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

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Effects of a small molecule R-spondin-1 substitute RS-246204 on a mouse intestinal organoid culture.

Organoids, a multi-cellular and organ-like structure cultured in vitro, can be used in a variety of fields such as disease modeling, drug discovery, or cell therapy development. When organoids derived from Lgr5 stem cells are cultured ex vivo, recombinant R-spondin-1 protein should be added at a high concentration for the initiation and maintenance of the organoids. Because the addition of large amounts of R-spondin-1 greatly increases the cost of organoids, the organoids grown with R-spondin-1 are not practical for large-scale drug screening and for the development of therapeutic agents. In this study, we tried to find a R-spondin-1 substitute compound that is able initiate small intestinal organoids without the use of the R-spondin-1 protein; thus, using organoid media that each included one compound from among an 8,364 compound library instead of R-spondin-1, we observed whether organoids were established from the crypts of the small intestine. As a result, we found one compound that could promote the initial formation and growth of enteroids in the medium without R-spondin-1 and named it RS-246204. The enteroids grown with RS-246204 had a similar differentiation capacity as well as self-renewal capacity as the enteroids grown with R-spondin-1. Furthermore, the RS-246204-derived enteroids could successfully produce the forskolin induced swelling and the organoid based epithelial to mesenchymal transition model. This compound could be used for developing a cost-efficient culturing method for intestinal organoids as well as for exploring Lgr5 signaling, intestinal stem cell physiology and therapeutics for GI tract diseases.

Anti-fibrogenic effect of PPAR-γ agonists in human intestinal myofibroblasts.

BACKGROUND: Intestinal fibrosis is a serious complication of inflammatory bowel disease, including Crohn's disease and ulcerative colitis. There is no specific treatment for intestinal fibrosis. Studies have indicated that peroxisome proliferator-activated receptor- γ (PPAR-γ) agonists have anti-fibrogenic properties in organs besides the gut; however, their effects on human intestinal fibrosis are poorly understood. This study investigated the anti-fibrogenic properties and mechanisms of PPAR-γ agonists on human primary intestinal myofibroblasts (HIFs). METHODS: HIFs were isolated from normal colonic tissue of patients undergoing resection due to colorectal cancer. HIFs were treated with TGF-ß1 and co-incubated with or without one of two synthetic PPAR-γ agonists, troglitazone or rosiglitazone. mRNA and protein expression of procollagen1A1, fibronectin, and α-smooth muscle actin were determined by semiquantitative reverse transcription-polymerase chain reaction and Western blot. LY294002 (Akt inhibitor) was used to examine whether Akt phosphorylation was a downstream mechanism of TGF-ß1 induced expression of procollagen1A1, fibronectin, and α-smooth muscle actin in HIFs. The irreversible PPAR-γ antagonist GW9662 was used to investigate whether the effect of PPAR-γ agonists was PPAR-γ dependent. RESULTS: Both PPAR-γ agonists reduced the TGF-ß1-induced expression of α-smooth muscle actin which was integrated into stress fibers in HIFs, as determined by actin microfilaments fluorescent staining and α-smooth muscle actin-specific immunocytochemistry. PPAR-γ agonists also inhibited TGF-ß1-induced mRNA and protein expressions of procollagen1A1, fibronectin, and α-smooth muscle actin. TGF-ß1 stimulation increased phosphorylation of downstream signaling molecules Smad2, Akt, and ERK. TGF-ß1 induced synthesis of procollagen1A1, fibronectin, and α-smooth muscle actin through a phosphatidylinositol 3-kinase/Akt-dependent mechanism. PPAR-γ agonists down regulated fibrogenesis, as shown by inhibition of Akt and Smad2 phosphorylation. This anti-fibrogenic effect was PPAR-γ independent. CONCLUSIONS: Troglitazone and rosiglitazone suppress TGF-ß1-induced synthesis of procollagen1A1, fibronectin, and α-smooth muscle actin in HIFs and may be useful in treating intestinal fibrosis.

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

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- ß1 (TGF-ß1) or Tumor necrosis factor-α (TNF-α) 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-ß1. TGF-ß1 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-ß1 stimulated intestinal organoids. TNF-α and TGF-ß1 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-α and TGF-ß synergistically induce type 2 EMT in IEOs. This 3D EMT model with in vivo physiology could be used to study EMT associated intestinal fibrosis.