Telocytes in human skin – are they involved in skin regeneration?

Abstract Telocytes (TCs), a particular interstitial cell type, have been recently described in a wide variety of mammalian organs (www.telocytes.com). The TCs are identified morphologically by a small cell body and extremely long (tens to hundreds of μm), thin prolongations (less than 100 nm in diameter, below the resolving power of light microscopy) called telopodes. Here, we demonstrated with electron microscopy and immunofluorescence that TCs were present in human dermis. In particular, TCs were found in the reticular dermis, around blood vessels, in the perifollicular sheath, outside the glassy membrane and surrounding sebaceous glands, arrector pili muscles and both the secretory and excretory portions of eccrine sweat glands. Immunofluorescence screening and laser scanning confocal microscopy showed two subpopulations of dermal TCs; one expressed c-kit/CD117 and the other was positive for CD34. Both subpopulations were also positive for vimentin. The TCs were connected to each other by homocellular junctions, and they formed an interstitial 3D network. We also found TCs adjoined to stem cells in the bulge region of hair follicles. Moreover, TCs established atypical heterocellular junctions with stem cells (clusters of undifferentiated cells). Given the frequency of allergic skin pathologies, we would like to emphasize the finding that close, planar junctions were frequently observed between TCs and mast cells. In conclusion, based on TC distribution and intercellular connections, our results suggested that TCs might be involved in skin homeostasis, skin remodelling, skin regeneration and skin repair.

The most specific ultrastructural feature of TCs is the presence of very long prolongations (several tens to hundreds of mm) called telopodes (Tps) [1]. A Tp comprises thin fibrillar-like segments (podomeres) in alternation with dilated, cistern-like regions (podoms), which accommodate mitochondria, elements of endoplasmic reticulum and caveolae. The podomere/podom structure gives Tps a moniliform aspect.
Typically, Tps interact with neighbouring cells, either directly, by cell-cell contact, which creates a 3D network, or indirectly, by shedding microvesicles or secreting paracrine signalling molecules, including microRNAs [6,29,30]. TCs cooperate with stem cells to form tandem cell structures [2], which are mostly found in stem cell niches of various organs [5,6,9,12]. These tandem cell structures have been implicated in tissue regeneration and/or repair.
The dermis contains a rich population of resident cells, as demonstrated by electron microscopy and immunohistochemistry. Fibroblasts represent the most prominent and heterogeneous population [31,32], followed by macrophages, mast cells and a network of mesenchymal cells with dendritic morphology. Cells different from dermal fibroblasts, but morphologically similar to TCs, were previously described in the human dermis. These included antigen-presenting dermal dendrocytes [33,34] and another intriguing 'dendritic-like' cell type, with a distinct CD34 positive phenotype, which, at that time, was called a 'dermal dendrite of type II' [35]. The precise nature and function of these cell types remain unclear, but they closely resemble TCs, both ultrastructurally and phenotypically.  In this study, we used electron microscopy and immunohistochemistry to elucidate the existence of TCs in human dermis and to examine the nature of their close contacts (junctions) with dermal stem cells.

Materials and methods
Biopsies of human skin were obtained from three patients (informed written consent). Normal skin samples were obtained from a re-excision procedure after removing a local melanoma. The second excisions were performed according to the Breslow index (tumoural depth), 14 days after primary excision. The samples of normal skin were taken at 1 cm distance from primary suture. Experiments were performed according to the Helsinki guidelines, in full compliance with the Bioethics Committee of the 'Victor Babeş' National Institute of Pathology, Bucharest regulations.

In situ immunostaining and confocal analysis
Paraffin embedded skin samples (7lm thick) were deparaffinised, washed for 30 min. in PBS, pH 7.4 and blocked with 2% BSA. The samples were incubated for 30 min. with 2% normal goat serum (Sigma-Aldrich Chemical, St. Louis, MO, USA). Samples were incubated overnight at 4°C in PBS with either rabbit anti c-Kit or one of the following mouse monoclonal antibodies: anti-vimentin (clone V9, 1:150), anti-CD34 (clone QBEnd-10, 1:25) (both from Dako, Glostrup, Denmark) or anti-nestin (clone 10C2, 1:100) (Millipore, Billerica, MA, USA). In addition, combinations were used in double labelling assays. After washing in PBS with 0.1% (vol/vol) Triton X-100, the sections were incubated with Alexa Fluor-conjugated, secondary goat anti-rabbit or goat antimouse antibodies (Invitrogen, Molecular Probes, Eugene, OR, USA) for another 2 hrs, at room temperature. Following an extensive washing step, the nuclei were stained with 1 lg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). Negative controls were performed by omitting the primary antibody from the same protocol. Epifluorescence was used to examine three to five immunolabelled sections from each biopsy on a Nikon Eclipse E600 microscope (Nikon Instruments Inc., Tokyo, Japan) with a Nikon Plan Apo 409 objective and the appropriate fluorescence filters. Digital pictures were acquired with a CCD Axiocam HRc Zeiss camera and AxioVision software (Carl Zeiss Imaging solution GmbH, Göttingen, Germany) or with confocal laser scanning microscopy, with a Nikon A1 laser microscope mounted on an ECLIPSE Ti-E inverted microscope. The confocal images were collected with a Plan Fluor 609 oil objective and 1.25-NA water (z-axis step 0.16 lm). The following lasers and emission filters were used: Ar laser at 488 nm (used for the excitation of Alexa Fluor 488) and emission filter 500-550 nm; 561.2 nm G-HeNe laser (for Alexa Fluor 546) and emission filter 570 -620 nm; and 405 nm Laser diode and 425-475 nm emission filter for DAPI. To improve the image quality, some original laser scanning microscopy data were subjected to digital deconvolution and three-dimensional reconstruction with an Imaris 9 64 (version 6.3.1.) from Bitplane AG (Zürich, Switzerland).

Transmission electron microscopy (TEM)
The TEM was performed on small (1 mm 3 ) tissue fragments, processed according to a routine Epon-embedding procedure, as previously described [36]. Thin sections (about 60 nm) were examined with a Morgagni 286 transmission microscope (FEI Company, Eindhoven, The Netherlands) at 60 kV. Digital electron micrographs were acquired with a MegaView III CCD and iTEM-SIS software (Olympus, Soft Imaging System GmbH, Münster, Germany). To highlight the TCs and Tps, TEM images were digitally coloured in blue with Adobe © Photoshop CS3.

Immunolabelling
Immunostaining revealed numerous CD34 positive cells in the connective tissue of human dermis. Most were endothelial cells, but there was also a large population of spindle-shaped cells, with long, very thin prolongations. These were presumably TCs, scattered between dermal collagen bundles. A few CD34 positive TCs were detected in the papillary dermis. These exhibited a bipolar shape with thin prolongations that extended parallel to the adjacent epidermis (Fig. 1A); they were concentrated around the spanning excretory ducts of eccrine sweat glands, blood vessels and the infundibulum of hair follicles ( Fig. 2A,B). Most CD34 positive TCs were identified in the reticular dermis, where they formed a network around the deep segments of hair follicles, in the bulge and sub-bulge areas ( Fig. 2A,C). They also surrounded sebaceous glands and arrector pili muscles (Fig. 2D). In addition, CD34 positive TCs were found in the deep dermis, around the secretory and the ductal parts of eccrine sweat glands (Fig. 3). The CD34 dermal TCs were frequently detected in the perivascular connective tissue, accompanying small and intermediate blood vessels (Fig. 4).  We also detected c-kit/CD117 expression in cells with TC morphology, located either in the papillary dermis (Fig. 5) or in the connective tissue surrounding skin adnexal structures, like the hair follicle (Fig. 6) and sweat glands (Fig. 7).
Double immunostaining for CD34 and c-kit clearly showed two distinct subsets of cells with TC morphology that expressed either CD34 or c-kit; these subsets had similar distributions in the papillary (Fig. 8) and reticular (Fig. 9) dermis, Fig. 10 Telocytes form an interstitial network in human dermis. Digitally enhanced (blue colour) TEM image shows telocytes (blue) with telopodes (Tp). Telocytes are connected by junctions (black arrows; see detailed image in Fig. 16A). The telocyte in the centre of image has three telopodes (Tp 1 -Tp 3 ) extended between arterioles and nerves. Note close contacts between a Tp and macrophages (M) (black arrowhead) and a Tp and mast cell (white arrows, see detailed image in Fig 17C). Mo -mononuclear cells; Ms -mesenchymal cell; NE -nerve ending.

TEM
The TEM analysis was focused on dermal connective tissue. Immunohistochemistry showed CD34 or c-kit positive cells around the hair follicles, blood vessels and the secretory and excretory parts of eccrine sweat glands. The TEM images showed that all these structures were surrounded by interstitial cells with TC morphology (Figs 10-15 (Figs 1, 10-12), was due to an irregular alternation between the thin podomere segments (diameter, 40-100 nm), and dilated podoms (diameter, 200-500 nm or more), which contained mitochondria, endoplasmic reticulum and caveolae (Fig. 1E).
The TCs were connected to each other by homocellular junctions to form an interstitial network (Fig. 16). These junctions were typically puncta adhaerentia minima or recessus adhaerens, but gap junctions were also observed. Heterocellular junctions (planar contacts or point contacts) were found connecting TCs and mast cells or mononuclear cells (Fig. 17).

TCs and stem cells
The TCs were found bordering a round cluster of undifferentiated cells (stem cells) in the bulge regions of hair follicles (Figs 13, 14). The stem cells showed large euchromatic nuclei, few mitochondria, few endoplasmic reticulum cisternae and numerous free ribosomes (Fig. 14B). The stem cells were not connected to each other by classical junctions (only small immature adhaerentes junctions were found); and they were not connected with cells from the outer root sheath. However, point contacts (Fig. 14A) and planar contacts (Fig. 14B) were observed between TCs and stem cells. We screened for stem cell clusters in the bulge areas by immunolabelling for nestin. This revealed small, oval-shaped, nestin-expressing cells in the outer root sheath of the bulge area. C-kit/CD117 positive TCs were spotted around the nestin positive stem cells, intercalated between the bulge areas of hair follicles and the adjoining nerve fibres (Fig. 13B).

Discussion
The present study showed that TCs represented a distinct population of cells. They were distinguished by their particular distribution and their immunophenotypes, which differentiated them from other skin cells. Two subpopulations of TCs were detected in the normal human dermis, based on specific marker expression of CD34 or c-kit. The TCs were present around vascular structures, nerves, smooth muscle bundles and adnexal structures, including the bulge regions of the hair follicles.
In the last two decades, several authors described the presence of CD34 positive stromal cells within the dermis [37]. At first, these studies were purely descriptive, and the histogenesis and putative function of this cell population remained enigmatic. Subsequent studies suggested that dermal CD34 positive interstitial cells were derived, at least partly, from circulating fibrocytes, were capable of tissue invasion and were implicated in wound healing [38].  Mouse hair follicle stem cells have been shown to express CD34, but in human, its expression pattern remains controversial. Ohyama et al. [39] showed that CD34 was expressed in the non-bulge outer root sheath; Raposio et al. [40] reported CD34 expression in the bulge area; Jiang et al. [41] reported expression in the basal layer of the interfollicular epidermis; and Trempus et al. showed expression in mouse keratinocytes in the hair follicle bulge [42]. These cells, identified by immunophenotype, were also found around microvessels of perifollicular regions, which represent a niche for mesenchymal stem cells, in human scalp skin [43]. Our data showed no CD34 positive staining in the epidermis, weak CD34 staining in the hair follicle outer sheath and some CD34 expression outside the outer root sheath in the infundibular, bulge and sub-bulge zones.
During embryogenesis, c-kit signalling is important for melanoblast and/or melanocyte migration, proliferation and differentiation; in addition, c-kit contributes to maintaining postnatal cutaneous melanogenesis [44,45]. However, its role in epidermal and hair follicle regeneration remains unknown. Consistent with previous studies [41], we detected c-kit in the rete ridges of the epidermis (stratum basale), melanocytes, mast cells, the outermost layer of the infundibulum and in the hair matrix.
The major obstacle in identifying TCs with light microscopy is that they are morphologically similar to different types of so called 'fibroblast-like' or 'dendritic-like' stromal cells. In this study, we used double immunohistochemical staining to distinguish TCs from other skin cells.  Unlike Langerhans cells, c-kit and CD34 positive TCs did not express CD1a, and they did not contain Birbeck granules. However, anti-CD1a antibodies labelled intraepidermal and rare dermal Langerhans cells (data not shown). Unlike other dermal dendritic cells, TCs did not express CD36 or CD11a, and they were predominantly located in the reticular dermis, rather than the papillary dermis. Furthermore, most c-kit positive cells with TC morphology did not express mast cell tryptase in double immunostaining experiments (data not shown). However, it was sometimes difficult to assess differential expression of c-kit and tryptase, most likely because mast cells and TCs were sometimes closely apposed, particularly in the papillary dermis (Fig. 12B). Unlike myofibroblasts, TCs did not express alpha smooth muscle actin, and they did not form myofilaments or fibronexus junctions with the extracellular matrix. Finally, TCs could be distinguished from ordinary fibroblasts, because they did not express procollagen or CD90.
The particular network-like arrangement of TCs, based on homotypic (TC-TC junctions) and heterocellular interactions, supported the notion that they may perform intercellular signalling. Thus, they may convert the interstitium into an integrated system that contributes to maintaining organ homeostasis.
The close apposition between TCs and mast cells was frequently observed in other organs that harboured TCs [6,[46][47][48]. In some studies, a stromal synapse was described [48]. This type of cooperation might suggest that TCs were involved in either activation or repression of mast cells during allergic reactions [49]. However, the precise nat-ure of this intercellular relationship and the pathogenic mechanism underlying different pathological states require further study.
The TCs have been spotted in the vicinity of several types of progenitors in various organs, like the heart [5], lungs [9] and skeletal muscles [12]. Our observations of TC distributions and interactions in human skin supported the notion that TCs are members of the stem cell niches, and may play the role of 'nurse' cells for adjacent mesenchymal and epithelial stem cells. Multiple stem and progenitor cell compartments have been identified in both dermal connective tissues and the epidermis. In the dermal layer, mesenchymal stem cells have been identified in the follicular connective sheath and the papilla [50][51][52]. In the human epidermis, there are at least two distinct stem cell compartments. Both compartments contain slow-cycling cells with high proliferative potential, and we found that both were guarded by TCs. One of these compartments was the basal layer of the interfollicular epidermis [53]; the other, was the bulge of the hair follicle. The latter is the best characterized stem cell compartment; it comprises a specific microenvironment, called the stem cell niche [54,55], located between the opening duct of the sebaceous gland and the attachment point of arrector pili muscle. This area is responsible for the regeneration of the pilosebaceous unit, but it does not possess a distinctive morphology in humans [41,56]. Thus, stem cells at this level are not morphologically distinct, but they exhibit a specific array of markers, including nestin [57]. According to the 'bulge activation hypothesis', bulge stem cells will proliferate and differentiate only after receiving signals from specialized adjacent stromal cells [58]. Potential sources of stem cells include the epithelial stem cells in the epidermis and hair follicle and those in other skin adnexa, like the sebaceous glands and sweat glands [59,60]. Sebaceous gland homeostasis requires a population of progenitor cells that constantly gives rise to differentiating cells that are eliminated through the hair canal. Lineage tracing suggested that a small population of cells near the base of the sebaceous gland might be stem cells [61]. We detected CD34 positive TCs in close contact with both sebaceous and sweat glands.
The existence of different skin stem cell compartments and the ability of these niches to respond differentially to environmental signals remain exciting topics in the field. The TC represents a new player in the composition of various niches, and they should not be overlooked. In our opinion, the presence of TCs represents potential indirect (chemical) and/or direct (junctional) contacts with resident stem cells that could increase the efficiency and efficacy of repair and regeneration processes.