Analyses on the quality control mechanism of mouse spermatogonial stem cells

• Project Area: The germline: its developmental cycle and epigenome network
• Project/Area Number: 20062006
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyoto University
• Principal Investigator: SHINOHARA Takashi 京都大学, 医学研究科, 教授 (30322770)
• Project Period (FY): 2008 – 2012
• Project Status: Completed (Fiscal Year 2012)
• Budget Amount: ¥95,200,000 (Direct Cost: ¥95,200,000); Fiscal Year 2012: ¥20,700,000 (Direct Cost: ¥20,700,000); Fiscal Year 2011: ¥20,700,000 (Direct Cost: ¥20,700,000); Fiscal Year 2010: ¥20,700,000 (Direct Cost: ¥20,700,000); Fiscal Year 2009: ¥20,700,000 (Direct Cost: ¥20,700,000); Fiscal Year 2008: ¥12,400,000 (Direct Cost: ¥12,400,000)
• Keywords: 精子形成 / 幹細胞 / 移植 / 培養 / エピジェネティックス / 自己複製 / 生殖細胞 / 遺伝学 / 発生・分化

Research Abstract

The purpose of this study is to understand the mechanism of quality control of spermatogonial stem cells. In our previous grant on priority area, we reported a long-term culture system for spermatogonial stem cells, which were designated as germline stem (GS) cells. We used GS cells to produce knockout animals by homologous recombination in these cells.
In the course of this study, we noticed that GS cells are very stable in their genetic and epigenetic properties. Unlike mbryonic stem (ES) cells that change their chromosome number and DNA methylation patterns in imprinted genes, GS cells maintained normal chromosome number and androgenetic DNA methylation patterns in imprinted genes for more than 2 years in vitro. Based on this observation, we hypothesized that GS cells have a unique mechanism to prevent germine transmission of genetic and epigenetic abnormalities to subsequent generations and that defects in these properties will lead to pluripotent cell derivation or abnormalities in spermatogenesis.
In the current grant, we were able to establish GS cells from fetal germ cells, which had defects in histone modification patterns in imprited genes. Offspring that were derived from these cells showed abnormal DNA methylation patterns in imprinted genes, which continued at least five generations. We also analyzed the impact of Dnmt genes or oncogenes in genetic and epigenetic properties of GS cells. Our analysis revealed that p53 is important in genetic integrity of GS cells and that DNMT3a/b are responsible for maintaining epigenetic integrity. We also established the first experimental system for in vitro tranformation of germ cells, which will be useful for understanding the mechanism of epigenetic regulation in SSCs.

Research Products

• [Journal Article] Hybridization of testis-derived stem cells with somatic cells and embryonic stem cells in Mice (2012)
  • Author(s): Takehashi, M., Tada, M., Kanatsu-Shinohara, M., Morimoto, H., Kazuki, Y., Oshimura, M., Tada, T. and Shinohara, T
  • Journal Title: Biol. Reprod Volume: 86(6) Issue: 6 Pages: 178-178
  • DOI: 10.1095/biolreprod.112.098988
  • Peer Reviewed
• [Journal Article] FGF2 mediates mouse spermatogonial stem cell self-renewal via upregulation of Etv5 and Bc16b genes through MAP2K1 activation. (2012)
  • Author(s): Ishii K, Kanatsu-Shinohara M, Toyokuni S, Shinohara T.
  • Journal Title: Development Volume: 139 Issue: 10 Pages: 1734-1743
  • DOI: 10.1242/dev.076539
  • Peer Reviewed
• [Journal Article] Reconstitution of mouse spermatogonial stem cell niches in culture (2012)
  • Author(s): Kanatsu-Shinohara, M., Inoue, K., Takashima, S.,Takehashi, M.,Ogonuki, N., Morimoto, H., Nagasawa, T., Ogura, A. and Shinohara, T
  • Journal Title: Cell Stem Cell Volume: 11(4) Issue: 4 Pages: 567-578
  • DOI: 10.1016/j.stem.2012.06.011
  • Peer Reviewed
• [Journal Article] Enrichment of mouse spermatogonial stem cells by melanoma cell adhesion molecule expression (2012)
  • Author(s): Kanatsu-Shinohara, M., Morimoto, H. and Shinohara, T
  • Journal Title: Biol. Reprod Volume: 87(6) Issue: 6 Pages: 139-139
  • DOI: 10.1095/biolreprod.112.103861
• [Journal Article] In Vitro Transformation of Mouse Testis Cells by Oncogene Transfection (2012)
  • Author(s): 森本裕子、李知英、田中敬、石井慧、豊國信哉、篠原美都、篠原隆司
  • Journal Title: Biology of Reproduction Volume: 86 Issue: 5 Pages: 1-11
  • DOI: 10.1095/biolreprod.111.095307
  • Peer Reviewed
• [Journal Article] In vitro transformation of mouse testis cells by oncogene transfection (2012)
  • Author(s): T., Ishii, K., Toyokuni, S., Kanat
  • Journal Title: Biology of Reproduction Volume: (印刷中)
  • Peer Reviewed
• [Journal Article] Homologous recombination in rat germline stenl cells (2011)
  • Author(s): Kanatsu-Shinohara, M., Kato-Itoh, M., Ikawa, M., Takehashi, M., Sanbo, M, Morioka, Y., Tanaka, T., Morimoto, H., Hirabayashi, M., and Shinohara, T
  • Journal Title: Biol. Reprod Volume: 85(1) Pages: 208-217
• [Journal Article] Dynamic changes in EPCAM expression during spermatogonial stem cell differentiation in the mouse testis (2011)
  • Author(s): Kanatsu-Shinohara, M., Takashima, S., and Shinohara, T
  • Journal Title: PLoS One Volume: 6(8)
  • NAID: 120003405532
• [Journal Article] Rac mediates spermatogonial stem cell homing to germline niches by regulating transmigration through the blood-testis barrier (2011)
  • Author(s): Takashima, S., Kanatsu-Shinohara, M., Tanaka, T., Takehashi, T., Morimoto, H. and Shinohara, T
  • Journal Title: Cell Stem Cell Volume: 9(5) Pages: 463-475
• [Journal Article] ges in EPCAM expression during spermatogonial stem cell differentiation in the (2011)
  • Author(s): -Shinohara M., Takashima S., Shino
  • Journal Title: PLoS One Volume: 6
  • Peer Reviewed
• [Journal Article] nial stem cell homing to germline niches by regulating transmigration through (2011)
  • Author(s): ohara, M., Tanaka, T., Takehashi, M.
  • Journal Title: Cell Stem Cell Volume: 9 Pages: 463-475
  • Peer Reviewed
• [Journal Article] Homologous recombination in rat germline stem cells. (2011)
  • Author(s): Kanatsu-Shinohara M., Kato-Itoh M., Ikawa M., Takehash iM., Sanbo M., Morioka Y., Tanaka T., Morimoto H., Hirabayashi M., Shinohara T.
  • Journal Title: Biol.Reprod. Volume: (In press)
  • Peer Reviewed
• [Journal Article] Unstable side population phenotype of mouse spermatogonial stem cells in vitro. (2011)
  • Author(s): Shinohara T., Ishii K., Kanatsu-Shinohara M.
  • Journal Title: J.Reprod.Dev. Volume: (In press)
  • NAID: 10028275625
  • Peer Reviewed
• [Journal Article] Serum- and feeder-free culture of mouse germline stem cells. (2011)
  • Author(s): Kanatsu-Shinohara M., Inoue K., Ogonuki N., Morimoto H., Ogura A., Shinohara T.
  • Journal Title: Biol.Reprod. Volume: 84 Pages: 97-105
  • Peer Reviewed
• [Journal Article] Genetic influences in mouse spermatogonial stem cell self-renewal (2010)
  • Author(s): Kanatsu-Shinohara, M… .and Shinohara T
  • Journal Title: J. Reprod. Dev Volume: 56(1) Issue: 3 Pages: 145-153
  • DOI: 10.1111/j.1440-169x.2009.01167.x
  • NAID: 10026323942
• [Journal Article] Regulation of germline transmission in the male mice by p21 and p27 cyclin-dependent kinase inhibitors (2010)
  • Author(s): Kanatsu-Shinohara, M., Takashima, S. and Shinohara T
  • Journal Title: Proc. Natl. Acad. Sci Volume: 107(14) Issue: 14 Pages: 6210-6215
  • DOI: 10.1073/pnas.0914448107
  • Peer Reviewed
• [Journal Article] Regulation of germline transmission in the male mice by p21 and p27 cyclin-dependent kinase inhibitors. (2010)
  • Author(s): Kanatsu-Shinohara, M., Takashima, S., Shinohara T.
  • Journal Title: Proc.Natl.Acad.Sci.USA 107 Pages: 6210-6215
• [Journal Article] Generation of genetically modified animals using spermatogonial stem cells. (2010)
  • Author(s): Takehashi, M., Kanatsu-Shinohara, M., Shinohara T.
  • Journal Title: Dev.Growth Differ. 52 Pages: 303-310
  • NAID: 10027564378
• [Journal Article] Genetic influences in mouse spermatogonial stem cell self-renewal. (2010)
  • Author(s): Kanatsu-Shinohara, M…., Shinohara T.
  • Journal Title: J.Reprod.Dev. 56 Pages: 145-153
  • NAID: 10026323942
• [Journal Article] Neonatal mouse testis-derived multipotent germline stem cells improve the cardiac function of acute ischemic heart mouse model (2010)
  • Author(s): Iwasa T., Baba S., Doi H., Kaichi S., Yokoo N., Miwa T., Kanatsu-Shinohara M., Shinohara T., Nakahata T., Heike T
  • Journal Title: Biochem.Biophys.Res.Commun. Volume: 400 Pages: 27-33
  • Peer Reviewed
• [Journal Article] Germline modification using mouse spermatogonial stem cells. (2010)
  • Author(s): Kanatsu-Shinohara M., Shinohara T.
  • Journal Title: Methods Enzymol. Volume: 477 Pages: 17-36
• [Journal Article] Heritable imprinting defect caused by epigenetic abnormalities in spermatogonial stem cells (2009)
  • Author(s): Lee, J., Kanatsu-Shinohara, M., Ogonuki, N., Miki, H., Inoue, K., Morimoto, T.,Ogura, A. and Shinohara, T
  • Journal Title: Biol. Reprod Volume: 80(3) Issue: 3 Pages: 518-527
  • DOI: 10.1095/biolreprod.108.072330
• [Journal Article] Abnormal DNA methyltransferase expression in mouse germline Stem Cells results in spermatogenic defects (2009)
  • Author(s): Takashima, S.,Takehashi, M.,Lee, J., Chuma, S., Okano, M., Hata, K., Suetake, I., Nakatsuji, N.,Miyoshi, H., Tajima, S., Sasaki, H., Kanatsu-Shinohara, M. and Shinohara, T
  • Journal Title: Biol. Reprod Volume: 81 Issue: 1 Pages: 155-164
  • DOI: 10.1095/biolreprod.108.074708
  • Peer Reviewed
• [Journal Article] Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation (2009)
  • Author(s): Lee, J., Kanatsu-Shinohara, M., Morimoto, H.,Kazuki, Y.,Takashima, S., Mitsuo Oshimura, Toyokuni, S. and Shinohara, T
  • Journal Title: Cell Stem Cell Volume: 5(1) Issue: 1 Pages: 76-86
  • DOI: 10.1016/j.stem.2009.04.020
  • NAID: 120001462495
• [Journal Article] Abnormal DNA methyltransferase expression in mouse germline stem cells results in spermatogenic defects. (2009)
  • Author(s): Takashima S., et al.
  • Journal Title: Biology of Reproduction 81 Pages: 155-164
  • Peer Reviewed
• [Journal Article] Genetic reconstruction of mouse spermatogonial stem cell self-renewal in vitro by Ras-cyclin D2 activation. (2009)
  • Author(s): Lee J., et al.
  • Journal Title: Cell Stem Cell 5 Pages: 76-86
  • Peer Reviewed
• [Journal Article] Phenotypic plasticity of mouse spermatogonial stem cells. (2009)
  • Author(s): Morimoto H., et al.
  • Journal Title: Plos One 4
  • Peer Reviewed
• [Journal Article] Genetic influences in mouse spermatogonial stem cell self-renewal. (2009)
  • Author(s): Kanatsu-Shinohara M., et al.
  • Journal Title: Journal of Reproduction and Development 56 Pages: 145-153
  • NAID: 10026323942
  • Peer Reviewed
• [Journal Article] Heritable imprinting defect caused by epigenetic abnormalities in mouse spermatogonial Stem cells (2009)
  • Author(s): Lee J., et.al.
  • Journal Title: Biol. Reprod 80 Pages: 518-27
  • Peer Reviewed
• [Journal Article] Homing of mouse spermatogonial stem cells to germline niche depends on b1-integrin (2008)
  • Author(s): Kanatsu-Shinohara, M.,Takehashi, M., Takashima, S., Lee, J., Chuma, S., Nakatsuji, N., Fassler R. and Shinohara, T
  • Journal Title: Cell Stem Cell Volume: 3(5) Issue: 5 Pages: 533-542
  • DOI: 10.1016/j.stem.2008.08.002
• [Journal Article] Long-term culture of male germline stem cells from hamster testes (2008)
  • Author(s): Kanatsu-Shinohara, M., Muneto, T., Lee, J., Takenaka, M., Chuma, S., Nakatsuji, N., Horiuchi, T. and Shinohara, T
  • Journal Title: Biol. Reprod Volume: 78(4) Pages: 611-617
• [Journal Article] Production of transgenic rats via lentiviral and xenogeneic transplantation of spermatogonial stem cells (2008)
  • Author(s): Kanatsu-Shinohara, M., Kato, M., Takehashi, M., Morimoto, H., Takashima, S., Chuma, S., Nakatsuji, N., Hirabayashi, M. and Shinohara, T
  • Journal Title: Biol. Reprod Volume: 79(6) Issue: 6 Pages: 1122-1128
  • DOI: 10.1095/biolreprod.108.071159
• [Journal Article] Production of transgenic rats following lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells (2008)
  • Author(s): Kanatsu-Shinohara M., et.al.
  • Journal Title: Biol. Reprod 79 Pages: 1121-8
  • Peer Reviewed
• [Journal Article] Homing of mouse spermatogonial stem cells to germline niche depends on β 1-integrin (2008)
  • Author(s): Kanatsu-Shinohara M., et.al.
  • Journal Title: Cell Stem Cell 3 Pages: 533-42
  • Peer Reviewed
• [Journal Article] spermatogonial stem cells.
  • Author(s): Morimoto, H…, Shinohara T.
  • Journal Title: Plos One 4
• [Presentation] Reconstitution of male germline niche in vitro (2013)
  • Organizer: 10^<th> International congress of Andrology
  • Place of Presentation: 豪州, Melbourne
  • Year and Date: 2013-02-23
• [Presentation] Reconstitution of male germline niche in vitro (2012)
  • Organizer: Brinster Symposium
  • Place of Presentation: 米国, Philadelphila
  • Year and Date: 2012-08-25
• [Presentation] In vltro transfbrmatlon of male germline stem cells (2012)
  • Organizer: The 58^<th>/60^<th> NIBB conference Germline-Specification, Sex, and Stem Cells-
  • Place of Presentation: 岡崎
  • Year and Date: 2012-07-20
• [Presentation] Positive and negative regulators of mouse spermatogonial stem cell self-renewal (2012)
  • Organizer: Keystone Meeting
  • Place of Presentation: 米国, Colorado
  • Year and Date: 2012-02-17
• [Presentation] Derivation of stem cell lines from the male germline (2012)
  • Organizer: International Workshop of Radiation Effects on Mutation in Somatic and Germline Stem Cells
  • Place of Presentation: 広島
  • Year and Date: 2012-01-18
• [Presentation] Derivation of stem cell lines from the male germline (2012)
  • Author(s): Takashi Shinohara
  • Organizer: International Workshop : Radiation Effects on Mutation in Somatic and Germline Stem Cell
  • Place of Presentation: 放影研(広島県)(招待講演)
  • Year and Date: 2012-01-18
• [Presentation] Homing of mouse spermatogonial stem cells to germline niche (2011)
  • Organizer: Developmental Control of Sex, Growth and Cellular Fate
  • Place of Presentation: 中国, 蘇州
  • Year and Date: 2011-10-12
• [Presentation] Positive and negative regulators of mouse spermatogonial stem cell self-renewal (2010)
  • Author(s): Takashi Shinohara, Mito Kanatsu-Shinohara
  • Organizer: 11^<th> International Symposium on Spematology
  • Place of Presentation: 沖縄コンベンションセンター(招待講演)
  • Year and Date: 2010-06-26
• [Presentation] Positive and negative regulators of mouse spermatogonial stem cell self-renewal (2010)
  • Author(s): Takashi Shinohara
  • Organizer: Keystone meeting
  • Place of Presentation: Keystone, Colorado, 米国
  • Year and Date: 2010-02-17
• [Presentation] CDKインヒビターp21とp27による精子幹細胞の制御 (2010)
  • Author(s): 篠原隆司
  • Organizer: CREST「iPS細胞」領域会議
  • Place of Presentation: 千里ライフサイエンスセンター(招待講演)
• [Presentation] Phenotypic plasticity of mouse spermatogonial stem cells (2010)
  • Author(s): 篠原隆司
  • Organizer: 特定領域研究「生殖細胞」班会議
  • Place of Presentation: 九州大学(招待講演)
• [Presentation] Derivation of embryonic germline stem cells (2009)
  • Organizer: The 36^<th> International Congress of Physiological Sciences
  • Place of Presentation: 京都
  • Year and Date: 2009-08-01
• [Presentation] (Kyoto University), "Derivation of embryonic germline stem cells" (2009)
  • Author(s): Takashi Shinohara
  • Organizer: The 36th International Congress of Physiological Sciences
  • Place of Presentation: Kyoto
  • Year and Date: 2009-08-01
• [Presentation] Derivation of embryonic germline stem cells (2009)
  • Author(s): Takashi Shinohara
  • Organizer: 36^<th> International Congress of Physiological Sciences
  • Place of Presentation: 京都
  • Year and Date: 2009-08-01
• [Presentation] Culture of Spermatogonial stem cells (2008)
  • Organizer: The 11^<th> Kyoto University International Symposium
  • Place of Presentation: 中国, 上海
  • Year and Date: 2008-10-09
• [Presentation] Culture of Spermatogonial stem cells (2008)
  • Organizer: Cornell University Stem Cell Lecture
  • Place of Presentation: 米国, New York
  • Year and Date: 2008-06-03
• [Presentation] Culture of Spermatogonial stem cells (2008)
  • Organizer: Cold Spring Harbor 73^<rd> Symposium: Control and Regulation of Stem Cells
  • Place of Presentation: 米国, New York
  • Year and Date: 2008-06-02
• [Presentation] Culture of Spermatogonial stem cells (2008)
  • Organizer: Society for the Study of Reproduction, Annual meeting
  • Place of Presentation: 米国, Hawaii
  • Year and Date: 2008-05-25
• [Presentation] Culture of Spermatogonial stem cells (2008)
  • Organizer: The 11th Kyoto University International Symposium.
  • Place of Presentation: 中国 上海
• [Presentation] Culture of Spermatogonial stem cells (2008)
  • Organizer: Cold Spring Harbor 73rd Symposium : Control and Regulation of Stem Cells
  • Place of Presentation: 米国 New York
• [Presentation] Culture of Spermatogonial stem cells (2008)
  • Organizer: Society for the Study of Reproduction, Annual meeting
  • Place of Presentation: 米国 Hawaii
• [Patent(Industrial Property Rights)] 生殖細胞からの多能性幹細胞様細胞の誘導 (2012)
  • Inventor(s): 高島誠司、篠原隆司
  • Industrial Property Rights Holder: 京都大学
  • Filing Date: 2012-12-28
  • Overseas