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A01-1 林班

多能性幹細胞による配偶子産生システムのin vitro 再構築

体外培養系で配偶子の産生過程を再現する「in vitro gametogenesis」は、配偶子の分化過程の解明や最終産物である機能的な配偶子の利用など、基礎生物学・医学・畜産学・水産学に与える影響は大きい。その中でも体外で増殖可能な多能性幹細胞を利用したin vitro gametogenesisは、試料の調整や遺伝子操作の簡便さから様々な目的への利用が見込まれる。研究代表者らはマウスの多能性幹細胞(ES/iPS細胞)から、始原生殖細胞や卵子を作製できることを報告したが、現行のES/iPS細胞を用いたin vitro gametogenesisには3つの大きな課題が残されている。すなわち、①in vitro gametogenesisで作られる配偶子のインテグリティ(機能的な完成度)が低い、②配偶子の分化に必須な体細胞環境を生体由来の細胞・組織に依存している、③成功例がマウスに限られている点である。

これらの課題を解決するために、本研究では他の計画研究班と緊密に連携して、①in vitro卵子におけるインテグリティの向上、②生殖腺の支持細胞の再構築、③他の動物への応用を可能にするロバストネスの検証を行う。これにより、in vitro gametogenesisを安定的に使いこなし、マウス以外の動物へ応用できる革新的技術として確立する。特に他の動物への応用を可能にするロバストネスの検証に関しては、他の計画研究(小林俊寛班)と共同でラット・ウサギを用いるほか、研究分担者とともにウシやニジマスへの適用を試みる。

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研究組織
主要論文

林 克彦

  • *Hayashi K, Hikabe O, Obata Y, Hirao Y.

    Reconstitution of mouse oogenesis in a dish from pluripotent stem cells.

    Nature Protoc. 12:1733-1744. doi: 10.1038/nprot.2017.070. (2017)

  • Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N, Shimamoto S, Imamura T, Nakashima K, Saitou M, *Hayashi K.

    Reconstitution in vitro of the entire cycle of the mouse female germ line.

    Nature. 539: 299–303. (2016)

  • Morohaku K, Tanimoto R, Sasaki K, Kawahara-Miki R, Kono T, Hayashi K, *Hirao Y, *Obata Y.

    Complete in vitro generation of fertile oocytes from mouse primordial germ cells.

    Proc Natl Acad Sci USA. 113(32):9021-9026. (2016)

  • Nakaki F, Hayashi K, Ohta H, Kurimoto K, Yabuta Y, *Saitou M.

    Induction of the mouse germ-cell fate by transcription factors in vitro.

    Nature 501 :222-226. (2013)

  • *Hayashi K and *Saitou M.

    Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.

    Nature Protoc. 8: 1513-1524. (2013)

  • *Hayashi K, Ogushi S, Kurimoto K, Shimamoto S, Ohta H, *Saitou M.

    Offspring from Oocytes Derived from in Vitro Primordial Germ Cell-Like Cells in Mice.

    Science. 338: 971-975. (2012)

  • Hayashi K, Ohta H, Kurimoto K, Aramaki S, *Saitou M.

    Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells.

    Cell. 146: 519-532. (2011)

  • Hayashi K and *Surani MA.

    Self-renewing epiblast stem cells exhibit continual delineation of germ cells with epigenetic reprogramming in vitro.

    Development. 136: 3549-3556. (2009)

  • Hayashi K, Chuva de Sousa Lopes SM, Tang F, *Surani MA.

    Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states.

    Cell Stem Cell. 3: 391-401. (2008)

吉崎 悟朗

  • Sato M, Hayashi M, *Yoshizaki G.

    Stem cell activity of type A spermatogonia is seasonally regulated in rainbow trout.

    Biol Reprod 96, 1303-1316 (2017)

  • *Seki S, Kusano K, Lee S, Iwasaki Y, Yagisawa M,Ishida M, Hiratsuka T, Sasado T, Naruse K, Yoshizaki G.

    Production of the medaka derived from vitrified whole testes by germ cell transplantation.

    Scientific Reports 7, 43185 (2017)

  • Lee S, Katayama N, *Yoshizaki G.

    Generation of juvenile rainbow trout derived from cryopreserved whole ovaries by intraperitoneal transplantation of ovarian germ cells.

    Biochem Biophys Res Commun 478, 1478-1483 (2016)

  • Katayama N, Kume S, Hattori-Ihara S, Sadaie S, Hayashi M, *Yoshizaki G.

    Germ cell-specific excision of loxP-flanked transgenes in rainbow trout Oncorhynchus mykiss.

    Biol Reprod 94, 79 (2016)

  • Lee S, Seki S, Katayama N, *Yoshizaki G.

    Production of viable trout offspring derived from frozen whole fish.

    Scientific Reports 5, 16045 (2015)

  • Hayashi M, Sato M, Nagasaka Y, Sadaie S, Kobayashi S, *Yoshizaki G.

    Enrichment of spermatogonial stem cells using side population in teleost.

    Biol Reprod 91,23 (2014)

  • Lee S, Iwasaki Y, Shikina S, *Yoshizaki G.

    Generation of functional eggs and sperm from cryopreserved whole testes.

    Proc Natl Acad Sci U S A. 110, 1640- 1645 (2013)

  • Shikina S, Nagasawa K, Hayashi M, Furuya M, Iwasaki Y, *Yoshizaki G.

    Short-term in vitro culturing improves transplantability of type A spermatogonia in rainbow trout (Oncorhynchus mykiss).

    Mol Reprod Dev 80, 763-773 (2013)

  • Morita T, Kumakura N, Morishima K, Mitsuboshi T, Ishida M, Hara T, Kudo S, Miwa M, Ihara S, Higuchi K, Takeuchi Y, *Yoshizaki G.

    Production of donor-derived offspring by allogeneic transplantation of spermatogonia in the yellowtail (Seriola quinqueradiata).

    Biol Reprod 86, 176 (2012)

  • Kise K, Yoshikawa H, Sato M, Tashiro M, Yazawa R, Nagasaka Y, Takeuchi Y, *Yoshizaki G.

    Flow-cytometric isolation and enrichment of teleost type A spermatogonia based on light-scattering properties.

    Biol Reprod 86,107 (2012)

  • *Yoshizaki G, Ichikawa M, Hayashi M, Iwasaki Y, Miwa M, Shikina S, Okutsu T.

    Sexual plasticity of ovarian germ cells in rainbow trout.

    Development 137, 1227-1230 (2010)

  • Okutsu T, Shikina S, Kanno M, Takeuchi Y, *Yoshizaki G.

    Production of trout offspring from triploid salmon parents.

    Science 317, 1517 (2007)

  • Okutsu T, Suzuki K, Takeuchi Y, Takeuchi T, *Yoshizaki G.

    Testicular germ cells can colonize sexually undifferentiated embryonic gonad and produce functional eggs in fish.

    Proc Natl Acad Sci U S A. 103, 2725- 2729 (2006)

  • Takeuchi Y, *Yoshizaki G, Takeuchi T.

    Biotechnology: surrogate broodstock produces salmonids.

    Nature 430, 629-630 (2004)

平尾 雄二

  • Mizumachi S, Aritomi T, Sasaki K, Matsubara K, *Hirao Y.

    Macromolecular crowded conditions strengthen contacts between mouse oocytes and companion granulosa cells during in vitro growth.

    J Reprod Dev. in press (2018)

  • Morohaku K, Hirao Y, *Obata Y.

    Development of fertile mouse oocytes from mitotic germ cells in vitro.

    Nature Protoc. 12: 1817-1829 (2017)

  • Morohaku K, Tanimoto R, Sasaki K, Kawahara-Miki R, Kono T, Hayashi K, *Hirao Y, Obata Y.

    Complete in vitro generation of fertile oocytes from mouse primordial germ cells.

    Proc Natl Acad Sci USA. 113: 9021–9026 (2016)

  • *Hirao Y, Somfai T, Naruse K.

    In vitro growth and maturation of vitrified–warmed bovine oocytes collected from early antral follicles.

    J Reprod Dev. 60: 68-72 (2014)

  • *Hirao Y, Naruse K, Kaneda M, Somfai T, Iga K, Shimizu M, Akagi S, Cao F, Kono T, Nagai T, Takenouchi N.

    Production of fertile offspring from oocytes grown in vitro by nuclear transfer in cattle.

    Biol Reprod. 89: 57, 1-11 (2013)

  • *Hirao Y, Shimizu M, Kosuke I, Takenouchi N.

    Optimization of oxygen concentration for growing bovine oocytes in vitro: constant low and high oxygen concentrations compromise the yield of fully grown oocytes.

    J Reprod Dev. 58: 204-211 (2012)

  • *Hirao Y, Shimizu M, Iga K, Takenouchi N.

    Growth of bovine oocyte-granulosa cell complexes cultured individually in microdrops of various sizes.

    J Reprod Dev 55: 88-93 (2009)

  • *Hirao Y, Takehiro Itoh T, Shimizu M, Iga K, Aoyagi K, Kobayashi M, Kacchi M, Hoshi H, Naoki Takenouchi.

    In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium.

    Biol Reprod. 70: 83-91 (2004)

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