Establishment and evaluation of bovine induced trophoblast cell line and contruction of in vitro implantation model using this cell line.

• Project/Area Number: 26292168
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Integrative animal science
• Research Institution: Okayama University
• Principal Investigator: Kimura Koji 岡山大学, 環境生命科学研究科, 教授 (50355070)
• Co-Investigator(Kenkyū-buntansha): 今井 裕 京都大学, 農学研究科, 教授 (10303869) ; 松山 秀一 国立研究開発法人農業・食品産業技術総合研究機構, 畜産研究部門, 主任研究員 (50455317)
• Project Period (FY): 2014-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥16,120,000 (Direct Cost: ¥12,400,000、Indirect Cost: ¥3,720,000); Fiscal Year 2017: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2016: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2015: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2014: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
• Keywords: ウシ / 着床・妊娠 / 栄養膜細胞 / iPS細胞 / 人工多能性幹細胞 / 着床 / 多能性 / 脱分化 / 幹細胞 / 畜産学 / 発生・分化 / 妊娠維持 / 妊娠

Outline of Final Research Achievements

It is necessary for improvement of pregnancy rate in cattle to clarify the mechanisms involved in its embryo development, implantation and establishment of pregnancy. The aims of the present study are to establish the bovine induced pluripotent stem cell and induced trophoblastic cells. Bovine amnion derived cells (bADC) were introduced with transposone expression vectors (piggyBac vectors) containing doxycycline (Dox)-inducible transcription factors. Colonies were selected by morphology criteria and then their characteristics were evaluated by expression of stem cell or trophectoderm markers. We established both of bovine iPS cell lines which can be differentiated to germ cells in vivo and trophoblast cell lines which processed trophoblast stem cell-like characteristics and the potential to differentiate into the extra-embryonic cell lineage.

Research Products

• [Journal Article] Long-term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes. (2018)
  • Author(s): Suyatno, Kitamura, Y., Minami, N., Yamada, M., Ikeda, S., and Imai H.
  • Journal Title: Mol. Reprod. Dev. Volume: 85 Issue: 3 Pages: 236-249
  • DOI: 10.1002/mrd.22958
  • NAID: 120006410739
  • Peer Reviewed
• [Journal Article] Recent advances of in vitro culture systems for spermatogonial stem cells in mammals. (2018)
  • Author(s): Sahare, MG, Suyatno and Imai H.
  • Journal Title: Reprod. Med. Biol. Volume: 17 Issue: 2 Pages: 134-142
  • DOI: 10.1002/rmb2.12087
  • NAID: 120006410733
  • Peer Reviewed
• [Journal Article] Generation of germ cells from pluripotent stem cells in mammals (2017)
  • Author(s): Hayashi M, Kawaguchi T, Durucova-Hills G. and Imai H.
  • Journal Title: Reprod. Med. Biol. Volume: 17 Pages: 107-114
  • NAID: 120006410735
  • Peer Reviewed
• [Journal Article] 哺乳動物における多能性幹細胞から生殖細胞系列への分化誘導 (2017)
  • Author(s): 林 将文・川口正高・今井 裕
  • Journal Title: 日本胚移植学雑誌 Volume: 39 Pages: 1-7
  • NAID: 40022347859
  • Peer Reviewed
• [Journal Article] Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors (2016)
  • Author(s): Kawaguchi T, Cho D, Hayashi M, Tsukiyama T, Kimura K, Matsuyama S, Minami N, Yamada M and Imai H
  • Journal Title: PloS One Volume: 11 Issue: 12 Pages: 1-15
  • DOI: 10.1371/journal.pone.0167550
  • NAID: 120005980901
  • Peer Reviewed / Open Access
• [Journal Article] Factors supporting long-term culture of bovine male germ cells (2016)
  • Author(s): Sahare M, Kim SM, Otomo A, Komatsu K, Minami N, Yamada M, Imai H.
  • Journal Title: Reprod. Fertil. Dev. Volume: in press
  • NAID: 120005678201
  • Peer Reviewed
• [Journal Article] ウシ多能性幹細胞株の樹立 (2016)
  • Author(s): 今井裕
  • Journal Title: 畜産技術 Volume: - Pages: 6-10
  • NAID: 40020736362
  • Peer Reviewed
• [Journal Article] The role of signaling pathways on proliferation and self-renewal of cultured bovine primitive germ cells (2015)
  • Author(s): Sahare M, Otomo A, Komatsu K, Minami N, Yamada M, Imai H.
  • Journal Title: Reproductive Medicine and Biology Volume: 14 Issue: 1 Pages: 17-25
  • DOI: 10.1007/s12522-014-0189-x
  • NAID: 120005678199
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Generation of naïve bovine induced pluripotent stem cells using PiggyBac transposition of doxycycline-inducible transcription factors. (2015)
  • Author(s): Kawaguchi T, Tsukiyama T, Kimura K, Matsuyama S, Minami N, Yamada M. and Imai H.
  • Journal Title: PlosOne Volume: 10
  • NAID: 120005649350
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Successful Nonsurgical Transfer of Bovine Elongating Conceptuses and Its Application to Sexing (2014)
  • Author(s): Koji Kimura and Shuichi Matsuyama
  • Journal Title: Journal of Reproduction and Development Volume: 60 Issue: 3 Pages: 210-215
  • DOI: 10.1262/jrd.2013-137
  • NAID: 130003390835
  • ISSN: 0916-8818, 1348-4400
  • Peer Reviewed / Open Access
• [Presentation] Induction of Differentiation of Bovine Male Germ Cells in Culture. (2017)
  • Author(s): Kitamura Y, Suyatno, Minami N, Yamada M. and Imai H.
  • Organizer: The 7th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2017).
  • Int'l Joint Research
• [Presentation] Characterisation of newly derived bovine iPSCs including their capacity to make PGC-like cells. (2017)
  • Author(s): Hayashi M, Durcova-Hills G, Minami N, Yamada M. and Imai H
  • Organizer: The International Research Symposium on Regulation of Germ Cell Development in Vivo and in Vitro.
  • Int'l Joint Research
• [Presentation] Isolation and long-term culture of adult bovine male germ cells in vitro. (2016)
  • Author(s): Suyatno, Kitamura Y, Minami N, Yamada M. and Imai H.
  • Organizer: International Symposium on the Future of Nuclear Transfer and Nuclear Reprogramming.
  • Place of Presentation: 甲府
  • Year and Date: 2016-03-10
  • Int'l Joint Research
• [Presentation] Reprogramming of bovine somatic cells into pluripotent stem cells (2016)
  • Author(s): Imai H
  • Organizer: International Symposium on the Future of Nuclear Transfer and Nuclear Reprogramming.
  • Place of Presentation: 甲府
  • Year and Date: 2016-03-10
  • Int'l Joint Research / Invited
• [Presentation] External pH affects the timing of mouse somatic cell reprogramming. (2015)
  • Author(s): Kim N, Yamada M, Minami N. and Imai H.
  • Organizer: International Society for Stem Cell Research
  • Place of Presentation: ストックホルム（スウェーデン）
  • Year and Date: 2015-06-24
  • Int'l Joint Research
• [Presentation] Induction of pluripotency in bovine cells using piggyBac transposition of doxycycline-inducible transcription factors. (2015)
  • Author(s): Kawaguchi T, Tsukiyama T, Kimura K, Matsuyama S, Minami N, Yamada M. and Imai H.
  • Organizer: International Gederation of Fertility Socities
  • Place of Presentation: 横浜
  • Year and Date: 2015-04-26
  • Int'l Joint Research
• [Presentation] piggyBacトランスポゾンを用いたリプログラミング因子導入によるウシ羊膜細胞からの栄養膜細胞株樹立の試み (2014)
  • Author(s): 川口高正、築山智之、松山秀一、木村康二、南直治郎、山田雅保、今井裕
  • Organizer: 日本繁殖生物学会
  • Place of Presentation: 帯広畜産大学
  • Year and Date: 2014-08-21 – 2014-08-23