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Differentiation 84 (2012) 41–53
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Differentiation
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The arterial and cardiac epicardium in development, disease and repair
Adriana C. Gittenberger-de Groot a,b,n, Elizabeth M. Winter b, Margot M. Bartelings b, Marie Jose
Goumans c, Marco C. DeRuiter b, Robert E. Poelmann b
a
Department of Cardiology, Leiden University Medical Center, Postal zone: S-5-24, P.O. Box 9600, 2300 RC Leiden, The Netherlands
Department of Anatomy and Embryology, Leiden University Medical Center, Postal zone: S-1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands
c
Department of Molecular Cell Biology, Leiden University Medical Center, Postal zone: S-1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands
b
a r t i c l e i n f o
abstract
Available online 30 May 2012
The importance of the epicardium covering the heart and the intrapericardial part of the great arteries
has reached a new summit. It has evolved as a major cellular component with impact both in
development, disease and more recently also repair potential. The role of the epicardium in
development, its differentiation from a proepicardial organ at the venous pole (vPEO) and the
differentiation capacities of the vPEO initiating cardiac epicardium (cEP) into epicardium derived cells
(EPDCs) have been extensively described in recent reviews on growth and transcription factor
pathways. In short, the epicardium is the source of the interstitial, the annulus fibrosus and the
adventitial fibroblasts, and differentiates into the coronary arterial smooth muscle cells. Furthermore,
EPDCs induce growth of the compact myocardium and differentiation of the Purkinje fibers. This review
includes an arterial pole located PEO (aPEO) that provides the epicardium covering the intrapericardial
great vessels. In avian and mouse models disturbance of epicardial outgrowth and maturation leads to a
broad spectrum of cardiac anomalies with main focus on non-compaction of the myocardium, deficient
annulus fibrosis, valve malformations and coronary artery abnormalities. The discovery that in disease
both arterial and cardiac epicardium can again differentiate into EPDCs and thus reactivate its
embryonic program and potential has highly broadened the scope of research interest. This reactivation
is seen after myocardial infarction and also in aneurysm formation of the ascending aorta. Use of EPDCs
for cell therapy show their positive function in paracrine mediated repair processes which can be
additive when combined with the cardiac progenitor stem cells that probably share the same
embryonic origin with EPDCs. Research into the many cell-autonomous and cell–cell-based capacities
of the adult epicardium will open up new realistic therapeutic avenues.
& 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.
Keywords:
Cardiac development
Epicardium derived cells (EPDCs)
Pericardium
Myocardial infarction
Vascular development
Epithelial-to mesenchymal-transition
Contents
1.
2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Origin of the epicardium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Epicardium derived cells (EPDCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Heterogeneity and differentiation of the EPDCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.1.
The cardiac fibroblast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.2.
The endocardial cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.3.
The coronary endothelial cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.4.
The coronary smooth muscle cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.5.
The cardiomyocyte. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.6.
The Purkinje fiber. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Abbreviations: aPEO, arterial pro-epicardial organ; CMPCs, cardiomyocyte progenitor
cells; ECs, endothelial cells; vPEO, venous pro-epicardial organ; VSMCs, vascular
smooth muscle cells
n
Corresponding author. Department of Cardiology, Leiden University Medical
Center, Postal zone: S-5-24, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
Tel.: þ31 71 526 3704(9306); fax: þ 31 71 526 6809.
E-mail address: [email protected] (A.C. Gittenberger-de Groot).
0301-4681/$ - see front matter & 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.
Join the International Society for Differentiation (www.isdifferentiation.org)
http://dx.doi.org/10.1016/j.diff.2012.05.002
42
5.
6.
7.
A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53
The epicardium in congenital and adult cardiac disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.1.
Non-compaction cardiomyopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
5.2.
Endocardial fibroelastosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.3.
Cardiac conduction system anomalies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.4.
Valvulopathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
5.5.
Coronary vascular anomalies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Cardiovascular repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
6.1.
Myocardial infarction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
6.2.
Thoracic aortic aneurysm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
1. Introduction
The epicardium covering both the heart and the intrapericardial part of the great arteries is subject to an intense revival of
interest. Research on both morphological and functional characteristics including effective gene signaling pathways is booming as
shown by a 10-fold increase in publications on the topic proepicardium between 2000 and 2011 and 43-fold on the topic
epicardium development. Many reviews are currently available
(Bollini et al., 2011; Lie-Venema et al., 2007; Limana et al., 2011;
Manner et al., 2001; Martin-Puig et al., 2008; Olivey and Svensson,
2010; Perez-Pomares and Pompa, 2011; Riley and Smart, 2011;
Smart et al., 2009; Smart and Riley, 2012; Wessels and PerezPomares, 2004; Winter and Gittenberger-de Groot, 2007a), presenting generally accepted data, but also aspects that are still controversial. Statements on the controversial issues are intriguing and
trigger new research. In the current review the focus on the origin,
fate, disease and repair provides novel insights in the potential of
the epicardium. The epicardium cannot be regarded as a separate
entity and is incorporated both structurally and functionally in the
cardiac and vessel wall components. To support insight and structure of this review schematic Fig. 1 is instrumental.
2. Origin of the epicardium
The epicardium develops from the epithelium of the coelomic
wall in close interaction with the underlying splanchnic mesoderm. With the formation of the intra-embryonic coelomic cavity,
separating the intra-embryonic mesoderm into a splanchnic and a
somatic layer, the splanchnic mesoderm lining the endoderm of
the foregut develops into the bilateral cardiogenic plates. These
are the precursors of the myocardial primary heart tube. This
cardiogenic mesoderm is referred to as first heart field, flanked
medially by second heart field (SHF) mesoderm (Buckingham
et al., 2005; Kelly, 2012). The addition of SHF derived cardiac
mesoderm at both the arterial and the venous pole of the heart
tube (Fig. 2a) enables the eventual formation of all cardiac
components (Cai et al., 2003). Research in this field is focused in
general on either the arterial pole or outflow tract (Mjaatvedt
et al., 2001; Waldo et al., 2001) or on the addition of myocardium
to the venous pole (Bax et al., 2010; Bleyl et al., 2010; Christoffels
et al., 2006; Gittenberger-de Groot et al., 2007; Mahtab et al.,
2009; Mommersteeg et al., 2010; van Wijk and van den Hoff,
2010). During addition of SHF mesoderm to the primary heart
tube, of which the myocardium is in direct contact with the
coelomic cavity (pericardial cavity) a secondary layer will cover
the complete heart and the developing roots and intrapericardial
part of the great arteries. This so-called epicardium grows from
both the venous and the arterial pole.
The venous pole-derived epicardium has received by far the
most attention. At the venous pole a bilateral cauliflower-like
mesothelial protrusion develops, which is reduced to a single
rightsided proepicardial organ in the chick (vPEO, Fig. 2b and c)
(Schlueter et al., 2006; Viragh and Challice, 1981; Viragh et al.,
1993) and a medial vPEO in the mouse (Schulte et al., 2007) from
which the epicardium (cEP) spreads over the cardiac tube. Several
mechanisms have been described to show how this cEP bridges
the gap between the PEO and the heart (Nahirney et al., 2003).
The cEP starts to spread dorsally at the atrioventricular canal and
inner curvature (Fig. 2b) and eventually covers the complete heart
tube up to the myocardial ventriculo-arterial junction at the
outflow tract (Manner et al., 2001; Perez-Pomares et al., 2003;
Vrancken Peeters et al., 1995). At this borderline the cEP meets
the epicardium covering and derived from the arterial pole,
earlier described as cephalic epicardium (Perez-Pomares et al.,
2003) and periarterial epicardium (Lie-Venema et al., 2003). We
here refer to this layer as the arterial epicardium (aEP) that is
continuous with the outer layer of the pericardial cavity the socalled pericardium. At this reflection site we see a structure in the
early embryo that is comparable to the PEO at the venous pole
(Fig. 2d–f). The protrusions arise in close proximity to a line of
perforations that marks the borderline between the splanchnic
and the somatic mesoderm (DeRuiter et al., 1991), being actually
the site of the underlying intermediary mesoderm and the
pronephros system. We postulate that this arterial PEO is analogous to the venous PEO which has been described to be an
evolutionary remnant of the pronephric system (Pombal et al.,
2008). As a consequence these PEO structures harbor a variety of
cells including endothelial and mesodermal cells. It explains the
expression of nephrogenic related genes such as Wilms tumor
1 supressor gene (WT-1), podoplanin and epicardin/Pod1 (Mahtab
et al., 2008; Moore et al., 1999; Perez-Pomares et al., 2002;
Pombal et al., 2008) in both the arterial and venous pole PEO as
well as the spreading cEP and aEP.
The aEP starts to spread from the arterial pole PEO over the
arteries around HH17 in the chicken embryo (Fig. 2d). The aEP has
structural and immunohistochemical differences compared to the
cEP (Perez-Pomares et al., 2003). The transient PEO structures at
both arterial and venous pole remain detectable even after
complete covering of the heart and great arteries in the chicken
by HH26 (Vrancken Peeters et al., 1995) and in the mouse by
E11.5 (Mahtab et al., 2008).
3. Epicardium derived cells (EPDCs)
After completed spreading of the epicardium over both the
myocardium and the arterial pole the first wave of epithelial-tomesenchymal transition (EMT) becomes apparent. Epicardial cells
lose their epithelial contacts and EPDCs migrate into the subepicardial space (Gittenberger-de Groot et al., 1998; Lie-Venema
et al., 2007; Manner, 1999). Many molecular pathways have been
described to be essential for EMT including E-cadherin in relation
A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53
43
Fig. 1. Schematic presentation of cellular contribution to heart development with special focus on role epicardium and epicardium derived cells (EPDCs) during normal
development, disease and repair processes. Four mesodermal cell lines (cardiomyocytes, endocardium, epicardium, and endothelium) are considered to form the main
building blocks of the heart. The differentiation of each line is depicted together with the main interactions with the other cell lines. The most frequent EPDC-related
congenital malformations and (acquired) disease processes are boxed in green, while three cardiac (stem) cell populations that may become reactivated are presented on
the far right side. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
to podoplanin (Mahtab et al., 2008), VCAM1 in relation to PDGFRa
(Bax et al., 2010; Kwee et al., 1995) and alpha6-beta4 integrin in
relation to fibronectin (Dettman et al., 2003; Sengbusch et al.,
2002). The progression of covering and inward migration has
been mapped for cEP in chicken–quail chimeras to start at HH19
(Lie-Venema et al., 2005) and in mouse embryos at E11.5 (Mahtab
et al., 2008). These early EPDCs invade the thin myocardial wall
starting in the inner curvature (Lie-Venema et al., 2005). Due to a
fenestrated atrial and ventricular myocardial layer providing
direct epicardial–endocardial contacts, a route exists for a small
population of EPDCs to reach a subendocardial position in the
avian (Fig. 3a) (Gittenberger-de Groot et al., 1998) and mouse
embryo (Fig. 3b, unpublished observations). Recent experiments
in the adult regenerating zebrafish heart have shown that
proliferation of the myocardium is dependent on both endocardial and epicardial derived signals, including Raldh2 (Kikuchi
et al., 2011). Moreover the epicardium is influenced by retinoic
acid (RA) induced liver endothelium-derived erythropoietin (EPO)
that stimulates insulin-like growth factor (Igf) in epicardial cells
which subsequently promote formation of the compact myocardium (Brade et al., 2011). As soon as EPDCs migrate into the
myocardium, with a spatio-temporal difference between the
developing right and left ventricle (unpublished observations), a
thick compact myocardial layer develops, which is more obvious
in the left ventricle next to the trabeculated myocardium
bordering the lumen. Disturbance of epicardial outgrowth, EMT
and migration leads to hypoplasia of the compact myocardium
which is described after mechanical inhibition of epicardial outgrowth (Gittenberger-de Groot et al., 2000) and genetic manipulations of a variety of genes influencing the cEP (Lie-Venema
et al., 2007) as exemplified by mutants of PDGFRa (Bax et al.,
2010; Mellgren et al., 2008), podoplanin (Mahtab et al., 2008) and
WT-1(von Gise et al., 2011). The aEP also shows EMT at stage
HH25 (chick) and ED12.5 (mouse) and EPDCs can be detected in
the outer layers of the developing great arteries (Fig. 3c) and at
the myocardial-to-endocardial cushion interface. It is still unclear
whether all covering epicardial cells have the potential to enter
EMT and form EPDCs at this stage. Alternatively, there is epicardial heterogeneity with only a subset of cells taking part in this
initial EMT wave providing the myocardium with the main
number of the future interstitial fibroblasts (Gittenberger-de
Groot et al., 1998; Krenning et al., 2010; Perez-Pomares and
Pompa, 2011).
The next wave of EMT in the atrioventricular and ventriculoarterial grooves correlates with the formation of the fibrous
atrioventricular annulus, separating atrial and ventricular myocardium and contributes to part of the population of the atrioventricular cushions (Gittenberger-de Groot et al., 1998; Kolditz
et al., 2008; Lie-Venema et al., 2008; Zhou et al., 2010). At the
ventriculo-arterial junction aEP is contiguous with EPDCs that
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A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53
Fig. 2. (a) Schematic representation of the looped heart tube indicating second heart field (SHF:ocher) that has contributed new myocardium (yellow) to the right ventricle
(RV) and the atrium (A). The first heart field derived myocardium of the left ventricle (LV) and atrioventricular canal (AVC) is brown. Neural crest cells (NCC) mainly
contribute a population to the arterial pole including the wall of the aortic sac (AoS) and the endocardial cushions (ECu) of the outflow tract (OFT). At the venous pole also
some NCCs are found in the area of the AVC endocardial cushions. In the pericardial cavity (PC) a proepicardial organ protrudes at the venous pole (vPEO) as well as a
homologous organ at the arterial pole (aPEO). Arterial epicardium (aEP) spreads over the AoS while the epicardium from the venous pole will eventually cover the
complete cardiac myocardium (cEP). (b)–(d) Scanning electronmicrographs of developing chicken hearts ((b) HH19, (c) HH18, (d) HH17). (b) Frontal view of the inner
curvature (asterisk) showing the spreading cEP (arrowheads) over the bare myocardium of the ventricles (V). The posteriorly located vPEO is visible in the inner curvature.
(c) Dorsal view of the vPEO positioned in the pericardial cavity (PC) between the sinus venosus (SV) and the liver primordium (L). (d) View into the PC surrounding the OFT.
At the connection of the AoS to the pharyngeal mesoderm of the SHF a rough area (asterisk and arrows) can be distinguished indicative of the site of an aPEO. (e) two
sections stained for WT-1 which is expressed in the aPE covering the vascular part of the arterial pole containing the aortic arch arteries (AoAA), the blebs of the bilateral
aPEO (arrows) as well as the pericardium (open arrows). The cEP has not yet covered the atrial (A) myocardium. (f) Section of the arterial pole of a mouse embryonic heart
showing the aEP covering the wall of the aorta (Ao), the posterior part of the PC, the bilateral aPEOs (arrows) and the pericardium. The cEP has reached the aEP at the aortic
side (arrowhead) but only a few cEP cells are seen at the pulmonary (Pu) side (open arrowhead). Bars: (e), (f): 200 mm. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
migrate into the border between the non-myocardial vessel wall
and the myocardium of the outflow tract (Fig. 3 c and d) and
into the endocardial outflow tract cushions where they may be
effective in the future formation of the arterial annuli and
semilunar valves (Fig. 3 c and d).
After ingrowth of the peritruncal coronary capillary plexus
into the aorta (Bogers et al., 1989; Waldo et al., 1990; Poelmann
et al., 1993) and the start of arterial perfusion of the coronary
microvascular system, EPDCs surround these main coronary
vessels and differentiate into smooth muscle cells essential for
A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53
45
Fig. 3. (a) Section of the thin compact (CM) and the trabeculated (TM) myocardium of the right ventricle (RV) of a chicken-quail chimera stained for the quail specific
antibody QCPN (brown). A layer of cardiac epicardium (cEP) is covering the CM. Many epicardium derived cells (EPDCs: arrows) of the first migration wave are in
subendocardial position. The nuclei of the endocardial cells (arrowheads) are not expressing the quail specific antibody QCPN. (b) Section of the RV myocardium of a mouse
embryo stained for WT-1 (brown). The surface cEP is stained as well as some subendocardial EPDCs (arrows). The endocardium (arrowheads) is non-stained. (c) Transverse
section of a mouse heart stained with the epicardial marker WT-1. The aorta (Ao) and pulmonary trunk (Pu) are lined on the outside by arterial epicardium (aEP). EPDCs
derived from this layer (open arrowheads) are found in the future adventitia and the outer media of these vessels. A number of aEP derived EPDCs can be followed to the
interface between the myocardium (M) and endocardial cushions (ECu) of the outflow tract (OFT). The borderline between the cuboid aEP and the squamous cEP is
indicated by arrowheads. (d) Section of the same mouse embryo as depicted in (c) at the level of the developing pulmonary semilunar valves. At the sites where arterial
derived EPDCs (arrows) are present the ECu and myocardium are adherent while this is not the case at sites where EPDCs are missing (asterisk). The myocardium is not yet
invaded by cEP derived EPDCs. Bars: (a)–(d): 50 mm.
the subsequent development into an arterial phenotype (Dettman
et al., 1998; Vrancken Peeters et al., 1999) in which PDGFB/
PDGFRb (Mellgren et al., 2008; Van Den Akker et al., 2008b) and
Notch signaling (del Monte et al., 2011) play an essential role.
Summarizing, we have now introduced the cEP and aEP that
merge at the ventriculo-arterial junction of the outflow tract. It is
also clear that cEP and aEP have their own spatio-temporal
migration and destination characteristics (Lie-Venema et al.,
2005; Perez-Pomares et al., 2003). The next step is to evaluate
their fate and functional capacities.
4. Heterogeneity and differentiation of the EPDCs
There is consensus on the differentiation potential of EPDCs
into the interstitial cardiac fibroblasts, the coronary vascular
smooth muscle cells and the adventitial fibroblasts. The initial
data were derived from the study of the avian embryo by
retroviral tracing and chicken–quail chimera studies (Dettman
et al., 1998; Manner et al., 2001; Mikawa and Gourdie, 1996;
Vrancken Peeters et al., 1999; Poelmann et al., 1993) and have
been confirmed by transgenic mouse tracing studies with Gata5
(Merki et al., 2005), WT-1(Zhou et al., 2008) and Tbx18 reporters
(Cai et al., 2008).
Discussion still exists on the potential of EPDCs to differentiate
into coronary endothelium, myocardial cells and their possible
inductive role in Purkinje fiber differentiation. Their potential to
remain in a relatively undifferentiated EPDC state (Chong et al.,
2011; Wessels and Perez-Pomares, 2004) also needs attention. As all
these cell types are essential for the maintenance of the developing
as well as the adult heart and as they may have a role in disease and
repair we will shortly formulate consensus and discussion points.
A question that has not been solved refers to whether the
epicardium covering the myocardium and the arterial pole consists of a heterogeneous population in which the various cell
types are already predestined or whether we are dealing with a
46
A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53
multipotent cell population that differentiates on the basis of
environmental clues and cell–cell interactions. More evidence is
accumulating for a heterogeneous population both in origin and
function (Katz et al., 2012). We will address this issue where it
has been remarked upon specifically in the literature.
4.1. The cardiac fibroblast
In a recent review the heterogeneous origin and multiple
functions of the cardiac fibroblast are discussed (Krenning et al.,
2010). During development the main source of the fibroblast is
the epicardium (Cai et al., 2008; Krenning et al., 2010; PerezPomares and Pompa, 2011) as demonstrated for the interstitial
fibroblast (Gittenberger-de Groot et al., 1998), the annulus fibrosis (Gittenberger-de Groot et al., 1998; Kolditz et al., 2008; Zhou
et al., 2010) and the adventitial coronary fibroblast (Dettman
et al., 1998; Manner et al., 2001; Vrancken Peeters et al., 1999).
Besides the addition of fibroblasts from the epicardium to the
atrioventricular (Wessels et al., in press) and semilunar valves in
relatively late stages, the endocardial cells lining the cushions are
the major source of the valve fibroblasts (de Lange et al., 2004),
although this population seems to be replaced in part by cells
from the circulating blood during adult life (Visconti et al., 2006).
There is a marked difference between the origin of the fibroblast
during normal development and disease as will be referred to in
the section on the various cardiac disease states.
4.2. The endocardial cell
The discussion on the origin of the endocardial cell that is
incorporated within the primary myocardial heart tube has died
down. The consensus is that the cardiomyogenic plate can
provide both myocardial cells as well as the endocardium (Sugi
and Markwald, 1996) that originate in the splanchnic mesoderm
flanking the endoderm of the developing foregut (DeRuiter et al.,
1992). With the acknowledgment of the relatively late addition of
SHF to the arterial pole, we now appreciate the endothelial
contribution from the pharyngeal mesoderm in the outflow tract,
elegantly proven by Noden et al. (1995). At those sites where the
endocardial cells line the endocardial cushions they have a high
potential for EMT. Common genetic pathways have been postulated in endocardial cushions and the subepicardial layer in the
atrioventricular sulcus (Perez-Pomares et al., 1997).
4.3. The coronary endothelial cell
The origin of the coronary endothelium remains a matter of
debate, which is probably due to its intimate spatial relationship
with the PEO and slightly later in development the cEP. Two major
opinions have dominated the field. The first one using a quail vPEO
transplanted into the isochronous chick pericardial cavity
(Poelmann et al., 1993; Vrancken Peeters et al., 1997; Winter and
Gittenberger-de Groot, 2007a) established that coronary endothelial cells do not derive from the coelomic lining, i.e., the vPEO, but
from microvasculature sprouting from the sinus venosus into the
stalk of the vPEO (Fig. 4a). This has been supported by several
mouse transgenic studies including the Gata5 and the WT-1
transgenic mouse (Merki et al., 2005; Zhou et al., 2008). The second
opinion is based on the finding in avian embryos that a small
number of endothelial cells co-stains for the Wilms tumor suppressor gene1 (WT-1), in this context used as an epicardial marker
(Perez-Pomares and Pompa, 2011). Using elegant mouse model
tracing techniques Red-Horse (Red-Horse et al., 2010) confirmed
that EC are derived from the sinus venosus. Subsequently, these
authors claimed that these venous endothelial cells dedifferentiate
and are stepwise converted (‘‘reprogrammed’’) into arteries, capillaries and veins. We have shown, however, that the sinus venosus
derived endothelial cells express the ‘‘arterial’’ Notch1 (Fig. 4 b–d)
underlining the plasticity of the embryonic microvascular endothelial cell (Perez-Pomares and Pompa, 2011; Van Den Akker et al.,
2008a; Van den Akker et al., 2012) rather than a dedifferentiation/
redifferentiation program driven by hypothetised local clues.
Fig. 4. Schematic view of the interrelationship of the liver primordium (L), the sinus venosus (SV), atrium (A), ventricle (V) and outflow tract (OFT). The proepicardial organ
at the venous pole (vPEO) allows future coronary endothelial cells to migrate from the L and SV to the surface of the ventricle (not by way of the atrium) under coverage of
the cardiac epicardium (cEP). (b) Sections of a ED10.5 mouse embryo in which the endothelium (arrows) of the SV (see magnification in (c.) and the lining of the liver
sinusoids (see magnification in (d)) are positive for Notch1, demonstrating that members of the Notch/Delta-like/Jagged pathway are unsuitable to discriminate at this
embryonic stage between arteries, microvasculature and veins. Bars: (b): 50 mm, (c), (d): 10 mm. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53
A recent report keeps the discussion on the origin of the coronary
ECs alive as distinct compartments of the pro-epicardial organ give
rise to small numbers of ECs ( Katz et al., 2012).
4.4. The coronary smooth muscle cells
There is consensus on the origin of the coronary SMCs from
differentiated EPDCs (Dettman et al., 1998; Mellgren et al., 2008;
Vrancken Peeters et al., 1999). Recent information sets the origin of
the pericyte and SMC apart (Chong et al., 2011). From quail–
chicken chimera studies the timing of EMT and required endothelial-EPDC cell–cell interaction has become evident. In this model
solitary SMCs have not been encountered (Vrancken Peeters et al.,
1999). Whether the differentiation into coronary artery and veins
is primarily driven by an autonomous endothelial differentiation
process or whether hemodynamic forces drive the arterial phenotype of the endothelial cells with subsequent recruitment of SMCs
needs further study. EPDC differentiation into SMCs is regulated by
many genes including serum response factor (Landerholm et al.,
1999), and PDGFRb and their ligands (Mellgren et al., 2008; Van
Den Akker et al., 2008b). Recent transgenic mouse studies support
a separate PDGFRa positive EPDC population to be sensitive to SMC
differentiation, but the role of local environmental factors cannot
be excluded yet (Smith et al., 2011). Remarkable is the high
plasticity of the endothelium and the underlying SMCs in the more
superficial venous and deeper located arterial network when the
PDGF pathway is disturbed (Van Den Akker et al., 2008b). Other
growth factors play an important role in coronary vascular differentiation like fibroblast growth factor (Carmeliet, 2000), VEGF
(Tomanek et al., 2002; Van Den Akker et al., 2008a) and Notch
(del Monte et al., 2011; Van Den Akker et al., 2008a), as recently
reviewed (Riley and Smart, 2011).
4.5. The cardiomyocyte
Conditional reporter mice studies (Cai et al., 2008; Smart et al.,
2011; Zhou et al., 2008), using WT-1 and Tbx18 as epicardial
reporters, support the differentiation of a subset of EPDCs into a
cell with a myocardial phenotype. It cannot be excluded that the
genetic tagging of epicardial cell lineages contains flaws. We and
others (Perez-Pomares and Pompa, 2011) argue that the conditional WT1 reporter mouse model cannot be used for selective
lineage tracing of WT1 positive epicardium as some of the SHF
progenitors of cardiomyocytes in the SHF are positive as well
(Jongbloed et al. 2011). Also the use of the Tbx18 gene as a
selective epicardial reporter gene has been refuted (Christoffels
et al., 2009). Several other data do not support the EPDCcardiomyocyte transition, including quail–chicken chimera studies
(Gittenberger-de Groot et al., 1998; Manner et al., 2001). Many
WT-1 positive cells can be found not only in the coelomic lining of
the pericardial coelomic cavity, but also in the underlying mesoderm of the SHF (Fig. 3c). This mesoderm, based on a BMP and FGF
balance can differentiate into both a myocardial and an epicardial
population (Kruithof et al., 2006); therefore, a common progenitor
of both cell lines is likely (van Wijk and van den Hoff, 2010). It can
not be excluded that relative undifferentiated cardiomyocyte
progenitor cells (CMPCs) (Smits et al., 2009; Timmers et al.,
2011) that are present within the venous pole SHF population
may have a WT-1 positive lineage background. Studies using adult
human EPDCs show that this population has mesenchymal stem
cell characteristics but is already more cardiac committed with
positive cardiac markers such as Gata4 and cTNT (van Tuyn et al.
2007). In the section on the potential of EPDCs in adult stages
during activation and disease this aspect of EPDC cardiomyocyte
transition will be further discussed.
47
4.6. The Purkinje fiber
The Purkinje fiber is a specialized cardiomyocyte induced
by endothelin produced by endothelin-converting enzyme
expressing endothelium and endocardium (Eid et al., 1994;
Gourdie et al., 1998; Mikawa et al., 2003). An instructive role
for the EPDC in Purkinje fiber differentiation in the cardiac wall
became evident during vPEO tracing and inhibition experiments.
We have postulated an essential interaction between EPDCs and
the endothelial/endocardial derived factors (Eralp et al., 2006;
Gittenberger-de Groot et al., 1998).
5. The epicardium in congenital and adult cardiac disease
The epicardium is an essential population for proper development of the heart and great vessels. Complete inhibition of the
outgrowth of the sinus venosus located vPEO leads to severe
cardiac malformations (Gittenberger-de Groot et al., 2000). These
include absence of vEP, aberrant and extensive outgrowth of aEP
over the myocardial outflow tract, deficient looping with a wide
inner curvature, absent ventricular and outflow tract septation
and atrioventricular cushion formation, combined with a thin
(2 layer) compact myocardium. This combination, due to lack of
epicardial covering, does not develop coronary vasculature and is
embryo-lethal. Experiments in which the PEO outgrowth is only
partially inhibited in chicken and quail embryos (Eralp et al.,
2005; Kolditz et al., 2007; Lie-Venema et al., 2003; Manner et al.,
2005) lead to a spectrum of cardiac malformations that are
reminiscent of congenital heart malformations seen in the human
population. The essential role of the epicardium can also be
deducted from (conditional) transgenic and knock out mouse
models in which genes and regulatory sequences, relevant for the
various stages of epicardial development, have been mutated.
These vary from inhibition of spreading as in the VCAM1 (Kwee
et al., 1995), alpha4-beta6 integrin (Yang et al., 1995) and FOG2
(Tevosian et al., 2000) mutants, or to disturbed EMT as in SP3
(Van Loo et al., 2007), podoplanin (Mahtab et al., 2009, 2008) and
PDGFRa and b (Bax et al., 2010; Mellgren et al., 2008) mutants. In
the majority of these models a concurrent hypoplasia of the vPEO
is apparent (Bax et al., 2010; Mahtab et al., 2008) which can
account for the smaller number of epicardial cells.
No epicardium specific gene, useful for lineage tracing or
specific knock-out strategies, has been determined, although large
screen microarray studies have been performed (Bochmann et al.,
2010). It is challenging to attribute specific cardiac diseases and
malformations to epicardial and PEO developmental defects. It is,
however, possible to describe the role of the epicardium in some
cardiac defects in avian and mouse models and postulate their
role in several human cardiac diseases. As will be clear from Fig. 1
we are dealing with composite structures in which, among other
cardiac cell types, epicardial cells play an essential role.
5.1. Non-compaction cardiomyopathy
In both, avian (Gittenberger-de Groot et al., 2000) and mouse
models (Bax et al., 2010; Mahtab et al., 2008) normal development
of the compact myocardium depends on proper interactions
between cardiomyocytes, EPDCs and the secreted extracellular
matrix. Primary epicardial derived abnormalities like in the WT-1
null mutant (Martinez-Estrada et al., 2010) exert their influence on
the myocardial cells through WT-1 dependent Raldh2, whereby RA
is not delivered to the myocardium as is elegantly proven for the
RxRa mutant (Guadix et al., 2011; Jenkins et al., 2005). Development of the normal compact myocardium, linked to the epicardium derived interstitial fibroblast population (Ieda et al., 2009),
48
A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53
can also be disturbed when the primary problem is cardiomyocyte
linked. Many cardiomyopathies, usually with a hypertrophic myocardium, are based on mutations of cardiomyocyte specific genes
(Maron et al., 2006). The most relevant human cardiomyopathy
that could result from a primary abnormal EPDC function is
the primary left ventricular non-compaction cardiomyopathy
(Lie-Venema et al., 2007). The morphological substrate consists
of a spongious myocardium often including the ventricular
septum. Differences in timing and amount of RV and LV invasion
by EPDCs might account for the preferential problem in the LV.
With respect to congenital heart disease a spongious structure
of the ventricular septum can be related to muscular VSDs varying
from small isolated VSDs, which tend to close spontaneously, to
multiple muscular VSDs. Recent data open the option that in the
hypoplastic left heart syndrome and the related borderline left
ventricles the epicardial to myocardial interaction is abnormal
leading to myocardial pathology (Mahtab et al., in press)
5.2. Endocardial fibroelastosis
The hypoplastic ventricle, with a restricted outflow and a
concomitant high pressure in cardiac anomalies such as pulmonary
atresia without VSD and the hypoplastic left heart syndrome,
presents with a thick layer of endocardial fibroelastosis. The layer
consists of fibroblasts and myofibroblasts that could develop from
cells derived from endocardial EMT possibly in combination with
the population of EPDCs that have been deposited subendocardially during development (Gittenberger-de Groot et al., 1998)
(Fig. 3a and b).
valve. Interestingly, in our avian model this is observed in
combination with accessory pathways (Lie-Venema et al., 2007),
an association also reported in human patients (Attenhofer Jost
et al., 2007).
EPDCs of aEP origin are encountered in the endocardial outflow tract cushions (Fig. 2c). We postulate that they act through
the important role of EPDCs in Notch signaling (del Monte et al.,
2011; Grieskamp et al., 2011; Van Den Akker et al. 2007; Van Den
Akker et al., in press) and thus influence bicuspid aortic valve
formation, which has been linked in patients to a Notch 1 mutation (McKellar et al., 2007).
5.5. Coronary vascular anomalies
Coronary vascular development from the undifferentiated microvascular endothelial plexus to the differentiated coronary arteries
and veins depends on epicardial development. Experimental studies
hampering normal coronary development and differentiation lead to
a number of anomalies that link congenital pattern variations to
abnormal ventriculo-coronary-arterial communications (VCAC or
fistulae) as described in the human fetus and neonate. PEO inhibition as well as delayed epicardial spreading in PEO ablation/rescue
experiments show single coronary ostia as well as pinpoint coronary
orifice formation. VCAC are found in animal models with complete
absent coronary arterial orifices in the aorta (Eralp et al., 2005; LieVenema et al., 2003). This resembles the human coronary malformations found in pulmonary atresia without VSD and VCAC, which
has been postulated to be a primary coronary vascular disease
(Gittenberger-de Groot et al., 2001).
5.3. Cardiac conduction system anomalies
6. Cardiovascular repair
With respect to the origin of conduction system disturbances,
we distinguish congenitally determined and acquired defects,
realizing that the main components of the cardiac conduction
system are myocardial in origin (Jongbloed et al., 2012). The role
of the epicardium and EPDCs is currently restricted to an
inductive influence on Purkinje fiber differentiation. Clinically, it
has been postulated that aspects of the genetically determined
long-QT syndrome might have a link to abnormal Purkinje fiber
function. Indirectly, the deficient formation of the annulus fibrosis
with persistent atrioventricular myocardial connections, so-called
accessory pathways, can lead to re-entry tachycardias. PEO
inhibition in avian embryos demonstrated deficient atrioventricular isolation. The required shift from a base-to-apex to an
apex-to-base conduction is delayed during development (Kolditz
et al., 2007). Mouse models in which normal epicardial spreading
and EPDC formation is disturbed in conjunction with deficient
annulus fibrosis formation have not been reported, whereas a
model with abnormal persistence of a myocardial phenotype has
been described for the Tbx2 mutant (Aanhaanen et al., 2011).
5.4. Valvulopathies
Cardiac valves can be distinguished in the arterial semilunar
and the atrioventricular valves. Anomalies consist of abnormal
anlage and morphology such as seen in bicuspid aortic valve and
common atrioventricular valves. Histological abnormalities with
dysplastic valves are considered a different entity which can be
found in combination with an abnormal morphology. EPDCs
migrate into the atrioventricular cushions (Gittenberger-de
Groot et al., 1998; Manner et al., 2001; Wessels et al., 2012)).
PEO inhibition can lead to complete absence of AV valve formation (Gittenberger-de Groot et al., 2000). Less severe cases with an
abnormal differentiation including deficient undermining of the
valve leaflet are reminiscent of Ebstein’s anomaly of the tricuspid
6.1. Myocardial infarction
Based on the potential of the epicardium and EPDCs during
normal development it is tempting to attribute a role in repair of
the cardiac wall and its vascularization in various adult cardiac
disease processes. The main cardiac disease studied in this
respect is ischemic heart disease with subsequent myocardial
fibrosis and heart failure. The primary cause is myocardial
infarction (MI) after a coronary obstruction or occlusion due to
atherosclerotic processes. If the epicardium and its EPDCs could
recapitulate their embryonic program and acquire multipotenital stem cell characteristics, benefit could be achieved to the
myocardial recuperation as well as stabilization of the novel
angiogenesis derived capillaries by addition of new SMCs.
Several research approaches have focused on this aspect of the
potential of the adult epicardial cell after myocardial infarction.
Various lines of research can be distinguished. A first cohort of
studies investigated the potential of the native epicardium after
MI. These studies were performed in mouse models following
experimental myocardial infarction. It has been shown that a
c-kit positive subepicardial EPDC population is found, indicating
renewed epicardial activity and acquisition of stem cell characteristics (Limana et al., 2010, 2011). Use of green fluorescent
protein tracing showed that the c-kit positive cells were epicardium derived. A different study using a retroviral fluorescent
Katushka labeling of quiescent epicardial cells that after MI
became cuboidal, showed EMT followed by EPDCs that migrated
into the myocardium. They differentiated after 4 day into a
myofibroblast phenotype (Gittenberger-de Groot et al., 2010)
but were not followed for further differentiation. The activated
epicardium and EPDCs became WT1 positive not only in the area
directly bordering the MI but also in more remote zones. It could
not be excluded that resident cardiac fibroblasts were
A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53
upregulating their WT1 expression. Further research is necessary
to explain this phenomenon. In a mouse model of MI the
reactivation of EMT of the adult epicardium was shown without
differentiation of the EPDCs into a myocardial or endothelial
phenotype; however, paracrine factors stimulating, e.g., angiogenesis were found (Zhou et al., 2011). Studies by Riley and coworkers emphasized the regenerative capacity of EPDCs after MI
by stimulation with Thymosin b4, showing renewed angiogenesis and arteriogenesis (Smart et al., 2007). Performing priming
by Thymosin b4 followed by myocardial infarction in the WT-1
reporter mouse, the EPDCs differentiate into a cardiomyocyte
phenotype (Smart et al., 2011). These findings could not be
confirmed in a MI mouse model in which Thymosin b4 was
provided concomitant with the MI induction. EPDCs did not
differentiate into a cardiomyocyte or endothelial phenotype
(Zhou et al., 2012). A recent study shows that Thymosin b4 is
not essential for proper cardiac development (Banerjee et al.,
2012). The role of Thymosin b4 needs careful evaluation.
A second approach is the in vitro work in which epicardial
cells are cultured in combination with cardiomyocytes. It has
been shown already in Eid et al. (1992) that activity of epicardial
cells modifies the cardiomyocyte phenotype. Recently, in a coculture combining chicken EPDCs and rat neonatal cardiomyocytes, this was further proven with a marked role for EPDCs on
myocardial alignment and contraction (Weeke-Klimp et al.,
2010). A very direct approach to influence repair of the heart
after MI was provided by using human adult epicardial cells
(Winter et al., 2007b). These cells were derived from the atrial
surface and cultured in vitro. The WT-1 positive epicardial cells
changed their phenotype from a cobblestone epithelium into
49
spindle shape. These human EPDCs acquired characteristics
resembling mesenchymal stem cells. Their cardiac commitment
was shown by expression of GATA4 and cTNT, while cardiomyocyte-specific genes were absent (van Tuyn et al., 2007). Injection
of these adult human EPDCs in immune incompetent mice into
the borderzone of the ischemic area resulted in a marked
improvement of cardiac function as measured by cardiac MRI.
This improvement was found already 2 day after injection
extending to 2 and 6 weeks (Gittenberger-de Groot et al.,
2010; Winter et al., 2007b). Furthermore, improved function,
marked angiogenesis and a widespread PCNA activation indicative of repair was observed. Experiments with an injection of
EPDCs combined with adult human cardiomyocyte progenitors
(CMPCs) aimed at induction of cardiomyocyte regeneration
(Winter et al., 2009). The results show an additive effect of the
separate injections on remodeling, although no new cardiac cell
types (endothelial cells, interstitial fibroblasts, smooth muscle
cells or cardiomyocytes) could be traced to human origin. These
data can now be supported by more recent studies on the
differentiation capacities of the EPDCs. The capacities, found
within the normal embryonic in vivo environment seem to be
retained in adult life and disease states The conclusion is that
many of the positive effects of EPDCs either after injection or by
stimulation of the native epicardial covering of the heart are due
to a paracrine mechanism (Limana et al., 2011; Winter et al.,
2007b, 2009; Zhou et al., 2011). These findings have great
potential for future therapeutic approaches, either drug or cell
based, that stimulate the native epicardium in repair of the
ischemic cardiac wall. An underdeveloped area is the priming of
pericardium based grafts for use in arterial or cardiac repair.
Fig. 5. (a)–(d) Tranverse sections of the wall of the adult ascending aorta just distal of the semilunar valve level. (a) Normal adult aortic wall of a case with a tricuspid aortic
valve. There is a regular staining of the smooth muscle cells with a smooth muscle actin (SMA) using the 1A4 antibody in intima (I) and the media (M). The adventitia (A) is
made up of loose fibrous tissue and contains vasa vasorum of which the wall of the small arteries also stain positive for 1A4. (b) Detail (see location in a.) of the arterial
epicardium (aEP), covering the adventitia. The aEP (blue) is negative for the epicardial marker WT-1. (c) Case with a tricuspid aortic valve and aneurysm formation of the
ascending aorta. Both in the intima and media there is marked loss of 1A4. The adventitia does not show marked differences as compared to the normal aortic wall. However
in detail (d) the overlying aEP is positive for WT-1 (brown) and there are signs of epithelial-mesenchymal-transition with WT-1 positive EPDCs (arrows) in the underlying
adventitia. Bars: (a)–(d): 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
50
A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53
6.2. Thoracic aortic aneurysm
Thoracic aortic aneurysm formation (Lindsay and Dietz, 2011)
is a disease of the ascending aorta with loss of vascular wall
structure and smooth muscle cells accompanied by cytolytic
necrosis and in severe cases to dissection. Based on our hypothesis that aEP might play a role in maintaining vessel wall integrity
also in adult life, comparable to the reactivation of the adult cEP
after myocardial infarction, we have set up a small study. We
compared the immunohistological characteristics for the epicardial marker WT-1 as well as TGFb and phospho-Smad2 of the
human adult thoracic aortic wall. We investigated the ascending
aorta with (1) a normal aortic wall and tricuspid aortic valve
(TAV) and (2) an aortic aneurysm and TAV. The results based on
five cases for each group showed a quiescent aEP (based on WT-1
staining) in group 1 and marked activation with EMT in group 2
(Fig. 5 a–d). These data were supported by TGFb expression that
was far more marked particularly in the intima in cases with TAV
and aneurysm (group2). These preliminary results are an indication that the epicardium of the arterial pole is capable of
reactivation and renewed EMT supporting repair in the adult
stage. Further research will reveal whether this repair phenomenon is also found in other groups of thoracic aneurysm formation
e.g. based on Loeys-Dietz and Marfan syndromes (Dietz and
Pyeritz, 1995) and in combination with bicuspid aortic valve
(McKellar et al., 2007).
7. Conclusion
In conclusion: The epicardium has acquired a dominant position in our understanding of proper cardiac development. Its
effect is visible in most major processes including looping,
myocardial maturation, septation, valve formation and coronary
vascular development and patterning. A complicating factor in
the study of the epicardium, using the current sophisticated
mouse models, is that no specific genes for epicardium or EPDCs
have been identified, as yet (Bochmann et al., 2010). Several
reviews appeared in the last year that focus on the signaling
pathways of the major growth factor families that all exert their
influence on the various aspects of EMT, migration and differentiation (Perez-Pomares and Pompa, 2011; Riley and Smart,
2011). Data are accumulating that in some forms of cardiac
disease the epicardium is capable of reactivating the embryonic
program (Gittenberger-de Groot et al., 2010; Limana et al., 2011;
Zhou et al., 2011) with beneficial effects on cardiac function. ^The
mechanism seems to be mainly cell-autonomous with supportive
actions for cell-cell interactions (Winter et al. 2009). These
findings invigorate the research into discovering the active
compounds that might replace or support cell transplantation
and biomedical devices in therapeutic approaches.
Acknowledgments
We would like to thank Bert J Wisse for preparation of the
figures and Ron Slagter for his excellent medical illustration work.
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