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Telocytes in human epicardium

Abstract The existence of the epicardial telocytes was previously documented by immunohistochemistry (IHC) or immunofluorescence. We have also demonstrated recently that telocytes are present in mice epicardium, within the cardiac stem-cell niches, and, possibly, they are acting as nurse cells for the cardiomyocyte progenitors. The rationale of this study was to show that telocytes do exist in human (sub)epicardium, too. Human autopsy hearts from 10 adults and 15 foetuses were used for conventional IHC for c-kit/CD117, CD34, vimentin, S-100, τ, Neurokinin 1, as well as using laser confocal microscopy. Tissue samples obtained by surgical biopsies from 10 adults were studied by digital transmission electron microscopy (TEM). Double immunolabelling for c-kit/CD34 and, for c-kit/vimentin suggests that in human beings, epicardial telocytes share similar immunophenotype features with myocardial telocytes. The presence of the telocytes in human epicardium is shown by TEM. Epicardial telocytes, like any of the telocytes are defined by telopodes, their cell prolongations, which are very long (several tens of μm), very thin (0.1–0.2 μm, below the resolving power of light microscopy) and with moniliform configuration. The interconnected epicardial telocytes create a 3D cellular network, connected with the 3D network of myocardial telocytes. TEM documented that telocytes release shed microvesicles or exocytotic multivesicular bodies in the intercellular space. The human epicardial telocytes have similar phenotype (TEM and IHC) with telocytes located among human working cardiomyocyte. It remains to be established the role(s) of telocytes in cardiac renewing/repair/regeneration processes, and also the pathological aspects induced by their ‘functional inhibition’, or by their variation in number. We consider telocytes as a real candidate for future developments of autologous cell-based therapy in heart diseases.


Introduction
Frequently over-passed and considered to be only a bare serosal cover for the myocardial pump, nowadays the epicardium is revealing some of its cellular and molecular secrets [1]. Recent studies documented the possible key role of the epicardial cells in myocardial development [2][3][4][5][6][7][8][9][10][11][12] and in cardiac regeneration and repair, eventually . However, insufficient attention was paid to fully characterize all cell types populating the epicardium. We previously found by serendipity within human and mammalian myocardium remained unknown. Here we show by transmission electron microscopy (TEM) (seeing is believing!) and immunofluorescence the presence of telocytes and telopodes in human beings. Their functional significance in epicardium remains to be explored.

Tissue specimens
For immunohistochemistry (IHC), cardiac tissue used in the study was obtained either from autopsy or from archived (paraffin embedded) material. We included cardiac samples from five individuals who died of non-cardiac diseases aged 55-

Confocal microscopy
The specimens were fixed for 4 hours in 4% freshly prepared paraformaldehyde, cryoprotected with 20% sucrose, quick-frozen in methylbutane at -130°C and stored at -80°C. Frozen sections 20 µm thick were placed on gelatin-coated slides and then microwaveprocessed in citrate buffer, pH 6.0. After repeated washes in PBS, the tissue sections were blocked and permeabilized with 100 mM glycine, 0.1% carboxylated bovine serum albumin and 0.01% Triton X-100 in PBS, pH 7.6. The cryosections were incubated overnight with the primary polyclonal antibody against c-kit (Dako, Glostrup, Denmark). After washing in PBS, the preparations were incubated with biotinylated donkey anti-rabbit IgG followed by streptavidin-Cy-2 (Biotrend, Cologne, Germany). The nuclei were stained with 0.001% DAPI (Molecular Probes, Carlsbad, CA, USA) and myofibrils were stained with phalloidin conjugated with TRITC (Sigma). Omission of the primary antibody served as a negative control.

Immunohistochemistry
Sections taken from epicardium to myocardium were fixed in 10% formalin and paraffin embedded. Sequential

Transmission electron microscopy
Small fragments of myocardium with epicardium were processed for TEM according to routine procedures, as we previously described [33, [36][37][38][39][40] Figure 1A shows by confocal microscopy c-kit ϩ cells in human subepicardial area. Figure 1B presents a higher magnification view. The epicardial localization of telocytes beneath the mesothelial cells is proven by TEM (Fig. 1C).  staining intensity, between epicardial sides ( Fig. 2A-C). c-kit positivity of some epicardial cells was already shown [41,[45][46][47][48]. High levels of CD34 positivity were noted in the epicardium, but due to the vicinity of a small vessels network, the discrimination between telocytes and endothelial cells was sometimes difficult. Telocytes morphology (peculiar telopodes) may be mimicked by the contiguous aspect of endothelial layer in different incidences (Fig. 3A). In order to individualize the CD34 signal from telocytes from that of endothelial cells, double labelling c-kit/CD34 was performed (Fig. 3B). Similarly, double labelling c-kit/vimentin is shown in Fig. 3C. The aspect reveals the telocytes location in the close vicinity of a capillary, an aspect similar with that previously observed in myocardium [36][37][38]41]. Figure 4A shows telocytes positive for S-100. Immunolabelling for protein was similar with that for S-100 (Fig. 3D) Immunolabelling for CD57 (NK1) was positive for telocytes. However, the results are difficult to interpret because, apparently, the reaction was less specific in cells with prolongations (Fig. 4B).

Transmission electron microscopy
Electron microscopy study unequivocally demonstrated the presence of the telocytes in human epicardium, having their distinctive ultrastructural features according to the TEM diagnostic criteria for telocytes, as described in myocardium [30,31,34,[36][37][38][39][40] and other organs [e.g. 35]. Figures 5-7 present distinctive ultrastructural details of telocytes. In order to make the telocytes more evident, images were digitally coloured in blue. In Fig. 5A it appears in the cell profile of an epicardial telocyte, in between collagen fascicles, in the neighbourhood of a cardiomyocyte.
The distinctive characteristics of telopodes are shown in Fig. 5A and B. Figure 5A shows a 2-telopode telocyte. Of course, the number of prolongations as well as the ramifications of telopodes depend on the site and angle of section, because TEM is essentially a 2D examination of an extremely thin section (~60 nm).
The telopodes shown in Fig. 5A have a length of about 25 and 20 mM, respectively. The calibre of telopodes is uneven, mostly below 0.2 mM. The aspect of telopodes is moniliform, with a dichotomous pattern of branching. Figure 5B presents, at a lower magnification, two very long (about 50 mM) and extremely thin (about 100 nm) telopodes, one with very convoluted profile (the cell process lying over the cardiomyocyte) and another having a surface without flexions or irregularities. For the last one the close vicinity with elastic fibres deserves to be mentioned. Figure 6 is illustrating the exchange of material between telocytes and the extracellular matrix or other cells in subepicardium. The labyrinthine shape of the telopodes seems to help this telocyte to deliver either single vesicles, the so-called 'shed vesicles' (Fig. 6A), or multivesicular bodies, as exosomes ( Fig. 6B and C). Figure 6B clearly shows the close vicinity (under 0.5 mM) of a nerve fibre with the telocyte and its telopode; this was repeatedly presented before as a diagnostic criterion for TEM identification of telocytes (as well as the vicinity with capillaries) [34,[37][38][39][40]. Figure 7 was digitally coloured, in order to differentially label myocytes (Sienna brown), nerve fibres (green) and telocytes (blue). Figure 7A is a typical aspect in epicardium, which presents relationships of a telopode with a large nerve bundle. Figure 7C reveals another typical aspect about telocytes: the frequent positioning at the 'meeting point' of different cell types composing cardiac tissue -myocytes, nerve fibres and adipocytes. The telopodes may establish close contacts with other interstitial cell ( Fig. 7B and C), previously named 'stromal synapses' [49].

Discussion
The cells identified as telocytes in human epicardium meet the characteristic ultrastructural criteria already established for telocyte: (1) (Figs 5A and B, 6A); (iii ) specialized cell-to-cell junctions (e.g. 'stromal synapses' [49]; (iv ) attachment plaques connect the telocytes to the extracellular matrix and the perivascular elastic material (Fig. 5A and B). The (sub)epicardial telocytes apparently make a mesh-like network, comparable with a fishing net woven by a 'short-sighted fisherman'. Similar data were previously reported for telocytes in myocardial sleeves of human pulmonary veins [50], as well as in mice epicardium [30,31,33,41]. Anyway, epicardial telopodes appear very similar with telopodes from myocardium [36][37][38][39][40]41] and other organs [50][51][52][53][54][55]. The screening of the literature revealed few studies evaluating c-kit immunoreactivity in the epicardium, and most of them have focused on the role of c-kit in cardiac development or repair [46][47][48]. Our data emphasize that IHC alone is not enough for the positive diagnosis of telocytes. However, it remains a useful tool for semi-quantitative data analysis, and evaluation of co-expression of antigens. As a consequence, the expression of c-kit alone is not sufficient to characterize an epicardial cell as telocyte. Coexpression of a panel of other markers, including CD34, vimentin, nestin, S-100 might be indicative for the telocytic nature of an interstitial c-kit ϩ cell.
A striking feature of epicardial telocytes is the releasing of shed microvesicles or multivesicular bodies as exosomes in the extracellular matrix (Fig. 6A-C). The nature and the functional meaning of material delivered (and processed?) by these microvesicles remain to be established. Of note, recent studies [57][58][59] have reported that microvesicles transfer tissue specific RNAs and proteins and this newly recognized system of intercellular communications might have a critical role in physiological and pathological processes and offer a new research direction for stem cell therapy [60][61][62][63]. One may speculate that such 'cargo' might be exploited as a therapeutic tool, as we have recently suggested [30].
In our study there were no significant differences of immunohistochemical profile in human beings, when tissue specimens from adults were compared with tissue specimens of foetal origin. Data presented here correlate well with studies approaching epicardial telocytes with similar or complementary methodologies (immunofluorescence or TEM), in human beings and/or murine species [30,31,33,41]. These results should be correlated with the fact that myocardium was explored much more extensively than epicardium, in both normal and pathological conditions. It is interesting to observe that even in studies centred on ultrastructural analysis of transmural biopsy specimens (during ischemiareperfusion injury studies) and 'no attempts were made to analyse endocardium and epicardium separately' [60].
A tempting idea is that this cell might have a therapeutic potential for the design of future cell-based cardiac repair strategies. At least apparently, 'the usual treatment' using mesenchymal stem cells (MSC) is not very encouraging: most recent data provided by Hu's group [64] suggest that only 3-4% of injected MSCs are remaining in the heart. It would be also very interesting to see what happens in a heart when a portion of epicardium is lacking, as well as in extreme situation of pericardiectomy [61,62]. Also, cell turnover should be monitored after protocols intending to transform pericardium in biological scaffolds during tissue engineering procedures (decellularized allografts after treatment with SDS) [63]. It also remains to be established if telocytes might be acknowledged as major a player in mesothelial-cell-induced cardiac repair.
In conclusion, at least for the moment, we strongly believe in our previous hypothesis that telocytes could be either tracks or are nursing cardiac progenitor cells for regeneration. It is attractive to    think that telocytes may nurse the endogenous/ exogenous stem cells for activation and commitment, but also they act as supporting cells for progenitor cells migration towards injured myocardium [30].