Generation of inner ear hair cells toward cell transplantation using pluripotent stem cells

• Project/Area Number: 16H05482
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Otorhinolaryngology
• Research Institution: Nara Medical University
• Principal Investigator: Yoshikawa Masahide 奈良県立医科大学, 医学部, 教授 (50230701)
• Co-Investigator(Kenkyū-buntansha): 北原 糺 奈良県立医科大学, 医学部, 教授 (30343255) ; 王寺 幸輝 奈良県立医科大学, 医学部, 准教授 (50343421) ; 和中 明生 奈良県立医科大学, 医学部, 教授 (90210989)
• Project Period (FY): 2016-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥17,290,000 (Direct Cost: ¥13,300,000、Indirect Cost: ¥3,990,000); Fiscal Year 2019: ¥3,250,000 (Direct Cost: ¥2,500,000、Indirect Cost: ¥750,000); Fiscal Year 2018: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000); Fiscal Year 2017: ¥5,200,000 (Direct Cost: ¥4,000,000、Indirect Cost: ¥1,200,000); Fiscal Year 2016: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
• Keywords: 内耳 / 再生医学 / 有毛細胞 / 幹細胞 / 耳科学 / 分化誘導 / 細胞培養 / 聴覚再生 / 難聴疾患 / ES細胞 / マウス / 転写因子 / 細胞移植 / iPS細胞 / 内耳再生

Outline of Final Research Achievements

The self-renewal capability of inner ear hair cells in mammals is extremely poor, thus stem cells harboring an ability to generate inner ear hair cells is a promising treatment option for damaged inner ear hair cells. We have successfully developed a new method for in vitro production of inner hair cells from pluripotent stem cells. In the present study, the mechanism of differentiation and possibility of cell transplantation using isolated cochlea tissue samples were investigated. The results showed that secretory factors related to resident cells in the inner ear facilitated differentiation of stem cells into inner ear hair cells, with several candidates determined based on RNA-sequence analysis. In addition, analysis of the co-culture with embryonic stem cell-derived inner ear hair cells and isolated cochlea tissues revealed cell settlement in the cochlea, suggesting the applicability of this in vivo method for cell transplantation therapy.

Academic Significance and Societal Importance of the Research Achievements

難聴治療の鍵となる内耳有毛細胞に着目し、様々な細胞に分化可能な多能性幹細胞から効率良く誘導する方法を開発した。本法は、これまでの方法に比べ簡便かつ高効率である。内耳細胞の再生研究および細胞移植治療に用いる細胞源確保に十分貢献できる成果である。本研究で開発した手法を、臨床応用が開始されたヒトiPS細胞へ適応することで、今後の耳科領域における研究を更に発展させる可能性がある。

Research Products

• [Journal Article] Differentiation of embryonic stem cells into inner ear vestibular hair cells using vestibular cell derived-conditioned medium (2019)
  • Author(s): Sakagami M, Ouji Y, Kawai N, Misu M, Yoshikawa M, Kitahara T.
  • Journal Title: Biochem Biophys Rep Volume: 19 Pages: 100649-100649
  • DOI: 10.1016/j.bbrep.2019.100649
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Isolation and culture of pleural mesothelial cells (2019)
  • Author(s): Kawai N, Ouji Y, Sakagami M, Tojo T, Sawabata N, Yoshikawa M, Taniguchi S.
  • Journal Title: Exp Lung Res Volume: 45 Issue: 5-6 Pages: 151-156
  • DOI: 10.1080/01902148.2018.1511002
  • Peer Reviewed
• [Journal Article] Effectiveness of cross-linked gelatin glue in canine lung surgery models (2019)
  • Author(s): Kawai No, Suzuki S, Yamashita K, Niwa K, Tojo T, Sawabata N, Nakai T, Ouji Y, Yoshikawa M, Ikada Y, Taniguchi S
  • Journal Title: J Nara Med Assoc Volume: 70 Pages: 35-43
  • NAID: 120006892014
  • Peer Reviewed
• [Journal Article] ここまで進んだ再生医学 多能性幹細胞から内耳有毛細胞への分化誘導法も含めて (2019)
  • Author(s): 吉川正英
  • Journal Title: 耳鼻咽喉科ニューロサイエンス Volume: 33 Pages: 1-5
• [Journal Article] インド帰国後の亜急性下痢、腹痛からセイロン鉤虫症と診断し、治療した症例 (2019)
  • Author(s): 宮里悠佑, 大場雄一郎, 吉川正英
  • Journal Title: Clinical Parasitolog Volume: 30 Pages: 113-115
• [Journal Article] アニサキス亜科線虫第4期幼虫感染の16例について (2019)
  • Author(s): 山田稔, 畑中秀亮, 石井明, 吉川正英
  • Journal Title: Clinical Parasitolog Volume: 30 Pages: 74-76
• [Journal Article] タイ帰国後に無鉤条虫を認めた1例 (2019)
  • Author(s): 平位暢康, 三須政康, 王寺幸輝,笠松丈人, 吉原真吾, 古川龍太郎, 西村知子, 奥田菜緒, 今北菜津子, 福盛達也, 小川拓, 笠原敬, 佐藤公俊, 前田光一, 三笠桂一, 吉川正英, 山崎浩, 森嶋康之
  • Journal Title: Clinical Parasitolog Volume: 30 Pages: 43-45
• [Journal Article] Differentiation of embryonic stem cells into inner ear vestibular hair cells using vestibular cell derived-conditioned medium (2019)
  • Author(s): Sakagami M, Ouji Y, Kawai N, Misu M, Yoshikawa M, Kitahara T.
  • Journal Title: Biochem Biophys Rep. Volume: in press
  • Peer Reviewed / Open Access
• [Journal Article] Effect of covering with cross-linked gelatin glue on tissue regeneration in a rat lung injury model. (2019)
  • Author(s): Kawai N, Suzuki S, Ouji Y, Takeda M, Sakagami M, Tojo T, Sawabata N, Yoshikawa M, Ikada Y, Taniguchi S.
  • Journal Title: Interact Cardiovasc Thorac Surg. Volume: ivy297 Issue: 1 Pages: 1-7
  • DOI: 10.1093/icvts/ivy297
  • Peer Reviewed
• [Journal Article] Induction of lung-like cells from mouse embryonic stem cells by decellularized lung matrix. (2018)
  • Author(s): Kawai N, Ouji Y, Sakagami M, Tojo T, Sawabata N, Yoshikawa M, Taniguchi S.
  • Journal Title: Biochem Biophys Rep. Volume: 15 Pages: 733-738
  • DOI: 10.1016/j.bbrep.2018.06.005
  • Peer Reviewed / Open Access
• [Journal Article] Molecular diagnosis and characterization of a culture-negative mycotic aneurysm due to ST54 Haemophilus influenzae type b with PBP 3 alterations. (2018)
  • Author(s): Hirai N, Kasahara K, Fujikura H, Yoshihara S, Ogawa T, Ogawa Y, Hishiya N, Suzuki Y, Nakano R, Yano H, Yoshikawa M, Mikasa K.
  • Journal Title: J Infect Chemother. Volume: 24 Issue: 7 Pages: 570-572
  • DOI: 10.1016/j.jiac.2017.12.013
  • Peer Reviewed
• [Journal Article] Ancylostoma ceylanicum, novel etiological agent for traveler's diarrhea-report of four Japanese patients who returned from Southeast Asia and Papua New Guinea. (2018)
  • Author(s): Yoshikawa M, Ouji Y, Hirai N, Nakamura-Uchiyama F, Yamada M, Arizono N, Akamatsu N, Yoh T, Kaya D, Nakatani T, Kikuchi E, Katanami Y, Satoh K, Maki R, Miyazato Y, Oba Y, Kasahara K, Mikasa K.
  • Journal Title: Trop Med Health Volume: 46 Issue: 1 Pages: 1-8
  • DOI: 10.1186/s41182-018-0087-8
  • Peer Reviewed
• [Journal Article] Efficient induction of inner ear hair cell-like cells from mouse ES cells using combination of Math1 transfection and conditioned medium from ST2 stromal cells. (2017)
  • Author(s): Ouji Y, Sakagami M, Omori H, Higashiyama S, Kawai N, Kitahara T, Wanaka A, Yoshikawa M.
  • Journal Title: Stem Cell Research Volume: 23 Pages: 50-56
  • DOI: 10.1016/j.scr.2017.06.013
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Antitumor effects of minodronate, a third-generation nitrogen-containing bisphosphonate, in synergy with γδT cells in human glioblastoma in vitro and in vivo. (2016)
  • Author(s): Nakazawa T, Nakamura M, Matsuda R, Nishimura F, Park YS, Motoyama Y, Hironaka Y, Nakagawa I, Yokota H, Yamada S, Tamura K, Takeshima Y, Omoto K, Tanaka Y, Ouji Y, Yoshikawa M, Tsujimura T, Nakase H.
  • Journal Title: J Neurooncol Volume: 129 Issue: 2 Pages: 231-241
  • DOI: 10.1007/s11060-016-2186-x
  • Peer Reviewed / Open Access
• [Journal Article] Ancylostoma ceylanicum hookworm infection in Japanese traveler who presented chronic diarrhea after return from Lao People's Democratic Republic. (2016)
  • Author(s): Kaya D, Yoshikawa M, Nakatani T, Tomo-Oka F, Fujimoto Y, Ishida K, Fujinaga Y, Aihara Y, Nagamatsu S, Matsuo E, Tokoro M, Ouji Y, Kikuchi E.
  • Journal Title: Parasitol Int. Volume: 65 Issue: 6 Pages: 737-740
  • DOI: 10.1016/j.parint.2016.07.001
  • Peer Reviewed
• [Presentation] 便検査を繰り返し施行することで診断に至ったランブル鞭毛虫症の一例 (2020)
  • Author(s): 村上紘一, 久保貴裕, 中谷敏也, 加知宏規, 尾山雅文, 元川雄貴, 森仁志, 芝本彰彦, 相原洋祐, 永松晋作, 上嶋昌和, 松尾英城, 菊池英亮, 吉川正英
  • Organizer: 日本消化器病学会近畿支部例会
• [Presentation] 卵形嚢由来細胞を用いたES細胞から内耳前庭有毛細胞への分化誘導法の開発 (2019)
  • Author(s): 阪上雅治、王寺幸輝、河合紀和、吉川正英、北原糺
  • Organizer: 第19回 日本再生医療学会総会
• [Presentation] ES細胞から内耳前庭有毛細胞への効率的分化誘導法の開発 (2018)
  • Author(s): 阪上雅治、吉川正英、北原糺
  • Organizer: 第28回日本耳科学会 総会・学術講演
• [Presentation] 脊髄炎で発症し肺肝病変を伴ったトキソカラ症の1例 (2018)
  • Author(s): 井口直彦、形岡博史、正畠良悟、桐山敬生、吉川正英、杉江和馬
  • Organizer: 第23回日本神経感染症学会総会・学術大会
• [Presentation] Characterization of murine hair follicle stem cells under long-term culture conditions (2018)
  • Author(s): Ouji Y, Kawai N, Sakagami M, Yoshikawa M.
  • Organizer: 16th International Society for Stem Cell Research Annual Meeting
  • Int'l Joint Research
• [Presentation] Differentiation of mouse embryonic stem cells into inner ear cell-like cells using vestibular cell-conditioned medium. (2018)
  • Author(s): Sakagami M, Ouji Y, Kawai N, Kitahara T, Yoshikawa M.
  • Organizer: 16th International Society for Stem Cell Research Annual Meeting
  • Int'l Joint Research
• [Presentation] 消化管に一隻のイヌ鉤虫が発見された日本在住のブラジル人症例 (2018)
  • Author(s): 赤松尚明、吉川正英、山田稔、楊孝治、平位暢康、王寺幸輝、有薗直樹
  • Organizer: 第29回 日本臨床寄生虫学会大会
• [Presentation] Effects of vestibular cell-conditioned medium on differentiation of embryonic stem cells into inner ear vestibular hair cells (2018)
  • Author(s): Sakagami M, Ouji Y, Kawai N, Kitahara T, Yoshikawa M.
  • Organizer: 41st Annual MidWinter Meeting, Association for research in otolaryngology (ARO)
  • Int'l Joint Research
• [Presentation] 長期培養維持した毛包上皮幹細胞のキャラクタリゼーション (2018)
  • Author(s): 王寺幸輝、河合紀和、阪上雅治、吉川正英
  • Organizer: 第17回 日本再生医療学会総会
• [Presentation] ES細胞から分化誘導した内耳有毛細胞のキャラクタリゼーション (2017)
  • Author(s): 阪上雅治、王寺幸輝、河合紀和、北原糺、吉川正英
  • Organizer: 第16回 日本再生医療学会総会
  • Place of Presentation: 仙台国際線センター（仙台市）
  • Year and Date: 2017-03-07
• [Presentation] 皮膚上皮幹細胞の長期培養条件の検討とWntシグナル (2017)
  • Author(s): 王寺幸輝、河合紀和、阪上雅治、三須政康、吉川正英
  • Organizer: 第16回 日本再生医療学会総会
  • Place of Presentation: 仙台国際線センター（仙台市）
  • Year and Date: 2017-03-07
• [Presentation] 無細胞化マトリックスを用いたES細胞から肺細胞への効率的分化誘導法の開発 (2017)
  • Author(s): 河合紀和、王寺幸輝、阪上雅治、東条尚、谷口繁樹、吉川正英
  • Organizer: 第16回 日本再生医療学会総会
  • Place of Presentation: 仙台国際線センター（仙台市）
  • Year and Date: 2017-03-07
• [Presentation] ES細胞から分化誘導した内耳有毛細胞のキャラクタリゼーション (2017)
  • Author(s): 阪上雅治、吉川正英、北原糺
  • Organizer: 第27回 日本耳科学会総会・学術講演会
• [Presentation] Maintenance and long-term expansion of murine skin epithelial stem cells by Wnt signaling pathway (2017)
  • Author(s): Ouji Y, Sakagami M, Kawai N, Yoshikawa M.
  • Organizer: 10th World Congress for Hair Research 2017
• [Presentation] セイロン鉤虫症は海外渡航時に注意すべき感染症であるー日本人4症例の検討ー (2017)
  • Author(s): 吉川正英、王寺幸輝、中村ー内山ふくみ、有薗直樹、山田稔、赤松尚明、賀屋大介、片浪雄一、眞木良祐、大場雄一郎
  • Organizer: 第58回 日本熱帯医学会大会
• [Presentation] 2009年にマレーシアにて感染したセイロン鉤虫症例 海外感染した日本人の同症第一確定例 (2017)
  • Author(s): 赤松尚明、山田稔、有薗直樹、楊孝治、王寺幸輝、吉川正英
  • Organizer: 第28回 日本臨床寄生虫学会大会
• [Presentation] 奈良医大感染症センター寄生虫専門外来10年の経験 (2017)
  • Author(s): 平位暢康、吉川正英、中村ふくみ、王寺幸輝、今井雄一郎、藤倉裕之、吉井誠也、吉原真吾、今北菜津子、小川拓、小川吉彦、米川真輔、平田一記、菱矢直邦、梶田明裕、白石直敬、佐藤公俊、福森達也、辻本和徳、中川智代、宇野健司、笠原敬、前田光一、古西満、武山雅博、大西智子、片浪雄一、忽那賢志、三笠桂一
  • Organizer: 第28回 日本臨床寄生虫学会大会
• [Book] 消化器内視鏡 【消化管感染症のすべて】十二指腸-小腸 寄生虫感染症 鉤虫症 (2019)
  • Author(s): 松尾英城, 吉川正英, 賀屋大介, 菊池英亮
  • Total Pages: 3
  • Publisher: 東京医学社
  • ISBN: 9784885636264
• [Book] 小児科、小児寄生虫感染症の対策・予防 (2018)
  • Author(s): 吉川正英、沢田敦、間瀬史絵、平位暢康、大西智子、武山雅博、笠原敬、王寺幸輝
  • Total Pages: 14
  • Publisher: 金原出版
• [Book] 日常検査で役立つ寄生虫・原虫検出法 抗原・抗体反応を利用した寄生虫検査 (2017)
  • Author(s): 王寺幸輝、吉川正英
  • Total Pages: 6
  • Publisher: Medical Technology
• [Book] Induction of Inner Ear Hair Cells from Mouse Embryonic Stem Cells In Vitro. (2016)
  • Author(s): Yoshikawa M, Ouji Y.
  • Total Pages: 11
  • Publisher: Methods Mol Biol
• [Book] Maintenance of Skin Epithelial Stem Cells by Wnt-3a In Vitro. (2016)
  • Author(s): Ouji Y, Yoshikawa M.
  • Total Pages: 10
  • Publisher: Methods Mol Biol
• [Book] Maintenance of Dermal Papilla Cells by Wnt-10b In Vitro. (2016)
  • Author(s): Ouji Y, Yoshikawa M.
  • Total Pages: 9
  • Publisher: Methods Mol Biol