Elizabeth M. Winter

Origin, Fate, and Function of Epicardium-Derived Cells (EPDCs) in Normal and Abnormal Cardiac Development

During heart development, cells of the primary and secondary heart field give rise to the myocardial component of the heart. The neural crest and epicardium provide the heart with a considerable amount of nonmyocardial cells that are indispensable for correct heart development. During the past 2 decades, the importance of epicardium-derived cells (EPDCs) in heart formation became increasingly clear. The epicardium is embryologically formed by the outgrowth of proepicardial cells over the naked heart tube. Following epithelial-mesenchymal transformation, EPDCs form the subepicardial mesenchyme and subsequently migrate into the myocardium, and differentiate into smooth muscle cells and fibroblasts. They contribute to the media of the coronary arteries, to the atrioventricular valves, and the fibrous heart skeleton. Furthermore, they are important for the myocardial architecture of the ventricular walls and for the induction of Purkinje fiber formation. Whereas the exact signaling cascades in EPDC migration and function still need to be elucidated, recent research has revealed several factors that are involved in EPDC migration and specialization, and in the cross-talk between EPDCs and other cells during heart development. Among these factors are the Ets transcription factors Ets-1 and Ets-2. New data obtained with lentiviral antisense constructs targeting Ets-1 and Ets-2 specifically in the epicardium indicate that both factors are independently involved in the migratory behavior of EPDCs. Ets-2 seems to be especially important for the migration of EPDCs into the myocardial wall, and to subendocardial positions in the atrioventricular cushions and the trabeculae. With respect to the clinical importance of correct EPDC development, the relation with coronary arteriogenesis has been noted well before. In this review, we also propose a role for EPDCs in cardiac looping, and emphasize their contribution to the development of the valves and myocardial architecture. Lastly, we focus on the congenital heart anomalies that might be caused primarily by an epicardial developmental defect.

Epicardial Cells of Human Adults Can Undergo an Epithelial‐to‐Mesenchymal Transition and Obtain Characteristics of Smooth Muscle Cells In Vitro

Myocardial and coronary development are both critically dependent on epicardial cells. During cardiomorphogenesis, a subset of epicardial cells undergoes an epithelial‐to‐mesenchymal transition (EMT) and invades the myocardium to differentiate into various cell types, including coronary smooth muscle cells and perivascular and cardiac interstitial fibroblasts. Our current knowledge of epicardial EMT and the ensuing epicardium‐derived cells (EPDCs) comes primarily from studies of chick and mouse embryonic development. Due to the absence of an in vitro culture system, very little is known about human EPDCs. Here, we report for the first time the establishment of cultures of primary epicardial cells from human adults and describe their immunophenotype, transcriptome, transducibility, and differentiation potential in vitro. Changes in morphology and β‐catenin staining pattern indicated that human epicardial cells spontaneously undergo EMT early during ex vivo culture. The surface antigen profile of the cells after EMT closely resembles that of subepithelial fibroblasts; however, only EPDCs express the cardiac marker genes GATA4 and cardiac troponin T. After infection with an adenovirus vector encoding the transcription factor myocardin or after treatment with transforming growth factor‐β1 or bone morphogenetic protein‐2, EPDCs obtain characteristics of smooth muscle cells. Moreover, EPDCs can undergo osteogenesis but fail to form adipocytes or endothelial cells in vitro. Cultured epicardial cells from human adults recapitulate at least part of the differentiation potential of their embryonic counterparts and represent an excellent model system to explore the biological properties and therapeutic potential of these cells.

Preservation of Left Ventricular Function and Attenuation of Remodeling After Transplantation of Human Epicardium-Derived Cells Into the Infarcted Mouse Heart

Background— Proper development of compact myocardium, coronary vessels, and Purkinje fibers depends on the presence of epicardium-derived cells (EPDCs) in embryonic myocardium. We hypothesized that adult human EPDCs might partly reactivate their embryonic program when transplanted into ischemic myocardium and improve cardiac performance after myocardial infarction. Methods and Results— EPDCs were isolated from human adult atrial tissue. Myocardial infarction was created in immunodeficient mice, followed by intramyocardial injection of 4×105 enhanced green fluorescent protein–labeled EPDCs (2-week survival, n=22; 6-week survival, n=15) or culture medium (n=24 and n=18, respectively). Left ventricular function was assessed with a 9.4T animal MRI unit. Ejection fraction was similar between groups on day 2 but was significantly higher in the EPDC-injected group at 2 weeks (short term), as well as after long-term survival at 6 weeks. End-systolic and end-diastolic volumes were significantly smaller in the EPDC-injected group than in the medium-injected group at all ages evaluated. At 2 weeks, vascularization was significantly increased in the EPDC-treated group, as was wall thickness, a development that might be explained by augmented DNA-damage repair activity in the infarcted area. Immunohistochemical analysis showed massive engraftment of injected EPDCs at 2 weeks, with expression of α-smooth muscle actin, von Willebrand factor, sarcoplasmic reticulum Ca2+-ATPase, and voltage-gated sodium channel (α-subunit; SCN5a). EPDCs were negative for cardiomyocyte markers. At 6-weeks survival, wall thickness was still increased, but only a few EPDCs could be detected. Conclusions— After transplantation into ischemic myocardium, adult human EPDCs preserve cardiac function and attenuate ventricular remodeling. Autologous human EPDCs are promising candidates for clinical application in infarcted hearts.

Cardiovascular development: towards biomedical applicability

Abstract.During cardiogenesis, the epicardium grows from the proepicardial organ to form the outermost layer of the early heart. Part of the epicardium undergoes epithelial-mesenchymal transformation, and migrates into the myocardium. These epicardium- derived cells differentiate into interstitial fibroblasts, coronary smooth muscle cells, and perivascular fibroblasts. Moreover, epicardium-derived cells are important regulators of formation of the compact myocardium, the coronary vasculature, and the Purkinje fiber network, thus being essential for proper cardiac development. The fibrous structures of the heart such as the fibrous heart skeleton and the semilunar and atrioventricular valves also depend on a contribution of these cells during development. We hypothesise that the essential properties of epicardium-derived cells can be recapitulated in adult diseased myocardium. These cells can therefore be considered as a novel source of adult stem cells useful in clinical cardiac regeneration therapy.

Epicardium-derived cells in cardiogenesis and cardiac regeneration

Abstract.During cardiogenesis, the epicardium grows from the proepicardial organ to form the outermost layer of the early heart. Part of the epicardium undergoes epithelial-mesenchymal transformation, and migrates into the myocardium. These epicardium- derived cells differentiate into interstitial fibroblasts, coronary smooth muscle cells, and perivascular fibroblasts. Moreover, epicardium-derived cells are important regulators of formation of the compact myocardium, the coronary vasculature, and the Purkinje fiber network, thus being essential for proper cardiac development. The fibrous structures of the heart such as the fibrous heart skeleton and the semilunar and atrioventricular valves also depend on a contribution of these cells during development. We hypothesise that the essential properties of epicardium-derived cells can be recapitulated in adult diseased myocardium. These cells can therefore be considered as a novel source of adult stem cells useful in clinical cardiac regeneration therapy.

The arterial and cardiac epicardium in development, disease and repair.

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.

A New Direction for Cardiac Regeneration Therapy Application of Synergistically Acting Epicardium-Derived Cells and Cardiomyocyte Progenitor Cells

Background— Adult human epicardium-derived cells (EPDCs), transplanted into the infarcted heart, are known to improve cardiac function, mainly through paracrine protection of the surrounding tissue. We hypothesized that this effect might be further improved if these supportive EPDCs were combined with cells that could possibly supply the ischemic heart with new cardiomyocytes. Therefore, we transplanted EPDCs together with cardiomyocyte progenitor cells that can generate mature cardiomyocytes in vitro. Methods and Results— EPDCs and cardiomyocyte progenitor cells were isolated from human adult atrial appendages, expanded in culture, and transplanted separately or together into the infarcted mouse myocardium (total cell number, 4×105). Cardiac function was determined 6 weeks later (9.4T MRI). Coculturing increased proliferation rate and production of several growth factors, indicating a mutual effect. Cotransplantation resulted in further improvement of cardiac function compared with single cell-type recipients ( P <0.05), which themselves demonstrated better function than vehicle-injected controls ( P <0.05). However, in contrast to our hypothesis, no graft-derived cardiomyocytes were observed within the 6-week survival, supporting that not only EPDCs but also cardiomyocyte progenitor cells acted in a paracrine manner. Because injected cell number and degree of engraftment were similar between groups, the additional functional improvement in the cotransplantation group cannot be explained by an increased amount of secreted factors but rather by an altered type of secretion. Conclusion— EPDCs and cardiomyocyte progenitor cells synergistically improve cardiac function after myocardial infarction, probably instigated by complementary paracrine actions. Our results demonstrate for the first time that synergistically acting cells hold great promise for future clinical regeneration therapy. Received January 7, 2009; accepted July 22, 2009.Background—Adult human epicardium-derived cells (EPDCs), transplanted into the infarcted heart, are known to improve cardiac function, mainly through paracrine protection of the surrounding tissue. We hypothesized that this effect might be further improved if these supportive EPDCs were combined with cells that could possibly supply the ischemic heart with new cardiomyocytes. Therefore, we transplanted EPDCs together with cardiomyocyte progenitor cells that can generate mature cardiomyocytes in vitro. Methods and Results—EPDCs and cardiomyocyte progenitor cells were isolated from human adult atrial appendages, expanded in culture, and transplanted separately or together into the infarcted mouse myocardium (total cell number, 4×105). Cardiac function was determined 6 weeks later (9.4T MRI). Coculturing increased proliferation rate and production of several growth factors, indicating a mutual effect. Cotransplantation resulted in further improvement of cardiac function compared with single cell-type recipients (P<0.05), which themselves demonstrated better function than vehicle-injected controls (P<0.05). However, in contrast to our hypothesis, no graft-derived cardiomyocytes were observed within the 6-week survival, supporting that not only EPDCs but also cardiomyocyte progenitor cells acted in a paracrine manner. Because injected cell number and degree of engraftment were similar between groups, the additional functional improvement in the cotransplantation group cannot be explained by an increased amount of secreted factors but rather by an altered type of secretion. Conclusion—EPDCs and cardiomyocyte progenitor cells synergistically improve cardiac function after myocardial infarction, probably instigated by complementary paracrine actions. Our results demonstrate for the first time that synergistically acting cells hold great promise for future clinical regeneration therapy.

Forced Myocardin Expression Enhances the Therapeutic Effect of Human Mesenchymal Stem Cells After Transplantation in Ischemic Mouse Hearts

Human mesenchymal stem cells (hMSCs) have only a limited differentiation potential toward cardiomyocytes. Forced expression of the cardiomyogenic transcription factor myocardin may stimulate hMSCs to acquire a cardiomyogenic phenotype, thereby improving their possible therapeutic potential. hMSCs were transduced with green fluorescent protein (GFP) and myocardin (hMSCmyoc) or GFP and empty vector (hMSC). After coronary ligation in immune‐compromised NOD/scid mice, hMSCmyoc (n = 10), hMSC (n = 10), or medium only (n = 12) was injected into the infarct area. Sham‐operated mice (n = 12) were used to determine baseline characteristics. Left ventricular (LV) volumes and ejection fraction (EF) were serially (days 2 and 14) assessed using 9.4‐T magnetic resonance imaging. LV pressure‐volume measurements were performed at day 15, followed by histological evaluation. At day 2, no differences in infarct size, LV volumes, or EF were observed among the myocardial infarction groups. At day 14, left ventricular ejection fraction in both cell‐treated groups was preserved compared with the nontreated group; in addition, hMSCmyoc injection also reduced LV volumes compared with medium injection (p < .05). Furthermore, pressure‐volume measurements revealed a significantly better LV function after hMSCmyoc injection compared with hMSC treatment. Immunohistochemistry at day 15 demonstrated that the engraftment rate was higher in the hMSCmyoc group compared with the hMSC group (p < .05). Furthermore, these cells expressed a number of cardiomyocyte‐specific markers not observed in the hMSC group. After myocardial infarction, injection of hMSCmyoc improved LV function and limited LV remodeling, effects not observed after injection of hMSC. Furthermore, forced myocardin expression improved engraftment and induced a cardiomyocyte‐like phenotype hMSC differentiation.

Cell tracking using iron oxide fails to distinguish dead from living transplanted cells in the infarcted heart

Recently, debate has arisen about the usefulness of cell tracking using iron oxide–labeled cells. Two important issues in determining the usefulness of cell tracking with MRI are generally overlooked; first, the effect of graft rejection in immunocompetent models, and second, the necessity for careful histological confirmation of the fate of the labeled cells in the presence of iron oxide. Therefore, both iron oxide–labeled living as well as dead epicardium‐derived cells (EPDCs) were investigated in ischemic myocardium of immunodeficient non‐obese diabetic (NOD)/acid: non‐obese diabetic severe combined immunodeficient (NOD/scid) mice with 9.4T MRI until 6 weeks after surgery, at which time immunohistochemical analysis was performed. In both groups, voids on MRI scans were observed that did not change in number, size, or localization over time. Based on MRI, no distinction could be made between living and dead injected cells. Prussian blue staining confirmed that the hypointense spots on MRI corresponded to iron‐loaded cells. However, in the dead‐EPDC recipients, all iron‐positive cells appeared to be macrophages, while the living‐EPDC recipients also contained engrafted iron‐loaded EPDCs. Iron labeling is inadequate for determining the fate of transplanted cells in the immunodeficient host, since dead cells produce an MRI signal indistinguishable from incorporated living cells. Magn Reson Med 63:817–821, 2010.

Epicardium-derived cells enhance proliferation, cellular maturation and alignment of cardiomyocytes

During heart development, cells from the proepicardial organ spread over the naked heart tube to form the epicardium. From here, epicardium-derived cells (EPDCs) migrate into the myocardium. EPDCs proved to be indispensable for the formation of the ventricular compact zone and myocardial maturation, by largely unknown mechanisms. In this study we investigated in vitro how EPDCs affect cardiomyocyte proliferation, cellular alignment and contraction, as well as the expression and cellular distribution of proteins involved in myocardial maturation. Embryonic quail EPDCs induced proliferation of neonatal mouse cardiomyocytes. This required cell-cell interactions, as proliferation was not observed in transwell cocultures. Western blot analysis showed elevated levels of electrical and mechanical junctions (connexin43, N-cadherin), sarcomeric proteins (Troponin-I, alpha-actinin), extracellular matrix (collagen I and periostin) in cocultures of EPDCs and cardiomyocytes. Immunohistochemistry indicated more membrane-bound expression of Cx43, N-cadherin, the mechanotransduction molecule focal adhesion kinase, and higher expression of the sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a). Newly developed software for analysis of directionality in immunofluorescent stainings showed a quantitatively determined enhanced cellular alignment of cardiomyocytes. This was functionally related to increased contraction. The in vitro effects of EPDCs on cardiomyocytes were confirmed in three reciprocal in vivo models for EPDC-depletion (chicken and mice) in which downregulation of myocardial N-cadherin, Cx43, and FAK were observed. In conclusion, direct interaction of EPDCs with cardiomyocytes induced proliferation, correct mechanical and electrical coupling of cardiomyocytes, ECM-deposition and concurrent establishment of cellular array. These findings implicate that EPDCs are ideal candidates as adjuvant cells for cardiomyocyte integration during cardiac (stem) cell therapy.