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First published online September 21, 2006
Stem Cells Vol. 25 No. 2 February 2007, pp. 271 -278
doi:10.1634/stemcells.2006-0366; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

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

John van Tuyna,b,c, Douwe E. Atsmaa, Elizabeth M. Winterc,d, Ietje van der Velde-van Dijkeb, Daniel A. Pijnappelsa, Noortje A.M. Baxa,d, Shoshan Knaän-Shanzerb, Adriana C. Gittenberger-de Grootd, Robert E. Poelmannd, Arnoud van der Laarsea, Ernst E. van der Walla, Martin J. Schalija, Antoine A.F. de Vriesb

aDepartment of Cardiology, Leiden University Medical Center, Leiden, The Netherlands;
bVirus and Stem Cell Biology Laboratory, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands;
cInteruniversity Cardiology Institute of the Netherlands, Utrecht, The Netherlands;
dDepartment of Anatomy, Leiden University Medical Center, Leiden, The Netherlands

Key Words. Osteoblast • Myogenesis • Mesenchymal stem cell • Adipogenesis • Adult stem cells • Angiogenesis • In vitro culture Epithelial-to-mesenchymal transition

Correspondence: Douwe E. Atsma, M.D., Ph.D., Department of Cardiology, Leiden University Medical Center, Leiden, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Telephone: +31715269245; Fax: +31715268270; e-mail: d.e.atsma{at}lumc.nl

Received June 16, 2006; accepted for publication September 14, 2006.
First published online in STEM CELLS EXPRESS   September 21, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 
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 ß-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-ß1 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 
Cardiomorphogenesis is an intricate process, involving cells from different embryonic origins that form distinct structures within the heart. The epicardium, which comprises the outermost layer of the heart and consists of mesothelial cells, is derived from a temporary structure of precursor cells with different developmental potentials termed the proepicardium (reviewed in [1]). Cells from the proepicardium migrate to and over the myocardial surface well after looping of the primitive heart tube has commenced to give rise to the epicardium ([2] and references therein). A fraction of these epicardial cells undergoes an epithelial-to-mesenchymal transition (EMT) and invades the subepicardial, myocardial, and subendocardial layers of the heart as well as the atrioventricular cushions [3–6]. These cells are referred to as epicardium-derived cells (EPDCs) [6] and have been shown by various labeling techniques to give rise to coronary smooth muscle cells (SMCs) and perivascular and cardiac interstitial fibroblasts [4, 5, 7–9]. The origin of coronary endothelial cells is still under debate. Quail-to-chick transplantation experiments [10, 11] as well as in ovo proepicardial labeling studies [8] support the presence of a separate population of (hem)angioblasts in the proepicardium, which originate from the primitive liver and/or the border area between the proepicardium and the liver. Aside from their material contribution to the different tissues of the heart, EPDCs have important regulatory functions in the development of the myocardium. Indeed, ablation or functional impairment of the proepicardium and/or its descendants by microsurgical or genetic manipulations resulted in myocardial (and coronary) dysmorphogenesis [12–16]. Finally, a role for cells of proepicardial origin in the morphogenesis of the atrioventricular valves, the formation of the interventricular septum, the differentiation of the Purkinje fibers, and the remodeling of the cardiac outflow tract has been proposed [6, 12, 13].

Recently, an epicardial cell line from rat was described [17]. These polarized mesothelial cells could be induced to undergo EMT and part of them had the capacity to form SMCs in vitro. This prompted us to investigate whether epicardial cells from human adults could be cultured as well and whether they displayed similar characteristics as embryonic EPDCs. The developmental cues that promote epicardial EMT and the subsequent differentiation of EPDCs are still unknown. Nonetheless, transforming growth factor-ß (TGF-ß) was recently shown to induce EMT and SMC differentiation in avian epicardial explants [18]. Another potential way to provoke SMC differentiation is by using the transcription factor (TF) myocardin [19]. Myocardin plays a pivotal role in both smooth and cardiac muscle differentiation, and ectopic expression of a recombinant myocardin gene in susceptible cell types results in the activation of a wide variety of heart and smooth muscle genes [19–22].

Here, we report the in vitro culture and subsequent characterization, using various cell biological, immunological, and molecular biological assays, of EPDCs derived from human adults. These studies reveal that primary human epicardial cells clearly differ from both primary human foreskin fibroblasts (FFBs) and mesenchymal stem cells (MSCs) in their surface antigen and gene expression profiles as well as in their differentiation potential. Moreover, our findings that the in vitro cultured primary human epicardial cells undergo EMT and, after stimulation with TGF-ß1 or bone morphogenetic protein-2 (BMP-2) or following forced expression of myocardin, obtain characteristics of SMCs indicate that these cells retain part of the differentiation capacity present in EPDCs during embryonic development.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 
Isolation and Culture of Human EPDCs
All experiments with human tissue specimens were carried out according to the official guidelines of the Leiden University Medical Center. EPDCs were cultured from right atrial appendages excised during cardiac surgery of adult patients. The layer of epicardium was stripped from the appendage, placed in a 2-cm2 culture dish (Greiner Bio-One, Frickenhausen, Germany, http://www.gbo.com/en) coated with porcine gelatin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and capped with a 14-mm round glass coverslip to keep the tissue from floating. The cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and medium 199 (M199) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10% fetal bovine serum (FBS) (all from Invitrogen, Carlsbad, CA, http://www.invitrogen.com), and 2 ng/ml basic fibroblast growth factor (bFGF) (Sigma-Aldrich). The culture medium was refreshed every 2 days. Four to seven days after initiation of the cultures, when outgrowths of epithelium-like cells were visible, the coverslips and the remaining tissue pieces were removed. These EPDCs were then either detached from the bottom of the culture dish with trypsin solution (Invitrogen) to establish subcultures or processed for immunofluorescent labeling of ß-catenin. Characterization experiments were performed with at least four different EPDC isolates, and differentiation experiments were confirmed with at least two different EPDC isolates.

Cell Culture
MSCs and FFBs were cultured in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin (DMEM-PS), and 10% FBS [22]. All cells were maintained at 37°C in a humidified air-5% CO2 atmosphere.

Immunophenotyping
The surface antigen expression profiles of EPDCs and FFBs were determined by flow cytometry as described previously for bone marrow-derived MSCs [23]. To detect Isl-1 and von Willebrand factor (vWF), the cells were rinsed with ice-cold phosphate-buffered saline (PBS), fixed in 100% ethanol for 1 hour at –20°C, and washed with PBS containing 5% FBS prior to incubation with primary antibodies.

RNA Extraction
Total cellular RNA was extracted from tissues and cultured cells using the Nucleospin II kit (Macherey-Nagel GmbH & Co. K.G., Düren, Germany, https://www.macherey-nagel.com). Human atrial RNA was derived from pooled right atrial appendages; human ventricular RNA was obtained from viable heart muscle tissue removed during partial left ventriculectomy. Skeletal muscle RNA was extracted from a specimen of vastus lateralis muscle. Arteries from human umbilical cords served as a source of vascular smooth muscle RNA.

Adenoviral Transduction
The conventional human adenovirus serotype 5 (hAd5) vector hAd5/F5.CMV.eGFP expressing the enhanced green fluorescent protein (eGFP) gene, the fiber-modified hAd5 vectors hAd5/F50.CMV.eGFP and hAd5/F50.CMV.myocL encoding eGFP and myocardin (respectively), and the control vector hAd5/F50.empty have been described previously [22, 24]. To determine the optimal adenovirus vector and its dose for the transduction of EPDCs, FFBs, and MSCs, they were exposed to 0, 12.5, 25, 50, 100, or 200 infectious units (IU) of hAd5/F5.CMV.eGFP or hAd5/F50.CMV.eGFP per cell and seeded in 2-cm2 wells at a concentration of 104 cells per cm2. The inoculum was removed after 16 hours, and at 72 hours postinfection, the cells were subjected to flow cytometric analyses. To analyze the effect of myocardin on EDPCs, FFBs, and MSCs, they were either mock-infected or infected with 50 (EPDCs) or 100 (FFBs, MSCs) IU of hAd5/F50.CMV.myocL or hAd5/F50.empty per cell and plated at a density of 104 cells per cm2 in 10-cm2 dishes for reverse transcription-polymerase chain reaction (RT-PCR) analysis or on glass coverslips for immunofluorescence microscopy (IFM). The culture medium was refreshed every other day until at 1 week after infection, when the cells were processed for RT-PCR analysis or IFM.

RT-PCR Analysis
RT was performed with 2 µg of total RNA as previously reported [22]. One hundredth of each 50-µl cDNA sample was used per amplification reaction, employing the PCR primers and conditions described by van Tuyn et al. [22] with the addition of primer pairs targeting transcripts encoding the {alpha}-subunit of the cardiac fast sodium channel (SCN5a) (5'-TGCTTGAGTATGCCGACAAG-3' and 5'-GTTGATGCACCTCCCAAACT-3') or vWF (5'-TCCTGGAGGAGCAGTGTCTT-3' and 5'-AGCTGCCTTCCAACATGAC-3') with an annealing temperature of 60°C and 38 or 35 cycles, respectively. As internal controls for the quantity and quality of the RNA specimens, RT-PCR amplifications targeting transcripts of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were performed in parallel. Total RNA samples derived from human atrium, ventricle, vascular smooth muscle, and skeletal muscle were also subjected to RT-PCR to provide positive controls and to determine the specificity of the marker genes. PCRs carried out with water instead of cDNA served as negative controls. For semiquantitative PCR, 5 µl of a 10-, 50-, 250-, 1,250-, and 6,250-fold dilution of the original cDNA sample in water was used.

Immunofluorescence Microscopy
Cells were processed for IFM as described previously [22]. The properties of the antibodies are listed in Table 1 of the supplemental online data. Nuclei were stained with Hoechst 33342 (Invitrogen). In double-labeling experiments, coverslips were simultaneously incubated with the rabbit anti-smooth muscle myosin heavy chain (smMHC) antiserum [25] in combination with sarcomeric {alpha}-actinin (sACTN)- or aortic smooth muscle actin (ASMA)-specific mouse monoclonal antibodies and visualized with a mixture of appropriate secondary antibodies.

In Vitro SMC, Adipocyte, and Osteoblast Differentiation
To study SMC differentiation, cells were seeded on glass coverslips at a density of 104 cells per cm2 and allowed to attach overnight. Next, the culture medium was replaced with DMEM-PS without growth factors or with recombinant human TGF-ß1 (PeproTech, Rocky Hill, NJ, http://www.peprotech.com) or BMP-2 (Sigma-Aldrich) at a final concentration of 50 ng/ml. The cells received fresh medium every 3 days and were processed for IFM at 11 days after the start of the experiment. In vitro adipogenesis and osteogenesis were induced as described before [22].


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 
Isolation of Human EPDCs
In vitro culture of epicardium dissected from right atrial appendages of human adults resulted in the outgrowth of cells with an epithelioid cobblestone-like morphology (Fig. 1A, left panel). These EPDCs displayed intense ß-catenin staining, especially at sites of cell-to-cell contact, confirming their epithelial nature (Fig. 1B, left panel). After trypsinization and seeding at a low density, the cells adopted a fibroid spindle-shaped appearance (Fig. 1A, right panel). These morphological changes were accompanied by a reduction in ß-catenin level and a redistribution of this component of adherent junctions to the cytoplasm, even at intercellular contact regions (Fig. 1B, right panel), suggesting that the EPDCs underwent EMT in vitro. Because it was not possible to maintain EPDCs as an epithelial sheet during prolonged culture, these cells were further characterized after they had undergone complete EMT, as determined by morphology and ß-catenin staining.


Figure 1
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  		Figure 1. Epithelial-to-mesenchymal transition of epicardium-derived cells (EPDCs). Left panels: EPDCs with epithelial morphology. Right panels: EPDCs with mesenchymal morphology. (A): Bright-field images. (B): Immunofluorescent images of ß-catenin (green) and Hoechst 33342 (nuclei; blue) staining.

 
Immunophenotypic Characterization of EPDCs, FFBs, and MSCs
MSCs, FFBs, and EPDCs from several human adults were expanded in culture, and their surface antigen profiles were compared by flow cytometry (Table 1). Consistent with previous reports [26, 27], the plasma membrane of MSCs was abundantly decorated with hyaluronate receptor (CD44), major T-cell antigen (Thy-1; CD90), endoglin (CD105), vascular cell adhesion molecule-1 (CD106), and human leukocyte class I (HLA-ABC) antigens. These multipotent stem cells also expressed low levels of transferrin receptor (CD71), P-selectin (CD62P), ß3 integrin (CD61), neural cell adhesion molecule (CD56), and membrane cofactor protein of the complement system (CD46) at their surface. Of the aforementioned proteins, EPDCs and FFBs only expressed high levels of CD44, CD90, CD105, and HLA-ABC and low levels of CD46 at the plasma membrane. Neither EPDCs nor FFBs showed detectable surface expression of CD56, CD61, CD62P, CD71, or CD106. In contrast to the EPDCs and MSCs, the FFBs stained positive for neutral endopeptidase (NEP; CD10). None of the three cell types expressed the hematopoietic markers CD11A, CD14, CD15, CD19, CD34, and CD45 or the endothelial markers platelet-endothelial cell adhesion molecule-1 (CD31), vascular endothelial growth factor receptor-2 (Flk-1), vWF, and VE (vascular endothelial)-cadherin at their surface. In addition, all cell types stained negative for the CXC motif chemokine receptor 4 (CXCR-4; fusin), human leukocyte class II subtype DR antigens (HLA-DR), the Coxsackie and adenovirus receptor (CAR), the cardiac progenitor cell marker Isl-1, and the human and murine stem cell markers CD133 and stem cell antigen (Sca)-1, respectively.


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  		Table 1. Surface marker profile of FFBs, MSCs, and EPDCs

 
EPDCs Express Several Cardiac and Smooth Muscle Genes
Using RT-PCR analysis, we investigated the expression of cardiac, smooth, and skeletal muscle genes in EPDCs, FFBs, and MSCs (Figs. 2 and 3). Only EPDCs expressed the cardiac marker genes encoding cardiac troponin T (cTnT) and GATA4, whereas all three cell types expressed dHand, Mef2C, and connexin 43 (Cx43; Fig. 2A) and low levels of the smooth muscle genes specifying ASMA, smooth muscle calponin (CNN1), and transgelin (SM22; Fig. 3A). Neither the EPDCs nor the control cells contained detectable amounts of mRNA for the cardiac sarcomeric components cardiac troponin I (cTnI), the atrial and ventricular forms of myosin light chain 2 (Mlc2a and Mlc2v, respectively), cardiac {alpha}- and ß-myosin heavy chain ({alpha}-MHC and ß-MHC, respectively), the cardiac TFs eHand and Nkx2.5, the atrial natriuretic peptide precursor A (ANF), or the ion channel proteins SCN5a or sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) (Fig. 2A). Finally, mRNA for smMHC, which is highly specific for SMCs, and transcripts for the skeletal muscle-specific sarcomeric proteins skeletal myosin heavy chain 2a (skMHC), fast-twitch skeletal troponin I (fsTnI), and slow-twitch skeletal troponin I (ssTnI) were absent in each of the three cell types (Figs. 2B and 3A). IFM confirmed the absence of GATA4 in FFBs and MSCs and revealed a nuclear localization of this TF in EPDCs (Fig. 2C). In contrast, Cx43 was found in the cytoplasm of all three cell types rather than at cell-to-cell contact areas as is typical for cardiac or skeletal muscle.


Figure 2
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  		Figure 2. Analysis of cardiac and skeletal muscle-specific gene transcription in FFBs, MSCs, and EPDCs. Reverse transcription-polymerase chain reaction analysis of cardiac (A) and skeletal muscle (B) marker genes. Controls include smooth muscle cells, human atrial tissue, human ventricular tissue, and human skeletal muscle tissue. Water served as negative control. (C): Immunofluorescent staining of FFBs, MSCs, and EPDCs for GATA4 (red) and Cx43 (green). Nuclei were stained with the blue-emitting fluorochrome Hoechst 33342. Control samples were incubated with secondary antibody only. (D): Cx43 staining of isolated neonatal cardiomyocytes from rat. Note the predominance of Cx43 at areas of cell-cell contact. Abbreviations: A, human atrial tissue; E and EPOC, epicardium-derived cells; F and FEB, foreskin fibroblast; M, mesenchymal stem cells; Nc, negative control; Sk, human skeletal muscle tissue; Sm, smooth muscle cells; V, human ventricular tissue.

 


Figure 3
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  		Figure 3. Smooth muscle cell differentiation of FFBs, MSCs, and EPDCs upon stimulation with vehicle (control), TGF-ß1, or BMP-2. Blue, nuclear staining with Hoechst 33342; green, ASMA; red, smooth muscle myosin heavy chain (smMHC). Images were taken at magnifications of x100 (upper part) and x400 (lower part). Abbreviations: ASMA, aortic smooth muscle actin; BMP, bone morphogenic protein; EPDC, epicardium-derived cells; FFB, foreskin fibroblast; smMHC, smooth muscle myosin heavy chain; TGF, transforming growth factor.

 
EPDCs Can Transdifferentiate into SMCs In Vitro
During cardiac development, part of the EPDCs that migrate into the subepicardial space and underlying myocardium differentiate into SMCs. Recent investigations showed that TGF-ß isoforms stimulate EMT and SMC differentiation in avian epicardial explants [18] and that pluripotent human embryocarcinoma cells acquire SMC characteristics after BMP-2 treatment [28]. Whereas the human EPDCs rapidly underwent EMT in culture without addition of specific growth factors, incubation with either TGF-ß1 or BMP-2 resulted in a flattened morphology of the cells and an intense (fibrillar) staining for ASMA and smMHC (Fig. 4), which are indicative of SMC differentiation. TGF-ß1 stimulation of FFBs led to the formation of ASMA filaments but did not induce synthesis of smMHC (Fig. 4), which is generally considered to be the most stringent SMC marker [22, 29]. BMP-2 treatment had no noticeable effect on these cells (Fig. 4). These findings indicate that the FFBs did not differentiate into SMCs. Interestingly, for MSCs, serum deprivation alone was sufficient to induce synthesis of both ASMA and smMHC (Fig. 4). This result, in combination with the effects of TGF-ß1 and BMP-2, leads us to conclude that MSCs have a high propensity to become SMCs in vitro. Under normal culture conditions, none of the three cell types contained ASMA or smMHC in amounts detectable by IFM (Fig. 3D and data not shown).


Figure 4
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  		Figure 4. Transduction of FFBs, MSCs, and EPDCs with enhanced eGFP-encoding adenovirus vectors. Dashed line, conventional hAd5 vector (hAd5/F5.CMV.eGFP); solid line, fiber-modified hAd5 vector (hAd5/F50.CMV.eGFP). Values are given as means ± SD (n = 4). Abbreviations: eGFP, enhanced green fluorescent protein; EPDC, epicardium-derived cells; FFB, foreskin fibroblast.

 
Efficient Transduction of EPDCs by Adenovirus Vectors
The usefulness of EPDCs as a model system is partially determined by the ability to efficiently transduce these cells for fundamental and applied research purposes. Genetic modification of EPDCs will allow knockdown of endogenous genes, ectopic expression of both endogenous and exogenous/therapeutic genes, and the analysis of mutant genes. Unfortunately, the in vitro cultured primary human epicardial cells were very hard to transfect using nonviral vector systems (data not shown). Wada et al. [17] reported that transfection of the rat epicardial cell line with plasmid DNA is equally difficult. We therefore tested the wild-type and fiber-modified hAd5 vectors hAd5/F5.CMV.eGFP and hAd5/F50.CMV.eGFP for their ability to transduce EPDCs. EPDCs were efficiently transduced with the conventional hAd5 vector hAd5/F5.CMV.eGFP, whereas the control cells (i.e., FFBs and MSCs) were not. The efficient transduction of EPDCs by hAd5/F5.CMV.eGFP is remarkable given that these cells do not contain detectable amounts of CAR (Table 1), which is the primary attachment receptor for hAd5, at their surface. Binding of hAd5 (vectors) to EPDCs thus occurs via an alternative receptor. The fiber-modified hAd5/F50.CMV.eGFP vector transduced all three cells types very well (Fig. 5). For the FFBs and MSCs, a dose of 100 IU of hAd5/F50.CMV.eGFP per cell was considered optimal because it resulted in high-level eGFP expression in virtually 100% of the cells without apparent toxicity. For the EPDCs, a dose of 50 IU of hAd5/F50.CMV.eGFP per cell resulted in a similar transduction level (Fig. 5).


Figure 5
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  		Figure 5. Analysis of smooth, cardiac, and skeletal muscle marker gene expression in FFBs, MSCs, and EPDCs transduced with Ad5/F50.CMV.myocL or with the control vector Ad5/F50.empty. (A): Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis of smooth muscle marker gene expression. The gray bar represents template amounts in fivefold increments from left to right. Depicted are the three template concentrations that best illustrate the relative abundance of a particular mRNA in the different samples. RT-PCR analysis of cardiac (B) and skeletal muscle (C) marker gene expression. (D): Double-labeling of sarcomeric {alpha}-actinin (green) and smMHC (red) in Ad5/F50.CMV.myocL- or Ad5/F50.empty-transduced EPDCs and MSCs. Nuclei were stained with the blue-emitting fluorochrome Hoechst 33342. Abbreviations: E, Ad5/F50.empty; EPDC, epicardium-derived cells; FFB, foreskin fibroblast; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, Ad5/F50.CMV.myocL; smMHC, smooth muscle myosin heavy chain.

 
Myocardin Induces Cardiac and Smooth Muscle Gene Expression in EPDCs
The TF myocardin is an important target in BMP and TGF-ß signaling cascades that lead to smooth and cardiac muscle cell differentiation [30, 31]. We therefore studied the effect of forced myocardin expression on the gene expression profile of EPDCs. Transduction of EPDCs with hAd5/F50.CMV.myocL caused activation of the smMHC gene and an increase in ASMA-, CNN1-, and SM22-specific mRNAs (Fig. 3A). However, as we reported before for MSCs [22], it also induced expression of the cardiac marker genes Mlc2a, Mlc2v, {alpha}-MHC, ß-MHC, eHand, ANF, SCN5a, SERCA2a, and ssTnI in EPDCs (Fig. 3B, 3C). Consistent with previous findings [22], myocardin did not activate the skeletal muscle-specific genes fsTnI and skMHC (Fig. 3C). Using IFM, we studied the coexpression of cardiac and smooth muscle-specific genes in EPDCs and MSCs. Figure 3D clearly shows costaining of sACTN and smMHC in both EPDCs and MSCs, demonstrating that cardiac and smooth muscle proteins are simultaneously produced in single cells. Remarkably, myocardin-dependent activation of cardiac and smooth muscle genes was much more effective in EDPCs than in MSCs (i.e., ±100% vs. ±10% of the cells; Fig. 3D).

EPDCs Can Undergo Osteogenesis In Vitro
MSCs can give rise to osteoblasts and adipocytes when cultured in appropriate differentiation media [26]. We tested whether EPDCs, which have undergone EMT and therefore have become a mesenchymal cell type, are also capable of differentiation into osteoblasts and adipocytes. This would indicate that EPDCs, like MSCs, have multipotent stem cell characteristics. Figure 6 shows that under conditions that promote osteogenesis and adipogenesis in MSCs, EPDCs differentiate into osteoblasts but not adipocytes. For FFBs, osteogenic and adipogenic stimulation did not lead to the formation of calcium deposits or lipid droplets.


Figure 6
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  		Figure 6. Adipogenesis and osteogenesis in FFBs, MSCs, and EPDCs. Red stain labels calcium deposits in the osteogenesis assay and fatty inclusions in the adipogenesis assay. Abbreviations: EPDC, epicardium-derived cells; FFB, foreskin fibroblast.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Summary
 Disclosures
 Acknowledgments
 References
 
This is the first study describing the isolation, ex vivo propagation, characterization, and differentiation potential of primary epicardial cells from human adults. The initially epithelioid cobblestone-like morphology of these cells indicates that true epicardial mesothelial cells, instead of subepicardial interstitial cells, were brought in culture. Unlike the previously described primary rat epicardial cells, which required stimulation with specific growth factors (e.g., bFGF or epidermal growth factor [EGF]) to undergo EMT in vitro [17], the human EPDCs spontaneously obtained a fibroblastic appearance after subculturing at low densities in serum-containing medium with or without (data not shown) supplemental bFGF. Nonetheless, both the human and rat epicardial cells could be transdifferentiated into SMCs. However, whereas the SMC differentiation of the rat epicardial cells was rather inefficient with less than or equal to 5% of the stimulated cells acquiring smooth muscle markers, nearly 100% of the human EPDCs differentiated into SMCs as determined by immunocytology using ASMA- or smMHC-specific antibodies. Whether these divergent results reflect species-related differences in the developmental potential of nonembryonic/adult epicardial cells or are caused by the use of different growth factors to induce SMC differentiation (i.e., EGF and platelet-derived growth factor in the rat experiments vs. BMP-2 and TGF-ß1 in our study) remains to be determined. In accordance with our findings, Compton et al. [18] showed that TGF-ß1, TGF-ß2, and TGF-ß3 cause loss of epithelial character and the expression of smooth muscle genes in chick proepicardium transplants in vitro. The effect of BMP on the phenotype of epicardial cells has not been investigated before. Importantly, BMP- or TGF-dependent SMC differentiation is not a general property of cultured human cells with a fibroblastic morphology given that treatment of FFBs with these growth factors did not induce an SMC phenotype, as was apparent from the absence of smMHC in these cells. The detection of ASMA in TGF-ß1-treated FFBs may be indicative of the differentiation of these cells into myofibroblasts [32]. The observation that serum deprivation alone is sufficient to induce formation of ASMA- and smMHC-positive filaments in MSCs lends support to the notion that SMC differentiation represents the default developmental pathway of these cells [33]. Infection of EPDCs with an adenovirus vector encoding myocardin, which is a master regulator of smooth muscle gene expression [20, 21], triggered smMHC synthesis as well. However, it did not lead to the assembly of a filamentous smMHC network and also caused the coexpression of numerous cardiac muscle genes. These results corroborate previous findings that myocardin alone cannot initiate a complete SMC differentiation program and activates heart muscle-specific genes at the same time [22, 34]. Interestingly, virtually all hAd5/F50.CMV.myocL-infected EPDCs coexpressed cardiac and smooth muscle genes, whereas at a comparable myocardin dose, only approximately 10% of the transduced MSCs stained positive for heart and smooth muscle proteins. This may indicate that, at least in humans, EPDCs are more receptive to stimuli for cardiac and smooth muscle differentiation than are MSCs. On the other hand, near-quantitative transduction of MSCs and EPDCs with a vesicular stomatitis virus G protein-pseudotyped lentivirus vector encoding myocardin in both instances led to the (co)expression of sACTN and smMHC in almost 100% of the cells (data not shown).

Given the controversy about the contribution of epicardial cells to the endothelium of coronary blood vessels [7, 10, 13], we also studied the postulated vasculogenic differentiation ability of EPDCs in vitro (supplemental online data). Although EPDCs could be induced to gather into tubular networks resembling those formed by human umbilical vein endothelial cells (HUVECs), both the low-level and diffuse intracellular distributions of vWF (data not shown) in the tube-forming cells indicate that the EPDCs did not adopt an endothelial phenotype. Viragh et al. [11] previously found in quail embryos that infolding of epicardial mesothelial cells into the subepicardial space resulted in the development of nonendothelial tubular structures. Whether these structures bear any relationship to the EPDC-derived meshworks that we observed in vitro requires further investigation. In agreement with our results, Wada et al. [17] did not succeed in inducing endothelial differentiation of primary rat epicardial cells in vitro. Taken together, these findings suggest that (genuine) epicardial cells either do not possess vasculogenic differentiation capacity or lose this ability at a certain stage of development and/or upon in vitro culture.

The differentiation potential of EPDCs was further explored by subjecting them to adipogenic or osteogenic stimuli in vitro. Like MSCs, EPDCs could be induced to form calcium deposits, whereas FFBs could not. However, under conditions that stimulated adipogenesis in MSCs, no lipid droplets were found in EPDCs or FFBs. These new findings are intriguing for several reasons. First, they demonstrate that a differentiated epithelial cell type after in vitro culture can give rise to various specialized types of non-epithelial cells. There is growing evidence that this remarkable plasticity is a general property of mesothelial cells [35]. Second, the fact that postembryonic epicardial cells have several developmental options adds to the discussion of whether EPDCs represent somatic stem cells [2]. A definitive answer to this question awaits the demonstration of clonality of phenotypically distinct EPDC-derived cells. On the basis of the currently available data, we favor the idea that the proepicardium is composed of a heterogenous population of more or less committed precursor cells with distinct differentation capacities. Third, their inability to differentiate into adipocytes may indicate that adult EPDCs are not the source of the subepicardial fat deposits common to human beings [36]. Perhaps, at some time during pre- or postembryonic life, the epicardial mesothelial cells lose the ability to become adipocytes and adipogenesis becomes restricted to the subepicardial interstitial cells. Fourth, the ease with which EPDCs can be converted into osteoblasts in vitro may provide an experimental framework for studying pericardial calcification, a condition strongly associated with constrictive pericarditis [36]. Whether the contribution of EPDCs to the cardiac valves [6] can explain valvular calcification and bone formation [37] remains to be determined. Furthermore, given (a) the strong tendency of SMCs to undergo osteogenesis under specific experimental and pathological conditions [38, 39] and (b) the smooth muscle differentiation ability that EPDCs display in vitro and in vivo, it would be interesting to investigate whether calcification of EPDCs is preceded by SMC differentiation.


   SUMMARY
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
Disclosures
 Acknowledgments
 References
 
We have shown that EPDCs of human adults can differentiate into various mesodermal cell types, including SMCs and osteoblasts, but have a more restricted differentiation capacity than MSCs. EPDCs thus best fit the description of a committed progenitor cell. Consistently, the surface marker profile of EPDCs is clearly distinct from that of MSCs. The immunophenotype of EPDCs also differs from that of other possible stem and progenitor cell populations in the heart, like human hematopoietic stem cells, endothelial progenitor cells, or cardiac progenitor cells. This corroborates that a novel progenitor cell type was isolated. The relatively straightforward method of isolating and culturing primary human epicardial cells, combined with the fact that these cells retain at least part of the properties of embryonic EPDCs, makes them an attractive model system for studying various aspects of normal and pathological cardiovascular development, including epicardial EMT and coronary SMC differentiation. Finally, these cells may prove valuable (a) as feeder layer for culturing human cardiomyocytes [40], (b) for screening cardiovascular drugs, (c) as vehicle to deliver therapeutic genes, and (d) for healing damaged hearts by restoring myocardial architecture and vascularization.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
References
 
We thank Binie Klein and Pieter Koolwijk for supplying the FFBs and HUVECs, respectively. Pieter Doevendans, Robbert Klautz, Pieter Voigt, and Rob Nelissen are gratefully acknowledged for providing leftover surgical material. We also thank Robert Adelstein for donating the smMHC-specific rabbit antiserum and Crucell N.V. (Leiden, The Netherlands, http://www.crucell.com) for sharing their adenovirus vector production system. Part of this work was financed by the Netherlands Organization for Health Research and Development (ZonMw; Grant MKG5942).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Summary
 Disclosures
 Acknowledgments
 References
 

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