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. 2015 Jun;98(3):346-51.
doi: 10.1016/j.yexmp.2015.03.033. Epub 2015 Mar 28.

Intestinal organoids: a model of intestinal fibrosis for evaluating anti-fibrotic drugs

Eva S Rodansky  1 , Laura A Johnson  1 , Sha Huang  1 , Jason R Spence  2 , Peter D R Higgins  3
Affiliations

Intestinal organoids: a model of intestinal fibrosis for evaluating anti-fibrotic drugs

Eva S Rodansky et al. Exp Mol Pathol. 2015 Jun.

Abstract

Background & aims: Intestinal fibrosis is a critical complication of Crohn's disease (CD). Current in vitro models of intestinal fibrosis cannot model the complex intestinal architecture, while in vivo rodent models do not fully recapitulate human disease and have limited utility for large-scale screening. Here, we exploit recent advances in stem cell derived human intestinal organoids (HIOs) as a new human model of fibrosis in CD.

Methods: Human pluripotent stem cells were differentiated into HIOs. We identified myofibroblasts, the key effector cells of fibrosis, by immunofluorescence staining for alpha-smooth muscle actin (αSMA), vimentin, and desmin. We examined the fibrogenic response of HIOs by treatment with transforming growth factor beta (TGFβ) in the presence or absence of the anti-fibrotic drug spironolactone. Fibrotic response was assayed by expression of fibrogenic genes (COL1A1 (collagen, type I, alpha 1), ACTA2 (alpha smooth muscle actin), FN1 (fibronectin 1), MYLK (myosin light chain kinase), and MKL1 (megakaryoblastic leukemia (translocation) 1)) and proteins (αSMA).

Results: Immunofluorescent staining of organoids identified a population of myofibroblasts within the HIO mesenchyme. TGFβ stimulation of HIOs produced a dose-dependent pro-fibrotic response. Spironolactone treatment blocked the fibrogenic response of HIOs to TGFβ.

Conclusions: HIOs contain myofibroblasts and respond to a pro-fibrotic stimulus in a manner that is consistent with isolated human myofibroblasts. HIOs are a promising model system that might bridge the gap between current in vitro and in vivo models of intestinal fibrosis in IBD.

Keywords: Drug screening; Fibrosis model; Myofibroblasts; Organoid; TGFβ.

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Conflict of interest statement

Disclosures: All authors have nothing to disclose. No conflicts of interest exist.

Figures

Figure 1
Figure 1
HIOs contain myofibroblasts and are activated by TGFβ. (A) Morphology (10× magnification) of representative HIO illustrating the presence of a well-defined lumen (asterisk) and a population αSMA+ cells (red staining, white arrows). (B,C) αSMA + (red) cells were identified as resident myofibroblasts (αSMA +, vimentin+, desmin−) by staining with vimentin (green in B), and desmin (green in C), 20× magnification. Individual representative vimentin+/desmin− myofibroblasts (circled) are shown in the insets. (D,E) Gene expression as determined by qPCR for pro-fibrotic genes FN1 (D) and MKL-1 (E) in HIOs treated with increasing amounts of TGFβ (0.5, 1, 2, 5 ng/mL) for 48 hrs. The expression of pro-fibrotic genes was normalized to GAPDH using the ΔΔCt method. Results are from 3 independent experiments. Statistical comparisons are made between the untreated and TGFβ-treated groups using the Paired Student’s t-test. (F,G) Activation of myofibroblasts within the HIOs by TGFβ. αSMA (red) and vimentin (green) was markedly induced in TGFβ-treated HIO (G). Vimentin is stained green in Figure 1 F and G. The scale bar represents 50 µM.
Figure 2
Figure 2
Induction of fibrogenic gene expression in HIO by TGFβ. Matrigel-embedded HIOs were cultured for 96 hours in the presence or absence of TGFβ (2 ng/mL). (A) Expression of COL1A1, (B) Expression of FN1, (C) Expression of ACTA2, (D) Expression of MYLK, (E) Expression of MKL1. Gene expression was normalized to GAPDH expression. Statistical comparisons are made between untreated and TGFβ-treated organoids using the Paired Student t test, *p < 0.05, ** p <0.01, ***p<0.001. Results are from 3 independent experiments performed in duplicate (total n=6).
Figure 3
Figure 3
Inhibition of TGFβ-mediated induction of fibrogenic genes by spironolactone (SPIR) in HIOs. Organoids were embedded in Matrigel and cultured for 96 hours in the presence or absence of TGFβ (2 ng/mL) and 0, 25, 50, 100, and 250 µM spironolactone. Gene expression analysis: A–D. (A) Expression of COL1A1, (B) Expression of FN1, (C) Expression of ACTA2, (D) Expression of MYLK. The expression of pro-fibrotic genes was normalized to GAPDH using the ΔΔCt method. Results are from 3 independent experiments. (E). Representative αSMA Western blot of HIOs treated with TGFβ (2 ng/mL) and 100 or 250 µM SPIR for 96 hours. (F) Quantitation of αSMA protein expression in response to co-treatment with TGFβ and either 100 or 250 µM SPIR. HIOs were treated for 96 hours with 2 ng/mL TGFβ. Western data from 3 independent HIO experiments were quantified using ImageJ. αSMA band density was normalized to GAPDH band density, and within each experiment, treatment groups were normalized to untreated. Horizontal bars represent the geometric mean of each group. Statistical comparisons are made between untreated HIO and TGFβ-treated or TGFβ and SPIR treated HIOs using the Paired Student t test are denoted with asterisks (*p < 0.05, ** p <0.01, ***p<0.001). Statistical comparisons between TGFβ-treated or TGFβ and SPIR treated HIOs are denoted with a pound sign (# p < 0.05, ## p <0.01, ### p<0.001).
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References

    1. Andres PG, Friedman LS. Epidemiology and the natural course of inflammatory bowel disease. Gastroenterol Clin North Am. 1999;28:255–281. vii. - PubMed
    1. Astashkina A, Grainger DW. Critical analysis of 3-D organoid in vitro cell culture models for high-throughput drug candidate toxicity assessments. Adv Drug Deliv Rev. 2014;69–70:1–18. - PubMed
    1. Brenna O, et al. Relevance of TNBS-colitis in rats: a methodological study with endoscopic, histologic and Transcriptomic [corrected] characterization and correlation to IBD. PLoS One. 2013;8:e54543. - PMC - PubMed
    1. Chen X, et al. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 2008;18:997–1006. - PubMed
    1. Chen YJ, et al. De novo formation of insulin-producing "neo-beta cell islets" from intestinal crypts. Cell Rep. 2014;6:1046–1058. - PMC - PubMed

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