Inflammation Research

, Volume 67, Issue 11–12, pp 975–984 | Cite as

Using human iPS cell-derived enterocytes as novel in vitro model for the evaluation of human intestinal mucosal damage

  • Satoshi Kondo
  • Shota Mizuno
  • Tadahiro Hashita
  • Takahiro Iwao
  • Tamihide MatsunagaEmail author
Original Research Paper


Objective and design

The primary component in gut mucus is mucin 2 (MUC2) secreted by goblet cells. Fluctuations in MUC2 expression are considered a useful indicator for evaluating mucosal damage and protective effect of various agents using animal studies. However, there are few in vitro studies evaluating mucosal damage using MUC2 as the indicator. Hence, we attempted to establish a novel in vitro model with MUC2 as the indicator for evaluating drug-induced mucosal damage and protective effect using enterocytes derived from human iPS cells.


Compounds were added into enterocytes derived from human iPS cells, and MUC2 mRNA and protein expression levels were evaluated. Further, the effect of compounds on membrane permeability was investigated.


Nonsteroidal anti-inflammatory drugs were found to decrease MUC2 mRNA expression in enterocytes, whereas mucosal protective agents increased mRNA levels. Changes in MUC2 protein expression were consistent with those of mRNA. Additionally, our results indicated that indomethacin caused mucosal damage, affecting membrane permeability of the drug. Moreover, we observed protective effect of rebamipide against the indomethacin-induced permeability increase.


The developed model could facilitate evaluating drug-induced mucosal damage and protective effects of various agents and could impact drug development studies regarding pharmacological efficacy and safety.


Human iPS cells Enterocytes Mucin 2 Nonsteroidal anti-inflammatory drugs Mucosal protective agents 



The authors are extremely grateful to Dr. Hidenori Akutsu, Dr. Yoshitaka Miyagawa, Dr. Hajime Okita, Dr. Nobutaka Kiyokawa, Dr. Masashi Toyoda, and Dr. Akihiro Umezawa for providing the human iPS cells.

Author contributions

SK, MS, TH, TI, and TM participated in research design; SK and MS conducted experiments and performed data analysis; SK, MS, TH, TI, and TM wrote or contributed to the writing of the manuscript.


This work was supported by Grant-in-Aid for Research in Nagoya City University in 2017, and Agency for Medical Research and Development (AMED) under Grant Number 17be0304203h0001.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.


  1. 1.
    Hayashi S, Kurata N, Kitahirachi E, Nishimura Y, Amagase K, et al. Cinacalcet, a calcimimetic, prevents nonsteroidal antiinflammatory drug-induced small intestinal damage in rats. J Physiol Pharmacol. 2013;64:453–63.PubMedGoogle Scholar
  2. 2.
    Yamamoto A, Itoh T, Nasu R, Nishida R. Sodium alginate ameliorates indomethacin-induced gastrointestinal mucosal injury via inhibiting translocation in rats. World J Gastroenterol. 2014;20:2641–52.CrossRefGoogle Scholar
  3. 3.
    Goldstein JL, Eisen GM, Lewis B, Gralnek IM, Zlotnick S, Fort JG. Video capsule endoscopy to prospectively assess small bowel injury with celecoxib, naproxen plus omeprazole, and placebo. Clin Gastroenterol Hepatol. 2005;3:133–41.CrossRefGoogle Scholar
  4. 4.
    Maiden L, Thjodleifsson B, Theodors A, Gonzalez J, Bjarnason I. A quantitative analysis of NSAID-induced small bowel pathology by capsule enteroscopy. Gastroenterology. 2005;128:1172–8.CrossRefGoogle Scholar
  5. 5.
    Fujimori S, Seo T, Gudis K, Ehara A, Kobayashi T, et al. Prevention of nonsteroidal anti-inflammatory drug-induced small-intestinal injury by prostaglandin: a pilot randomized controlled trial evaluated by capsule endoscopy. Gastrointest Endosc. 2009;69:1339–46.CrossRefGoogle Scholar
  6. 6.
    Amagase K, Kimura Y, Wada A, Yukishige T, Murakami T, et al. Prophylactic effect of monosodium glutamate on NSAID-induced enteropathy in rats. Curr Pharm Des. 2014;20:2783–90.CrossRefGoogle Scholar
  7. 7.
    Urashima H, Okamoto T, Takeji Y, Shinohara H, Fujisawa S. Rebamipide increases the amount of mucin-like substances on the conjunctiva and cornea in the N-acetylcysteine-treated in vivo model. Cornea. 2004;23:613–9.CrossRefGoogle Scholar
  8. 8.
    Urashima H, Takeji Y, Okamoto T, Fujisawa S, Shinohara H. Rebamipide increases mucin-like substance contents and periodic acid Schiff reagent-positive cells density in normal rabbits. J Ocul Pharmacol Ther. 2012;28:264–70.CrossRefGoogle Scholar
  9. 9.
    Yasuda-Onozawa Y, Handa O, Naito Y, Ushiroda C, Suyama Y, et al. Rebamipide upregulates mucin secretion of intestinal goblet cells via Akt phosphorylation. Mol Med Rep. 2017;16:8216–22.CrossRefGoogle Scholar
  10. 10.
    Bu XD, Li N, Tian XQ, Huang PL. Caco-2 and LS174T cell lines provide different models for studying mucin expression in colon cancer. Tissue Cell. 2011;43:201–6.CrossRefGoogle Scholar
  11. 11.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;30:861–72.CrossRefGoogle Scholar
  12. 12.
    Iwao T, Toyota M, Miyagawa Y, Okita H, Kiyokawa N, Akutsu H, Umezawa A, Nagata K, Matsunaga T. Differentiation of human induced pluripotent stem cells into functional enterocyte-like cells using a simple method. Drug Metab Pharmacokinet. 2014;29:44–51.CrossRefGoogle Scholar
  13. 13.
    Iwao T, Kodama N, Kondo Y, Kabeya T, Nakamura K, Horikawa T, Niwa T, Kurose K, Matsunaga T. Generation of enterocyte-like cells with pharmacokinetic functions from human induced pluripotent stem cells using small-molecule compounds. Drug Metab Dispos. 2015;43:603–10.CrossRefGoogle Scholar
  14. 14.
    Kodama N, Iwao T, Katano T, Ohta K, Yuasa H, Matsunaga T. Characteristic analysis of intestinal transport in enterocyte-like cells differentiated from human induced pluripotent stem cells. Drug Metab Dispos. 2016;44:662–7.CrossRefGoogle Scholar
  15. 15.
    Kabeya T, Qiu S, Hibino M, Nagasaki M, Kodama N, Iwao T, Matsunaga T. cAMP signaling promotes the differentiation of human induced pluripotent stem cells into intestinal epithelial cells. Drug Metab Dispos. 2018;46:1411–9.CrossRefGoogle Scholar
  16. 16.
    Dorofeyev AE, Vasilenko IV, Rassokhina OA, Kondratiuk RB. Mucosal barrier in ulcerative colitis and Crohn’s disease. Gastroenterol Res Pract. 2013;2013:431231.CrossRefGoogle Scholar
  17. 17.
    Kim YD, Jeon JY, Woo HJ, Lee JC, Chung JH, et al. Interleukin-1beta induced MUC2 gene expression and mucin secretion via activation of PKC-MEK/ERK, and PI3K in human airway epithelial cells. J Korean Med Sci. 2002;17:765–71.CrossRefGoogle Scholar
  18. 18.
    Enss ML, Cornberg M, Wagner S, Gebert A, Henrichs M, et al. Proinflammatory cytokines trigger MUC gene expression and mucin release in the intestinal cancer cell line LS180. Inflamm Res. 2000;49:162–9.CrossRefGoogle Scholar
  19. 19.
    Van Seuningen I, Pigny P, Perrais M, Porchet N, Aubert JP. Transcriptional regulation of the 11p15 mucin genes. Towards new biological tools in human therapy, in inflammatory diseases and cancer? Front Biosci. 2001;6:d1216–34.PubMedGoogle Scholar
  20. 20.
    Mejías-Luque R, Lindén SK, Garrido M, Tye H, Najdovska M, et al. Inflammation modulates the expression of the intestinal mucins MUC2 and MUC4 in gastric tumors. Oncogene. 2010;29:1753–62.CrossRefGoogle Scholar
  21. 21.
    Fischer BM, Rochelle LG, Voynow JA, Akley NJ, Adler KB. Tumor necrosis factor-alpha stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro. Am J Respir Cell Mol Biol. 1999;20:413–22.CrossRefGoogle Scholar
  22. 22.
    Ahn DH, Crawley SC, Hokari R, Kato S, Yang SC, et al. TNF-alpha activates MUC2 transcription via NF-kappaB but inhibits via JNK activation. Cell Physiol Biochem. 2005;15:29–40.CrossRefGoogle Scholar
  23. 23.
    Wu J, Gong J, Geng J, Song Y. Deoxycholic acid induces the overexpression of intestinal mucin, MUC2, via NF-kB signaling pathway in human esophageal adenocarcinoma cells. BMC Cancer. 2008;8:333.CrossRefGoogle Scholar
  24. 24.
    Vieira MJ, Perosa SR, Argaãaraz GA, Silva JA Jr, Cavalheiro EA. Graça Naffah-Mazzacoratti M. Indomethacin can downregulate the levels of inflammatory mediators in the hippocampus of rats submitted to pilocarpine-induced status epilepticus. Clinics. 2014;69:621–6.CrossRefGoogle Scholar
  25. 25.
    Franklin IJ, Walton LJ, Greenhalgh RM, Powell JT. The influence of indomethacin on the metabolism and cytokine secretion of human aneurysmal aorta. Eur J Vasc Endovasc Surg. 1999;18:35–42.CrossRefGoogle Scholar
  26. 26.
    Bour AM, Westendorp RG, Laterveer JC, Bollen EL, Remarque EJ. Interaction of indomethacin with cytokine production in whole blood. Potential mechanism for a brain-protective effect. Exp Gerontol. 2000;35:1017–24.CrossRefGoogle Scholar
  27. 27.
    Takeuchi K, Tanaka A, Hayashi Y, Kubo Y. Functional mechanism underlying COX-2 expression following administration of indomethacin in rat stomachs: importance of gastric hypermotility. Dig Dis Sci. 2004;49:180–7.CrossRefGoogle Scholar
  28. 28.
    Tanaka A, Araki H, Hase S, Komoike Y, Takeuchi K. Up-regulation of COX-2 by inhibition of COX-1 in the rat: a key to NSAID-induced gastric injury. Aliment Pharmacol Ther. 2002;16:90–101.CrossRefGoogle Scholar
  29. 29.
    Hatazawa R, Ohno R, Tanigami M, Tanaka A, Takeuchi K. Roles of endogenous prostaglandins and cyclooxygenase isozymes in healing of indomethacin-induced small intestinal lesions in rats. J Pharmacol Exp Ther. 2006;318:691–9.CrossRefGoogle Scholar
  30. 30.
    Simmons DL, Wagner D, Westover K. Nonsteroidal anti-inflammatory drugs, acetaminophen, cyclooxygenase 2, and fever. Clin Infect Dis. 2000;31:211–8.CrossRefGoogle Scholar
  31. 31.
    Diao L, Mei Q, Xu JM, Liu XC, Hu J, et al. Rebamipide suppresses diclofenac induced intestinal permeability via mitochondrial protection in mice. World J Gastroenterol. 2012;18:1059–66.CrossRefGoogle Scholar
  32. 32.
    Matysiak-Budnik T, de Mascarel A, Abely M, Mayo K, Heyman M, Mégraud F. Positive effect of rebamipide on gastric permeability in mice after eradication of Helicobacter felis. Scand J Gastroenterol. 2000;35:470–5.CrossRefGoogle Scholar
  33. 33.
    Banan A, Fitzpatrick L, Zhang Y, Keshavarzian A. OPC-compounds prevent oxidant-induced carbonylation and depolymerization of the F-actin cytoskeleton and intestinal barrier hyperpermeability. Free Radic Biol Med. 2001;30:287–98.CrossRefGoogle Scholar
  34. 34.
    Nagano Y, Matsui H, Muramatsu M, Shimokawa O, Shibahara T, et al. Rebamipide significantly inhibits indomethacin-induced mitochondrial damage, lipid peroxidation, and apoptosis in gastric epithelial RGM-1 cells. Dig Dis Sci. 2005;50:76–83.CrossRefGoogle Scholar
  35. 35.
    Murakami K, Okajima K, Harada N, Isobe H, Okabe H. Rebamipide prevents indomethacin-induced gastric mucosal lesion formation by inhibiting activation of neutrophils in rats. Dig Dis Sci. 1998;43:139–42.Google Scholar
  36. 36.
    Naito Y, Yoshikawa T, Iinuma S, Yagi N, Matsuyama K, et al. Rebamipide protects against indomethacin-induced gastric mucosal injury in healthy volunteers in a double-blind, placebo-controlled study. Dig Dis Sci. 1998;43:83S–9S.PubMedGoogle Scholar
  37. 37.
    Niwa Y, Nakamura M, Ohmiya N, Maeda O, Ando T, et al. Efficacy of rebamipide for diclofenac-induced small-intestinal mucosal injuries in healthy subjects: a prospective, randomized, double-blinded, placebo-controlled, cross-over study. J Gastroenterol. 2008;43:270–6.CrossRefGoogle Scholar
  38. 38.
    Kurata S, Nakashima T, Osaki T, Uematsu N, Shibamori M, et al. Rebamipide protects small intestinal mucosal injuries caused by indomethacin by modulating intestinal microbiota and the gene expression in intestinal mucosa in a rat model. J Clin Biochem Nutr. 2015;56:20–7.CrossRefGoogle Scholar
  39. 39.
    Mizoguchi H, Ogawa Y, Kanatsu K, Tanaka A, Kato S, Takeuchi K. Protective effect of rebamipide on indomethacin-induced intestinal damage in rats. J Gastroenterol Hepatol. 2001;16:1112–9.CrossRefGoogle Scholar
  40. 40.
    Yamao J, Kikuchi E, Matsumoto M, Nakayama M, Ann T, et al. Assessing the efficacy of famotidine and rebamipide in the treatment of gastric mucosal lesions in patients receiving long-term NSAID therapy (FORCE—famotidine or rebamipide in comparison by endoscopy). J Gastroenterol. 2006;41:1178–85.CrossRefGoogle Scholar
  41. 41.
    Gagliano-Jucá T, Moreno RA, Zaminelli T, Napolitano M, Magalhães AF, et al. Rebamipide does not protect against naproxen-induced gastric damage: a randomized double-blind controlled trial. BMC Gastroenterol. 2016;16:58.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  1. 1.Department of Drug Safety Research, Nonclinical Research CenterTokushima Research Institute, Otsuka Pharmaceutical Co., Ltd.TokushimaJapan
  2. 2.Department of Clinical Pharmacy, Graduate School of Pharmaceutical SciencesNagoya City UniversityNagoyaJapan

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