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Organoid-based epithelial to mesenchymal transition (OEMT) model: from an intestinal fibrosis perspective.

ABSTRACT: The current in vitro or in vivo intestinal fibrosis models have many limitations. Recent advancements in the isolation and culturing of organoids has led to development of various three-dimensional (3D) intestinal disease models with in vivo physiology. In this study, we generated an organoid-based epithelial to mesenchymal transition (OEMT) model, which could be used as a novel intestinal fibrosis model. Intestinal epithelial organoids (IEOs) were isolated and cultured from the small intestines of normal mice. IEOs were treated with transforming growth factor- ?1 (TGF-?1) or Tumor necrosis factor-? (TNF-?) to evaluate their phenotypic change. Raw 264.7 cells (macrophage) stimulated with lipopolysaccharide were co-cultured with IEOs in growth media with or without TGF-?1. TGF-?1 alone slightly induced epithelial to mesenchymal transition (EMT) in the IEOs but mainly disrupted them. Macrophage released cytokines synergistically induced mesenchymal phenotypic changes in TGF-?1 stimulated intestinal organoids. TNF-? and TGF-?1 synergistically induced proliferation of mesenchymal cells as well as EMT in the IEOs. We generated a novel OEMT model based on our finding that TNF-? and TGF-? synergistically induce type 2 EMT in IEOs. This 3D EMT model with in vivo physiology could be used to study EMT associated intestinal fibrosis.

SUBMITTER: Hahn S

PROVIDER: S-EPMC5446415 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Organoid-based epithelial to mesenchymal transition (OEMT) model: from an intestinal fibrosis perspective.

Hahn Soojung S Nam Myeong-Ok MO Noh Jung Hyun JH Lee Dong Hyeon DH Han Hyun Wook HW Kim Duk Hwan DH Hahm Ki Baik KB Hong Sung Pyo SP Yoo Jun-Hwan JH Yoo Jongman J

Scientific reports 20170526 1

The current in vitro or in vivo intestinal fibrosis models have many limitations. Recent advancements in the isolation and culturing of organoids has led to development of various three-dimensional (3D) intestinal disease models with in vivo physiology. In this study, we generated an organoid-based epithelial to mesenchymal transition (OEMT) model, which could be used as a novel intestinal fibrosis model. Intestinal epithelial organoids (IEOs) were isolated and cultured from the small intestines ...[more]

PMID: 28550311

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Project description:Background and aimsRecent evidences reveal that epithelial to mesenchymal transition (EMT) exacerbates the process of intestinal fibrosis. Tumor necrosis factor-like ligand 1A (TL1A) is a member of the tumor necrosis family (TNF), which can take part in the development of colonic inflammation and fibrosis by regulating immune response or inflammatory factors. The purpose of this study was to elucidate the possible contribution of TL1A in onset and progression of intestinal inflammation and fibrosis through EMT.MethodsColonic specimens were obtained from patients with inflammatory bowel disease (IBD) and control individuals. The expression levels of TL1A and EMT-related markers in intestinal tissues were evaluated. Furthermore, the human colorectal adenocarcinoma cell line, HT-29, was stimulated with TL1A, anti-TL1A antibody, or BMP-7 to assess EMT process. In addition, transgenic mice expressing high levels of TL1A in lymphoid cells were used to further investigate the mechanism of TL1A in intestinal fibrosis.ResultsHigh levels of TL1A expression were detected in the intestinal specimens of patients with ulcerative colitis and Crohn's disease and were negatively associated with the expression of an epithelial marker (E-cadherin), while it was positively associated with the expression of interstitial markers (FSP1 and α-SMA). Transgenic mice with high expression of TL1A were more sensitive to dextran sodium sulfate and exhibited severe intestinal inflammation and fibrosis. Additionally, the TGF-β1/Smad3 pathway may be involved in TL1A-induced EMT, and the expression of IL-13 and EMT-related transcriptional molecules (e.g., ZEB1 and Snail1) was increased in the intestinal specimens of the transgenic mice. Furthermore, TL1A-induced EMT can be influenced by anti-TL1A antibody or BMP-7 in vitro.ConclusionsTL1A participates in the formation and process of EMT in intestinal fibrosis. This new knowledge enables us to better understand the pathogenesis of intestinal fibrosis and identify new therapeutic targets for its treatment.

Project description:DCLK1 and Lgr5 have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. Epithelial-mesenchymal transition (EMT) is a key development program that is often activated during cancer invasion and metastasis, and also imparts a self-renewal capability to disseminating cancer cells. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we observed a relative decrease in DCLK1 expression in the colonic crypts, with significant shift towards stromal staining at peak (12 days post infection) hyperplasia, whereas staining for Lgr5 and Msi-1 increased several fold. When hyperplasia was regressing (days 20-34), an expansion of DCLK1+ve cells in the CR-infected crypts compared with that seen in uninfected control was recorded. Purified colonic crypt cells exhibiting epigenetic modulation of the transforming growth factor-? (TGF?), Wnt and Notch pathways on 12 or 34 days post infection formed monolayers in vitro, and underwent trans-differentiation into fibroblast-like cells that stained positive for vimentin, fibronectin and DCLK1. These cells when trypsinized and regrown in soft agar, formed colonospheres/organoids that developed into crypt-like structures (colonoids) in Matrigel and stained positive for DCLK1. Mice exhibiting 12 or 34 days of TMCH were given azoxymethane once for 8?h (Gp1) or weekly for 3 weeks (Gp2), and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as colonospheres in soft agar and nodules/tumors in nude mice. Crypt cells isolated from Gp2 animals failed to form the monolayers, but developed into colonospheres in soft agar and nodules/tumors in nude mice. Thus, both hyperplasia and increased presence of DCLK1+ve cells promote cellular transformation in response to a second hit. The TMCH model, therefore, provides an excellent template to study how alterations in intestinal stem cells promote trans-differentiation, crypt regeneration or colon carcinogenesis following bacterial infection.

Project description:Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease with no effective treatment. The epithelial-mesenchymal transition (EMT) is a critical stage during the development of fibrosis. To assess the effect of sulforaphane (SFN) on the EMT and fibrosis using an in vitro transforming growth factor (TGF)-?1-induced model and an in vivo bleomycin (BLM)-induced model.In vitro studies, cell viability, and cytotoxicity were measured using a Cell Counting Kit-8. The functional TGF-?1-induced EMT and fibrosis were assessed using western blotting and a quantitative real-time polymerase chain reaction. The lungs were analyzed histopathologically in vivo using hematoxylin and eosin and Masson's trichrome staining. The BLM-induced fibrosis was characterized by western blotting and immunohistochemical analyses for fibronectin, TGF-?1, E-cadherin (E-cad), and ?-smooth muscle actin (SMA) in lung tissues.SFN reversed mesenchymal-like changes induced by TGF-?1 and restored cells to their epithelial-like morphology. The results confirmed that the expression of the epithelial marker, E-cadherin, increased after SFN treatment, while expression of the mesenchymal markers, N-cadherin, vimentin, and ?-SMA decreased in A549 cells after SFN treatment. In addition, SFN inhibited TGF-?1-induced mRNA expression of the EMT-related transcription factors, Slug, Snail, and Twist. The SFN treatment attenuated TGF-?1-induced expression of fibrosis-related proteins, such as fibronection, collagen I, collagen IV, and ?-SMA in MRC-5 cells. Furthermore, SFN reduced the TGF-?1-induced phosphorylation of SMAD2/3 protein in A549 cells and MRC-5 cells. BLM induced fibrosis in mouse lungs that was also attenuated by SFN treatment, and SFN treatment decreased BLM-induced fibronectin expression, TGF-?1 expression, and the levels of collagen I in the lungs of mice.SFN showed a significant anti-fibrotic effect in TGF-?-treated cell lines and BLM-induced fibrosis in mice. These findings showed that SFN has anti-fibrotic activity that may be considered in the treatment of IPF.

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Organoid-based epithelial to mesenchymal transition (OEMT) model: from an intestinal fibrosis perspective.

Publications

Organoid-based epithelial to mesenchymal transition (OEMT) model: from an intestinal fibrosis perspective.

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