RAPID COMMUNICATION

Re-Activated Adult Epicardial Progenitor CellsAre a Heterogeneous Population Molecularly Distinct

from Their Embryonic Counterparts

Sveva Bollini,1 Joaquim Miguel Nunes Vieira,1 Sara Howard,2 Karina Natasha Dube,2

Gemma Mary Balmer,2 Nicola Smart,1 and Paul Richard Riley1

Cardiovascular disease remains the major cause of mortality, and cardiac cell therapy has recently emerged as aparadigm for heart repair. The epicardium is a layer of mesothelial cells covering the heart that during devel-opment contributes to different cardiovascular lineages, including cardiomyocytes, but which becomes quiescentafter birth. We previously revealed that the peptide thymosin beta 4 (Tb4) can reactivate adult epicardium-derivedcells (EPDCs) after myocardial infarction (MI), to proliferate, and differentiate into cardiovascular derivatives.The aim of this study was to provide a lineage characterization of the adult EPDCs relative to the embryonicepicardial lineage and to determine prospective cell fate biases within the activated adult population duringcardiovascular repair. Wt1GFPCre/ + mice were primed with Tb4 and MI induced by ligation of the left anteriordescending coronary artery. Adult WT1+ GFP+ EPDCs were fluorescence-activated cell sorted (FACS) at 2, 4,and 7 days after MI. Embryonic WT1+ GFP+ EPDCs were isolated from embryonic hearts (E12.5) by FACS, andsorted cells were characterized by real-time quantitative reverse transcriptase–polymerase chain reaction (RT-qPCR) and immunostaining. Adult WT1 + GFP + EPDCs were highly heterogeneous, expressing cardiac pro-genitor and mesenchymal stem markers. Based on the expression of stem cell antigen-1 (Sca-1), CD44, and CD90,we identified different subpopulations of EPDCs of varying cardiovascular potential, according to marker geneprofiles, with a molecular phenotype distinct from the source embryonic epicardial cells at E12.5. Thus, adultWT1 + GFP + cells are a heterogeneous population that when activated can restore an embryonic gene programme,but do not revert entirely to adopt an embryonic phenotype. Potential biases in cardiovascular cell fate suggest thatdiscrete subpopulations of EPDCs might be clinically relevant for regenerative therapy.

Introduction

Modern clinical interventions mean that fewerpeople die due to a myocardial infarct (heart attack),

but an increasing number live with a scarred heart, whichcompromises cardiac function and, subsequently, results inprogressive heart failure [1]. Despite the important advancesachieved over recent years in treating heart failure [2], theultimate resolution remains heart transplantation [3], which iscompounded by the shortage of organ donors and compli-cations associated with potential immune rejection by thepatient host [4].

Regenerative medicine has emerged as an alternativestrategy to facilitate cardiac repair, which includes stem celltherapy, reprogramming and tissue engineering. With regardto transplantation of stem cells, there has been rapid pro-gression to the clinical trials reported [5–8]; however, pa-

tient benefit has been modest at best. Moreover, a generalconsensus on the most suitable stem cell source remains tobe elucidated, and the mechanisms of action remains to befully determined. Several studies have recently demon-strated that functional improvements obtained after en-graftment of stem cells into the heart can be largelyattributable to the secretion of paracrine-acting factorsstimulating cardio-protection, angiogenesis, and modulationof inflammation, rather than via direct trans-differentiationinto new cardiovascular cells [9–11]. In support of thesestudies, recent work has confirmed that exogenous trans-planted stem cells can mediate heart repair by acting onendogenous cardiac progenitor cells (CPCs) residing in theheart, by activating them to differentiate into new cardio-vascular cells, through the modulation of the local microen-vironment and the paracrine release of specific trophic factors[12–14]. Thus, regenerative medicine is now exploring

1Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.2Molecular Medicine Unit, UCL Institute of Child Health, London, United Kingdom.

STEM CELLS AND DEVELOPMENT

Volume 23, Number 15, 2014

� Mary Ann Liebert, Inc.

DOI: 10.1089/scd.2014.0019

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strategies based on the stimulation and activation of the localCPCs, in parallel with the transplantation of exogenous stemcells, in order to reactivate inherent repair mechanisms of theadult mammalian heart.

Several types of CPCs have been broadly described in theliterature and mainly characterized according to the expres-sion of specific stem cell markers, such as stem cell antigen-1(Sca-1) or c-kit, or based on their in vitro culture properties[15–17] (extensively reviewed in Bollini et al. [18]). Thesedifferent CPCs have been described as independent popula-tions, although it is not clear whether they are derived fromdifferent cell sources during development or whether theyrepresent cells derived from a common precursor subse-quently isolated at different stages of differentiation andmaturation. Among all the different CPC sources defined sofar, there are two distinct populations, the origin of which canbe specifically defined and traced back to embryonic devel-opment: notably Isl1+ CPCs [19] and epicardium-derivedcells (EPDCs) [20,21].

While the Isl1 + population appears to represent remnantsof cardioblasts arising during development and are rarelyobserved at postnatal stages, the EPDCs are derived from alayer of mesothelial cells covering the surface of the heart,which, during development, contributes to the majority ofthe major cardiovascular lineages. The epicardium developsinitially from an outgrowth of cells called the pro-epicardialorgan (PEO), a primordial cluster of epithelial cells locatedat the inflow region of the forming heart. PEO cells migrateto envelop the muscular heart tube and form the mesothelialepicardium. EPDCs then undergo epithelial-to-mesenchymal(EMT) transformation and migrate into the underlyingmyocardium, where they differentiate into adventitial fi-broblasts, give rise to vascular smooth muscle cells, andcontribute to the endothelium of the coronary vasculature[22,23]. EPDCs have also been proposed to contribute tothe cardiomyocyte lineage in the developing heart [24,25],albeit this remains a controversial finding [26]. EPDCs,therefore, appear to represent a font of progenitors withpotential for cardiac regeneration and heart repair; how-ever, their clinical translation is hampered by the fact thatthe adult epicardium becomes quiescent soon after birth.We previously revealed that stimulation by the same fac-tors which promote the activation and plasticity of theembryonic EPDCs can restore and sustain re-activation oftheir postnatal counterparts, as an exemplary paradigm ofreinstating embryonic potential in putative therapeuticprogenitor cell populations in the adult heart. The peptidethymosin beta 4 (Tb4) has been identified as a regulator ofEPDC activation and subsequent differentiation duringcoronary vasculature formation throughout embryogenesis,as well as a potent inducer of adult EPDC proliferation anddifferentiation into smooth muscle, endothelium, and fi-broblast lineages, after ex vivo outgrowth from adult heartexplants [27]. Translating this paracrine approach intomurine preclinical models in vivo, we demonstrated thatTb4 stimulation supported neovascularization in both theintact and ischemic heart and instructed adult EPDCs torecapitulate their developmental program with reactivationof specific embryonic epicardial genes, such as Wt1, fol-lowed by limited differentiation into new functional maturecardiomyocytes, after myocardial infarction (MI) [28,29].Therefore, the adult epicardium can be considered a source

of dormant cardiac progenitor cells, which, upon appro-priate paracrine stimulation, can become re-activated, suchthat adult EPDCs have the potential to contribute to cardiachomeostasis and repair. While progress around under-standing how to restore the cardiovascular potential ofEPDCs has been recently realized, much remains to bedetermined regarding a detailed characterization of theadult lineage and most notably, in comparison to the em-bryonic source.

Here, we present a detailed fluorescence-activated cellsorting (FACS)-based molecular phenotyping of murineadult EPDCs as activated after Tb4-treatment and MI anddraw direct comparisons with the embryonic epicardiallineage. We reveal a unique cell surface marker signatureand heterogeneity in cellular composition within the adultepicardium, which is reflected in a range of cardiovascularcell fate potential as determined by surrogate marker geneexpression.

Materials and Methods

Mouse lines and MI protocol

Wt1GFPCre/ + mice have been previously described [29]and were generated by crossing transgenic Wt1GFPCre/ +

male mice with wild-type C57BL/6 females purchased fromCharles River. Adult mice were primed with daily intra-peritoneal injections of Tb4 [12 mg/kg solution in phosphate-buffered saline (PBS) 1 · ; RegeneRX] for 7 days [29]. Forthe MI model, Wt1GFPCre/ + mice (n = 145) weighing be-tween 25 and 30 g and 8 weeks old were used, as previouslyreported. Briefly, mice were anesthetized with isofluoraneunder assisted external ventilation through the insertionof an endotracheal tube and underwent thoracotomy in orderto perform a permanent ligation of the left anterior des-cending artery. Buprenorphine (buprenorphine hydrochlo-ride; Vetergesic) was delivered as a 0.015 mg/mL solutionvia intra-peritoneal injections at 20 min before the procedureto provide analgesia. After recovery, animals received fol-low-up intra-peritoneal injections of Tb4 on alternate days.Two, 4, and 7 days after MI, hearts were assessed using acombination of FACS, immunofluorescence, and real-timereverse transcriptase–polymerase chain reaction (RT-PCR)analysis.

All animal experiments were carried out according to theUK Home Office project licence PPL 30/2987 compliantwith the UK Animals (Scientific Procedures) Act 1986 andapproved by the University College London BiologicalServices Ethical Review Process.

WT1 + GFP + EPDCs isolation

Wt1GFPCre/ + embryos were harvested at embryonic dayE12.5 postcoitum and hearts were immediately processedby enzymatic digestion, as previously reported [19]. Briefly,hearts were washed in ice-cold Hank’s balanced salt solution(HBSS) buffer (Gibco) and incubated in 0.1% collagenase II(Worthington Biochemical) and 0.25% trypsin-EDTA (In-vitrogen) solution for 30 min at 37�C until a single-cellsuspension was obtained. WT1 + GFP + embryonic EPDCswere subsequently isolated via FACS sorting on GFP expres-sion and characterized by FACS. Adult Wt1GFPCre/ + heartswere collected at 2, 4, and 7 days after MI was induced and

1720 BOLLINI ET AL.

processed to isolate adult epicardial cells according to [30].Dissociated cardiac cells were isolated via multiple enzymaticincubations at 37�C in a 1% collagenase IV (Sigma) and 2.5%trypsin (Invitrogen) solution in HBSS. The enzymatic diges-tion was carried out in order to detach only the activatedepicardial and sub-epicardial space, leaving the underlyingmyocardium intact. The obtained cell solution was subse-quently depleted from any hematopoietic contamination usingthe Lineage Cell Depletion kit (Miltenyi Biotec), followingthe manufacturer’s instructions and further characterized bymeans of flow cytometry.

Flow cytometry characterization and FACS sortingof embryonic and adult EPDCs

The WT1 + GFP + embryonic (E12.5) EPDCs and the adultWT1 + GFP + EPDCs (2, 4, and 7 days after MI) were char-acterized by FACS using a panel of epicardial, mesenchymal,and cardiac stem cell markers. The EPDCs immunopheno-type was assessed using the following antibodies: PerCP-Cy5.5-conjugated anti-mouse Sca-1 (clone D7; eBioscience);Alexa Fluor 647-conjugated anti-mouse c-kit antibody (clone2B8; Biolegend); PE-conjugated anti-mouse CD29 (Integrinb1, clone HMb1-1; Biolegend); APC-Cy7- and Alexa Fluor647-conjugated anti-mouse CD90.2 (clone 30-H12; Biole-gend), APC-conjugated anti-mouse PDGFRb (clone AP85;eBioscience), PE-Cy7-conjugated anti-mouse CD44 (cloneIM7; eBioscience), Alexa Fluor 647-conjugated anti-mouseCD45 (clone 30-F11; Biolegend), APC-conjugated anti-mouse CD184 (CXCR4, clone 2B11; eBioscience), and anti-mouse CD105 primary antibody (R&D System) with AlexaFluor 647-conjugated anti-rat secondary antibody (MolecularProbes, Life Tech). Compensation was set manually on theanalyzer using CompBeads Plus compensation beads (BDBiosciences). Cells were acquired using a Beckam CoulterCyAn analyzer that was equipped with 488 nm blue and 635 nmred diode lasers and running Summit V3.4 software. Data wereanalyzed using the FlowJo software. Embryonic WT1+ GFP+

EPDCs and subpopulations of adult WT1+ GFP+ EPDCs wereisolated by FACS sorting using a Beckam Coulter MoFlo XDPsorter, equipped with UV 355 nm, blue 488 nm, and red 647 nmlasers and running Summit V5.2 software.

Immunofluorescence staining of EPDCs

Hearts were collected at 4 days after MI, fixed in 4% PFAsolution for 2 h on ice, and snap frozen in methylbutane andliquid nitrogen for cryosectioning. Eight-micrometer-thicksections were processed for immunofluorescence stainingusing the following primary antibodies: anti-mouse GFP,WT1, CD29, CD45, Sca-1, c-kit, CD90, CD44 (all Abcam),and PDGFRb (R&D System). Anti-chicken Alexa Fluor594-conjugated and anti-rat and anti-rabbit Alexa Fluor 488-conjugated secondary antibodies were used (Molecular Probes,Life Tech). Images were acquired using a Zeiss Apotomemiscoscope and a Leica structural illumination DM 6000Bmicroscope with AxioVision and Leica MMAF acquisitionsoftware.

Gene expression profile of adult EPDCs

Total RNA was isolated from WT1 + GFP + embryonic(E12.5) EPDCs, from GFP - epicardial cells, and from WT1 +

GFP + EPDCs FACS sorted at 4 days after MI using theRNeasy Micro kit (Qiagen). cDNA was obtained using theWhole Transcriptome kit (Qiagen), following the manufac-turer’s instructions and used for real-time quantitative RT-PCR using SYBR Green on an ABI 7900 for the followinggenes: Wt1, Tbx18, Raldh2, Pdgfrb, Isl1, Gata4, Flk1, Sma,SM22a, Pecam, and Fapa. Hprt1 was used as an endogenouscontrol. Fold change was determined by applying the 2 -DDCT

method. Primer sequences are available on request.

Statistical analysis

All values are expressed as mean – standard error. Statis-tical difference between the considered groups was evaluatedby one-way ANOVA multiple-comparison test and Student’st-test, using Graph Pad Prism 6.0 software. A P-value < 0.05was considered significant.

Results

Isolation and immunophenotypic characterizationof embryonic E12.5 and adult WT1 + GFP + EPDCs

To restore the embryonic program in the adult EPDCs viareactivation of the expression of Wt1, we primed transgenicWt1GFPCre/ + mice with a daily injection of Tb4, followed byinduction of MI as previously described [29]. WT1+ GFP +

EPDCs were subsequently analyzed by FACS, based on theexpression of GFP, avoiding contamination from the under-lying myocardium. The adult activated WT1+ GFP + EPDCswere isolated after 2, 4, and 7 days post-MI and Tb4 priming.FACS analyses revealed that the GFP + fraction within thetotal adult-activated epicardial population was 9.31% –2.36% (n = 12 hearts), 15.15% – 1.51% (n = 12 hearts), and9.23% – 2.50% (n = 12 hearts), respectively (Fig. 1A, B).Control treatment with PBS (vehicle) before MI revealed thatinjury alone stimulated 11.0% – 2.7% (n = 18 hearts) at 4 dayspost-MI (Supplementary Fig. 1A; Supplementary Data areavailable online at www.liebertpub.com/scd).

The phenotype of the adult-reactivated WT1 + GFP +

EPDCs was then evaluated by FACS using a panel of car-diac and mesenchymal stem cell markers, as well as epi-cardial cell antigens (Table 1 and in Fig. 1C). A proportionof the adult WT1 + GFP + EPDCs cells were found to bepositive for the expression of the cardiac stem cell markerSca-1 [31] ranging from 64.0% – 2.1% at day 2 post-MI to54.0% – 2.8% at day 4 and 58.0% – 4.9% at day 7 post-MI,with no significant differences across the time points ana-lyzed; c-kit antigen [15] was hardly detected in these cells,being expressed in no more than 1.9% – 0.2% of the GFP +

EPDCs at 4 days post-MI. A significant proportion of theadult WT1 + GFP + EPDCs (90%) were of mesenchymalorigin, as confirmed by the expression of CD29, with neg-ligible expression of the hematopoietic marker CD45 [32,33].While the mesenchymal marker CD90 [32] increased from day2 to 7 post-MI (47.3% – 4.6%, 65.5% – 4.7%, and 78.1% –3.4%, respectively), the expression of CD44 [33–35] remainedconstant over the 7 days after injury at about 20% of theWT1 + GFP + cells. Chemokine (C-X-C motif) receptor 4(CXCR4), the receptor for the stromal-derived factor-1(SDF-1) playing a critical role in stem cell recruitment af-ter MI [36], was expressed at relatively low levels in theadult-reactivated EPDCs, being present in 4.9% – 2.2% of

ADULT EPICARDIAL PROGENITOR CELLS ARE HETEROGENEOUS 1721

the WT1 + GFP + cells at 2 days after MI and increasedto 12.3% – 4.2% 7 days after injury. CD105, a marker ofmesenchymal stem cells [32], endogenous cardiac progeni-tors, such as the human cardiospheres [37] and the recentlydescribed proepicardial-derived murine cardiac-residentMSC-like stem cells [38], was expressed in 13.2% – 3.8% ofthe adult WT1 + GFP + EPDCs at 2 days after MI and in-creased to 22.7% – 1.4% a week from injury. The expressionof the epicardial marker PDGFRb [39] increased from23.0% – 12.8% of the WT1 + GFP + cells at 2 days post-MI

to 51.0% – 7.5% by day 7. The variable distribution of thesemarkers among the adult-reactivated EPDCs, therefore, re-vealed a highly heterogeneous phenotype. We identified aconsensus signature for the adult-reactivated WT1 + GFP +

EPDCs according to the combination of markers mosthighly expressed at 4 days after injury, which was Sca-1 + /CD90 + /CD44 + /PDGFRb+ . The presence of EPDCs ac-cording to these markers was confirmed in situ by im-munostaining in heart sections (n = 15 hearts, Fig. 2A–I).Control (PBS-treated; n = 18) mice also revealed an equivalent

Table 1. Immunophenotype of Embryonic and Adult WT1 + GFP +EPDCs

eEPDCs E12.5 aEPDCs 2D MI aEPDCs 4D MI aEPDCs 7D MI

Sca-1 2.2% – 1.3% 64.0% – 2.1% 54.0% – 2.8% 58.0% – 4.9%c-Kit 4.5% – 3.8% 0.3% – 0.1% 1.9% – 0.2% 1.1% – 1.1%CD29 91.9% – 4.8% 89.0% – 4.4% 91.0% – 0.9% 91.2% – 3.8%CD90 5.4% – 0.9% 47.3% – 4.6% 65.5% – 4.7% 78.1% – 3.4%PDGFRb 23.0% – 12.8% 19.0% – 5% 51.0% – 7.5% 45.7% – 5.6%CD44 0.1% – 0.1% 19.8% – 4.7% 17.5% – 4.8% 14.5% – 5.0%CD105 0.22% – 0.01% 13.2% – 3.8% 12.5% – 2.1% 22.7% – 1.4%CXCR4 4.6% – 2.3% 4.9% – 2.2% 4.7% – 0.8% 12.3% – 4.2%CD45 0.1% – 0.1% 2.0% – 0.1% 1.2% – 1.2% 2.0% – 0.2%

eEPDCS E12.5, WT1 + GFP + embryonic EPDCs isolated at E12.5; aEPDCs 2D MI, WT1 + GFP + adult EPDCs isolated 2 days post-MI;aEPDCs 4D MI, WT1 + GFP + adult EPDCs isolated 4 days post-MI; aEPDCs 7D MI, WT1 + GFP + adult EPDCs isolated 7 days post-MI;EPDC, epicardium-derived cell; MI, myocardial infarction.

FIG. 1. Isolation of adult-reactivated WT1 + GFP + epicardium-derived cells (EPDCs) after priming with thymosin beta 4(Tb4) at day 2, 4, and 7 after myocardial infarction (MI). (A) The efficiency of isolation is expressed as a percentage of totalepicardial cells isolated via enzymatic digestion and processed by fluorescence-activated cell sorting (FACS); (B) repre-sentative dot plots of adult WT1 + GFP + EPDCs isolated by FACS at day 4 post-MI compared with control conditions(CTRL, uninjured heart). GFP + cells are detected as the small population on the side, highlighted in the gate; (C)immunophenotype analysis of adult-reactivated WT1 + GFP + EPDCs (adult EPDCs, top row) and of embryonic E12.5WT1 + GFP + EPDCs (E12.5 EPDCs, bottom row): green peaks correspond to stained cells, and black peaks correspond toisotype controls. Color images available online at www.liebertpub.com/scd

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Sca-1 + /CD90 + /CD44 + /PDGFRb + cell surface marker pro-file at day 4 post-MI (Supplementary Fig. 1B), although therewas a reduced incidence of both Sca-1 (49.3% – 3.1% vs.54.0% – 2.8%) and CD44 (8.7% – 2.0% vs. 17.5% – 4.8%)between PBS and Tb4-treated animals (compare plots in Fig.1C with Supplementary Fig. 1B).

Given that a hallmark of adult-reactivated EPDCs afterTb4-priming and MI is the re-expression of embryonic epi-cardial genes, including Wt1, Tbx18, and Raldh2 [29,40], wecompared the phenotype of the adult WT1+ GFP + EPDCswith their embryonic E12.5 WT1 + GFP + counterparts (Table1 and Fig. 1C). Embryonic WT1 + GFP + EPDCs expressedCD29, PDGFRb, and CXCR4 at similar levels comparedwith the adult population and were similarly negative forCD45, with equivalent low c-kit expression; however, theirexpression of Sca-1, CD90 (P < 0.0001), CD44, and CD105(P < 0.01) was significantly lower as compared with the adult-reactivated WT1 + population.

Identification of subpopulations of adult-activatedWT1 + GFP + EPDCs after Tb4 priming and injury

Given the heterogeneous profile of the adult-reactivatedEPDCS, we further evaluated the presence of different sub-populations of progenitor cells based on the expression ofSca-1, as the most abundant cell surface marker (present inmore than 50% of the WT1 + GFP + EPDCs after MI). Sca-1also clearly demarcated two sub-populations within the con-trol (PBS-treated, n = 12) animals (Supplementary Fig. 1C).Within the Sca-1 + cell fraction, we distinguished four dif-ferent subpopulations according to the expression of CD90and CD44. CD90+ /CD44 + cells were 10.1% – 1.5% (n = 9hearts), 5.3% – 0.8% (n = 12 hearts), and 5.4% – 0.9% (n = 9hearts) of the Sca-1 + fraction at day 2, 4, and 7 post-MI,respectively. CD90+ /CD44 - cells were 44.7% – 5.2% (n = 9hearts), 50.0% – 3.2% (n = 12 hearts), and 66.2% – 4.4%(n = 12 hearts) at day 2, 4, and 7 post-MI. CD90- /CD44 +

FIG. 2. The adult-reactivated WT1 + GFP + EPDCs reveal a highly heterogeneous phenotype with expression of specificcardiac progenitor and mesenchymal stem cell markers. In (A) representative image of MI at day 4, highlighted in red, LV,left ventricle; RV, right ventricle, scale bar 50 mm. The adult-reactivated WT1 + GFP + EPDCs were analyzed at 4 days afterMI and co-stained for GFP (red) and the markers (green) mostly expressed in their immunophenotype, such as WT1 (Bmerge; single staining at higher magnification in B¢, B† and merge in B%), CD45 (C merge; single staining at highermagnification in C¢, C† and merge in C%), stem cell antigen-1 (Sca-1) (D merge; single staining at higher magnificationin D¢, D† and merge in D%; D† highlights specific membrane localised Sca-1 + staining, against background of auto-fluorescence in D), c-kit (E merge; single staining at higher magnification in E¢, E† and merge in E%), CD29 (F merge;single staining at higher magnification in F¢, F† and merge in F%), CD90 (G merge; single staining at higher magnificationin G¢, G† and merge in G%), PDGFRb (H merge; single staining at higher magnification in H¢, H† and merge in H%), andCD44 (I merge; single staining at higher magnification in I¢, I† and merge in I%). Scale bar in main picture is 50 and 10 mmin the inlets at higher magnification. Color images available online at www.liebertpub.com/scd

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cells were 4.8% – 2.1% (n = 9 hearts), 3.7% – 0.9% (n = 12hearts), and 0.9% – 0.2% (n = 9 hearts) at day 2, 4, and 7 post-MI. CD90- /CD44 - cells were 40.4% – 4.1% (n = 9 hearts),37.4% – 2.3% (n = 12 hearts), and 27.6% – 4.4% (n = 9 hearts)of the Sca-1 + fraction at day 2, 4, and 7 post-MI. The ma-jority of adult-reactivated WT1 + GFP + Sca-1 + EPDCs weredistributed according to the expression of CD90, with a minorproportion of cells characterized by the expression of CD44(Fig. 3).

After Tb4 priming and injury (Supplementary Fig. 2A), weisolated a sub-fraction of WT1 + GFP + Sca-1 + cells that werehighly positive for CD90 and CD44, here defined as CD90hiCD44hi and represented by the right-shifted population fol-lowing flow cytometry analysis (Fig. 3A and SupplementaryFig. 2B). Comparing the CD90hi CD44hi subpopulation withthe remaining CD90+ and low CD44+ cells within the WT1 +

GFP + Sca-1 + EPDCs, here defined as CD90+ CD44lo(Supplementary Fig. 2B, C), it was evident that the CD90+

CD44lo population constituted a more significant proportionof the total WT1 + GFP + Sca-1 + population at 40% acrossdays 2–7 post-MI, versus the 10% of CD90hi CD44hi at day 2,which was reduced to 5% by days 4 and 7 (Supplementary Fig.2C); again supporting the conclusion of a heterogeneous-re-activated adult epicardial lineage.

Gene expression profile of adult-activated WT1 +

GFP + EPDCs subpopulations after Tb4priming and injury

We subsequently analyzed the potential of the adult-activated WT1 + GFP + EPDCs isolated 4 days after MI, astage at which they still retain a cardiac progenitor pheno-type [29], by comparing the gene expression profiles forcandidate lineage markers with embryonic epicardium atE12.5.

Comparisons were made between the adult WT1 + GFP +

Sca-1 + EPDCs, the Sca-1 - sub-population, the remainingadult GFP - epicardial cells, and the embryonic E12.5WT1 + GFP + EPDCs (n = 17 hearts, Fig. 4). Relative to theembryonic WT1 + GFP + E12.5 cells, the adult WT1 + GFP +

Sca-1 + EPDCs revealed a significantly elevated expres-sion of embryonic epicardial genes: Wt1 (7.6-fold increase,P < 0.0001), Tbx18 (2.4-fold increase; P < 0.05), Raldh2(2.1-fold increase; P < 0.05), and Pdgfrb (16.5-fold increase;P < 0.0001); whereas the expression of Tcf21/Epicardin wasonly elevated in the adult GFP - cells relative to the E12.5WT1 + GFP + cells (4.9-fold increase; P < 0.001).

We further investigated the profile of the adult-activatedWT1 + GFP + Sca-1 + EPDCs with regard to the expression

FIG. 3. Sca-1 defines the majority of adult-reactivated WT1 + GFP + EPDCs with cells equally distributed according tothe expression of CD90 and a minor proportion of progenitors characterized by the high expression of CD44. WT1 + GFP +

were isolated from the total epicardial cells harvested by enzimatic digestion and subsequently, sorted on Sca-1 expression.Within the WT1 + GFP + Sca-1 + EPDCs fraction, cells were further discriminated into four subpopulations based on theexpression of CD90 and CD44. According to the level of reactivation, within the WT1 + GFP + Sca-1 + CD90 + CD44 +

population, it was possible to discriminate a subfraction of progenitor cells highly expressing CD90 and CD44, representedby the shifted population in green in the dot plot on the right (A), compared with the situation illustrated in (B). Colorimages available online at www.liebertpub.com/scd

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of Isl1, as a marker of heart precursors from the secondaryheart field during development, and of postnatal cardioblasts[19,41,42], along with Flk1, which defines multipotentcardiovascular progenitors during development that report-edly give rise to cardiomyocyte, endothelial, and vascularsmooth muscle lineages [43,44]. Comparing the Sca-1 + andSca-1 - subpopulations of the WT1 + GFP + EPDCs with theadult GFP - fraction after MI, we observed that Sca-1 + cellsexpressed Isl1 and Flk1 at higher levels compared with theGFP - population (155- and 16.6-fold increase, respectively;P < 0.01). In particular, while Isl1 expression was not sig-nificantly different between the Sca-1 + and Sca-1 - frac-tions, Flk1 was differentially up-regulated in Sca-1 + cells(1,762-fold increase; P < 0.01). Likewise, Gata4, which isan early cardiac transcription factor that is essential duringcardiogenesis [45] and a marker of myocardial and vascularprecursors in human and mouse epicardium [41,46], was

more up-regulated in the Sca-1 + cells than in the Sca-1 -

(10-fold increase, P < 0.0001), but expressed at relativelylower levels than in the GFP - epicardial cells (0.6- and0.06-fold, respectively, P < 0.0001).

An examination of more mature cardiovascular markerswas performed to define the adult-activated WT1 + GFP +

EPDCs commitment to fibroblast, smooth muscle, and en-dothelial lineages. a-smooth muscle actin (Sma) was up-regulated in the adult WT1 + GFP + Sca-1 + EPDCs comparedwith Sca-1- counterparts (4-fold increase, P < 0.0001) but com-parable to levels in the adult GFP - epicardial cells, (0.9-fold, not statistically different). The fibroblast activationprotein alpha (Fapa) was more up-regulated in the Sca-1 +

cells compared with the Sca-1 - WT1 + GFP + EPDCs andcompared with the GFP - epicardial cells (47- and 7.7-foldincrease respectively, P < 0.0001). The vascular smooth mus-cle marker Sm22a was down-regulated in the adult WT1 +

FIG. 4. WT1 + GFP + Sca-1 + progenitors represent the EPDC subpopulation with the strongest capacity to re-activate thedevelopmental programme within the adult epicardium and with evidence of cardiac progenitor potential. (A) Dot plots ofFACS-sorted cells representing the gating strategy; (B) real-time quantitative reverse transcriptase–polymerase chain re-action (RT-PCR) analysis for the expression of the epicardial genes Wt1, Tbx18, Raldh2, Tcf21/Epicardin, and Pdgfrb,comparing the adult WT1 + GFP + Sca-1 - and Sca-1 + EPDCs (in purple and red) with the adult GFP - epicardial cells (inblue) by calibrating the results against the embryonic E12.5 WT1 + GFP + EPDCs (in black). *P < 0.05, ***P < 0.001, and****P < 0.0001. (C) Real-time quantitative RT-PCR analysis for the expression of the cardiac progenitor and cardiovasculargenes Isl1, Flk1, Gata4, Sma, Fapa, Sm22a, and Pecam, comparing the adult WT1 + GFP + Sca-1 - and Sca-1 + EPDCs (inpurple and red) with the adult GFP - epicardial cells (in blue) as calibrator; **P < 0.01, ***P < 0.001, and ****P < 0.0001.Color images available online at www.liebertpub.com/scd

ADULT EPICARDIAL PROGENITOR CELLS ARE HETEROGENEOUS 1725

GFP + Sca-1 + and Sca-1 - EPDC fractions compared with theGFP - cells (0.4- and 0.3-fold, P < 0.001 and P < 0.0001), aswas the endothelial cell marker Pecam, which was signifi-cantly less expressed in the two WT1 + GFP + EPDCs pop-ulations (0.8- and 0.0007-fold, respectively, P < 0.05 andP < 0.0001), although the Sca-1 + fraction showed higher ex-pression than the Sca-1- (approximately a 1,000-fold increase,P < 0.0001). These results suggest a myo-fibroblast potentialin the Sca-1 + fraction within the adult-activated WT1 +

GFP + EPDCs by virtue of the expression of Sma and Fapaat the expense of coronary vascular potential, given the lowexpression of Sm22a and Pecam when compared with theGFP - epicardial cell population. Notably, this potential wasenhanced in Tb4-treated animals, as compared with PBS-treated controls (n = 12), by which Sma and Fapa expressionwas significantly elevated in Tb4-treated GFP + Sca-1 + andSca-1 - populations (Supplementary Fig. 1D).

Given our identification of a sub-fraction of WT1 + GFP +

Sca-1 + cells, which was highly positive for CD90 and CD44at 4 days after MI, we further investigated the gene ex-pression profile of these CD90hi CD44hi cells comparingthem with the remaining WT1 + GFP + Sca-1 - fraction andcalibrated against the adult GFP - epicardial cells (n = 17hearts; Fig. 5). These WT1 + GFP + Sca-1 + CD90hi CD44hiprogenitors showed a significant up-regulation of the spe-

cific embryonic epicardial genes Wt1 (10- and 4-fold in-crease relative to the WT1 + GFP + Sca-1 - and the Sca-1 +

CD90 + CD44lo cells, P < 0.05 and 26.2-fold more than theGFP - cells, P < 0.01), Tbx18 (15- and 12.3-fold increase,respectively, compared to the WT1 + GFP + Sca-1 - and theSca-1 + CD90 + CD44lo cells and 3.3-fold more than GFP -

cells, P < 0.01), Raldh2 (3- and 2.3-folds more than WT1 +

GFP + Sca-1 - and GFP - cells; P < 0.05), and Pdgfrb (1.7-fold more than GFP - cells; P < 0.05), as well as of thecardiac progenitor markers Gata4 (1,900- and 681-fold in-crease compared with WT1 + GFP + Sca-1 - and the Sca-1 +

CD90 + CD44lo cells and 27.7-fold more than GFP - cells,respectively; P < 0.01), Flk1 (57- and 67-fold increase whenrelated to WT1 + GFP + Sca-1 - and the Sca-1 + CD90 +

CD44lo cells, P < 0.01 and 2.8-fold increase compared withGFP - cells, P < 0.05) when compared with the other sub-populations of adult-activated cells analyzed here.

These comparative expression data suggest that the pop-ulation of WT1 + GFP + Sca-1 + adult-activated EPDCs,further stratified by CD90hi CD44hi cells, is the most highlyactivated subpopulation of EPDCs after Tb4-priming andinjury, according to the relative expression of embryonicepicardial genes. Moreover, this sub-population appears toretain multipotency by virtue of the expression of earlycardiac progenitor markers Isl1, Gata4, and Flk1.

FIG. 5. After Tb4 priming and injury, the Sca-1 + CD90hi CD44hi fraction is the most highly activated WT1 + GFP +

EPDC population that retains cardiovascular multipotency via the expression of early cardiac progenitor markers. (A) Dotplots of FACS-sorted cells representing the gating strategy; (B) real-time quantitative RT-PCR analysis for the expression ofthe epicardial and cardiovascular genes Wt1, Tbx18, Raldh2, Pdgfrb, Gata4, and Flk1, comparing the two subpopulations ofadult-activated WT1 + GFP + Sca-1 + CD90 + CD44lo (in red) and CD90hi CD44hi (in green) EPDCs with the WT1 + GFP +

Sca-1 - counterpart (in purple) and calibrating the fold change against the adult GFP - epicardial cells (in blue). *P < 0.05,**P < 0.01. Color images available online at www.liebertpub.com/scd

1726 BOLLINI ET AL.

Discussion

Regenerative medicine strategies based on CPC therapyhave emerged as a therapeutic opportunity to facilitate heartrepair. Much attention has been focused on the identificationof the most suitable source of endogenous CPCs to mediatetissue regeneration after ischemic heart disease.

Of the different CPC populations described to date [18],EPDCs have emerged as a source with significant potentialarising from their embryonic origin. During development,EPDCs are multipotent, giving rise to all the cardiovascularlineages [21,22,24,25]. However, in contrast, the adult epi-cardium is effectively dormant, having virtually lost alldevelopmental plasticity in the mature heart [20]. A workinghypothesis is that in order to realize the cardiovascular po-tential of adult EPDCs, it is necessary to reinstate theirdevelopmental program, through stimulation with the samemolecules that regulate the lineage potential of the epicar-dium during embryogenesis. While the embryonic epicar-dium has been well characterized with distinct subsets ofcells arising from the PEO, as defined by markers such asWT1, Tbx18, Tcf21, Sema3D, and Scleraxis, [21,25,27,47–49], virtually nothing is known about the detailed compo-sition of the adult lineage.

Using FACS analyses, we characterized the adult-reactivatedWT1 + EPDCs after MI and demonstrated that these cellsreveal a highly heterogeneous molecular phenotype. In par-ticular, we focused our analysis on the expression of specificcardiac progenitor and mesenchymal stem cell markerswithin the adult-activated epicardium, such as Sca-1, c-kit,CD29, CD90, CD44, and CD105. Sca-1 has been widelyreported to discriminate different subsets of cardiac resi-dent stem/progenitor cells in the murine heart, with a sig-nificant role in cardiac repair [18,50–52]. While Sca-1 isexpressed by 50%–60% of the adult WT1 + EPDCs, anotherwell-characterized cardiac progenitor/stem cell marker, c-kit, appeared to be present at extremely low level ( < 1%of the population were c-kit + ), suggesting that the adult-reactivated EPDCs are a source of Sca-1 + CPCs with c-kitlikely identifying a different progenitor compartment withinthe epicardium [46,48]. The adult WT1 + EPDCs appearedto have undergone EMT after injury, as confirmed by theirbroad expression for the mesenchymal antigen CD29. TheWT1 + progenitors were further delineated as CD90 + ,CD44 + , and CD105 + , confirming a mesenchymal-like stemcell phenotype analogous to adult cardiac-resident stemcells of proepicardial origin [38]. While Sca-1, c-kit,CD29, and CD44 expression was equivalent across days 2,4, and 7 post-MI; CD90, CD105, and additional markersPDGFRb and CXCR4 revealed an increasing trend in theirexpression, most notably between days 4 and 7 after injury,suggesting the maturation, within a week after injury, ofan activated mesenchymal phenotype, and one that is po-tentially more responsive in terms of proliferation andmigration.

Reactivation of the developmental program in the dormantadult epicardium suggested that the adult lineage might revertentirely to an embryonic phenotype. Here, we reveal that theadult-reactivated WT1+ GFP + EPDCs are, in fact, molecu-larly distinct from their embryonic counterparts at E12.5; astage in development when embryonic EPDCs begin to ac-tively contribute cardiovascular derivatives. The embryonic

WT1 + epicardium revealed a significantly reduced expres-sion for markers characterizing the adult WT1 + EPDCs, withthe exception of CD29, PDGFRb, and CXCR4, which weresimilarly expressed in both populations. This observationsuggested that, despite the reactivation of the develop-mental program via re-expression of the embryonic geneWt1, the adult EPDCs adopt a different phenotype in re-sponse to cardiac injury relative to embryonic EPDCs whichcontribute to the developing heart. Gene expression profilingrevealed that differences in cell surface markers were alsoreflected at a transcriptional level. Our analysis focused onthe Sca-1 + sub-population of WT1 + GFP + adult progeni-tors at day 4 after MI. At this stage, EPDCs are still re-stricted to the epicardium and sub-epicardial space andpresent with an immature progenitor phenotype, after whichthey subsequently migrate toward the site of injury withinthe underlying myocardium and give rise to mature car-diovascular cells [29]. We first calibrated the gene expres-sion fold changes against the embryonic E12.5 WT1 +

EPDCs and compared the values obtained among the Sca-1 +

and Sca-1 - subsets of the adult equivalent versus the re-maining GFP - cells populating the sub-epicardial space.Remarkably, the Sca-1 + subset of WT1 + progenitors re-vealed expression of developmental epicardial genes atlevels significantly higher than their E12.5 counterparts,suggesting that the expression of Sca-1 might discriminate afraction of cardiac progenitors which retained the strongestpotential to re-activate the developmental program withinthe adult epicardium. We isolated the expanded sub-epi-cardial cell population after MI via enzymatic digestion andcompared the myogenic and vascular profiles of the adultWT1 + GFP + Sca-1 + and WT1 + GFP + Sca-1 - EPDCsagainst the GFP - epicardial cells after Tb4 priming and MI.This analysis revealed that the Sca-1 + subpopulation wasnot only the most active in terms of initiating the develop-mental epicardial gene program, but also pre-empted car-diovascular lineage commitment, via significantly higherexpression of both Isl1 and Flk1, the transcriptional signa-ture of common primordial/multipotent cardiac progenitors,previously described during development [19,42,44]. Inaddition, the Sca-1 + EPDCs revealed a significant up-reg-ulation of the fibroblast marker Fapa and the smooth muscleactin marker Sma when compared with the WT1 + GFP +

Sca-1 - EPDCS and the other GFP - epicardial cells, con-sistent with a bias toward the (myo-) fibroblast lineage afterinjury [53,54]. Furthermore, the increased expression of(myo-) fibroblast gene expression was also further aug-mented by Tb4 treatment, as compared with injury alone.The early cardiac marker Gata4 was down-regulated com-pared with the GFP - epicardial cells, suggesting that thislatter population might retain a higher myogenic potential.Notably, smooth muscle and endothelial vascular markersSm22a and Pecam were down-regulated in both subsetsstratified by Sca-1 expression within the WT1 + progenitors,confirming that neovascularization, which is augmented afterinjury by Tb4 priming, appears to be triggered by a distinctTb4-activated subset of epicardial progenitors, with limitedinvolvement of the WT1 + GFP + EPDCs.

We further investigated the distribution of mesenchymalmarkers CD90 and CD44 within the Sca-1 + fraction of theadult WT1 + epicardium. Tb4 priming and reactivation afterinjury facilitated isolation of a fraction of cells with high

ADULT EPICARDIAL PROGENITOR CELLS ARE HETEROGENEOUS 1727

levels of CD44 and CD90, identifying a sub-populationfated for potential roles in cell–cell interaction, cell adhe-sion, or migration. This Sca-1 + fraction of WT1 + progen-itors retained the most embryonic potential via surrogateexpression of the developmental epicardial genes. In addi-tion, the same sub-population revealed expression of thecardiovascular genes Gata4, Isl1, and Flk1, suggesting thatit may constitute derivatives of the embryonic Isl1 + /Flk1 +

population and an equivalent bipotential toward the vascularsmooth muscle and cardiomyocyte lineages [44].

Understanding the composition of the adult-activatedepicardium alongside the cardiomyogenic or cardiovascularpotential of the resident sub-populations provides importantinsight into the deployment of resident CPCs for cardiacrepair. The potential to instigate tissue repair has to be off-set by the fact that injury promoted a (myo-) fibroblast fatewithin the Sca-1 + sub-population, which could result inincreased collagen deposition and scarring. While fibrosisand scar formation is the default wound-healing response inthe injured hearts of adult mammals, including humans, thisestablishes a local environment that is incompatible withcell replacement. Thus, a (myo-) fibroblast fate would needto be modulated to establish more conducive conditions forcardiovascular regeneration. That said, an important balancehas to be struck to prevent cardiac rupture at the site ofinjury and, moreover, to utilize important reciprocal sig-naling between Sca-1 + epicardium-derived fibroblasts andcoronary vascular cells and cardiomyocytes, as is known tooccur during developmental stages [55].

It remains to be determined whether the epicardium inhuman patients offers an analogous potential to contributetoward tissue regeneration after ischemic injury. Studies thatrealize a therapeutic application of epicardium-derived CPCswill be required to exploit aged animal models, given theprevalence of cardiovascular disease and heart failure in theaging human population, notwithstanding the risk that CPCpotential itself may be compromised with age [56]. More-over, in vivo studies on human patients are essentially limitedto functional imaging (eg, via MRI), which has yet to reachcellular resolution for tracking CPCs during ischemic events.Instead, studies that can take advantage of surgical biopsies(eg, after right coronary artery bypass) may pave the wayfor ex vivo analyses of human primary epicardial CPCs tofacilitate a future pathway toward drug discovery.

Acknowledgments

This work was funded by the British Heart Foundation (RG08/003/25264). The authors are grateful to Ayad Eddaoudifrom the UCL Institute of Child Health Flow Core Facility forassistance in flow cytometry analysis and to RegeneRx Bio-pharmaceuticals for providing clinical-grade Tb4.

Author Disclosure Statement

The authors have no conflict of interests or any disclosureto declare. No competing financial interests exist.

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Address correspondence to:Prof. Paul Richard Riley

Department of Physiology, Anatomy and GeneticsUniversity of OxfordSherrington Building

South Parks RoadOxford OX1 3PTUnited Kingdom

E-mail: paul.riley@dpag.ox.ac.uk

Received for publication January 8, 2014Accepted after revision April 3, 2014

Prepublished on Liebert Instant Online April 4, 2014

1730 BOLLINI ET AL.