Establishment of cellular bank of critically endangered animals and challenge to the iPS cell transformation

• Project/Area Number: 25640117
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Conservation of biological resources
• Research Institution: Iwate University (2016); Tohoku University (2013-2015)
• Principal Investigator: Tomokazu Fukuda 岩手大学, 理工学部, 教授 (40321640)
• Co-Investigator(Kenkyū-buntansha): 村山 美穂 京都大学, 野生動物研究センター, 教授 (60293552)
• Project Period (FY): 2013-04-01 – 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 2015: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2014: ¥780,000 (Direct Cost: ¥600,000、Indirect Cost: ¥180,000); Fiscal Year 2013: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: 資源保存 / 生物多様性 / 遺伝学 / 細胞生物学 / 絶滅危惧種 / 無限分裂 / 幹細胞 / 培養細胞 / 絶滅危惧動物 / 細胞バンク

Outline of Final Research Achievements

In this study, we tried to establish the functional new cell lines (iPS cell lines and immortalized cell line) from critically endangered animals. We used the sea turtles, and lowland anoa, and african savanna elephant. The primary cells shows enlarged cytoplasm after the specific times of cell divisions. In these cell lines, senescence associated protein, p16 is expected to accumulated in its cytoplasm. In general, senescence cells shows positive staining for SA-beta galactosidase staining.
For the induction of immortalized cell lines, the genetic introduction by lentiviruses were used. The primary cells of the critically endangered animals were immortalized with the expression of mutant cyclin dependent kinase (CDK4) and Cyclin D, and enzymatic subunit of human derived telomerase.

Research Products

• [Int'l Joint Research] University of Southern Maine/Environmental Toxicology/Dr. John Wise(米国)
• [Journal Article] Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators. (2016)
  • Author(s): Fukuda T, Iino Y, Eitsuka T, Onuma M, Katayama M, Murata K, Inoue-Murayama M, Hara K, Isogai E, Kiyono T
  • Journal Title: Cytotechnology Volume: 68 Issue: 5 Pages: 1937-1947
  • DOI: 10.1007/s10616-016-0004-0
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Expression of human cell cycle regulators in the primary cell line of the African savannah elephant (loxodonta africana) increases proliferation until senescence, but does not induce immortalization. (2015)
  • Author(s): Fukuda T, Iino Y, Onuma M, Gen B, Inoue-Murayama M, Kiyono T
  • Journal Title: In Vitro Cell Dev Biol Anim Volume: 52 Issue: 1 Pages: 20-26
  • DOI: 10.1007/s11626-015-9943-6
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Hexavalent chromium is cytotoxic and genotoxic to hawksbill sea turtle cells (2014)
  • Author(s): Wise, S. S. Xie, H. Fukuda, T. Douglas Thompson, W. Wise, J. P.
  • Journal Title: Toxicol Appl Pharmacol Volume: 279 Pages: 113-118
  • Peer Reviewed
• [Journal Article] Primary fibroblast cultures and karyotype analysis for the olive ridley sea turtle (Lepidochelys olivacea) (2014)
  • Author(s): Fukuda, T. Katayama, M. Kinoshita, K. Kasugai, T. Okamoto, H. Kobayashi, K. Kurita, M. Soichi, M. Donai, K. Uchida, T. Onuma, M. Sone, H. Isogai, E. Inoue-Murayama, M.
  • Journal Title: In Vitro Cell Dev Biol Anim Volume: 50 Pages: 381-383
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Primary fibroblast cultures from the olive ridley sea turtle (Lepidochelys olivacea) and the determination of its karyotype (2014)
  • Author(s): Fukuda, T. Katayama, M. Kinoshita, K. Kasugai, T. Okamoto, H. Kobayashi, K. Kurita, M. Soichi, M. Donai, K. Uchida, T. Onuma, M. Sone, H. Isogai, E. Inoue-Murayama, M.
  • Journal Title: In Vitro Cellular & Developmental Biology -Animal Volume: in press
  • Peer Reviewed
• [Presentation] ヤンバルクイナ線維芽細胞からの不死化細胞の樹立 (2016)
  • Author(s): 片山 雅史, 清野 透,大沼 学,村山 美穂, 福田 智一
  • Organizer: 野生動物医学会
  • Place of Presentation: 宮崎市民プラザ(宮崎県宮崎市)
  • Year and Date: 2016-09-16
• [Presentation] アメリカ平原ハタネズミ由来の人工多能性幹細胞の作成 (2016)
  • Author(s): 片山 雅史, 平山 貴士, ,堀江 健吾, 清野 透, ,土内 憲一郎, 谷 哲弥, 竹田 省, 西森 克彦 , 福田 智一
  • Organizer: 日本繁殖生物学会
  • Place of Presentation: 麻布大学(神奈川県相模原市)
  • Year and Date: 2016-09-11
• [Presentation] ウシiPS細胞の樹立を目指したiPS細胞誘導ベクターとウシLIF発現フィーダー細胞の開発 (2016)
  • Author(s): 平出 美鈴, 熊谷 友希 ,佐々木 玲 ,福田 智一 ,小林 正之
  • Organizer: 日本繁殖生物学会
  • Place of Presentation: 麻布大学(神奈川県相模原市)
  • Year and Date: 2016-09-11
• [Presentation] 絶滅危惧スイギュウの一種、ローランド アノア由来の無限分裂細胞の作成 (2016)
  • Author(s): 福田智一・飯野佑佳・永塚貴弘・大沼学・片山雅史・村田浩一・ 村山美穂・原久美子・清野透
  • Organizer: 東北畜産学会2016年大会
  • Place of Presentation: アイーナ(岩手県盛岡市)
  • Year and Date: 2016-09-06
• [Presentation] ウシiPS細胞誘導ベクター、およびウシiPS細胞樹立に要するサイトカインLIFを発現するフィーダー細胞の開発 (2016)
  • Author(s): 平出美鈴、熊谷友希、佐藤卓、古舘千秋、佐々木玲、福田智一、小林正之
  • Organizer: 東北畜産学会2016年大会
  • Place of Presentation: アイーナ(岩手県盛岡市)
  • Year and Date: 2016-09-06
• [Presentation] ヒト由来遺伝子を使用した鳥類の不死化細胞の樹立の試み (2015)
  • Author(s): 片山 雅史, 清野 透, 大沼 学, 西森 克彦, 村山 美穂, 福田 智一
  • Organizer: 第21回野生動物医学会
  • Place of Presentation: 北海道江別市
  • Year and Date: 2015-08-02
• [Presentation] CDK4, Cyclin D, TERTの発現はアフリカサバンナゾウ由来の培養細胞の分裂回数を増加させるが無限分裂化までは至らない (2015)
  • Author(s): 福田智一，飯野佑佳，大沼学，村山美穂，清野透
  • Organizer: 第21回野生動物医学会
  • Place of Presentation: 北海道江別市
  • Year and Date: 2015-08-02
• [Presentation] 染色体異常を伴わないローランドアノア線維芽細胞由来の不死化細胞樹立の試み (2015)
  • Author(s): 飯野佑佳，清野透，永塚貴弘，片山雅史，土内憲一郎，黒田健吾，村田浩一, 大沼学，福田智一
  • Organizer: 第21回野生動物医学会
  • Place of Presentation: 北海道江別市
  • Year and Date: 2015-08-02
• [Presentation] Establishment of primary cell culture and immortalized cells from Loggerhead sea turtle, an ideal material for the genomic analysis with next generation sequencing. (2014)
  • Author(s): Fukuda T, Donai K, Eitsuka T, Kurita M, Saito T, Okamoto H, Kinoshita K, Katayama M, Soichi M, Uchida T, Onuma M, Sone H, Inoue-Murayama M, Kiyono T
  • Organizer: Unraveling Biodiversity from DNA- From the Management of Databases to the Use of Next Generation Sequencers
  • Place of Presentation: 国立環境研究所(茨城県つくば市)
  • Year and Date: 2014-07-01 – 2014-07-07