FIB-SEM tomography of human skin telocytes and their extracellular vesicles

We have shown in 2012 the existence of telocytes (TCs) in human dermis. TCs were described by transmission electron microscopy (TEM) as interstitial cells located in non-epithelial spaces (stroma) of many organs (see www.telocytes.com). TCs have very long prolongations (tens to hundreds micrometers) named Telopodes (Tps). These Tps have a special conformation with dilated portions named podoms (containing mitochondria, endoplasmic reticulum and caveolae) and very thin segments (below resolving power of light microscopy), called podomers. To show the real 3D architecture of TC network, we used the most advanced available electron microscope technology: focused ion beam scanning electron microscopy (FIB-SEM) tomography. Generally, 3D reconstruction of dermal TCs by FIB-SEM tomography revealed the existence of Tps with various conformations: (i) long, flattened irregular veils (ribbon-like segments) with knobs, corresponding to podoms, and (ii) tubular structures (podomers) with uneven calibre because of irregular dilations (knobs) – the podoms. FIB-SEM tomography also showed numerous extracellular vesicles (diameter 438.6 ± 149.1 nm, n = 30) released by a human dermal TC. Our data might be useful for understanding the role(s) of TCs in intercellular signalling and communication, as well as for comprehension of pathologies like scleroderma, multiple sclerosis, psoriasis, etc.

Here, we present FIB-SEM tomography of human papillary dermis TCs showing their complex 3D architecture, as well as the budding and shedding of extracellular vesicles. FIB-SEM tomography does not contradict TEM, but provides additional important details.  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 [24]. Experiments were performed according to the Helsinki guidelines, in full compliance with the Bioethics Committee of the 'Victor Babes ß' National Institute of Pathology, Bucharest regulations. The small samples of skin were processed as described previously [12]. Briefly, the 1-mm-cube fragments were fixed by immersion in 4% glutaraldehyde, and post-fixed in 1% OsO 4 with 1.5% K 4 Fe(CN) 6 (potassium ferrocyanide -reduced osmium) to increase the membranes contrast. Subsequently, the samples were dehydrated through increasing graded ethanol series and embedded in epoxy resin (Agar 100 from Agar Scientific, Essex, UK) at 60°C for 48 hrs.

FIB/SEM image stack acquisition
Focused ion beam milling and SEM imaging were carried out with a ZE-ISS Auriga Crossbeam system (from Carl Zeiss Microscopy, M€ unchen, Germany). FIB milling was performed with 600 pA to 20 nA for the given samples. SEM-Imaging current was 220 pA. To achieve the best signal contrast, the mixed Inlens and energy-selective backscattered detector signals were used. FIB milling steps was 10 nm/slice and each 5th slice was imaged. Accordingly, each image represents 50 nm of the stack, at 9k9 magnification. Image pixel size was 10.27 nm.

Results and discussion
The specific morphological features of human dermal TCs are the telopodes, as demonstrated previously using TEM [24]. Figure 1A depicts a TC from the papillary dermis situated just beneath the basement membrane of epidermis in close proximity with a Merkel cell. This 2D image reveals the 'classic' morphology of a TC: a stellate body and very thin and narrow TPs, some which appear discontinuous because of the limitations of a single plane of section. For this reason, FIB-SEM technology (ZEISS Auriga Crossbeam system) was used, which allowed imaging of several hundred serial sections and accurate reconstruction of the TC 3D volume. Automated serial sectioning and imaging of human dermis provided a total of 350 micrographs. Sixty-six images were serially removed from the block face. A stack of 275 serial images (Fig. 2) were assembled to obtain a 3D reconstruction, 360°orthogonal rotation and a 3D digitally-coloured volume rendering of the TC in dermis. FIB-SEM images from Figure 2 revealed the presence of a typical TC in human dermis. The same cell was clearly visible from section 67-342 allowing the reconstruction of 2270 lm 3 (Fig. 3). The TC appearance shows a cell with different extensions: a 'ribbon-like' telopode, a telopode with classical morphology and a telopode with anfractuous shape (details in Fig. 4).
The surface-to-volume ratio is increased several folds in flat Tps compared to tubular Tps. This means, inter alia, a larger surface for receiving signals from extracellular space or vice versa. Interestingly, the dynamics of telopodes in cell culture depend on the extracellular type of matrix proteins. The stronger adherence and spreading were noted for TC seeded on fibronectin, while the lowest were on laminin [54]. Moreover, in cell cultures, low-level laser stimulation (using neodymiumdoped yttrium aluminium garnet laser) determines a maximum growth rate of Tp lateral extensions of 10.3 AE 1.0 lm/min. [55]. This raises the possibility of using low-level laser stimulation for therapeutic purposes.
The 3D reconstruction by FIB-SEM tomography of human dermal TCs allowed also the identification of extracellular vesicles (Figs. 3-6, Video S1), as shown previously for cardiac TCs [56].
In fact, a rough estimation of the number of extracellular vesicles (n = 30 for one cell) showed a vesicle diameter of 438.6 AE 149.1 nm. Considering the international standards e.g. (i) dimensions over 100 nm, (ii) origin by budding and shedding of plasma membrane and (iii) the monovesicular ultrastructure) [57,58], we think that the extracellular vesicles we found by FIB-SEM are microvesicles or ectovesicles or shed vesicles, rather than exosomes.
As previously shown, TCs were found in human dermis having a strategic position: around blood vessels, in the perifollicular sheath, outside the glassy membrane and surrounding sebaceous glands, arrector pili muscles and both the secretory and excretory segments of eccrine sweat glands [24]. Moreover, TCs frequently co-exist in close contacts with stem cells, for example, in skin dermis [24], lungs [2], skeletal muscle [59], meninges and choroid plexus [4] or liver [7]. Therefore, we consider that TCs together stem cells form a structural and functional unit, a 'tandem' [18]. This opinion is supported by the fact that TCs transfer extracellular vesicles loaded with microRNAs to stem cells [21], as well as the fact that extracellular vesicles have potential roles in regenerative medicine [60].
Last but not least, very recent data suggest that TCs through their Tps could be regarded as a primitive nervous system [61] or being involved in morphogenetic bioelectrical signalling [62,63]. Telocytes are expected to contribute to age-intervention protocols [64].