation of bone marrow mononuclear cells
using injectable fibrin matrix enhances neovascularization in
infarcted myocardium. Biomaterials, 26(3), 319326. doi:10.1016/
j.biomaterials.2004.02.058.
Christman, K. L., & Lee, R. J. (2006). Biomaterials for the treatment
of myocardial infarction. Journal of the American College of
Cardiology, 48(5), 907913. doi:10.1016/j.jacc.2006.06.005.
Simpson, D., Liu, H., Fan, T. H., Nerem, R., & Dudley, S. C., Jr.
(2007). A tissue engineering approach to progenitor cell delivery
results in significant cell engraftment and improved myocardial
remodeling. Stem Cells, 25(9), 23502357. doi:10.1634/stemcells.
2007-0132.
Zimmermann, W. H., Melnychenko, I., Wasmeier, G., Didie, M.,
Naito, H., Nixdorff, U., et al. (2006). Engineered heart tissue
grafts improve systolic and diastolic function in infarcted rat
hearts. Natural Medicines, 12(4), 452458. doi:10.1038/nm1394.

52. Caspi, O., Lesman, A., Basevitch, Y., Gepstein, A., Arbel, G., Habib,
I. H., et al. (2007). Tissue engineering of vascularized cardiac muscle
from human embryonic stem cells. Circulation Research, 100(2),
263272. doi:10.1161/01.RES.0000257776.05673.ff.
53. Leor, J., Aboulafia-Etzion, S., Dar, A., Shapiro, L., Barbash, I. M.,
Battler, A., et al. (2000). Bioengineered cardiac grafts: a new
approach to repair the infarcted myocardium? Circulation, 102(19
Suppl 3), III56III61.
54. Madden, L. R., Mortisen, D. J., Sussman, E. M., Dupras, S. K.,
Fugate, J. A., Cuy, J. L., et al. (2010). Proangiogenic scaffolds as
functional templates for cardiac tissue engineering. Proceedings of
the National Academy of Sciences of the United States of America,
107(34), 1521115216. doi:10.1073/pnas.1006442107.
55. Miyahara, Y., Nagaya, N., Kataoka, M., Yanagawa, B., Tanaka,
K., Hao, H., et al. (2006). Monolayered mesenchymal stem cells
repair scarred myocardium after myocardial infarction. Natural
Medicines, 12(4), 459465.
56. Shimizu, T., Yamato, M., Isoi, Y., Akutsu, T., Setomaru, T., Abe,
K., et al. (2002). Fabrication of pulsatile cardiac tissue grafts using
a novel 3-dimensional cell sheet manipulation technique and
temperature-responsive cell culture surfaces. Circulation Research, 90(3), e40.
57. Bartosh, T. J., Wang, Z., Rosales, A. A., Dimitrijevich, S. D., &
Roque, R. S. (2008). 3D-model of adult cardiac stem cells promotes
cardiac differentiation and resistance to oxidative stress. Journal of
Cellular Biochemistry, 105(2), 612623. doi:10.1002/jcb.21862.
58. Stevens, K. R., Pabon, L., Muskheli, V., & Murry, C. E. (2009).
Scaffold-free human cardiac tissue patch created from embryonic
stem cells. Tissue Engineering. Part A, 15(6), 12111222.
doi:10.1089/ten.tea.2008.0151.
59. Tan, M. Y., Zhi, W., Wei, R. Q., Huang, Y. C., Zhou, K. P., Tan, B.,
et al. (2009). Repair of infarcted myocardium using mesenchymal
stem cell seeded small intestinal submucosa in rabbits. Biomaterials, 30(19), 32343240. doi:10.1016/j.biomaterials.2009.02.013.
60. Wei, H. J., Chen, C. H., Lee, W. Y., Chiu, I., Hwang, S. M., Lin, W.
W., et al. (2008). Bioengineered cardiac patch constructed from
multilayered mesenchymal stem cells for myocardial repair. Biomaterials, 29(26), 35473556. doi:10.1016/j.biomaterials.2008.05.009.
61. Ueyama, K., Bing, G., Tabata, Y., Ozeki, M., Doi, K., Nishimura,
K., et al. (2004). Development of biologic coronary artery bypass
grafting in a rabbit model: revival of a classic concept with
modern biotechnology. J Thorac Cardiovasc Surg, 127(6), 1608
1615. doi:10.1016/j.jtcvs.2003.08.043.
62. Eschenhagen, T., Fink, C., Remmers, U., Scholz, H., Wattchow,
J., Weil, J., et al. (1997). Three-dimensional reconstitution of
embryonic cardiomyocytes in a collagen matrix: a new heart
muscle model system. The FASEB Journal, 11(8), 683694.
63. Ott, H. C., Matthiesen, T. S., Goh, S. K., Black, L. D., Kren, S.
M., Netoff, T. I., et al. (2008). Perfusion-decellularized matrix:
using natures platform to engineer a bioartificial heart. Natural
Medicines, 14(2), 213221. doi:10.1038/nm1684.
64. Atala, A. (2009). Engineering organs. Current Opinion in Biotechnology, 20(5), 575592. doi:10.1016/j.copbio.2009.10.003.