Telocytes in liver: electron microscopic and immunofluorescent evidence

Hepatic interstitial cells play a vital role in regulating essential biological processes of the liver. Telocytes (TCs), a novel type of interstitial cells firstly identified by Popescu and his coworkers, have been reported in many tissues and organs, but not yet in liver (go to http://www.telocytes.com). We used transmission electron microscopy and immunofluorescence (double labelling for CD34 and c-kit/CD117, or vimentin, or PDGF Receptor-α, or β) to provide evidence for the existence of TCs in mice liver. The distribution of TCs in liver was found to be of similar density in the four hepatic lobes. In conclusion, here we show the presence of TCs in mice liver. It remains to be determined the possible roles of TCs in the control of liver homeostasis and regeneration, the more so as a close special relationship was found between TCs and hepatic putative stem (progenitor) cells.

This study was aimed to investigate the existence of TCs in the liver by transmission electron microscopy (TEM) as this technique assures the precise identification of TCs [30]. In addition, we used immunofluorescence methods, particularly the double labelling for CD34 and PDGFR-a considered at present as the immunohistochemical marker for TCs in gastrointestinal tract [31].

Transmission electron microscopy
Tissues were cut into 1 mm 3 fragments and fixed by immersion in 5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.4) overnight at 4°C. After that, it was washed in phosphate buffer for four times followed by post-fixation with 1% osmium tetroxide in 0.1 M phosphate buffer for 2 hrs at 4°C. Tissues were dehydrated through graded alcohols (50, 70, 90 and 100%) for 30 min. each and embedded in Epon 812. Semithin sections were cut at 1.5 lm and stained with toluidine blue, and histologically analysed by light microscopy. Ultrathin sections were cut at 70 nm and contrasted with uranyl acetate and lead citrate, and they were examined with a JEM-1010 electron microscope (JEOL, Tokyo, Japan). Snap-shots were taken using a video camera Veleta and the iTEM Olympus Soft Imaging System (Tokyo, Japan).

Semi-quantification of hepatic TCs
Representative sections of the left lateral, right, median and caudate lobes of mice livers were used for immuofluorescent staining. For the semi-quantification of hepatic TCs, double staining for c-kit and CD34 was used. Each lobe of the liver was randomly obtained of 20 images (4009) in the central area using confocal laser scanning microscope (LSM 710; Carl Zeiss MicroImaging GmbH, Jena, Germany). The anti-ckit and CD34 images from the same field were merged by Zen 2011 software (Carl Zeiss MicroImaging GmbH). Three mice were used in this experiment. Counting of the hepatic TCs was performed in a double-blinded method. The density of TCs was expressed as TCs number/ number of DAPI-stained nuclei.

Statistical analysis
Data were presented as mean AE SD. A one-way ANOVA was conducted to evaluate the one-way layout data. If a significant difference was observed, Bonferroni's post hoc test was conducted to identify groups with significant differences. All analyses were performed with SPSS 17.0, (IBM SPSS Statistics, Armonk, NY, USA) and all statistical tests were two-sided. P-values that were less than 0.05 were considered to be statistically significant.

Discussion
This study shows TCs as a distinct population of cells, distinguished from other interstitial cells (mainly Kupffer cells and hepatic stellate cells) in liver by their location, morphology and immunophenotypes. Kupffer cells are located inside the sinusoids [32] while the cells identified in the present study are in the space of Disse. Hepatic stellate cells, also known as Ito cells are pericytes found in the space of Disse [32]. Although the cells described in this study are also located in the space of Disse, they have characteristic very long prolongations (Tps) and specific biomarkers (double-positive for CD34 and c-kit/CD117, or vimentin, or PDGF-a, or PDGF-b), making them different from hepatic stellate cells in both morphology and immunophenotype [30].
The precise functions of TCs in liver remain to be established. However, based on the literature, at least three relevant and potential roles could be proposed: (i) intercellular connections via Tps [1,30], (ii) intercellular signalling via shedding vesicles or paracrine secretion [1,30] and (iii) liver regeneration as was supported for heart [9,22,30,33]. It is highly needed to explore the potential functions of TCs in the pathological conditions of the liver and the interaction between TCs and other cells. Further studies are required to investigate the role of TCs in liver fibrosis as reported in systemic sclerosis [24].
In conclusion, this study firstly demonstrated the presence of TCs in liver based on the specific ultrastructural and immunofluorescent characteristics. The presence of TCs in the liver opens a new window for better understanding responses that have not been determined in hepatic biology. Telocytes may be a new kind of target cells for the treatment and prevention of liver diseases.