Real-time imaging analysis of proliferation/differentiation of colonic epithelial cells in primary culture

• Project/Area Number: 21590803
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: NAKAMURA Tetsuya 東京医科歯科大学, 大学院・医歯学総合研究科, 寄附講座教員 (70265809)
• Co-Investigator(Kenkyū-buntansha): WATANABE Mamoru 東京医科歯科大学, 医歯学総合研究科, 教授 (10175127)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2011: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2010: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2009: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: 消化管上皮幹細胞 / ライブセルイメージング / 初代培養技術 / 細胞移植治療 / 大腸上皮幹細胞 / ライブイメージング / 細胞増殖 / 細胞分化 / 初代培養 / 幹細胞移植 / 再生治療 / 炎症性腸疾患

Research Abstract

We have developed a novel culture method for colonic epithelial cells that allows long-term maintenance and efficient expansion of stem cells in vitro. Colonic crypts from normal adult mice were isolated and three-dimensionally cultured in serum-free medium supplemented with a combination of growth factors. The crypt cells formed a round cystic organoid consisting of epithelial monolayer of multi-lineage cells, continuously grew with increasing size of organoid structure, and could be propagated for months without losing their properties. Importantly, Lgr5+colonic stem cells were constantly maintained for a long time period in our culture. In order to test transplantability of these cultured cells, GFP+colonic cells were re-introduced into the lumen of mouse colon superficially damaged by DSS treatment. As observed 4 weeks post-transplantation, the infused organoids readily integrated into the epithelium to form self-renewing GFP+epithelial patches that were histologically normal. Similar results were obtained with colon organoids that were derived from a single Lgr5+colon stem cell after extensive in vitro expansion.
The techniques that we have developed would be of significant help to build a basis to develop a novel colon stem cell therapy based on in vitro expansion of a single adult colonic stem cell.

Research Products

• [Journal Article] Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+stem cell (2012)
  • Author(s): Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Clevers H, and Watanabe M
  • Journal Title: Nature Medicine Volume: 18 Issue: 4 Pages: 618-623
  • DOI: 10.1038/nm.2695
  • Peer Reviewed
• [Journal Article] Real-time analysis of P-glycoprotein-mediated drug transport across primary intestinal epithelium three-dimensionally cultured in vitro (2012)
  • Author(s): Mizutani T, Nakamura T, Morikawa R, Fukuda M, Mochizuki W, Yamauchi Y, Nozaki K, Yui S, Nemoto Y, Nagaishi T, Okamoto R, Tsuchiya K, and Watanabe M
  • Journal Title: Biochem Biophys Res Commun Volume: 419(2) Pages: 238-243
• [Journal Article] Real-time analysis of P-glycoprotein-mediated drug transport across primary intestinal epithelium three-dimensionally cultured in vitro (2012)
  • Author(s): Mizutani T, Nakamura T, et al
  • Journal Title: Biochem Biophys Res Commun Volume: 419 Issue: 2 Pages: 238-243
  • DOI: 10.1016/j.bbrc.2012.01.155
  • Peer Reviewed
• [Journal Article] Gene bookmarking accelerates the kinetics of post-mitotic transcriptional activation (2011)
  • Author(s): Zhao R, Nakamura T, Fu Y, Lazar Z, and Spector DL
  • Journal Title: Nature Cell Biology Volume: 13(11) Pages: 1295-1304
• [Journal Article] Suppression of Hath1 gene expression directly regulated by Hes1 via Notch signaling is associated with goblet cell depletion in ulcerative colitis (2011)
  • Author(s): Zheng X, Tsuchiya K, Okamoto R, Iwasaki M, Kano Y, Sakamoto N, Nakamura T, Watanabe M
  • Journal Title: Inflamm Bowel Dis Volume: 17(11) Pages: 2251-2260
• [Journal Article] Longitudinal cell formation in the entire human small intestine is correlated with the localization of Hath1 and Klf4 (2011)
  • Author(s): Iwasaki M, Tsuchiya K, Okamoto R, Zheng X, Kano Y, Okamoto E, Okada E, Araki A, Suzuki S, Sakamoto N, Kitagaki K, Akashi T, Eishi Y, Nakamura T, Watanabe M
  • Journal Title: J Gastroenterol Volume: 46(2) Pages: 191-201
  • NAID: 10029027237
• [Journal Article] Nuclear organization and dynamics of 7SK RNA in regulating gene expression (2010)
  • Author(s): Prasanth KV, Camiolo M, Chan G, Tripathi V, Denis L, Nakamura T, Hubner M, and Spector DL
  • Journal Title: Mol BiolCell Volume: 21(23) Pages: 4184-4196
• [Journal Article] A long nuclear retained non-coding RNA regulates synaptogenesis by modulating gene expression (2010)
  • Author(s): Bernard D, Prasanth KV, Tripathi V, Colasse S, Nakamura T, Xuan Z, Zhang MQ, Sedel F, Jourdren L, Coulpier F, Triller A, Spector DL, and Bessis A
  • Journal Title: EMBO J Volume: 29(18) Pages: 3082-3093
• [Journal Article] Delta-like 1 expression promotes goblet cell differentiation in Notch-inactivated human colonic epithelial cells (2010)
  • Author(s): Akiyama J, Okamoto R, Iwasaki M, Zheng X, Yui S, Tsuchiya K, Nakamura T, and Watanabe M
  • Journal Title: Biochem Biophys Res Commun Volume: 393(4) Pages: 662-667
• [Journal Article] Delta-like 1 expression promotes goblet cell differentiation in Notch-inactivated human colonic epithelial cells. (2010)
  • Author(s): Akiyama J, Nakamura T, et al.
  • Journal Title: Biochemical and Biophysical Research Communications. (in press)
  • Peer Reviewed
• [Journal Article] Signaling pathway via TNF-a/NF-kB in intestinal epithelial cells may be directly involved in colitis-associated carcinogenesis (2009)
  • Author(s): Onizawa M, Nagaishi T, Kanai T, Nagano KI, Oshima S, Nemoto Y, Yoshioka A, Totsuka T, Okamoto R, Nakamura T, Sakamoto N, Tsuchiya K, Aoki K, Ohya K, Yagita H, Watanabe M
  • Journal Title: Am J Physiol Gastrointest Liver Physiol Volume: 296(4) Pages: 850-859
• [Journal Article] Requirement of Notch activation during regeneration of the intestinal epithelia (2009)
  • Author(s): Okamoto R, Tsuchiya K, Nemoto Y, Akiyama J, Nakamura T, Kanai T, Watanabe M
  • Journal Title: Am J Physiol Gastrointest Liver Physiol Volume: 296(1) Pages: 23-35
• [Journal Article] Requirement of Notch activation during regeneration of the intestinal epithelia. (2009)
  • Author(s): Okamoto R, Nakamura T, et al.
  • Journal Title: Am J Physiol GI & Liver. 296
  • Peer Reviewed
• [Presentation] Epithelial regeneration by cultured colonic cells expanded from a single adult LGR5+stem cell (2012)
  • Author(s): Tetsuya Nakamura(Invited Speaker)
  • Organizer: The 6th Korea-JapanIBD Symposium
  • Place of Presentation: Tokyo, Japan
  • Year and Date: 2012-01-28
• [Presentation] Epithelial regeneration by cultured colonic cells expanded from a single adult Lgr5+ stem cell (2012)
  • Author(s): Nakamura T
  • Organizer: The 6th Korea-Japan IBD Symposium
  • Place of Presentation: Tokyo
  • Year and Date: 2012-01-28
• [Presentation] Regeneration of damaged colon epithelium by transplanted colon Lgr5+stem cells maintained and expanded in vitro (2011)
  • Author(s): Shiro Yui, Tetsuya Nakamura, Toshiro Sato, Yasuhiro Nemoto, Tomohiro Mizutani, Xiu Zheng, Shizuko Ichinose, Takashi Nagaishi, Ryuichi Okamoto, Kiichiro Tsuchiya, Hans Clevers, and Mamoru Watanabe
  • Organizer: The5^<th> Japan & US Collaboration Conference in Gastroenterology
  • Place of Presentation: Tokyo, Japan
  • Year and Date: 2011-11-10
• [Presentation] Flagellin via TLR5 on basolateral membrane of primary intestinal epithelial cells(IEC) shows the role of IEC in the response to bacteria. (2011)
  • Author(s): Tsuchiya K, Zheng X, Kano Y, Okamoto R, Nakamura T, Watanabe M
  • Organizer: United European Gastroenterology Week 2011
  • Place of Presentation: Stockholm, Sweden
  • Year and Date: 2011-10-26
• [Presentation] Regeneration of damaged colonic tissue by transplanted colonic epithelial stem cells maintained and expanded in vitro (2011)
  • Author(s): Yui S, Nakamura T, Nemoto Y, Mizutani T, Zheng X, Nagaishi T, Tsuchiya K, Watanabe M, Okamoto R, Ichinose S, Sato T, Clevers H
  • Organizer: GI Research Academy 2011
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2011-06-17
• [Presentation] 再生医療へ向けた腸管上皮研究～幹細胞体外培養と細胞移植～ (2011)
  • Author(s): 中村哲也
  • Organizer: 日本消化器病学会総会シンポジウム指定口演
  • Place of Presentation: 東京
  • Year and Date: 2011-05-14
• [Presentation] Regeneration of Damaged Colonic Tissue by Transplantation of Colonic Epithelial Stem Cells Maintained and Expanded In Vitro (2011)
  • Author(s): Shiro Yui, Tetsuya Nakamura, Yasuhiro Nemoto, Tomohiro Mizutani, Xiu Zheng, Shizuko Ichinose, Takashi Nagaishi, Ryuichi Okamoto, Kiichiro Tsuchiya, Toshiro Sato, Hans Clevers, and Mamoru Watanabe
  • Organizer: Basic Science Plenary Symposium, Digestive DiseaseWeek 2011
  • Place of Presentation: Chicago, USA
• [Presentation] A well-defined culture system for colonic epithelial cells thet allows efficient enrichment of LGR5+stem cells. (2010)
  • Author(s): Yui S, Nakamura T, Nemoto Y, Mizutani T, Zheng X, Ichinose S, Nagaishi T, Okamoto R, Tsuchiya K, Watanabe M
  • Organizer: The 1st JSGE International Topic Conference-Stem Cells in Digestive Organs-
  • Place of Presentation: Kamakura, Japan
  • Year and Date: 2010-09-26
• [Presentation] Up-reguration of DELTA1 expression is required for goblet cell differentiation in human intestinal epithelial cells (2010)
  • Author(s): Akiyama J, Okamoto R, Tsuchiya K, Nakamura T, Watanabe M
  • Organizer: The 1st JSGE International Topic Conference-Stem Cells in Digestive Organs-
  • Place of Presentation: Kamakura, Japan
  • Year and Date: 2010-09-26
• [Presentation] HES1 via notch signaling directly suppresses ATOH1/HATH1 gene transcription, resulting in the undifferentiated state of intestinal epithelial cells. (2010)
  • Author(s): Zheng X, Tsuchiya K, Okamoto R, Okamoto E, Iwasaki M, Kano Yoshihito, Nakamura T, Watanabe M
  • Organizer: The 1st JSGE International Topic Conference-Stem Cells in Digestive Organs-
  • Place of Presentation: Kamakura, Japan
  • Year and Date: 2010-09-26
• [Presentation] TNF-αand notch signaling synergistically up-regulate OLFM4 expression in human intestinal epithelial cells (2010)
  • Author(s): Murano T, Okamoto R, Shimizu H, Akiyama J, Tsuchiya K, Nakamura T, Watanabe M
  • Organizer: The 1st JSGE International Topic Conference-Stem Cells in Digestive Organs-
  • Place of Presentation: Kamakura, Japan
  • Year and Date: 2010-09-26
• [Presentation] A long-term, fully-deffined culture system for colonic epithelial cells that alows efficient expansion of stem cell compartment (2010)
  • Author(s): Tetsuya Nakamura(Invited speaker), Watanabe M
  • Organizer: The 1st JSGE International Topic Conference-Stem Cells in Digestive Organs-
  • Place of Presentation: Kamakura, Japan
  • Year and Date: 2010-09-25
• [Presentation] Hes1 via Notch signaling directly suppresses Atoh1/Hath1 gene transcription, resulting in the goblet cell depletion of Ulcerative Colitis (2010)
  • Author(s): Zheng X, Tsuchiya K, Okamoto R, Iwasaki M, Kano Y, Nakamura T, Watanabe M
  • Organizer: Digestive Disease Week 2010
  • Place of Presentation: New Orleans, USA
  • Year and Date: 2010-05-03
• [Presentation] A long-term, mesenchyme-free in vitro culture system of colonic epithelial cells retaining stem cell compartment with proliferation/differentiation properties (2010)
  • Author(s): Tetsuya Nakamura, Shiro Yui, Tomohiro Mizutani, Yasuhiro Nemoto, Ryuichi Okamoto, Kiichiro Tsuchiya, Shizuko Ichinose, and Mamoru Watanabe
  • Organizer: Digestive Disease Week 2010
  • Place of Presentation: New Orleans, USA
• [Presentation] A long-term, mesenchyme-free in vitro culture system of colonic epithelial cells retaining stem cell compartment with proliferation/differentiation properties. (2010)
  • Author(s): Nakamura T, Yui S, Mizutani K, Nemoto Y, Okamoto R, Tsuchiya K, Ichinose S, Watanabe M
  • Organizer: Digestive Disease Week2010
  • Place of Presentation: New Orleans, USAS
• [Presentation] A Long-term, fully-defined culture system for colonic epithelial cells that allows efficient expansion of stem cell (2010)
  • Author(s): Nakamura T, Watanabe M
  • Organizer: The 1st JSGE International Topic Conference
  • Place of Presentation: Kamakura, Japan
• [Presentation] GSK3 inhibitor induces the intestinal differen tiation by the proteinstabilization of Atoh1 (2009)
  • Author(s): Tsuchiya K, Okamoto R, Nakamura T, Watanabe M
  • Organizer: Digestive Disease Week 2009
  • Place of Presentation: Chicago, USA
  • Year and Date: 2009-06-02
• [Patent(Industrial Property Rights)] 大腸上皮幹細胞の単離・培養技術とこれを用いた大腸上皮移植技術 (2011)
  • Inventor(s): 渡辺守・中村哲也
  • Industrial Property Rights Holder: 国立大学法人東京医科歯科大学
  • Filing Date: 2011-10-27
• [Patent(Industrial Property Rights)] 大腸上皮幹細胞の単離・培養技術とこれを用いた大腸上皮移植技術 (2011)
  • Inventor(s): 渡辺守・中村哲也
  • Industrial Property Rights Holder: 国立大学法人東京医科歯科大学
  • Industrial Property Number: 2011-236469
  • Filing Date: 2011-10-27