再生腸上皮幹細胞ニッチの誘導・再構築による炎症腸管粘膜再生治療の確立

2015 Fiscal Year Annual Research Report

• Project/Area Number: 25293170
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: 岡本 隆一 東京医科歯科大学, 再生医療研究センター, 教授 (50451935)
• Co-Investigator(Kenkyū-buntansha): 土屋 輝一郎 東京医科歯科大学, 医学部附属病院, 准教授 (40376786) ; 中村 哲也 東京医科歯科大学, 医歯(薬)学総合研究科, 教授 (70265809)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Keywords: 腸管上皮 / 炎症性腸疾患 / Notchシグナル / オルガノイド / ニッチ

Outline of Annual Research Achievements

本年度の研究により、以下の成果が得られた
1) 「再生腸上皮」構成細胞で発現が誘導される候補遺伝子群の同定：上記の如く、炎症粘膜で誘導される再生上皮とこれを構成する細胞内の遺伝子発現について、網羅的に探索を行った。この結果、両シグナルの活性化により32倍以上の発現増加が誘導される遺伝子群として31遺伝子が同定された。さらにこのうちNotch活性化またはTNF-α添加のいずれか一方のみ条件と比較し1.5倍以上の発現増加を認めた遺伝子群として21遺伝子を同定した。この遺伝子群の中には幹細胞特異的遺伝子OLFM4が含まれ、同遺伝子は炎症粘膜の再生上皮で広範な発現が誘導される事が確認済みであることから、これを除く20遺伝子(PLA2G2A,LAMA3,PTGES等)についても「再生腸上皮」を構成する上皮細胞で広範かつ特異的に発現が誘導される可能性があるものと考えられた。
2) 「再生腸上皮」における特殊な幹細胞分画の同定：再生腸上皮において構成される幹細胞ニッチはAtoh1陽性(かつDll1陽性またはDll4陽性)細胞が主たる役割を担っているものと考えられたことから、同細胞の動態を追跡可能なAtoh1陽性細胞系譜追跡(Lineage tracing)マウスを作製し、腸上皮におけるAtoh1陽性細胞の分布と動態の解析を実施した。その結果、正常腸上皮においては分泌型細胞に一致してAtoh1陽性細胞がレポーター蛍光タンパクにより標識される一方、短期の解析に於いては幹細胞の形質を有する細胞には標識されず、通常、幹細胞ではAtoh1の発現は誘導されないものと考えられた。しかしながら長期の解析や大腸炎モデルにおける再生腸上皮の解析に於いては、一部のAtoh1陽性細胞が分泌型上皮以外の形質を獲得し、特殊な幹細胞分画を構成することにより腸上皮の再生に貢献している可能性を示す結果が得られている。

Research Progress Status

27年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

27年度が最終年度であるため、記入しない。

Causes of Carryover

27年度が最終年度であるため、記入しない。

Expenditure Plan for Carryover Budget

27年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Role of epithelial cells in the pathogenesis and treatment of inflammatory bowel disease. (2016)
  • Author(s): Ryuichi Okamoto, Mamoru Watanabe
  • Journal Title: J. Gastroenterol Volume: 51 Pages: 11-21
  • DOI: 10.1007/s00535-015-1098-4
  • Peer Reviewed / Open Access
• [Journal Article] Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes. (2016)
  • Author(s): Kengo Nozaki, Tomohiro Mizutani, Mamoru Watanabe, Tetsuya Nakamura. et al
  • Journal Title: J. Gastroenterol. Volume: 51 Pages: 206-213
  • DOI: 10.1007/s00535-016-1170-8
  • Peer Reviewed / Open Access
• [Journal Article] Matrix Metalloproteinase-14 Mediates Formation of Bile Ducts and Hepatic Maturation of Fetal Hepatic Progenitor Cells. (2015)
  • Author(s): Satoshi Otani, Sei Kakinuma, Ryuichi Okamoto, Mamoru Watanabe.et al
  • Journal Title: Biochem. Biophys. Res. Commun Volume: 469 Pages: 1062-1068
  • DOI: 10.1016/j.bbrc.2015.12.105
  • Peer Reviewed / Open Access
• [Journal Article] Perspectives for Regenerative Medicine in the Treatment of Inflammatory Bowel Diseases. (2015)
  • Author(s): Ryuichi Okamoto, Mamoru Watanabe
  • Journal Title: Digestion Volume: 92 Pages: 73-77
  • DOI: 10.1159/000438663
  • Peer Reviewed
• [Journal Article] TNFAIP3 promotes survival of CD4 T cells by restricting MTOR and promoting autophagy. (2015)
  • Author(s): Yu Matsuzawa, Shigeru Oshima, Ryuichi Okamoto, Mamoru Watanabe.et al
  • Journal Title: Autophagy Volume: 11 Pages: 1052-1062
  • DOI: 10.1080/15548627.2015.1055439
  • Peer Reviewed / Open Access
• [Journal Article] Atonal homolog 1 protein stabilized by tumor necrosis factor α induces high malignant potential in colon cancer cell line. (2015)
  • Author(s): Keita Fukushima, Kiichiro Tsuchiya, Okamoto Ryuichi, Mamoru Watanabe. et al
  • Journal Title: Cancer Sci Volume: 106 Pages: 1000-1007
  • DOI: 10.1111/cas.12703
  • Peer Reviewed / Open Access
• [Journal Article] Reduced human α-defensin 6 in non-inflamed jejunal tissue of Crohn’s disease patients (2015)
  • Author(s): Hayashi R , Tsuchiya K, Okamoto R, Watanabe M.et al.
  • Journal Title: Inflammatory Bowel Diseases
  • Peer Reviewed
• [Presentation] Establishment of a 3D cell culture-based screening platform to identify natural products that can regulate transepithelial water transport of the human gastrointestinal tract. (2015)
  • Author(s): Satoru Fujii, Ryuichi Okamoto, Toru Nakata, Kohei Suzuki, Fumiaki Ishibashi, Ami Kawamoto,Sayaka Ohashi Segawa, Tomohiro Mizutani, Kiichiro Tsuchiya, Tetsuya Nakamura, Mamoru Watanabe.
  • Organizer: Pacifichem2015
  • Place of Presentation: ハワイ（米国）
  • Year and Date: 2015-12-17 – 2015-12-17
  • Int'l Joint Research
• [Presentation] TNF-α stabilizes atoh1 protein in colitis-associated colorectal cancer resulting in enhanced malignant potential. (2015)
  • Author(s): Tomoaki Shirasaki, Keita Fukushima, Kiichiro Tsuchiya, Shuuji Hibiya, Ryohei Hayashi, Nobukatsu Horita, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe.
  • Organizer: UEGW2015
  • Place of Presentation: バルセロナ（スペイン）
  • Year and Date: 2015-10-28 – 2015-10-28
  • Int'l Joint Research
• [Presentation] Contiuous stimulation with cytokines leads to irreversible activation of NF-γb signaling in colonic epithelial cells by organoid culture. (2015)
  • Author(s): Shuji Hibiya, Kiichiro Tsuchiya, Tomoaki Shirasaki, Keita Fukushima, Ryohei Hayashi, Nobukatsu Horita, Shigeru Oshima ,Ryuichi Okamoto, Tetsuya Nakamura,Mamoru Watanabe
  • Organizer: UEGW2015
  • Place of Presentation: バルセロナ（スペイン）
  • Year and Date: 2015-10-27 – 2015-10-27
  • Int'l Joint Research
• [Presentation] Atoh1 protein stabilized by TNF-α acquires enhanced malignant potential in colitis-associated colorectal cancer. (2015)
  • Author(s): Tomoaki Shirasaki, Keita Fukushima, Kiichiro Tsuchiya, Shuuji Hibiya, Ryohei Hayashi, Nobukatsu Horita, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe.
  • Organizer: AOCC2015
  • Place of Presentation: 北京（中国）
  • Year and Date: 2015-06-19 – 2015-06-19
  • Int'l Joint Research
• [Presentation] A novel fluorescent labelling system into small intestinal organoid reveals independent long-lived intestinal stem cells in a crypt. (2015)
  • Author(s): Horita N,Tsuchiya K,Hayashi R,Fukushima K,Hibiya S,Fukuda M,Mizutani T,Okamoto R,Nakamura T and Watanabe M.
  • Organizer: AOCC2015
  • Place of Presentation: 北京（中国）
  • Year and Date: 2015-06-19 – 2015-06-19
  • Int'l Joint Research
• [Presentation] Enrichment of Intestinal Stem Cells by the 3D-Culture of IBD Patient Derived Intestinal epithelium. (2015)
  • Author(s): Kohei Suzuki,Satoru Fujii,Toru Nakata,Ami Kawamoto,Fumiaki Ishibashi,Sayaka Ohashi Segawa, Tomohiro Mizutani, Kiichiro Tsuchiya,Kazuo Otsuka,Tetsuya Nakamura,Ryuichi Okamoto,Mamoru Watanabe.
  • Organizer: AOCC2015
  • Place of Presentation: 北京（中国）
  • Year and Date: 2015-06-19 – 2015-06-19
  • Int'l Joint Research
• [Presentation] Continuous Stimulation With Cytokines Leads to Irreversible Accumulation of NF-kB Signaling in Colonic Epithelial Cells. (2015)
  • Author(s): Shuji Hibiya, Kiichiro Tsuchiya, Keita Fukushima, Ryohei Hayashi, Nobukatsu Horita, Shigeru Oshima, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe.
  • Organizer: DDW 2015
  • Place of Presentation: ワシントン（米国）
  • Year and Date: 2015-05-19 – 2015-05-19
  • Int'l Joint Research
• [Presentation] Distinct Role of Notch Ligands, DLL1 and Dll4, in Normal and in Tumor Intestinal Epithelium. (2015)
  • Author(s): Toru Nakata, Hiromichi Shimizu, Kohei Suzuki, Satoru Fujii, Go Ito, Kiichiro Tsuchiya, Tetsuya Nakamura, Ryuichi Okamoto, Katsuto Hozumi, Mamoru Watanabe.
  • Organizer: DDW 2015
  • Place of Presentation: ワシントン（米国）
  • Year and Date: 2015-05-19 – 2015-05-19
  • Int'l Joint Research
• [Presentation] Atoh1 Protein Expression by TNF-a and the Acquisition of Malignant Potential in Colitis- Associated Colorectal Cancer. (2015)
  • Author(s): Keita Fukushima, Kiichiro Tsuchiya, Shuji Hibiya, Ryohei Hayashi, Nobukatsu Horita, Yoshihito Kano, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe.
  • Organizer: DDW 2015
  • Place of Presentation: ワシントン（米国）
  • Year and Date: 2015-05-17 – 2015-05-17
  • Int'l Joint Research
• [Presentation] Notch Ligands DLL1 and Dll4 Are Expressed by Distinct Population of Epithelial Cells in the Mice Intestine. (2015)
  • Author(s): Hiromichi Shimizu, Kohei Suzuki, Satoru Fujii, Toru Nakata, Go Ito, Tatsuro Murano, Kiichiro Tsuchiya, Tetsuya Nakamura, Ryuichi Okamoto, Katsuto Hozumi, Mamoru Watanabe.
  • Organizer: DDW 2015
  • Place of Presentation: ワシントン；米国
  • Year and Date: 2015-05-16 – 2015-05-16
  • Int'l Joint Research
• [Presentation] Live imaging analysis of intraepithelial lymphocytes (IELs) co-cultured with intestinal epithelial organoids. (2015)
  • Author(s): Nozaki K, Mochizuki W, Matsumoto Y, Matsumoto T, Fukuda M, Mizutani T, Watanabe M, Nakamura T
  • Organizer: Cold Spring Harbor Meeting "Fundamental Immunology and Its Therapeutic Applications"
  • Place of Presentation: ニューヨーク（米国）
  • Year and Date: 2015-04-16 – 2015-04-16
  • Int'l Joint Research