Telocytes in gastric lamina propria of the Chinese giant salamander, Andrias davidianus

In this study, we attempt to identify gastric telocytes (TCs) of the Chinese giant salamander Andrias davidianus, by light microscopy, immunohistochemistry and transmission electron microscopy (TEM) methods. Toluidine blue staining showed TCs with one to two very thin and long telopodes (Tps) that were located in gastric lamina propria. Tps had characteristic structures, including podoms, podomers and dichotomous branching. Immunohistochemistry showed the existence of CD34+/PDGFRα+ TCs with moniliform Tps in stroma and were close to gastric glands and blood vessels. TEM micrographs also demonstrated the presence of TCs in interstitium between gastric glands. TCs/Tps were located in close proximity to gastric glands, blood vessels, endocrine cells and stem cells. In particular, Tps frequently surrounded stem cells. TCs and Tps, Tps and stem cells established close contacts. Moreover, the exosomes were also found near TCs/Tps. Our data confirmed the presence of TCs in gastric lamina propria of the amphibian, and suggested that TCs cooperate with resident stem cells to regulate endocrine cells and gastric glands regeneration and homeostasis.

Transmission electron microscopy (TEM). TCs and their long Tps were observed in the connective tissue between gastric glands, which contained many electron-dense and round secretory granules (Figs 4, 5, 6, 7, 8 and 9).  The cell bodies of TCs appeared as pyriform (Fig. 4A), spindle-shaped (Figs 5, 6, 7A and 8) and quadrangle (Fig. 9A). The average length of Tps was 27.41 ± 13.10 μ m by measured in the TEM images. Some Tps were found in close proximity to gastric glands (Figs 4 and 5). Significantly, a TC with two long Tps was observed adjacent to an endocrine cell, which contained many electron-dense, small and round secretory granules (Fig. 6). Additionally, TCs were frequently observed in close proximity to stem cells (Figs 7, 8 and 9). TCs and stem cells were observed around blood vessel (Fig. 7). Some TCs/Tps closely surrounded stem cells. Moreover, Tps and stem cell established heterocellular close contacts (Figs 7C and 8B). TCs and Tps established homocellular close contacts (Fig. 9B). The exosomes were found between TCs and Tps ( Fig. 9B-D).

Discussion
Previous studies have demonstrated the presence of TCs in the human stomach 26,27 . However, in other animals, including lower animals, gastric TCs were not identified. Furthermore, previous TCs identification in human stomach only employed immunohistochemistry and immunofluorescence methods, but the most key diagnostic technique for TCs-TEM was not performed. The ultrastructural illustrations of gastric TCs remain absent regardless of higher and lower animals. In the present study, TCs had extremely long and thin cell processes, i.e. Tps, which appeared as moniliform in morphology due to their characteristic structures, podoms and podomeres. Also, Tps had dichotomous branching. Moreover, TCs/Tps frequently established structure contacts with other TCs/Tps and surrounding cell types. Although fibroblasts and ICC had also cell processes, they were short, thick and cone shaped, and lacked other structures of TCs 3,4 . In addition, TCs were CD34 + and PDGFRα + , while fibroblasts and ICC were PDGFRα − and CD34 − 26,28 . Therefore, we can confirm the existence of TCs in the gastric lamina propria of the lower animal, A. davidianus, according to their immunophenotypes and ultrastructural characteristics.
A previous study revealed that the presence of TCs in the mucosa, submucosa and muscle coat of human stomach 26 . Moreover, TCs encircled funds of gastric glands, muscle bundles, blood vessels and nerve structures. The study suggested that TCs might correspond to the fibroblast-like cells. In the other study, Manetti et al. 27 documented the existence of TCs mainly in the muscle layers and myenteric plexus, few TCs in the muscularis mucosae and submucosa of human systemic sclerosis gastric wall. They proposed that TCs loss might have important pathophysiological implications in systemic sclerosis of human organs. These results suggested TCs play physiological and pathological roles in the human stomach. In the present study, TCs/Tps were widely located in interstitium of gastric lamina propria of A. davidianus. Furthermore, they frequently were observed in close proximity to gastric gland and blood vessels as well as the reports of Vannucchi et al. 26 . Exclusively, by TEM methods, we observed the TCs location frequently close to stem cells. Moreover, we unexpectedly found the TCs/ Tps adjacent to endocrine cells. All results suggested TCs and these cell types may be correlative in physiological functions in the gastric lamina propria.
Previous studies also demonstrated that TCs were located in the lamina propria of several animal organs, such as rat duodenum and jejunum, and A. davidianus ileum 4,11,25 . These investigations suggested that TCs may be involved in immune response, intercellular signaling, tissue homeostasis maintenance, and glandular cells secretion and renewal 2,4,11,25,29 . Generally, TCs are suggested to control growth and differentiation of other cell types, mainly stem/progenitor cells, and further regulate surrounding cells regeneration and tissue repair in a particular niche 1,9,30 . In pathological state, they can initiate immune response and trigger tissue inflammation to induce pathogenesis under some challenges 31 . A previous study confirmed that several highly expressed molecules, such as IL-6, VEGF, MIP-1α , MIP-2 and MCP-1, were present in the mouse/rat cardiac TC secretory profile and proteome 32 . These data suggested that the TCs secretome plays a modulatory role in stem cell proliferation and differentiation. In the present study, the TCs were observed around the basal portion of the gastric glands and endocrine cells, where epithelial stem cells are located 33 . Moreover, TCs/Tps were really observed in close proximity to stem cells and they established heterocellular close contacts. Therefore, TCs might be act as nurse cells for stem cells and further to regulate surrounding gastric glands and endocrine cells generation and renewal 1,26 . Additionally, TCs might be involved in the glands/hormones synthesis and release of gastric glands/endocrine cells and maintain homeostasis. The homocellular close contacts between TCs and Tps were also present. It is suggested that TCs and Tps can form a 3D network and play a mechanical support role in gastric lamina propria. Furthermore, Tps and stem cells may directly perform intercellular communication by heterocellular close contacts. Additionally, the exosomes were also found near TCs/Tps and stem cells in this study. TCs/Tps and stem cells may also indirectly execute intercellular communication by exosomes 34 , which transfer macromolecules, such as mRNA, microRNAs, proteins and lipids, and participate in obsolete proteins eradication, mediate antigen presentation, stimulate T lymphocyte proliferation, and modulate immune responses 35,36 . Accordingly, TCs may utilize exosomes to play various roles 4,37 . Whichever communication methods, TCs may communicate with stem cells, nurse resident stem cells, and cooperate with them to regulate gastric glands repair and regeneration 3,4 , as well as in another digestive gland-liver 38,39 . In addition, TCs and stem cells frequently establish structural connection. The close contact of TCs established with stem cells and the exosomes containing paracrine factors released from TCs might be necessary for stem cells to survive and play roles. It is also suggested that TCs co-culture and co-transplantation with stem cells may more effective and reliable than mono-cellular therapies in regenerative medicine 40 .

Conclusion
This is the first report to confirm the presence of TCs in an amphibian stomach. Especially, we identified gastric TCs by TEM and disclosed the ultrastructural relation of TCs and other resident cell types, such as gastric gland cells, stem cells and endocrine cells. These results suggest that TCs might play a role in regulating other cell types regeneration and homeostasis. Our data will improve the insight of TCs functions in amphibian stomach. Light microscopy. The light microscopy technique was conducted according to Zhang et al. 41 . The stomachs samples of A. davidianus were fixed in 10% neutral buffered formalin for 24 h at room temperature, and then the samples were washed with 0.01 M phosphate-buffered saline (PBS) at pH 7.4, dehydrated in series of graded ethanol, and embedded in paraffin. Sections (5 μ m) were cut on a microtome (Yidi, Jinhua, China). After deparaffinization, the sections were stained with toluidine blue solution (0.8% toluidine blue + 0.6% KMnO 4 ) for 40 s. After washing in distilled water, the stained sections were dehydrated and mounted. Finally, they were examined and photographed by using a BM 2000 light microscopy with ScopeImage 9.0 (H3D) software (Yongxin, Nanjing, China).

Immunohistochemistry (IHC). IHC was performed according to the protocol provided by the anti-CD34
and anti-PDGFRα antibody manufacturer (Abcam, Cambridge, England). Briefly, after deparaffination, the sections (5 μ m) were incubated in 3% H 2 O 2 /methanol for 15 min. After washing the sections in PBS (0.01 M pH 7.4) for 3 × 5 min, they were pre-treated using heat mediated antigen retrieval with 0.01 M sodium citrate buffer (pH 6.0) for 15 min, and then washed in PBS for 3 × 5 min. All following steps were carried out in a moist chamber. The sections were incubated in normal goat serum at room temperature for 15 min. After discarding the goat serum, the sections were incubated in the anti-CD34 and anti-PDGFRα antibody (Abcam, Cambridge, England) with diluted in PBS (1:200) at 4 °C overnight, respectively. After rinsing in PBS for 3 × 5 min, the sections were incubated with biotinylated anti-rabbit IgG at 37 °C for 30 min. Afterwards, they were detected using an HRP conjugated compact polymer system at 37 °C for 30 min. The 3,3-diaminobenzidin (DAB) was used as the chromogen. The colouration was terminated with PBS when positive cells were clearly visible. The sections were counterstained with haematoxylin and mounted. Finally, the sections were also photographed by using BM 2000 light microscopy.
Transmission electron microscopy (TEM). TEM was conducted according to methods of Yang et al. 23 and Zhang et al. 25  using an ultramicrotome (ReichertJung, Wien, Austria), and then the sections were mounted on cooper coated grids. Finally, these sections were stained with 1% uranyl acetate and Reynold' s lead citrate for 20 min. The stained sections were observed and photographed by a high resolution digital camera connected to the TEM (Hitachi H-7650, Tokyo, Japan).

Measurement and Statistics.
The length of Tps was measured in the light microscopical and TEM images by using Image Pro-plus 7.0 software (Media Cybernetics, Rockville, MD, USA), respectively. Data were analyzed by using Excel software (Microsoft, Redmond, USA).