Differentiation 84 (2012) 41–53

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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 JoseGoumans 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 Netherlandsb Department of Anatomy and Embryology, Leiden University Medical Center, Postal zone: S-1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlandsc Department of Molecular Cell Biology, Leiden University Medical Center, Postal zone: S-1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands

a r t i c l e i n f o

Available online 30 May 2012

Keywords:

Cardiac development

Epicardium derived cells (EPDCs)

Pericardium

Myocardial infarction

Vascular development

Epithelial-to mesenchymal-transition

81/$ - see front matter & 2012 International

International Society for Differentiation (ww

x.doi.org/10.1016/j.diff.2012.05.002

viations: aPEO, arterial pro-epicardial organ; CM

s, endothelial cells; vPEO, venous pro-epicardia

muscle cells

esponding author. Department of Cardiology

Postal zone: S-5-24, P.O. Box 9600, 2300 R

1 71 526 3704(9306); fax: þ31 71 526 6809.

ail address: acgitten@lumc.nl (A.C. Gittenberg

a b s t r a c t

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.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2. Origin of the epicardium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

3. Epicardium derived cells (EPDCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

4. 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

Society of Differentiation. Publish

w.isdifferentiation.org)

PCs, cardiomyocyte progenitor

l organ; VSMCs, vascular

, Leiden University Medical

C Leiden, The Netherlands.

er-de Groot).

ed by Elsevier B.V. All rights reserved.

A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–5342

5. 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

6. Cardiovascular repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

6.1. Myocardial infarction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

6.2. Thoracic aortic aneurysm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

1. Introduction

The epicardium covering both the heart and the intraperi-cardial part of the great arteries is subject to an intense revival ofinterest. Research on both morphological and functional character-istics including effective gene signaling pathways is booming asshown by a 10-fold increase in publications on the topic pro-epicardium between 2000 and 2011 and 43-fold on the topicepicardium 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 Perez-Pomares, 2004; Winter and Gittenberger-de Groot, 2007a), present-ing generally accepted data, but also aspects that are still contro-versial. Statements on the controversial issues are intriguing andtrigger new research. In the current review the focus on the origin,fate, disease and repair provides novel insights in the potential ofthe epicardium. The epicardium cannot be regarded as a separateentity and is incorporated both structurally and functionally in thecardiac and vessel wall components. To support insight and struc-ture of this review schematic Fig. 1 is instrumental.

2. Origin of the epicardium

The epicardium develops from the epithelium of the coelomicwall in close interaction with the underlying splanchnic meso-derm. With the formation of the intra-embryonic coelomic cavity,separating the intra-embryonic mesoderm into a splanchnic and asomatic layer, the splanchnic mesoderm lining the endoderm ofthe foregut develops into the bilateral cardiogenic plates. Theseare the precursors of the myocardial primary heart tube. Thiscardiogenic mesoderm is referred to as first heart field, flankedmedially by second heart field (SHF) mesoderm (Buckinghamet al., 2005; Kelly, 2012). The addition of SHF derived cardiacmesoderm at both the arterial and the venous pole of the hearttube (Fig. 2a) enables the eventual formation of all cardiaccomponents (Cai et al., 2003). Research in this field is focused ingeneral on either the arterial pole or outflow tract (Mjaatvedtet al., 2001; Waldo et al., 2001) or on the addition of myocardiumto the venous pole (Bax et al., 2010; Bleyl et al., 2010; Christoffelset 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 hearttube, of which the myocardium is in direct contact with thecoelomic cavity (pericardial cavity) a secondary layer will coverthe complete heart and the developing roots and intrapericardialpart of the great arteries. This so-called epicardium grows fromboth the venous and the arterial pole.

The venous pole-derived epicardium has received by far themost attention. At the venous pole a bilateral cauliflower-like

mesothelial protrusion develops, which is reduced to a singlerightsided 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) fromwhich the epicardium (cEP) spreads over the cardiac tube. Severalmechanisms have been described to show how this cEP bridgesthe gap between the PEO and the heart (Nahirney et al., 2003).The cEP starts to spread dorsally at the atrioventricular canal andinner curvature (Fig. 2b) and eventually covers the complete hearttube up to the myocardial ventriculo-arterial junction at theoutflow tract (Manner et al., 2001; Perez-Pomares et al., 2003;Vrancken Peeters et al., 1995). At this borderline the cEP meetsthe 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). Wehere refer to this layer as the arterial epicardium (aEP) that iscontinuous with the outer layer of the pericardial cavity the so-called pericardium. At this reflection site we see a structure in theearly embryo that is comparable to the PEO at the venous pole(Fig. 2d–f). The protrusions arise in close proximity to a line ofperforations that marks the borderline between the splanchnicand the somatic mesoderm (DeRuiter et al., 1991), being actuallythe site of the underlying intermediary mesoderm and thepronephros system. We postulate that this arterial PEO is ana-logous to the venous PEO which has been described to be anevolutionary remnant of the pronephric system (Pombal et al.,2008). As a consequence these PEO structures harbor a variety ofcells including endothelial and mesodermal cells. It explains theexpression of nephrogenic related genes such as Wilms tumor1 supressor gene (WT-1), podoplanin and epicardin/Pod1 (Mahtabet al., 2008; Moore et al., 1999; Perez-Pomares et al., 2002;Pombal et al., 2008) in both the arterial and venous pole PEO aswell as the spreading cEP and aEP.

The aEP starts to spread from the arterial pole PEO over thearteries around HH17 in the chicken embryo (Fig. 2d). The aEP hasstructural and immunohistochemical differences compared to thecEP (Perez-Pomares et al., 2003). The transient PEO structures atboth arterial and venous pole remain detectable even aftercomplete covering of the heart and great arteries in the chickenby HH26 (Vrancken Peeters et al., 1995) and in the mouse byE11.5 (Mahtab et al., 2008).

3. Epicardium derived cells (EPDCs)

After completed spreading of the epicardium over both themyocardium and the arterial pole the first wave of epithelial-to-mesenchymal transition (EMT) becomes apparent. Epicardial cellslose their epithelial contacts and EPDCs migrate into the sub-epicardial space (Gittenberger-de Groot et al., 1998; Lie-Venemaet al., 2007; Manner, 1999). Many molecular pathways have beendescribed to be essential for EMT including E-cadherin in relation

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.)

A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53 43

to podoplanin (Mahtab et al., 2008), VCAM1 in relation to PDGFRa(Bax et al., 2010; Kwee et al., 1995) and alpha6-beta4 integrin inrelation to fibronectin (Dettman et al., 2003; Sengbusch et al.,2002). The progression of covering and inward migration hasbeen mapped for cEP in chicken–quail chimeras to start at HH19(Lie-Venema et al., 2005) and in mouse embryos at E11.5 (Mahtabet al., 2008). These early EPDCs invade the thin myocardial wallstarting in the inner curvature (Lie-Venema et al., 2005). Due to afenestrated atrial and ventricular myocardial layer providingdirect epicardial–endocardial contacts, a route exists for a smallpopulation of EPDCs to reach a subendocardial position in theavian (Fig. 3a) (Gittenberger-de Groot et al., 1998) and mouseembryo (Fig. 3b, unpublished observations). Recent experimentsin the adult regenerating zebrafish heart have shown thatproliferation of the myocardium is dependent on both endo-cardial and epicardial derived signals, including Raldh2 (Kikuchiet al., 2011). Moreover the epicardium is influenced by retinoicacid (RA) induced liver endothelium-derived erythropoietin (EPO)that stimulates insulin-like growth factor (Igf) in epicardial cellswhich subsequently promote formation of the compact myo-cardium (Brade et al., 2011). As soon as EPDCs migrate into themyocardium, with a spatio-temporal difference between thedeveloping right and left ventricle (unpublished observations), athick compact myocardial layer develops, which is more obviousin the left ventricle next to the trabeculated myocardium

bordering the lumen. Disturbance of epicardial outgrowth, EMTand migration leads to hypoplasia of the compact myocardiumwhich is described after mechanical inhibition of epicardial out-growth (Gittenberger-de Groot et al., 2000) and genetic manip-ulations of a variety of genes influencing the cEP (Lie-Venemaet al., 2007) as exemplified by mutants of PDGFRa (Bax et al.,2010; Mellgren et al., 2008), podoplanin (Mahtab et al., 2008) andWT-1(von Gise et al., 2011). The aEP also shows EMT at stageHH25 (chick) and ED12.5 (mouse) and EPDCs can be detected inthe outer layers of the developing great arteries (Fig. 3c) and atthe myocardial-to-endocardial cushion interface. It is still unclearwhether all covering epicardial cells have the potential to enterEMT and form EPDCs at this stage. Alternatively, there is epicar-dial heterogeneity with only a subset of cells taking part in thisinitial EMT wave providing the myocardium with the mainnumber of the future interstitial fibroblasts (Gittenberger-deGroot et al., 1998; Krenning et al., 2010; Perez-Pomares andPompa, 2011).

The next wave of EMT in the atrioventricular and ventriculo-arterial grooves correlates with the formation of the fibrousatrioventricular annulus, separating atrial and ventricular myo-cardium and contributes to part of the population of the atrio-ventricular cushions (Gittenberger-de Groot et al., 1998; Kolditzet al., 2008; Lie-Venema et al., 2008; Zhou et al., 2010). At theventriculo-arterial junction aEP is contiguous with EPDCs that

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.)

A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–5344

migrate into the border between the non-myocardial vessel walland the myocardium of the outflow tract (Fig. 3 c and d) andinto the endocardial outflow tract cushions where they may beeffective in the future formation of the arterial annuli andsemilunar valves (Fig. 3 c and d).

After ingrowth of the peritruncal coronary capillary plexusinto the aorta (Bogers et al., 1989; Waldo et al., 1990; Poelmannet al., 1993) and the start of arterial perfusion of the coronarymicrovascular system, EPDCs surround these main coronaryvessels and differentiate into smooth muscle cells essential for

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.

A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53 45

the subsequent development into an arterial phenotype (Dettmanet al., 1998; Vrancken Peeters et al., 1999) in which PDGFB/PDGFRb (Mellgren et al., 2008; Van Den Akker et al., 2008b) andNotch signaling (del Monte et al., 2011) play an essential role.

Summarizing, we have now introduced the cEP and aEP thatmerge at the ventriculo-arterial junction of the outflow tract. It isalso clear that cEP and aEP have their own spatio-temporalmigration and destination characteristics (Lie-Venema et al.,2005; Perez-Pomares et al., 2003). The next step is to evaluatetheir fate and functional capacities.

4. Heterogeneity and differentiation of the EPDCs

There is consensus on the differentiation potential of EPDCsinto the interstitial cardiac fibroblasts, the coronary vascularsmooth muscle cells and the adventitial fibroblasts. The initialdata were derived from the study of the avian embryo by

retroviral tracing and chicken–quail chimera studies (Dettmanet al., 1998; Manner et al., 2001; Mikawa and Gourdie, 1996;Vrancken Peeters et al., 1999; Poelmann et al., 1993) and havebeen 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 differentiateinto coronary endothelium, myocardial cells and their possibleinductive role in Purkinje fiber differentiation. Their potential toremain in a relatively undifferentiated EPDC state (Chong et al.,2011; Wessels and Perez-Pomares, 2004) also needs attention. As allthese cell types are essential for the maintenance of the developingas well as the adult heart and as they may have a role in disease andrepair we will shortly formulate consensus and discussion points.

A question that has not been solved refers to whether theepicardium covering the myocardium and the arterial pole con-sists of a heterogeneous population in which the various celltypes are already predestined or whether we are dealing with a

A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–5346

multipotent cell population that differentiates on the basis ofenvironmental clues and cell–cell interactions. More evidence isaccumulating for a heterogeneous population both in origin andfunction (Katz et al., 2012). We will address this issue where ithas been remarked upon specifically in the literature.

4.1. The cardiac fibroblast

In a recent review the heterogeneous origin and multiplefunctions of the cardiac fibroblast are discussed (Krenning et al.,2010). During development the main source of the fibroblast isthe epicardium (Cai et al., 2008; Krenning et al., 2010; Perez-Pomares and Pompa, 2011) as demonstrated for the interstitialfibroblast (Gittenberger-de Groot et al., 1998), the annulus fibro-sis (Gittenberger-de Groot et al., 1998; Kolditz et al., 2008; Zhouet al., 2010) and the adventitial coronary fibroblast (Dettmanet al., 1998; Manner et al., 2001; Vrancken Peeters et al., 1999).

Besides the addition of fibroblasts from the epicardium to theatrioventricular (Wessels et al., in press) and semilunar valves inrelatively late stages, the endocardial cells lining the cushions arethe major source of the valve fibroblasts (de Lange et al., 2004),although this population seems to be replaced in part by cellsfrom the circulating blood during adult life (Visconti et al., 2006).There is a marked difference between the origin of the fibroblastduring normal development and disease as will be referred to inthe section on the various cardiac disease states.

4.2. The endocardial cell

The discussion on the origin of the endocardial cell that isincorporated within the primary myocardial heart tube has dieddown. The consensus is that the cardiomyogenic plate canprovide both myocardial cells as well as the endocardium (Sugiand Markwald, 1996) that originate in the splanchnic mesodermflanking the endoderm of the developing foregut (DeRuiter et al.,1992). With the acknowledgment of the relatively late addition of

Fig. 4. Schematic view of the interrelationship of the liver primordium (L), the sinus ven

at the venous pole (vPEO) allows future coronary endothelial cells to migrate from the L

the cardiac epicardium (cEP). (b) Sections of a ED10.5 mouse embryo in which the en

sinusoids (see magnification in (d)) are positive for Notch1, demonstrating that memb

embryonic stage between arteries, microvasculature and veins. Bars: (b): 50 mm, (c), (

reader is referred to the web version of this article.)

SHF to the arterial pole, we now appreciate the endothelialcontribution from the pharyngeal mesoderm in the outflow tract,elegantly proven by Noden et al. (1995). At those sites where theendocardial cells line the endocardial cushions they have a highpotential for EMT. Common genetic pathways have been postu-lated in endocardial cushions and the subepicardial layer in theatrioventricular sulcus (Perez-Pomares et al., 1997).

4.3. The coronary endothelial cell

The origin of the coronary endothelium remains a matter ofdebate, which is probably due to its intimate spatial relationshipwith the PEO and slightly later in development the cEP. Two majoropinions have dominated the field. The first one using a quail vPEOtransplanted into the isochronous chick pericardial cavity(Poelmann et al., 1993; Vrancken Peeters et al., 1997; Winter andGittenberger-de Groot, 2007a) established that coronary endothe-lial cells do not derive from the coelomic lining, i.e., the vPEO, butfrom microvasculature sprouting from the sinus venosus into thestalk of the vPEO (Fig. 4a). This has been supported by severalmouse transgenic studies including the Gata5 and the WT-1transgenic mouse (Merki et al., 2005; Zhou et al., 2008). The secondopinion is based on the finding in avian embryos that a smallnumber of endothelial cells co-stains for the Wilms tumor sup-pressor gene1 (WT-1), in this context used as an epicardial marker(Perez-Pomares and Pompa, 2011). Using elegant mouse modeltracing techniques Red-Horse (Red-Horse et al., 2010) confirmedthat EC are derived from the sinus venosus. Subsequently, theseauthors claimed that these venous endothelial cells dedifferentiateand are stepwise converted (‘‘reprogrammed’’) into arteries, capil-laries and veins. We have shown, however, that the sinus venosusderived endothelial cells express the ‘‘arterial’’ Notch1 (Fig. 4 b–d)underlining the plasticity of the embryonic microvascular endothe-lial 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.

osus (SV), atrium (A), ventricle (V) and outflow tract (OFT). The proepicardial organ

and SV to the surface of the ventricle (not by way of the atrium) under coverage of

dothelium (arrows) of the SV (see magnification in (c.) and the lining of the liver

ers of the Notch/Delta-like/Jagged pathway are unsuitable to discriminate at this

d): 10 mm. (For interpretation of the references to color in this figure legend, the

A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53 47

A recent report keeps the discussion on the origin of the coronaryECs alive as distinct compartments of the pro-epicardial organ giverise 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 fromdifferentiated EPDCs (Dettman et al., 1998; Mellgren et al., 2008;Vrancken Peeters et al., 1999). Recent information sets the origin ofthe pericyte and SMC apart (Chong et al., 2011). From quail–chicken chimera studies the timing of EMT and required endothe-lial-EPDC cell–cell interaction has become evident. In this modelsolitary SMCs have not been encountered (Vrancken Peeters et al.,1999). Whether the differentiation into coronary artery and veinsis primarily driven by an autonomous endothelial differentiationprocess or whether hemodynamic forces drive the arterial pheno-type of the endothelial cells with subsequent recruitment of SMCsneeds further study. EPDC differentiation into SMCs is regulated bymany genes including serum response factor (Landerholm et al.,1999), and PDGFRb and their ligands (Mellgren et al., 2008; VanDen Akker et al., 2008b). Recent transgenic mouse studies supporta separate PDGFRa positive EPDC population to be sensitive to SMCdifferentiation, but the role of local environmental factors cannotbe excluded yet (Smith et al., 2011). Remarkable is the highplasticity of the endothelium and the underlying SMCs in the moresuperficial venous and deeper located arterial network when thePDGF pathway is disturbed (Van Den Akker et al., 2008b). Othergrowth factors play an important role in coronary vascular differ-entiation 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 recentlyreviewed (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 epicardialreporters, support the differentiation of a subset of EPDCs into acell with a myocardial phenotype. It cannot be excluded that thegenetic tagging of epicardial cell lineages contains flaws. We andothers (Perez-Pomares and Pompa, 2011) argue that the condi-tional WT1 reporter mouse model cannot be used for selectivelineage tracing of WT1 positive epicardium as some of the SHFprogenitors of cardiomyocytes in the SHF are positive as well(Jongbloed et al. 2011). Also the use of the Tbx18 gene as aselective epicardial reporter gene has been refuted (Christoffelset al., 2009). Several other data do not support the EPDC-cardiomyocyte transition, including quail–chicken chimera studies(Gittenberger-de Groot et al., 1998; Manner et al., 2001). ManyWT-1 positive cells can be found not only in the coelomic lining ofthe pericardial coelomic cavity, but also in the underlying meso-derm of the SHF (Fig. 3c). This mesoderm, based on a BMP and FGFbalance can differentiate into both a myocardial and an epicardialpopulation (Kruithof et al., 2006); therefore, a common progenitorof both cell lines is likely (van Wijk and van den Hoff, 2010). It cannot be excluded that relative undifferentiated cardiomyocyteprogenitor cells (CMPCs) (Smits et al., 2009; Timmers et al.,2011) that are present within the venous pole SHF populationmay have a WT-1 positive lineage background. Studies using adulthuman EPDCs show that this population has mesenchymal stemcell characteristics but is already more cardiac committed withpositive cardiac markers such as Gata4 and cTNT (van Tuyn et al.2007). In the section on the potential of EPDCs in adult stagesduring activation and disease this aspect of EPDC cardiomyocytetransition will be further discussed.

4.6. The Purkinje fiber

The Purkinje fiber is a specialized cardiomyocyte inducedby endothelin produced by endothelin-converting enzymeexpressing endothelium and endocardium (Eid et al., 1994;Gourdie et al., 1998; Mikawa et al., 2003). An instructive rolefor the EPDC in Purkinje fiber differentiation in the cardiac wallbecame evident during vPEO tracing and inhibition experiments.We have postulated an essential interaction between EPDCs andthe 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 develop-ment of the heart and great vessels. Complete inhibition of theoutgrowth of the sinus venosus located vPEO leads to severecardiac malformations (Gittenberger-de Groot et al., 2000). Theseinclude absence of vEP, aberrant and extensive outgrowth of aEPover the myocardial outflow tract, deficient looping with a wideinner curvature, absent ventricular and outflow tract septationand atrioventricular cushion formation, combined with a thin(2 layer) compact myocardium. This combination, due to lack ofepicardial covering, does not develop coronary vasculature and isembryo-lethal. Experiments in which the PEO outgrowth is onlypartially 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 arereminiscent of congenital heart malformations seen in the humanpopulation. The essential role of the epicardium can also bededucted from (conditional) transgenic and knock out mousemodels in which genes and regulatory sequences, relevant for thevarious stages of epicardial development, have been mutated.These vary from inhibition of spreading as in the VCAM1 (Kweeet 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) andPDGFRa and b (Bax et al., 2010; Mellgren et al., 2008) mutants. Inthe majority of these models a concurrent hypoplasia of the vPEOis apparent (Bax et al., 2010; Mahtab et al., 2008) which canaccount for the smaller number of epicardial cells.

No epicardium specific gene, useful for lineage tracing orspecific knock-out strategies, has been determined, although largescreen microarray studies have been performed (Bochmann et al.,2010). It is challenging to attribute specific cardiac diseases andmalformations to epicardial and PEO developmental defects. It is,however, possible to describe the role of the epicardium in somecardiac defects in avian and mouse models and postulate theirrole in several human cardiac diseases. As will be clear from Fig. 1we are dealing with composite structures in which, among othercardiac cell types, epicardial cells play an essential role.

5.1. Non-compaction cardiomyopathy

In both, avian (Gittenberger-de Groot et al., 2000) and mousemodels (Bax et al., 2010; Mahtab et al., 2008) normal developmentof the compact myocardium depends on proper interactionsbetween cardiomyocytes, EPDCs and the secreted extracellularmatrix. Primary epicardial derived abnormalities like in the WT-1null mutant (Martinez-Estrada et al., 2010) exert their influence onthe myocardial cells through WT-1 dependent Raldh2, whereby RAis not delivered to the myocardium as is elegantly proven for theRxRa mutant (Guadix et al., 2011; Jenkins et al., 2005). Develop-ment of the normal compact myocardium, linked to the epi-cardium derived interstitial fibroblast population (Ieda et al., 2009),

A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–5348

can also be disturbed when the primary problem is cardiomyocytelinked. Many cardiomyopathies, usually with a hypertrophic myo-cardium, are based on mutations of cardiomyocyte specific genes(Maron et al., 2006). The most relevant human cardiomyopathythat could result from a primary abnormal EPDC function isthe primary left ventricular non-compaction cardiomyopathy(Lie-Venema et al., 2007). The morphological substrate consistsof a spongious myocardium often including the ventricularseptum. Differences in timing and amount of RV and LV invasionby EPDCs might account for the preferential problem in the LV.

With respect to congenital heart disease a spongious structureof the ventricular septum can be related to muscular VSDs varyingfrom small isolated VSDs, which tend to close spontaneously, tomultiple muscular VSDs. Recent data open the option that in thehypoplastic left heart syndrome and the related borderline leftventricles the epicardial to myocardial interaction is abnormalleading to myocardial pathology (Mahtab et al., in press)

5.2. Endocardial fibroelastosis

The hypoplastic ventricle, with a restricted outflow and aconcomitant high pressure in cardiac anomalies such as pulmonaryatresia without VSD and the hypoplastic left heart syndrome,presents with a thick layer of endocardial fibroelastosis. The layerconsists of fibroblasts and myofibroblasts that could develop fromcells derived from endocardial EMT possibly in combination withthe population of EPDCs that have been deposited subendo-cardially during development (Gittenberger-de Groot et al., 1998)(Fig. 3a and b).

5.3. Cardiac conduction system anomalies

With respect to the origin of conduction system disturbances,we distinguish congenitally determined and acquired defects,realizing that the main components of the cardiac conductionsystem are myocardial in origin (Jongbloed et al., 2012). The roleof the epicardium and EPDCs is currently restricted to aninductive influence on Purkinje fiber differentiation. Clinically, ithas been postulated that aspects of the genetically determinedlong-QT syndrome might have a link to abnormal Purkinje fiberfunction. Indirectly, the deficient formation of the annulus fibrosiswith persistent atrioventricular myocardial connections, so-calledaccessory pathways, can lead to re-entry tachycardias. PEOinhibition in avian embryos demonstrated deficient atrioventri-cular isolation. The required shift from a base-to-apex to anapex-to-base conduction is delayed during development (Kolditzet al., 2007). Mouse models in which normal epicardial spreadingand EPDC formation is disturbed in conjunction with deficientannulus fibrosis formation have not been reported, whereas amodel with abnormal persistence of a myocardial phenotype hasbeen described for the Tbx2 mutant (Aanhaanen et al., 2011).

5.4. Valvulopathies

Cardiac valves can be distinguished in the arterial semilunarand the atrioventricular valves. Anomalies consist of abnormalanlage and morphology such as seen in bicuspid aortic valve andcommon atrioventricular valves. Histological abnormalities withdysplastic valves are considered a different entity which can befound in combination with an abnormal morphology. EPDCsmigrate into the atrioventricular cushions (Gittenberger-deGroot et al., 1998; Manner et al., 2001; Wessels et al., 2012)).PEO inhibition can lead to complete absence of AV valve forma-tion (Gittenberger-de Groot et al., 2000). Less severe cases with anabnormal differentiation including deficient undermining of thevalve leaflet are reminiscent of Ebstein’s anomaly of the tricuspid

valve. Interestingly, in our avian model this is observed incombination with accessory pathways (Lie-Venema et al., 2007),an association also reported in human patients (Attenhofer Jostet al., 2007).

EPDCs of aEP origin are encountered in the endocardial out-flow tract cushions (Fig. 2c). We postulate that they act throughthe important role of EPDCs in Notch signaling (del Monte et al.,2011; Grieskamp et al., 2011; Van Den Akker et al. 2007; Van DenAkker et al., in press) and thus influence bicuspid aortic valveformation, which has been linked in patients to a Notch 1 muta-tion (McKellar et al., 2007).

5.5. Coronary vascular anomalies

Coronary vascular development from the undifferentiated micro-vascular endothelial plexus to the differentiated coronary arteriesand veins depends on epicardial development. Experimental studieshampering normal coronary development and differentiation lead toa number of anomalies that link congenital pattern variations toabnormal ventriculo-coronary-arterial communications (VCAC orfistulae) as described in the human fetus and neonate. PEO inhibi-tion as well as delayed epicardial spreading in PEO ablation/rescueexperiments show single coronary ostia as well as pinpoint coronaryorifice formation. VCAC are found in animal models with completeabsent coronary arterial orifices in the aorta (Eralp et al., 2005; Lie-Venema et al., 2003). This resembles the human coronary malfor-mations found in pulmonary atresia without VSD and VCAC, whichhas been postulated to be a primary coronary vascular disease(Gittenberger-de Groot et al., 2001).

6. Cardiovascular repair

6.1. Myocardial infarction

Based on the potential of the epicardium and EPDCs duringnormal development it is tempting to attribute a role in repair ofthe cardiac wall and its vascularization in various adult cardiacdisease processes. The main cardiac disease studied in thisrespect is ischemic heart disease with subsequent myocardialfibrosis and heart failure. The primary cause is myocardialinfarction (MI) after a coronary obstruction or occlusion due toatherosclerotic processes. If the epicardium and its EPDCs couldrecapitulate their embryonic program and acquire multipoteni-tal stem cell characteristics, benefit could be achieved to themyocardial recuperation as well as stabilization of the novelangiogenesis derived capillaries by addition of new SMCs.Several research approaches have focused on this aspect of thepotential of the adult epicardial cell after myocardial infarction.Various lines of research can be distinguished. A first cohort ofstudies investigated the potential of the native epicardium afterMI. These studies were performed in mouse models followingexperimental myocardial infarction. It has been shown that ac-kit positive subepicardial EPDC population is found, indicatingrenewed epicardial activity and acquisition of stem cell char-acteristics (Limana et al., 2010, 2011). Use of green fluorescentprotein tracing showed that the c-kit positive cells were epicar-dium derived. A different study using a retroviral fluorescentKatushka labeling of quiescent epicardial cells that after MIbecame cuboidal, showed EMT followed by EPDCs that migratedinto the myocardium. They differentiated after 4 day into amyofibroblast phenotype (Gittenberger-de Groot et al., 2010)but were not followed for further differentiation. The activatedepicardium and EPDCs became WT1 positive not only in the areadirectly bordering the MI but also in more remote zones. It couldnot be excluded that resident cardiac fibroblasts were

A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–53 49

upregulating their WT1 expression. Further research is necessaryto explain this phenomenon. In a mouse model of MI thereactivation of EMT of the adult epicardium was shown withoutdifferentiation of the EPDCs into a myocardial or endothelialphenotype; however, paracrine factors stimulating, e.g., angio-genesis were found (Zhou et al., 2011). Studies by Riley and co-workers emphasized the regenerative capacity of EPDCs after MIby stimulation with Thymosin b4, showing renewed angiogen-esis and arteriogenesis (Smart et al., 2007). Performing primingby Thymosin b4 followed by myocardial infarction in the WT-1reporter mouse, the EPDCs differentiate into a cardiomyocytephenotype (Smart et al., 2011). These findings could not beconfirmed in a MI mouse model in which Thymosin b4 wasprovided concomitant with the MI induction. EPDCs did notdifferentiate into a cardiomyocyte or endothelial phenotype(Zhou et al., 2012). A recent study shows that Thymosin b4 isnot 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 epicardialcells are cultured in combination with cardiomyocytes. It hasbeen shown already in Eid et al. (1992) that activity of epicardialcells modifies the cardiomyocyte phenotype. Recently, in a co-culture combining chicken EPDCs and rat neonatal cardiomyo-cytes, this was further proven with a marked role for EPDCs onmyocardial alignment and contraction (Weeke-Klimp et al.,2010). A very direct approach to influence repair of the heartafter MI was provided by using human adult epicardial cells(Winter et al., 2007b). These cells were derived from the atrialsurface and cultured in vitro. The WT-1 positive epicardial cellschanged their phenotype from a cobblestone epithelium into

Fig. 5. (a)–(d) Tranverse sections of the wall of the adult ascending aorta just distal of th

valve. There is a regular staining of the smooth muscle cells with a smooth muscle actin

made up of loose fibrous tissue and contains vasa vasorum of which the wall of the sm

epicardium (aEP), covering the adventitia. The aEP (blue) is negative for the epicardial m

ascending aorta. Both in the intima and media there is marked loss of 1A4. The adventitia

in detail (d) the overlying aEP is positive for WT-1 (brown) and there are signs of epith

adventitia. Bars: (a)–(d): 50 mm. (For interpretation of the references to color in this figu

spindle shape. These human EPDCs acquired characteristicsresembling mesenchymal stem cells. Their cardiac commitmentwas shown by expression of GATA4 and cTNT, while cardiomyo-cyte-specific genes were absent (van Tuyn et al., 2007). Injectionof these adult human EPDCs in immune incompetent mice intothe borderzone of the ischemic area resulted in a markedimprovement of cardiac function as measured by cardiac MRI.This improvement was found already 2 day after injectionextending 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 indica-tive of repair was observed. Experiments with an injection ofEPDCs combined with adult human cardiomyocyte progenitors(CMPCs) aimed at induction of cardiomyocyte regeneration(Winter et al., 2009). The results show an additive effect of theseparate injections on remodeling, although no new cardiac celltypes (endothelial cells, interstitial fibroblasts, smooth musclecells or cardiomyocytes) could be traced to human origin. Thesedata can now be supported by more recent studies on thedifferentiation capacities of the EPDCs. The capacities, foundwithin the normal embryonic in vivo environment seem to beretained in adult life and disease states The conclusion is thatmany of the positive effects of EPDCs either after injection or bystimulation of the native epicardial covering of the heart are dueto a paracrine mechanism (Limana et al., 2011; Winter et al.,2007b, 2009; Zhou et al., 2011). These findings have greatpotential for future therapeutic approaches, either drug or cellbased, that stimulate the native epicardium in repair of theischemic cardiac wall. An underdeveloped area is the priming ofpericardium based grafts for use in arterial or cardiac repair.

e semilunar valve level. (a) Normal adult aortic wall of a case with a tricuspid aortic

(SMA) using the 1A4 antibody in intima (I) and the media (M). The adventitia (A) is

all arteries also stain positive for 1A4. (b) Detail (see location in a.) of the arterial

arker WT-1. (c) Case with a tricuspid aortic valve and aneurysm formation of the

does not show marked differences as compared to the normal aortic wall. However

elial-mesenchymal-transition with WT-1 positive EPDCs (arrows) in the underlying

re legend, the reader is referred to the web version of this article.)

A.C. Gittenberger-de Groot et al. / Differentiation 84 (2012) 41–5350

6.2. Thoracic aortic aneurysm

Thoracic aortic aneurysm formation (Lindsay and Dietz, 2011)is a disease of the ascending aorta with loss of vascular wallstructure and smooth muscle cells accompanied by cytolyticnecrosis and in severe cases to dissection. Based on our hypoth-esis that aEP might play a role in maintaining vessel wall integrityalso in adult life, comparable to the reactivation of the adult cEPafter myocardial infarction, we have set up a small study. Wecompared the immunohistological characteristics for the epicar-dial marker WT-1 as well as TGFb and phospho-Smad2 of thehuman adult thoracic aortic wall. We investigated the ascendingaorta with (1) a normal aortic wall and tricuspid aortic valve(TAV) and (2) an aortic aneurysm and TAV. The results based onfive cases for each group showed a quiescent aEP (based on WT-1staining) in group 1 and marked activation with EMT in group 2(Fig. 5 a–d). These data were supported by TGFb expression thatwas far more marked particularly in the intima in cases with TAVand aneurysm (group2). These preliminary results are an indica-tion that the epicardium of the arterial pole is capable ofreactivation and renewed EMT supporting repair in the adultstage. Further research will reveal whether this repair phenom-enon is also found in other groups of thoracic aneurysm formatione.g. based on Loeys-Dietz and Marfan syndromes (Dietz andPyeritz, 1995) and in combination with bicuspid aortic valve(McKellar et al., 2007).

7. Conclusion

In conclusion: The epicardium has acquired a dominant posi-tion in our understanding of proper cardiac development. Itseffect is visible in most major processes including looping,myocardial maturation, septation, valve formation and coronaryvascular development and patterning. A complicating factor inthe study of the epicardium, using the current sophisticatedmouse models, is that no specific genes for epicardium or EPDCshave been identified, as yet (Bochmann et al., 2010). Severalreviews appeared in the last year that focus on the signalingpathways of the major growth factor families that all exert theirinfluence on the various aspects of EMT, migration and differ-entiation (Perez-Pomares and Pompa, 2011; Riley and Smart,2011). Data are accumulating that in some forms of cardiacdisease the epicardium is capable of reactivating the embryonicprogram (Gittenberger-de Groot et al., 2010; Limana et al., 2011;Zhou et al., 2011) with beneficial effects on cardiac function. ^Themechanism seems to be mainly cell-autonomous with supportiveactions for cell-cell interactions (Winter et al. 2009). Thesefindings invigorate the research into discovering the activecompounds that might replace or support cell transplantationand biomedical devices in therapeutic approaches.

Acknowledgments

We would like to thank Bert J Wisse for preparation of thefigures and Ron Slagter for his excellent medical illustration work.

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