Generation of inner ear cells from mouse iPS cells and its application to Age-related hearing loss

• Project/Area Number: 15K20229
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Otorhinolaryngology
• Research Institution: Juntendo University
• Principal Investigator: FUKUNAGA Ichiro 順天堂大学, 医学(系)研究科(研究院), 博士研究員 (20746581)
• Research Collaborator: KAMIYA Kazusaku 順天堂大学, 医学部, 准教授 (10374159)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2015: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: iPS細胞 / 蝸牛支持細胞 / Connexin26 / ギャップ結合プラーク / 加齢性難聴 / 遺伝性難聴 / GJB2 / 分化誘導 / 鳥類胚 / 蝸牛線維細胞 / 有毛細胞

Outline of Final Research Achievements

In this study, we generated Cx26 expressing cells with GJP formation from mouse iPS cells (iCx26GJC). To investigate whether the iCx26GJCs were functional, we performed scrape-loading assay with Lucifer yellow (LY) and Ca2+imaging.　In the iCx26GJC culture, we observed that LY diffused beyond the wounded parental cells. In contrast, such extent of dye transfer was not observed in undifferentiated iPSCs. Furthermore, iCx26GJC exhibited spontaneous Ca2+ transients and their propagation. The spontaneous Ca2+ signaling activity was clearly inhibited by the p2x receptor antagonist (PPADS) and connexin hemichannel blocker (FFA), and the activity was restored after removal of the inhibitors.
on the other hand, to enhance MSC invasion to cochlea tissue, we developed a novel transplant strategy by induction of SDF-1 /MCP-1 expression in host cochlear tissue and enhanced expression of their receptors, chemokine (C-C motif) receptor 2 (CCR2) and C-X-C chemokine receptor type 4 (CXCR4) in MSC.

Research Products

• [Journal Article] In vitro models of GJB2-related hearing loss recapitulate Ca2+ transients via a gap junction characteristic of developing cochlea. (2016)
  • Author(s): Ichiro Fukunaga, Ayumi Fujimoto, Kaori Hatakeyama, Toru Aoki, Atena Nishikawa, Tetsuo Noda, Osamu Minowa, Nagomi Kurebayashi, Katsuhisa Ikeda, Kazusaku Kamiya.
  • Journal Title: Stem Cell Reports Volume: 7 Issue: 6 Pages: 1023-1036
  • DOI: 10.1016/j.stemcr.2016.10.005
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Connexin26 regulates assembly and maintenance of cochlear gap junction macromolecular complex for normal hearing (2015)
  • Author(s): Kazusaku Kamiya, Ichiro Fukunaga, Kaori Hatakeyama and Katsuhisa Ikeda
  • Journal Title: AIP Conference Proceedings Volume: 1703 Pages: 030018-030018
  • DOI: 10.1063/1.4939333
  • Acknowledgement Compliant
• [Journal Article] Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice. (2015)
  • Author(s): Anzai, T., Fukunaga, I., Hatakeyama, K., Fujimoto, A., Kobayashi, K., Nishikawa, A., Aoki, T., Noda, T., Minowa, O., Ikeda, K., and Kamiya, K
  • Journal Title: PloS one Volume: 10 Issue: 10 Pages: e0141258-e0141258
  • DOI: 10.1371/journal.pone.0141258
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] iPS細胞からConnexin26ギャップ結合プラークを構築する細胞への分化誘導. (2017)
  • Author(s): 福永一朗, 畠山佳欧里, 青木 徹, 藤本あゆみ, 西川貴菜, 美野輪治, 池田勝久, 神谷和作
  • Organizer: 第16回日本再生医療学会
  • Place of Presentation: 仙台
  • Year and Date: 2017-03-07
• [Presentation] Gene therapy and cell therapy targeting cochlear gap junction formation for GJB2 associated hearing loss. (2016)
  • Author(s): Ichiro Fukunaga, Takashi Iizuka, Kaori Hatakeyama, Ayumi Fujimoto, Osamu Minowa, Katsuhisa Ikeda, Kazusaku Kamiya.
  • Organizer: Inner Ear Biology
  • Place of Presentation: Montpellier, France.
  • Year and Date: 2016-09-17
  • Int'l Joint Research
• [Presentation] Generation of Cx26-gap junction plaque forming cell from iPS cell. (2016)
  • Author(s): Ichiro Fukunaga, Ayumi Fujimoto, Kaori Hatakeyama, Toru Aoki, Atena Nishikawa, Osamu Minowa, Katsuhisa Ikeda, Kazusaku Kamiya.
  • Organizer: Inner Ear Biology
  • Place of Presentation: Montpellier, France.
  • Year and Date: 2016-09-17
  • Int'l Joint Research
• [Presentation] iPS細胞からのConnexin26発現細胞への分化誘導と細胞間ギャップ結合プラークの構築 (2016)
  • Author(s): 福永一朗、畠山佳欧里、 青木徹、藤本あゆみ、 西川貴菜、美野輪治、 池田勝久、神谷和作
  • Organizer: 第15回日本再生医療学会総会
  • Place of Presentation: 大阪国際会議場(大阪府 大阪市)
  • Year and Date: 2016-03-17
• [Presentation] コネキシン26欠損マウスからのiPS細胞の樹立 (2016)
  • Author(s): 青木徹、福永一朗、 畠山佳欧里、藤本あゆみ、西川貴菜、美野輪治、 池田勝久、神谷和作
  • Organizer: 第15回日本再生医療学会総会
  • Place of Presentation: 大阪国際会議場(大阪府 大阪市)
  • Year and Date: 2016-03-17
• [Presentation] 内耳フィーダー細胞を用いたiPS細胞の分化誘導によるCx26ギャップ結合形成細胞の樹立 (2016)
  • Author(s): 藤本あゆみ、福永一朗、 畠山佳欧里、青木徹、 西川貴菜、池田勝久、 神谷和作
  • Organizer: 第15回日本再生医療学会総会
  • Place of Presentation: 大阪国際会議場(大阪府 大阪市)
  • Year and Date: 2016-03-17
• [Presentation] Differentiation of mouse iPS cell into Cx26-positive cell and formation of inter cellular Cx26-gap junction plaque (2016)
  • Author(s): Ichiro Fukunaga, Kaori Hatakeyama,Toru Aoki, Ayumi Fujimoto, Atena Nishikawa,Katsuhisa Ikeda, Kazusaku Kamiya
  • Organizer: Association for Research in Otolaryngology (ARO), 39th Annual MidWinter Meeting
  • Place of Presentation: 米国, サンディエゴ
  • Year and Date: 2016-02-20
  • Int'l Joint Research
• [Presentation] Restoration of Cochlear Gap Junction for GJB2 (2016)
  • Author(s): Kazusaku Kamiya, Ichiro Fukunaｇa
  • Organizer: Association for Research in Otolaryngology (ARO), 39th Annual MidWinter Meeting
  • Place of Presentation: 米国, サンディエゴ
  • Year and Date: 2016-02-20
  • Int'l Joint Research
• [Presentation] Induction of iPS cell differetiation into Connexin26 positive cells with gap junction plaques using cochlear feeder cell (2016)
  • Author(s): Ayumi Fujimoto, Ichiro Fukunaga, Kaori Hatakeyama, Toru Aoki, Atena Nishikawa, Katsuhisa Ikeda, Kazusaku Kamiya
  • Organizer: Association for Research in Otolaryngology (ARO), 39th Annual MidWinter Meeting
  • Place of Presentation: 米国, サンディエゴ
  • Year and Date: 2016-02-20
  • Int'l Joint Research
• [Presentation] Differentiation of mouse iPS cell into Cx26-positive cell and formation of inter cellular Cx26-gap junction plaque (2015)
  • Author(s): Ichiro Fukunaga, Kaori Hatakeyama,Toru Aoki, Atena Nishikawa, Ayumi Fujimoto, Osamu Minowa, Katsuhisa Ikeda, Kazusaku Kamiya
  • Organizer: 52nd Inner Ear Biology Workshop
  • Place of Presentation: イタリア,ローマ
  • Year and Date: 2015-09-12
  • Int'l Joint Research
• [Presentation] Cochlear gap junction plaque, stabilized macromolecular complex composed of specific connexins (2015)
  • Author(s): Kazusaku Kamiya, Ichiro Fukunaga, Kaori Hatakeyama, Toru Aoki, Ayumi Fujimoto, Atena Nishikawa ,Takashi Anzai, Osamu Minowa, Katsuhisa Ikeda
  • Organizer: 52nd Inner Ear Biology Workshop
  • Place of Presentation: イタリア,ローマ
  • Year and Date: 2015-09-12
  • Int'l Joint Research
• [Remarks] 内耳再生医療・遺伝子治療研究グループ
  • URL: http://www.juntendojibi.com/?page_id=3251
• [Patent(Industrial Property Rights)] 内耳細胞の製造法 (2017)
  • Inventor(s): 神谷和作, 福永一朗
  • Industrial Property Rights Holder: 順天堂大学
  • Industrial Property Rights Type: 特許
  • Filing Date: 2017-02-22
  • Overseas
• [Patent(Industrial Property Rights)] 内耳前駆細胞の製造法 (2016)
  • Inventor(s): 神谷和作, 福永一朗
  • Industrial Property Rights Holder: 学校法人 順天堂
  • Industrial Property Rights Type: 特許
  • Filing Date: 2016-02-22