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Received 20 Sep 2013 | Accepted 7 May 2014 | Published 6 Jun 2014

vessels and encased by extracellular matrix (ECM). This complex tertiary structure is established during the regenerative window between post-natal days 1 and 7. We show that these HCs proliferate within the rst 24 h and are released between days 2 and 7 after myocardial infarction. The ECM subsequently reforms to encapsulate HCs after 21 days. Vav1-tdTomato labelling reveals an integral contribution of CD45 HCs to the developing

epicardium, which is not derived from the proepicardial organ. Transplantation experiments with either whole bone marrow or a Vav1 subpopulation of cells conrm a contribution of

HCs to the intact adult epicardium, which is elevated during the rst 24 weeks of adult life but depleted in aged mice.

DOI: 10.1038/ncomms5054 OPEN

Dynamic haematopoietic cell contribution to the developing and adult epicardium

Gemma M. Balmer1,*, Sveva Bollini2,*, Karina N. Dub1, Juan Pedro Martinez-Barbera3, Owen Williams4

& Paul R. Riley5

1 Molecular Medicine Unit, UCL-Institute of Child Health, London WC1N 1EH, UK. 2 Regenerative Medicine Laboratory, University of Genoa & IRCCS AOU San Martino-IST, Largo Rosanna Benzi, 16132 Genoa, Italy. 3 Neural Development Unit, UCL-Institute of Child Health, London WC1N 1EH, UK. 4 Molecular Haematology and Cancer Biology Unit, UCL-Institute of Child Health, London WC1N 1EH, UK. 5 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to P.R.R. (email: mailto:[email protected]

Web End [email protected] ).

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In the adult mammalian heart, the existence of resident stem cells, which can act to restore lost tissue after a heart attack (myocardial infarction (MI))1, is the subject of ongoing

investigation. The adult epicardium, classically described as a single cell layer epithelium (mesothelium) lining the chamber myocardium of the heart, has emerged in recent times as a putative resident source of cardiovascular progenitors, which when appropriately activated, for example, by pharmacological treatment with factors such as thymosin b4, can contribute to neovascularisation and myocardial regeneration of the injured adult mouse heart2,3. In addition, the adult epicardium also acts as an important source of trophic signals and has been shown to secrete a number of key angiogenic signals to instruct new coronary vessel growth following MI4. The application of epicardial cell biology to the treatment of cardiovascular injury originates from the epicardiums developmental potential and from the ability to reactivate these properties in the adult heart. During heart development, the epicardium arises from a transient structure called the proepicardial organ (PEO), of mesoderm origin, located at the inow region of the developing heart tube above the primordial liver (reviewed in ref. 5). As the heart begins to loop (E8.59.0 in mouse), PEO cells undergo epithelial mesenchymal transition to migrate towards the developing myocardium and envelop it to form the epicardium proper. This is then followed by a successive round of epithelial mesenchymal transition, whereby epicardium-derived cells (EPDCs) migrate into the sub-epicardial region and contribute interstitial broblasts, vascular smooth muscle cells and, to a lesser extent, coronary endothelial cells and cardiomyocytes to the embryonic heart. The retained fetal epicardial layer also provides growth factors and cytokines to nurture the growth of the underlying myocardium (reviewed in ref. 6). A re-expression of embryonic epicardial genes (characterized by the signature genes Wt1, Tbx18 and Raldh2) initiates adult EPDC activation3,4 and is a hallmark of the downstream contribution of EPDCs in response to MI. The plasticity of EPDCs in the injury setting24,7 prompted us to investigate whether the adult epicardium might be a more complex structure than the previously described simple mesothelium, and whether it might house heterogeneous cell types to act as a regenerative reserve during cardiovascular homeostasis and in response to stress or pathology.

Thus far, the potential of the adult epicardium to be reactivated and contribute to cardiac repair has been realized entirely in the context of reactivation of a dormant, homogeneous single epithelial cell layer. Our ndings suggest the epicardium is not a simple mesothelium but is, in fact, made up of discrete clusters of heterogeneous cell types, which include a novel haematopoietic contribution of distinct origin, encased by extracellular matrix (ECM) that anatomically resembles a stem or progenitor cell niche. This establishes the basis for a re-evaluation of the reparative potential of the adult epicardial lineage. Moreover, a haematopoietic cell (HC) contribution to the epicardium during development and adult homeostasis contrasts with the canonical view of a single PEO source maintained in steady state throughout adulthood. Consequently, the complex architecture of the adult epicardium and turnover of diverse constituent cell types offers the possibility of pharmacological targeting in cardiovascular repair.

ResultsAdult epicardial cell clusters encased by extracellular matrix. We rst analysed intact hearts and, contrary to the prevailing view of the epicardium as a single cell layer, observed small cells with large nuclei-cytoplasmic ratios in situ, at intervals throughout the ventricular chambers and contiguous with the

epicardium, as dened by the surface glycoprotein podoplanin3 (Fig. 1ac). These cell clusters were held in suspension via basement membrane proteins and key components of the ECM such as bronectin (Fig. 1d,e; Supplementary Fig. 1af), b1-integrin (CD29; Fig. 1f), collagen IV (Supplementary Movie 1 of a confocal Z-stack projection) and hyaluronic acid (Supplementary Fig. 1gr). The cells within these apparent microenvironments contained a population dened by specic expression of the cell surface glycoprotein CD44 (ref. 8), previously shown to mark a population of PEO origin that was proposed to give rise to mesenchymal-like stem cells residing within the adult epicardium9 (Fig. 1g). Further characterization revealed the presence of distinct CD45 HCs (Fig. 1h), which

represented the more predominant resident population. Epicardium cell clusters were localized in close proximity to supercial coronary vessels (Fig. 1i,j), including those that contained CD45 HCs (Fig. 1k). Each cluster consisted of

between 3 and 15 CD45 cells; the mean number of cells per

cluster was 6.61.8 s.e.m. (n 25 sections across 5 hearts, at

8 weeks of age) and there were 2.120.26 clusters per mm of epicardium (mean circumference of epicardium/total number of cell clusters/sections.e.m.; n 25 sections analysed across

5 hearts, at 8 weeks of age; Po0.01; Students t-test). These data suggest the epicardium is not a simple mesothelium10, but contains structures that represent a heterogeneous mix of cell populations, including HCs, with vascular support and a non-cellular contribution of ECM.

Epicardial clusters form during early neonatal stages. The three-dimensional geometry of the clusters (Supplementary Movie 1) was indicative of tertiary structure within the adult epicardium, over and above the primary cell constituents and secondary two-dimensional organization of the surface epithelium. To determine when this tertiary structure might be established, we focused on early post-natal stages (P) and specically between days 1 to 7 after birth. This represents a critical window of time on two counts: rst, the potential of the epicardium during development is lost at around P4 in terms of an inherent ability of EPDCs to migrate, contribute cardiovascular derivatives and trophic activity11, and second, the rst week post birth is consistent with loss of regenerative capacity of the heart during P1 to P7. Myocardium was regenerated following ventricular resection injury at P1, but brosis and scarring ensued at P7, and although this switch was accounted for by a loss of proliferative potential of resident cardiomyocytes during this time frame, a contribution from a stem/progenitor cell component could not be excluded12. At P1, ECM components, such as bronectin, were expressed widely throughout the heart and there were no apparent distinct clusters of cells within the epicardium (Fig. 2a), rather a dispersed incidence of CD44 and CD45

cells, which lacked any anatomical constraint (Fig. 2b,c). In contrast, by P7 we observed evidence of nascent epicardial ECM, encapsulating small numbers of CD44 and CD45 cells

(Fig. 2df). There was no evidence of increased CD44 or

CD45 cells during this post-natal window, but simply a re

organization into discrete ECM-dened clusters. Thus, the relatively complex epicardial topology appeared to be initiated during the rst week after birth, putatively reecting a more limited stem cell potential, which may in turn contribute to the transient regenerative capacity of the neonatal mouse heart.

A dynamic CD45 cell and ECM response to ischaemic

injury. We next determined whether the adult epicardium modulated cell function through structural remodelling under conditions of physiological challenge or pathology13. Our focus

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DAPI Pdpn

DAPI Fn

DAPI Pdpn

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DAPI CD29

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ep ep ep

cv cv

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Figure 1 | Clusters of cells constrained by ECM exist in the intact adult epicardium. Immunouorescent staining (IMF) for podoplanin (Pdpn; green) in the intact adult epicardium identies clusters of cells within basement membrane (white arrowheads; ac). Clusters in the epicardium (white arrowheads) are encapsulated by ECM as indicated by IMF for bronectin (Fn; green; d). Inset box in d is shown at higher magnication in e, and CD29 (b1-integrin; f). IMF for CD44 and CD45 revealed the clusters were a heterogeneous population of mesenchymal (CD44; g) and HCs (CD45; h).

The clusters resided in close proximity to coronary vessels (i,j) as conrmed by immunostaining on a Tie2Cre;R26R-tdTomato background (k). cv, coronary vessel; ep, epicardium; my, myocardium. Scale bars, 40 mm (a); 20 mm (be); 10 mm (fk).

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here was on the novel CD45 population given the previous

description of epicardium-derived CD44-derived mesenchymal stem cell-like cells9. MI can cause extensive epicardial brosis14 and, consequently, analyses were carried out on neighbouring intact epicardium, as previously investigated3,4,15. After MI, we observed that resident CD45 cells began to proliferate at 24 h

as detected by pulse chase (injections at 4, 8 and 22 h post MI) with 5-bromo-2-deoxyuridine (BrdU; Fig. 3a,b) and co-staining with Ki67 (Fig. 3cf). The numbers of proliferating cells per cluster ranged from 2 to 5 and the mean number of BrdU or

Ki67 cells was 3.20.6 s.e.m. (n 15 sections, across 6 hearts

at 8 weeks of age). Subsequently, the ECM boundary appeared to break down, within the rst 2 days, as best illustrated by a downregulation of bronectin protein expression leading to loss of epicardial integrity (Fig. 3g). ECM breakdown was supported by co-localization of matrix metalloproteinase 2 and 9 expression at corresponding stages post MI (Supplementary Fig. 2ad). This breakdown persisted to day 7 post MI and was accompanied by concomitant expansion of CD45 cells (Fig. 3i,k;) and their

release into the subepicardial region and underlying myocardium (Fig. 3k, right hand panel). Interestingly, regional differences in epicardial response were observed, with disassembly of ECM structures proximal to the site of injury in the left ventricle (LV), whereas those more distal in the right ventricle remained relatively intact, even up to 7 days after injury (Fig. 3h,j,l), indicating a spatial gradient in response to injury signalling. After 21 days post MI, the tertiary structure reformed in the LV with restoration of CD44 cells (Fig. 3m) and CD45 cells (Fig. 3n),

such that by day 42 there was a return to the pre-injury quiescent state (compare Figs 1ac and 3o) and comparable to that

observed throughout in the right ventricle (Fig. 3p). Collectively, these data suggest that the adult epicardium is a dynamic structure, which responds to injury signals from the infarcted LV to break down the local ECM and release proliferative progenitors into the underlying injured myocardium.

Canonical Wt1 epicardial cells are distinct from HC clusters.

We next investigated the relative spatial localization of reactivated Wt1 cells, previously identied as an important subpopulation

of epicardial progenitors following MI3,4, within the higher-order structure of the adult epicardium. Injury-activated Wt1 cells

were distinct from the resident CD45 HC population

(Fig. 4a,b) and, although they were not localized within the clusters they resided immediately proximal (Fig. 4c,d). The response of the Wt1 population varied considerably depending

on the extent of infarction and injury, as previously reported3,4. Following injury (day 4 post MI), the breakdown of the ECM clusters and expansion of the sub-epicardial region contained some Wt1 cells, as reported by Wt1CreERT2;R26R-tdTomato

lineage tracing from developmental stages alongside immunostaining for Wt1 (Fig. 4e,f). Efcient recombination of the R26R-tdTomato, by tamoxifen-induced Wt1CreERT2, was observed with specic tdTomato reporter labelling of the

epicardium throughout development and into adulthood (Supplementary Fig. 3al), as well as efcient tracing of Wt1

cell derivatives, such as smooth muscle cells, within the adult heart (Supplementary Fig. 4ad). Wt1-tdTomato cells were

also evident throughout the myocardium (Fig. 4e), reecting the increased contribution of Wt1 derivatives over time (Supplementary Fig. 3hl). The Wt1-negative cells in the

ep ep

DAPI Fn

DAPI Fn CD44

DAPI Fn CD45

DAPI Fn CD44

DAPI Fn CD45

Figure 2 | Tertiary epicardial structure forms during early post-natal stages. At post-natal day 1 (P1), there is no evidence of cell clusters or higher-order structure in the epicardium; Fn staining is diffused throughout (a), and the CD44 and CD45 cells that are present do not occupy

discrete locations within the epicardium (b,c). By P7, there is evidence of Fn surrounding the forming clusters containing CD44 (d) and CD45

cells (e,f; clusters highlighted by white arrowheads). White inset boxes in the left panels are highlighted at higher magnication in neighbouring right panels. ep, epicardium; my, myocardium. Scale bars,: 50 mm (a, left panel); 20 mm (a, right panel); 30 mm (bf, left panel); 20 mm (f, right panel).

Figure 3 | Epicardial HC clusters respond dynamically to myocardial injury. Following MI, there is an initial proliferative response as determined by BrdU pulse labelling of cluster cells 1 day post injury (1 d.p.i.) in both the left (LV) and right (RV) ventricles (white arrowheads highlight BrdU cells;

a,b). CD45 cells located within the epicardial layer were conrmed as undergoing cell cycle activity via Ki67 co-staining in both the LV (c,d) and RV (e,f;

white arrowheads highlight CD45 /Ki67 cells). As early as day 2 post MI (2 d.p.i.) in the LV, the clusters begin to break down (g). Disassembly of

ECM-encapsulated cell clusters persists through day 4 (4 d.p.i.; i) and progresses to day 7 (7 d.p.i.; k) to release CD45 cells (g,i,k) This is not the case in

the RV where the clusters remain intact over the same time course of injury constraining the resident cell types (h,j,l); white arrowhead in l highlights intact cluster 7 d.p.i. By day 21 post MI (21 d.p.i.), the clusters have reformed (white arrowheads in higher magnication view highlight Fn at the apical surface) in the LV (m) to resemble the persistent intact clusters of the RV (n) and pre-injury state (Fig. 1); the reinstated niche is evident 42 days post MI (42 d.p.i.; o) and is comparable to that in the RV as highlighted by white arrowheads (p). White inset boxes in the left panels are highlighted at higher magnication in neighbouring right panels. ep, epicardium; my, myocardium. Scale bars, 50 mm (ap, left panels); 20 mm (g,i,j,l,n); 10 mm (a,b,h,k,mp, right panels).

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sub-epicardial region were predominantly CD45 (Fig. 4g,h),

suggesting an inux of HCs consistent with an early pro-inammatory response16. The extent of CD45 cellular

contribution to the expanded epicardium at day 4 post MI was comparable to that observed within the bone marrow (Supplementary Fig. 5).

1 d.p.i.

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D2 MI

D21 MI

DAPI CD45 Wt1

D4 MI

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ep ep

DAPI Wt1 Tom.

DAPI CD45 Wt1

DAPI Tom.

Vav1-tdTomato

Vav1-tdTomato D4 MI

ep sc

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Figure 4 | Injury-activated Wt1 epicardial cells are mutually exclusive from CD45/Vav1 HCs in the expanded epicardium. At 2 days post MI

(D2 MI), cells re-expressing Wt1 (highlighted by white arrowheads) are distinct from the CD45 population residing in the expanding epicardium

(a,b; white inset box in a, shown at higher magnication in b). At day 21 post MI (D21 MI), Wt1 cells reside proximal to the niche-like clusters (c,d; white inset box in c, shown at higher magnication in d). Wt1CreERT2;R26R-tdTomato labelling and Wt1-antibody staining revealed Wt1 /tdTomato cells

residing in the expanded epicardium and tdTomato-labelled derivatives within the underlying myocardium (e,f; arrowheads indicate double-positive Wt1 /tdTomato cells) relative to an abundance of inltrating CD45 cells (g,h; white inset box in g shown in higher magnication in h; arrows

in h indicate CD45 cells and arrowheads indicate Wt1 cells) and HCs labelled in a Vav1-Cre; R26R-tdTomato reporter line (i,j; white inset box in i,

shown at higher magnication in j). At day 4 post MI (D4 MI), Wt1 and tdTomato are expressed in distinct cell populations with a few notable exceptions highlighted by white arrowheads (kn; white inset boxes in k and m are highlighted at higher magnication in l and n, respectively). ep, epicardium; my, myocardium; sc, scar. Scale bars, 50 mm, (a,c,e,g,i); 100 mm (k,m); 20 mm (b,d); 30 mm (f,h,j,l,n).

CD45 epicardial cells arise from the haemagenic endothelium.

To conrm a haematopoietic origin for the CD45 population,

we traced cells residing in the sub-epicardium using Vav1-Cre; R26R-tdTomato mice. Vav1-Cre labels developmental

haematopoietic progenitors arising from the yolk sac blood islands (Supplementary Fig. 6a,b) and the aorta-gonadmesonephros region (sites of primitive and denitive haematopoiesis, respectively; Supplementary Fig. 6c), the fetal liver

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Vav1-Cre/+;R26R-tdTomato/+

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E9.5

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peo

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Figure 5 | Vav1 /CD45 HCs contribute to the developing epicardium and do not originate from the PEO. In Vav1-Cre;R26R-tdTomato embryonic

hearts, tdTomato (Vav1 ) haematopoietic stem cells (HSCs) contribute to the developing epicardium from E12.5 to E16.5. At E14.5 (a,b) and E16.5 (c,d),

tdTomato (Tom) cells reside in the epicardium and underlying myocardium (white inset boxes in a and c are highlighted at higher magnication in b and

d, respectively; white arrowheads in b and d highlight tdTomato cells specically in the epicardium). At E14.5, further immunostaining for combined

a-CD45 and tdTomato (e) revealed that all CD45 cells residing within the developing epicardium were Vav1-tdTomato (fh; white arrowheads

highlight double-positive cells), whereas Vav1-tdTomato cells negative for CD45 were localized within the underlying myocardium (fh). The CD45

HCs were distinct from the Wt1 contribution as indicated by CD45 immunostaining in conjunction with lineage tracing in WtCreERT2;R26R-tdTomato

developing hearts at E14.5 (il; white inset boxes in i and k are highlighted at higher magnication in j and l, respectively; white arrowheads in j and l highlight CD45 cells specically in the epicardium). The number of CD45 cells that contributes to the epicardium increases from E10.5, coincident

with formation of the epicardium, through to E16.5 with a signicant increase between E12.5 and 14.5 (mean % CD45 cells s.e.m.; Pr0.001; n 6

hearts; m). In Vav1-tdTomato-labelled embryos, tdTomato cells do not contribute to the proepicardial organ (peo; indicated in blue in n; rest of heart

indicated in green via Nkx2.5Cre;R26R-EYFP) at E9.5 (o; peo is demarcated by Wt1 immunostaining) and CD45 cells do not localize to the peo (p),

suggesting tdTomato/CD45 HCs that contribute to the forming epicardium arise from an alternate source, previously described as the haemagenic

endothelium and/or fetal liver (Supplementary Fig. 6). ep, epicardium; lv, left ventricle; my, myocardium; peo, proepicardial organ; rv, right ventricle. Scale bars, 200 mm (a,i); 20 mm (b,d,fh); 400 mm (c,e); 50 mm (j,l); 100 mm (i,o,p); 600 mm (n). All statistics are calculated by Students t-test; *Pr0.001.

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(a second site of denitive haematopoiesis; Supplementary Fig. 6d,e), as well as adult pan-circulatory HCs1719. Signicant numbers of Vav1-tdTomato cells were observed in the

expanded sub-epicardium (Fig. 4i,j) by day 4 post MI and these were largely negative for Wt1 protein expression aside from isolated examples of tdTomato /Wt1 cells (Fig. 3kn). The

relative proportion of single tdTomato (Vav1 ) versus

single Wt1 cells was 77.616.3% versus 22.44.8% (mean

%s.e.m.; Pr0.001; n 6 hearts; Students t-test). Importantly,

we excluded the endothelial markers, CD31 and Endomucin, from the CD45 sub-population located within the epicardial

and sub-epicardial regions at day 4 post MI, in support of an HC phenotype (Supplementary Fig. 7ac).

Given the prevalence of CD45 and Vav1 cells in both the

intact epicardium and in response to injury, we traced Vav1-tdTomato cells through developmental stages to determine

whether there may be an embryonic haematopoietic contribution to the forming epicardium (Fig. 5). We observed tdTomato

cells residing in the epicardium and underlying myocardium from E12.5, with a prominent contribution at both E14.5 (Fig. 5a,b) and E16.5 (Fig. 5c,d). The presence of HCs within the embryonic epicardium was conrmed by immunostaining for CD45 at E14.5 in Vav1-Cre;R26R-tdTomato mice (Fig. 5eh), which revealed CD45 /m-tomato cells integrated within the

outer epicardial layer (Fig. 5fh) that were negative for the endothelial cell marker CD31 (Supplementary Fig. 7d). Not all Vav1 cells were CD45 , consistent with the broader Vav1

lineage17, but all CD45 cells localized within the epicardium

were Vav1 , as evident in the lineage-traced hearts at E14.5

(Fig. 5fh). At higher magnication (Fig. 5g), the CD45 /

tdTomato cells were located on the apical surface of the heart,

contiguous with the outer epicardium, suggesting they contributed intrinsically to the polarized epithelium. In contrast, immunostaining for CD45 in Wt1CreERT2;R26R-tdTomato mice (Fig. 5il), revealed that the HCs were distinct from Wt1-tdTomato epicardial cells (Fig. 5l). The incidence of

CD45 cells residing in the epicardium signicantly increased

from 7.90.01% at E12.5 to a maximum of 17.0 1.2% at E14.5 (Pr0.001; n 6 hearts; Students t-test; Fig. 5m). To exclude the

possibility that the CD45 cells might have arisen from the PEO

(Fig. 5n), we stained Vav1-tdTomato mice with a-CD45 and observed that both tdTomato and CD45 were not expressed within the Wt1 PEO (Fig. 5o,p). In addition, lineage tracing

with Gata5Cre;R26R-tdTomato and Wt1CreERT2;R26R-tdTomato mice revealed that neither Gata5 nor Wt1

epicardial lineages gave rise to CD45 cells (Supplementary Fig. 8). In contrast, a proportion of Gata5-tdTomato cells

(o10%) were also CD44 , consistent with a mesenchymal-like

cell origin from the PEO9. These data suggest that HCs contribute to the forming epicardium during development and arise from a Vav1 haemogenic (haemangiogenic) source, which is distinct

from the PEO.

Bone marrow-derived HCs integrate into the adult epicardium. To explore a potential haematopoietic contribution to the epicardium of the intact heart during adult homeostasis, we carried out cell transplantation of either labelled bone marrow (BM) from b-actin-Cre;R26R-EYFP or HCs from Vav1-Cre;R26R-tdTomato donors into lethally or sub-lethally irradiated unlabelled hosts (Supplementary Fig. 9a,b). Ionizing radiation targets mitotically active cells, which in the intact adult epicardium are likely to be represented by the previously described CD44

cCFU-Fs9. Haematopoietic reconstitution occurs in mouse in waves, with transient reconstituting cells detectable after 24 weeks, but which disappear after 810 weeks, and longer-term

reconstituting cells that contribute at least 6 months after transplantation20. Thus, a 6-month follow-up herein was employed to negate the possibility of HC inux due to irradiation-induced inammation and, consequently, to investigate potential long-term (homeostatic) BM contribution and epicardial cell turnover. Following irradiation, we were unable to detect either CD44 cells, consistent with ablation of

the cCFU-Fs as previously described9 or host CD45 cells, even

after 6 months. YFP BM cells were observed in the epicardium

and sub-epicardial region after 2 months, residing both within the single epicardial cell layer overlying the myocardium and epicardial cell clusters (Fig. 6ad). YFP cells were also

observed to trafc into adult epicardial clusters at 2 months (Supplementary 10a,b). At 6 months, a YFP contribution was

still evident in the epicardium with some contribution to the adjacent myocardium (Fig. 6eh). Quantitation revealed the overall numbers of YFP BM cells signicantly increased in the

epicardium over the 4-month duration of the analyses (Pr0.001; n 6 hearts; Students t-test; Fig. 6i). Although early on,

following host g-irradiation, donor BM-derived cells may inltrate due to radiation-induced injury and inammation, the signicant YFP BM presence at 6 months represents an

injury-independent contribution. Podoplanin immunostaining conrmed the localization of YFP BM derivatives to the

epicardium at both 2 and 6 months (Fig. 6j,n); although there was signicant variation in the abundance of transplanted cells depending on location within the chambers (compare Fig. 6k,m with Fig. 6n,q), after 2 and 6 months. The YFP cells were all

CD45 indicative of the HC compartment of the BM inoculum

(Fig. 6k,o). The majority of those residing within the epicardium itself (490%) were negative for the monocyte marker Ly-6C (Fig. 6l,p); however, a more detailed characterization of their immuno-phenotype revealed a minor subpopulation (o10%) of transplanted tomato /CD45 cells, which were positive for the

macrophage marker F4/80 (Supplementary Fig. 11a,b); in contrast, none of the transplanted cells co-stained for the neutrophil marker myeloperoxidase (Supplementary Fig. 11c). Evidence of subsequent YFP BM cell differentiation within the

myocardium was observed by virtue of cells co-staining for the pericyte marker NG2 (Fig. 6m,q; Supplementary Fig. 12a).

Further analyses on the lineage potential of the transplanted BM cells at 2 months revealed co-staining of YFP cells with an

additional pericyte marker CD146 (ref. 21 (Supplementary Fig. 12b) and the exclusion of the broblast marker PDGFRb

Supplementary Fig. 12c,d).

Transplanted Vav1 CD45 cells become depleted with age.

The BM ndings were supported by transplantation with lineage-negative HCs as labelled by Vav1-Cre;R26R-tdTomato. Tomato cells were observed in the epicardium at 2 months as

demarcated by podoplanin (Fig. 6r) and were localized into clusters (Supplementary Fig. 10b). These cells were CD45 , and

consistent with the whole BM YFP transplantation data the

majority were Ly-6C (Fig. 6s,t), of which a proportion had

differentiated into NG2 pericytes located in the underlying

myocardium (Fig. 6u), consistent with the previous observations for whole BM. Tomato cells post-BM transplantation either

residing within the epicardial or underlying myocardium were also negative for the endothelial cell markers CD31 and Endomucin (Supplementary Fig. 7eh). We subsequently investigated the status of the CD45 compartment in older animals, given

that aging is proposed to act as a negative modier of cellular efcacy (reviewed in ref. 22). A time course from post-natal day 7 (P7) through to 18 months suggested the epicardium is turned over via HC contribution, with increasing recruitment of CD45

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-actin-YFP

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Figure 6 | Bone marrow transplantation reveals a haematopoietic contribution to the adult epicardium that alters with age. Two months after whole bone marrow (BM) transplantation of b-actin-Cre;R26R-EYFP labelled BM into lethally irradiated unlabelled hosts (Supplementary Fig. 9a), YFP cells are

localized to the adult epicardium (a,b; as highlighted by white arrowheads) and the YFP BM cells contribute to epicardial clusters (c,d). The YFP cells

persist at 6 months post transplantation and are localized to the epicardium (eh; as highlighted by white arrowheads) and there was a signicant increase in epicardial YFP cells over the intervening 4 months (mean %YFP cells s.e.m.; Pr0.001; n 6 hearts; i). At 2 and 6 months, podoplanin

immunostaining conrmed the epicardial localization of YFP BM cells (j,n) and these were CD45 (k,o; white arrowheads; white asterisk highlights a

YFP /CD45 cell), suggesting they represented the haematopoietic compartment of the whole BM inoculum, but were negative for Ly6C, thus excluding

the mature myeloid lineage (l,p; white arrowheads indicate YFP cells in red which do not co-express Ly6C in green). A sub-population of YFP BM cells

within the underlying myocardium expressed NG2, suggesting they had differentiated into pericytes (m,q; highlighted by white arrowheads). Two months after transplantation of ow-sorted Vav1 cells, from Vav1-Cre;R26R-tdTomato-labelled bone marrow into lethally irradiated unlabelled hosts

(Supplementary Fig. 9b), tdTomato cells are localized to the podoplanin adult epicardium (r; as highlighted by white arrowheads). The tdTomato

BM cells were CD45 (s; white arrowheads), conrming them as representative of the haematopoietic compartment of the whole BM inoculum. The

tdTomato donor cells were negative for Ly6C, thus excluding the mature myeloid lineage (t; white arrowheads indicate tdTomato cells in red, which do

not co-express Ly6C in green). A sub-population of tdTomato BM cells within the underlying myocardium expressed NG2, suggesting they had

differentiated into pericytes (u; highlighted by white arrowheads). The number of CD45 cells increased in the adult epicardium with age up to 24 weeks

(v,w) but decreased thereafter as measured at 52 and 78 weeks (x,y). The increases between 1 and 8, and 8 and 24 weeks were signicant, as was the decrease between 24 and 52 weeks (mean % CD45 cellss.e.m. of n 3 hearts, 6 sections analysed per heart; Pr0.001; z), suggesting an increased

haematopoietic epicardial reserve with adulthood to maturity but depletion or impaired replenishment with advanced age (12 and 18 months). ep, epicardium; my, myocardium. Scale bars, 50 mm (a,b,e,f,vy); 20 mm (c,d,j,ko,qs,u); 10 mm; (g,h); 10 mm (i,p,t). All statistics are calculated by Students t-test; *Pr0.001.

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cells between P7 and 6 months (Fig. 6vy). In older mice, the number of CD45 cells was signicantly reduced from

20.32.1% at 6 months down to 12.22.2% at 12 months and 9.671.3% at 18 months (means.e.m. of n 3 hearts,

6 sections analysed per heart; Pr0.001; Students t-test; Fig. 6z), suggesting that either HC contribution to the epicardium is compromised with age or the resident CD45 population is

depleted over time.

DiscussionThe characterization of the adult epicardium as a complex anatomical structure is in stark contrast to the widely held view of the epicardium as a simple mesothelium and single cell layer epithelium. The adult epicardial topology contained features associated with a canonical stem cell niche, as have been characterized in the BM, skin (hair follicle bulge), intestine (villi and crypts) and subventricular zone of the brain2326. Most notably, the existence of clusters of cells, within the epicardium, encased by basement membrane and with associated vascular support meets several anatomical niche criteria (reviewed in ref.27). In the adult heart, cell turnover and renewal based on stem cell deployment has been demonstrated9,28; yet, occupancy of a cardiogenic stem cell niche29 remains to be determined30,31. Whether the epicardium can be regarded as a classical niche requires functional disruption of the microenvironment described, so as to directly affect the resident stem/progenitor cell population in terms of homing, multipotency or differentiation of progeny during homeostasis or injury. These represent ongoing, but technically challenging, experiments that lie outside the scope of the current study.

The niche-like structure is formed during a critical post-natal window when the inherent regenerative capacity of the heart is lost within the rst week after birth12,32. This may reect the limited reparative capacity of the maturing heart, at least in part, via the constraint of epicardial cells within the ECM (Supplementary Movie 1). Following myocardial injury, an initial breakdown of epicardial basement membrane was accompanied by release of constituent cells and subsequent reformation via assembly of ECM. The ECM plays a key role in a niche setting, as it is thought to protect the progenitor compartment from depletion while simultaneously protecting the host tissue from overproliferation. Although the resident cells remain in a state of quiescent under normal conditions, the ECM is also important for transmitting signals to induce self-renewal and/or differentiation during physiological stress or in response to injury (reviewed in ref. 33). The resident cellular compartments in the epicardium consist of both CD44 mesenchymal-like

stem cells (cCFU-Fs), previously demonstrated to be of epicardial origin9, and a novel BM-derived CD45 haematopoietic

population, which appeared to participate in homeostatic EPDC turnover with age. Both cell types are incorporated into the clusters irrespective of origin during the rst week of life and the CD45 population is subsequently replenished from BM.

Although tissue monocytes of BM origin are known to migrate into almost every solid organ of the body, including the adult heart (reviewed in ref. 34), an HC contribution to the epicardium represents a novel nding. The contribution to adult epicardium by HCs was injury independent, with long-term reconstitution of donor HCs following BM transplantation well beyond any potential inammatory response arising from host irradiation. Although we were unable to denitively discriminate the response of a resident CD45 /Vav1 population from BM inltration

during injury, the remodelling of the ECM surrounding the cell clusters and resident CD45 cell proliferation was distinct from

an inux of BM HCs post MI. Moreover, the long-term

reconstitution of BM/Vav1 transplanted cells suggested the

BM can act as a source of replenishment of epicardial HCs, which in turn are then able to respond to injury independently of an acute BM response.

Previously, whole BM transplantation revealed that engraftment of BM-derived cells occurred as part of normal cardiac valve function, setting a precedent for a BM contribution to cardiovascular homeostasis per se35. Our study supports a role for BM in adult heart homeostasis and the HC turnover, contrasts with the current steady-state view of a single source of epicardium maintained throughout adulthood; this in turn reinforces the concept that the adult mammalian heart should no longer be considered a post-mitotic organ3638.

CD45 HCs were labelled by a Vav1-Cre-induced reporter,

which, when subsequently traced through developmental stages, revealed a novel contribution to the embryonic epicardium from the haemagenic endothelium or fetal liver18,19. A distinct sub-population of HCs was recently characterized within the developing heart as arising from the endocardial lineage, and contributing an additional reservoir for denitive haematopoiesis39. In contrast, we report here the integration of CD45 HCs into the developing epicardium, arising from

classical sites of both primitive (yolk sac blood islands) and denitive (aorta-gonad-mesonephros, fetal liver) haematopoiesis within the embryo. Notably, the CD45 /Vav1 population was

entirely distinct from Wt1-expressing cells of the PEO. Cellular heterogeneity within the epicardium has been reported in previous studies, which focused on the lineage composition of the PEO, as the canonical source of epicardium progenitors9,40,41. Although a number of sub-compartments within the PEO itself have been well characterized, notably those labelled as Wt1

and Tbx18 , as well as partially overlapping Sema3D and

Scleraxis sub-populations40, a novel source of epicardial cells of

alternate embryological origin has not hitherto been reported. Our ndings challenge current dogma that the epicardium arises exclusively from the transient, mesoderm-derived PEO, located at the inow region of the developing heart5.

Understanding the developmental biology of the epicardium is instrumental for developing novel cell therapies for heart regeneration. We and others have previously demonstrated that the adult epicardium, can be reactivated, via induction of an embryonic gene programme, to act as a potential resident cell source, which can be tapped to reconstitute damaged heart tissue24,7,42. This potential of the adult epicardium was realized entirely in the context of a homogeneous Wt1 epicardial

cell response. The integral contribution of CD45 HCs to

both embryonic and adult epicardium, and the turnover of a BM-derived HC source during adult homeostasis, should yield important insight into the potential for both self-renewal and resident cell-based repair within the heart. It will be of interest to determine whether the HC contribution to the epicardium and complex tertiary structure of constraining ECM identied here in mouse translates to humans, and whether it might be targeted for activation and contribution to tissue regeneration following ischaemic heart disease.

Methods

Generation of epicardial trace mice. Wt1CreERT2/ , Gata5Cre/ and Vav1Cre/ ; R26RtdTomsto/ mice were generated crossing Rosa26R-tdTomato43 reporter mice with Wt1 CreERT2/ (ref. 44), Gata5Cre/ (ref. 45) and Vav1Cre/ (ref. 46) mice, and genotyping as previously described. Wt1 expression in the developing epicardium was achieved through double intraperitoneal tamoxifen injections. Toxic effects of tamoxifen were countered by co-injection of progesterone. Pregnant dams received two 2-mg tamoxifen injections and simultaneous 1 mg progesterone injections at E9 and E11. Embryos were collected at specic time points and prepared for immunouorescence. Images were obtained using a using Zeiss LSM 710 confocal microscope equipped with argon and helium neon lasers using 20, 40 and 63/1.4 (oil immersion) objectives. All animal experiments

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were carried out according to the UK Home Ofce project licences PPL30/2987 Compliant with the UK Animals (Scientic Procedures) Act 1986 and approved by the University College London Biological Services Ethical Review Process. Animal husbandry at UCL Biological Services was in accordance with the UK Home Ofce Certicate of Designation.

Immunodetection. Immunouorescence was performed on cryosections of embryonic, uninjured and post-MI hearts using standard protocols with the following antibodies: green uorescent protein (GFP; Clontech and Abcam, which detect EYFP), Fibronectin, CD44, CD45, BrdU, Hyaluronic acid, Ly-6C and Wt1 (all from Abcam), podoplanin (Novus Europe), Collagen IV (AbD Serotec), NG2 (Chemicon) and CD29 (Millipore). To rule out the possibility of autouorescence accounting for the detection of either GFP or yellow uorescent protein (YFP) protein expression, sections through the LV were stained with a polyclonal anti-GFP antibody (which detects both uorescent proteins). The specicity of the anti-GFP antibody was ascertained by immunouorescence on non-primed, intact hearts, which detected neither labelled cells in the epicardial region, nor their derivatives (no signal).

To detect BrdU-positive nuclei, sections were treated with 2 N HCl for 30 min at room temperature (22 C) to denature the DNA, and neutralized in 0.1 M sodium borate pH 8.5 for 12 min before incubation with the anti-BrdU antibody. Owing to the destruction of cellular antigens resulting from acid treatment, these steps were performed after the incubation with antibodies to GFP and SaA. Images were acquired using a Zeiss LSM 710 confocal microscope equipped with argon and helium neon lasers using 20, 40 and 63/1.4 (oil immersion) objectives.

mTomato uorescent protein used to test the Cre-lox recombination of Gata5, Wt1 and Vav1 Cre driver strains was visualized directly without secondary staining protocols at its emission wavelength of 581 nm.

MI surgery. Mice were housed and maintained in a controlled environment. All surgical and pharmacological procedures were performed in accordance with the Animals (Scientic Procedures) Act 1986, (Home Ofce, UK). MI was induced in isourane-anaesthetized mice by permanent ligation of the left anterior descending artery. On recovery, animals received intraperitoneal injection of BrdU(80 mg kg 1; Invitrogen) if required. Further injections were administered at2, 4 and 8 h post surgery. Hearts were harvested at 2, 4, 7, 21 and 42 days after ligation and bisected transversely midway through the scar and xed in 4% paraformaldehyde for cryosectioning and immunostaining analyses.

BM collection. All mice were culled before tissue dissection in accordance with the Home Ofce regulation. Hind limbs were dissected to isolate the femur and knee joint. Femurs were ushed using a 23-G needle and 1-ml syringe (both from Becton Dickinson) with PBS, and cells were collected and kept on ice. Cells were then ltered using a 70-mm cell strainer (Becton Dickinson) and centrifuged at 200 g for 5 min. Five millilitres of Red Cell Lysis Buffer was used to resuspend the pellet and incubated for 3 min before centrifuging at 200 g for 5 min to pellet the cells. Cells were then used for RNA extraction or transplantation.

BM irradiation and transplantation. Mice are housed and maintained in a controlled environment. All procedures are performed in accordance with the Animals (Scientic Procedures) Act 1986, (Home Ofce, UK). Mice were sublethally (6 Gy) irradiated or lethally irradiated, using split dose (4 5 Gy) radiation,

24 h before cell transfer.

Donor mice were culled before tissue dissection in accordance with the Home Ofce regulation. Hind limbs were dissected to isolate the femur and tibia bones. Single-cell suspensions were isolated in Hepes-buffered HBSS and either transplanted directly into recipient mice or lineage-negative cells were rst puried using the mouse Lineage Cell Depletion kit (Miltenyi Biotec). In total, 1 107 or

2.5 105 lineage-negative cells were injected via the lateral tail vein.

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Acknowledgements

We thank Bertrand Vernay at the UCL-ICH Light Microscopy Core Facility for technical assistance with imaging and Cynthia Andoniadou for early embryo dissections. This work was generously supported by a Wellcome Trust PhD studentship 089592/Z/09/A (G.M.B.) and British Heart Foundation programme grant RG/ 08/003/25264 (P.R.R.).

Author contributions

G.M.B. carried out the experiments, analysed the data and assisted in compiling gures; S.B. carried out immunostaining analyses, analysed data and provided MI heart injury samples; K.N.D. provided MI heart samples; J.P.M.-B. co-supervised experiments and

provided technical input into lineage tracing; O.W. carried out the bone marrow and Vav1 cell transplantations; P.R.R. established the project, analysed data and wrote the

manuscript.

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Abstract

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The epicardium is a cellular source with the potential to reconstitute lost cardiovascular tissue following myocardial infarction. Here we show that the adult epicardium contains a population of CD45+ haematopoietic cells (HCs), which are located proximal to coronary vessels and encased by extracellular matrix (ECM). This complex tertiary structure is established during the regenerative window between post-natal days 1 and 7. We show that these HCs proliferate within the first 24 h and are released between days 2 and 7 after myocardial infarction. The ECM subsequently reforms to encapsulate HCs after 21 days. Vav1-tdTomato labelling reveals an integral contribution of CD45+ HCs to the developing epicardium, which is not derived from the proepicardial organ. Transplantation experiments with either whole bone marrow or a Vav1+ subpopulation of cells confirm a contribution of HCs to the intact adult epicardium, which is elevated during the first 24 weeks of adult life but depleted in aged mice.

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Title

Dynamic haematopoietic cell contribution to the developing and adult epicardium

Author

Balmer, Gemma M; Bollini, Sveva; Dubé, Karina N; Martinez-barbera, Juan Pedro; Williams, Owen; Riley, Paul R

Pages

4054

Publication year

2014

Publication date

Jun 2014

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms5054

ProQuest document ID

1532971914

Dynamic haematopoietic cell contribution to the developing and adult epicardium

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