Song et al. J Transl Med (2019) 17:158 https://doi.org/10.1186/s12967-019-1870-y Journal of Translational Medicine RESEARCH Open Access Infuence of gene modifcation in biological behaviors and responses of mouse lung telocytes to infammation Dongli Song1†, Menglin Xu1†, Ruixue Qi1†, Ruihua Ma1, Yile Zhou1, Duojiao Wu1,2*, Hao Fang3,4* and Xiangdong Wang1,2* Abstract Background: Telocytes play key roles in maintenance of organ/tissue function and prevention of organ injury. However, there are great challenges to investigate telocytes functions using primary telocytes, due to the difculties of isolation, identifcation, and stability. The present study aims at constructing continuous cell strain of mouse lung telocyte cell line with stable characters by gene modifcation and investigating biological behaviors and responses of gene-modifed telocytes to infammation. Methods: Mouse primary lung telocytes were isolated and identifed using immune-labeling markers and immuno- electron microscopy. Primary telocytes were transformed with Simian vacuolating virus 40 small and large T antigen (SV40). Biological characters, behaviors morphology, and proliferation of those gene-modifed telocytes were defned and monitored dynamically for 50 generations, as compared with primary lung telocytes. Cell cycle of mouse primary lung telocytes or gene-modifed telocytes was detected by fow cytometry. Results: Gene modifed telocytes of generations 5, 10, 30 and 50 were observed with telopodes and also showed CD34 and ckit positive. Multiple cellular morphology were also observed on telocyte cell-line under monitor of celliq and enhanced cell proliferation were showed. SV40 transduction was also reduced apoptosis and increased the ratio of S and G2 phases in telocyte cell-line. Conclusion: We successfully constructed mouse lung telocyte cell-line which maintained the biological properties and behaviors as primary telocytes and could responses to infammation induced by LPS. Thus, gene-modifed lung telocytes, Telocyte Line, would provide a cell tool for researchers exploring the roles and applications of telocytes involved in physiological and pathological states in future. Keywords: Telocytes, Gene modifcation, Lung, SV40, Transformation Background including atrial and ventricular myocardium, bladder, Telocytes (TCs) have been found widely spreaded in lung skeletal muscle, gastrointestinal tract, eyes, and a large number of organs and tissues of mammals, others. TCs communicate with neighboring cells by homo- and heterocellular contacts and transfer genetic information and signaling molecules to infuence other *Correspondence: [email protected]; [email protected]; cells [1, 2]. Shoshkes-Carme et al. [3] recently demon- [email protected] †Dongli Song, Menglin Xu and Ruixue Qi contributed equally as co-frst strated that TCs can be the major source of Wnt signal- authors. ing, and play dependent roles in proliferation of intestinal 1 Zhongshan Hospital Institute of Clinical Science, Shanghai Institute stem cells and epithelial renewal. Tis particular study of Clinical Bioinformatics, Shanghai Engineering Research for AI Technology for Cardiopulmonary Diseases, Shanghai, China provides solid evidence that forkhead box L1-positive 3 Department of Anesthesiology, Zhongshan Hospital, Shanghai, China TCs contribute to the formation of the subepithelial Full list of author information is available at the end of the article plexus of the intestine, support the entire epithelium, and © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Song et al. J Transl Med (2019) 17:158 Page 2 of 58 provide niche signals to intestinal stem cells, by produc- and cultured in a incubator, with 5% CO2 in air, at 37 °C, ing Wnt ligands and maintaining Wnt signal pathway. for 30 min, to wipe of most fbroblasts. Cell suspension Although TCs are often allocated within the stem cell was transferred to other fakes and cultured for another niche, pulmonary TCs have diferent genetic profles and 12 h before changing culture medium. Culture medium biological functions from stem cells, fbroblasts, alveo- was changed every 48 h. Cells were examined by phase lar type II cells, airway basal cells, proximal airway cells, contrast microscope, under an inverted Olympus phase and T cells from bronchial lymph nodes or from lungs, contrast microscope (1 × 51; Tokyo, Japan). respectively [4–7]. TCs functionally and constructively support and connect tissue cells, function as signal mas- Lentivirus construction and infection sage carriers, and beneft gene and cell therapies [8–12]. Lentivirus particles containing the small and large T anti- Gene modifcation is considered as a potential therapy gen of anti-aging gene from Simian vacuolating virus 40 to genetically prevent and treat diseases, even though a (SV40) gene were constructed (HanyinCo., Shanghai, large number of factors still need to furthermore clarifed, China) according to the former articles [15]. Te oligo e.g. safety, efcacy, and complexity. With rapid develop- (listed 5′–3′) SV40-F: CCG GAA TTC ATG GAT AAA ment of gene editing technology, the precision and ef- GTT TTA AAC AGA GAG GAATC and SV40-R: GCT CTA cacy of gene modifcation are improved signifcantly [13, GAT TAC AAG TCC TCT TCA GAA ATG AG. TCs were 14]. Safety profles of gene modifcation as part of limita- infected on 6-well plates for 12 h at 37 °C, replaced fresh tions require more preclinical and clinical evidence. Te medium, and harvested for qPCR analysis to determine present study investigates potential efects of gene modi- the SV40 mRNA expression level after being cultured for fcation on morphological phenomes, biological behav- 96 h at 37 °C. iors and functions, as well as responses to infammation in lung TCs. An anti-aging gene from Simian vacuolating RNA extraction and PCR virus 40 was transferred as the target gene into primary Primary TCs and TCs transformed with SV40 (TCs SV40) TCs isolated from mouse lungs to improve the longevity were seeded in 24-well plates with a density of 104 cells/ of cells, since primary TCs hardly survive in the in vitro well and cultured for 24 h at 37 °C. Cells were washed system for a few weeks or months [5–7]. thrice with PBS, and total RNA was isolated and tran- scribed into single-stranded cDNA using the 1st Strand Materials and methods cDNA Synthesis Kit for RT-PCR (AMV, Roche) following TCs isolation and primary cell culture the recommendations of the manufacturer. Mice were provided by Animal Facility in Biomedical cDNA was synthesized from 1 µg of total RNA using Research Center of Zhongshan Hospital, Fudan Univer- PrimeScript® RT reagent Kit (Takara Bio Inc., Shiga, sity. Tis study was approved by the Fudan University Japan). PCR was performed with 1 µl of cDNA using Ethical Committee for animal experiments. GoTaq polymerase (Promega) for 25 cycles with specifc Mouse lung TCs were isolated and prepared as escribed primers for SV40. PCR reaction products were resolved previously [4]. In brief, female BABL/c mice aged and stained with Gelred (Biotium Inc., Newark, USA). 6–8 weeks were used and anaesthetized. Te trachea and lung tissues were isolated and collected into sterile tubes, Detection of cell bio‑behaviors containing Dulbecco’s Modifed Eagle’s Medium plus Te bio-behaviors of TCs and TCs SV40 were recorded and F12 (DMEM/F12) integration (Gibco, NY, USA), supple- analyzed using a Cell-IQ cell culturing platform (Chip- mented with 100 UI/ml penicillin, 0.1 mg/ml streptomy- Man Technologies, Tampere, Finland), equipped with a cin (Sigma-Aldrich Shanghai Trading Co Ltd., Shanghai, phase-contrast microscope (Nikon CFI Achromat phase China). Samples were placed on ice and transported to contrast objective with 10 magnifcation) and a cam- the cell culture laboratory within 30 min. Te tissues were era20. Te bio-behaviors, including cell proliferation, 3 cut into about 1 × mm in sterile DMEM/F12 and incu- division, death, cell morphology, and cell movement, can bated in 10 mg/ml collagenase type II (Sigma-Aldrich, be monitored and recorded as time-lapse data by this St. Louis, MO, USA) and 2000 U/ml deoxyribonuclease I Cell-IQ system uses machine vision technology. Images (Sigma-Aldrich) on an orbital shaker for 4 h, at 37 °C. Cell were captured at about 30 min intervals for 48 h. Analy- suspension was separated by 40-m-diameter cell strainer sis was carried out with a freely distributed Image soft- (BD Falcon, NJ, USA), and dispersed cells were collected ware (McMaster Biophotonics Facility, Hamilton, ON), by centrifugation. Cells were resuspended in DMEM/ using the Manual Tracking plugin created by Fabrice F12, supplemented with 10% fetal calf serum, 100 UI/ Cordelie´res (Institute Curie, Orsay, France). ml penicillin, 0.1 mg/ml streptomycin (Sigma-Aldrich), Song et al. J Transl Med (2019) 17:158 Page 3 of 58 RNA microarrays and long non‑coding RNA (lncRNA) stained with uranyl acetate and lead citrate, and observed classifcation pipeline at an acceleration voltage of 80 kV electron microscope RNA microarrays and lncRNA classifcation pipeline (JEOL JEM-1230, Tokyo, Japan). were tested in primary TCs or TCs SV40. Briefy, total ® RNA was collected using NucleoSpin RNA Plus accord- Immunoelectron microscopy ing the manufacturer’s protocol (Macherey–Nagel, Inc., Ultrathin sections were prepared and collected on nickel Düren, Germany). Microarray and quality controls of grids. Immunolabeling staining for CD34/Vimentin and gene expression profling were performed after RNA ckit/platelet-derived growth factor receptor α (PDGFR-α) and cDNA amplifcations, using the GeneChip® Human was used as previously reported [22].