神経幹細胞の未分化性維持機構の解析

• Project/Area Number: 14081207
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Keio University
• Principal Investigator: 島崎 琢也 慶應義塾大学, 医学部, 助手 (00324749)
• Project Period (FY): 2002 – 2006
• Project Status: Completed (Fiscal Year 2006)
• Budget Amount: ¥46,300,000 (Direct Cost: ¥46,300,000); Fiscal Year 2006: ¥7,300,000 (Direct Cost: ¥7,300,000); Fiscal Year 2005: ¥5,800,000 (Direct Cost: ¥5,800,000); Fiscal Year 2004: ¥11,600,000 (Direct Cost: ¥11,600,000); Fiscal Year 2003: ¥10,800,000 (Direct Cost: ¥10,800,000); Fiscal Year 2002: ¥10,800,000 (Direct Cost: ¥10,800,000)
• Keywords: 神経幹細胞 / ES細胞 / PA6細胞 / 培養上清 / 生存 / Gab1 / PI3-Kinase / Akt / Olig2 / EGF / 増殖 / 脊髄 / EGFレセプター / MAP kinase / PI3-kinase / オリゴデンドロサイト前駆細胞 / gp130 / Galectin-1 / GFAP / 自己維持 / olig2 / アストロサイト

Research Abstract

神経幹細胞は、その発生において、分化能が時間的・空間的な特異性を持つように制御され、可塑性が失われていくと考えられており、それが応用面においての障害の1つとなっている。したがって、特に可塑性の高いと考えられる発生早期の神経幹細胞の維持、分化および時間的・空間的特異性の制御機構の解明を進める必要があるが、発生早期の中枢神経系が小さいために細胞数が得にくく、これまで解析が困難であった。そこで発生早期の神経幹細胞/神経系前駆細胞(NS/PCs)と同等の性質を示すES細胞由来NS/PCsを用い、その分化、増殖、生存および領域特異性に影響を与える因子の探索を行った。
我々の研究室では、これまで、ES細胞から胚様体(EB)形成により分化誘導したNS/PCsを、分散後に浮遊培養させることによって、neurosphereとして選択的に増殖させるシステムを構築してきた(Okada et al.,unpublished)。このin vitro分化系を用い、様々な細胞の培養上清をこのES細胞由来neurosphere形成時に添加して、その形成効率に与える影響を検討した。その結果、ストローマ細胞であるPA6細胞の培養上清(PA6CM)が、neurosphere形成効率を、低密度培養下で顕著に上昇させることを見出した。次に、胚様体を分散した直後(Day0)または3日目(Day3)にPA6CMを加えてneurosphereを形成させ、1週間後に生細胞数の定量を行ったところ、Day0から添加したものでは非添加時に比べて著しい生細胞の増加がみられたが、Day3に添加したものでは差が見られなかった。さらに、分散したEBをPA6CM存在下で4時間培養後、それぞれ死細胞あるいはアポトーシスを起こしている細胞を標識するPIとAnnexin Vの二重染色を行い、フローサイトメトリーにより解析したところ、PA6CMはアポトーシスを起こしていない生細胞の割合を増加させていた。これらのことから、PA6CMは、神経幹細胞の、増殖よりはむしろ低細胞密度下での生存を促進している可能性が示唆された。

Research Products

• [Journal Article] EGF-signaling mediated by Gabl is required for the spatiotemporally regulated proliferation of Olig2-expressing progenitors in the embryonic spinal cord (2007)
  • Author(s): Hayakawa-Yano Y., Nishida K., Fukami S., Gotoh Y., Hirano T., Nakagawa T., Shimazaki T, Okano H
  • Journal Title: Stem Cells (In press)
• [Journal Article] Leptin suppresses ghrelin-induced activation of neuropeptide Y neurons in the arcuate nucleus via phosphatidylinositol 3-kinase- and phosphodiesterase 3-mediated pathway. (2007)
  • Author(s): Kohno D., Nakata M., Maekawa F., Fujiwara K., Maejima Y., Kuramochi M., Shimazaki T, Okano H., Onaka T., Yada T
  • Journal Title: Endocrinology (In press)
• [Journal Article] SOCS3 is required to temporally fine-tune photoreceptor cell differentiation. (2007)
  • Author(s): Ozawa Y., Nakao K., Shimazaki T., Shinmura S., Kurihara T., Ishida S., Yoshimura A., Tsubota K., Okano H
  • Journal Title: Developmental Biology 303 Pages: 591-600
• [Journal Article] Conditional ablation of Stat3 or Socs3 discloses a dual role for reactive astrocytes after spinal cord injury. (2006)
  • Author(s): Okada S, Nakamura M, Katoh H, Miyao T, Shimazaki T, Ishii K, Yamane J, Yoshimura A, Iwamoto Y, Toyama Y, Okano H
  • Journal Title: Nature Medicine 12 Pages: 829-834
• [Journal Article] A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells. (2006)
  • Author(s): Sakaguchi M, Shingo T, Shimazaki T, Okano HJ, Shiwa M, Ishibashi S, Oguro H, Ninomiya M, Kadoya T, Horie H, Shibuya A, Mizusawa H, Poirier F, Nakauchi H, Sawamoto K, Okano H
  • Journal Title: Proceedings of the National Academy of Sciences of the United States of America 103 Pages: 7112-7117
• [Journal Article] Histopathological and behavioral improvement of murine Mucopolysaccharidosis type VII by intra-cerebral transplantation of neural stem cells. (2006)
  • Author(s): Fukuhara Y, Li X-K, Kitazawa Y, Inagaki M, Matsuoka K, Kosuga M, Kosaki R, Shimazaki T, et al.
  • Journal Title: Molecular Therapy 13 Pages: 548-555
• [Journal Article] Essential role of Shp2-binding sites on the docking protein FRS2α for mammalian corticogenesis and for FGF2-dependent proliferation of cultured neural progenitor cells. (2005)
  • Author(s): Yamamoto S, Yoshino I, Shimazaki T, Murohashi M, Hevner RF, Lax I, Okano H, Shibuya M, Schlessinger J, Gotoh N.
  • Journal Title: Proc.Natl.Acad.Sci.USA 102 Pages: 15983-15988
• [Journal Article] Inhibition of glioma cell proliferation by neural stem cell factor. (2005)
  • Author(s): Suzuki T, Izumoto S, Wada K, Fujimoto Y, Maruno M, Yamasaki M, Kanemura Y, Shimazaki T, Okano H, Yoshimine T.
  • Journal Title: J.Neurooncol. 74 Pages: 233-239
• [Journal Article] Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart. (2005)
  • Author(s): Tomita Y, Matsumura K, Wakamatsu Y, Matsuzaki Y, Shibuya I, Kawaguchi H, Ieda M, Kanakubo S, Shimazaki T. et al.
  • Journal Title: J.Cell Biol. 170 Pages: 1135-1146
• [Journal Article] Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells. (2005)
  • Author(s): Yuasa S, Itabashi Y, Koshimizu U, Tanaka T, Sugimura K, Fukami S, Itabashi Y, Hattori F, Shimazaki T, Ogawa S, Okano H, Fukuda K.
  • Journal Title: Nature Biotech. 23 Pages: 607-611
• [Journal Article] Prospective characterization of neural stem cells by flow cytometry analysis using a combination of surface markers. (2005)
  • Author(s): Nagato M, Heike T, Kato T, Yamanaka Y, Yoshimoto M, Shimazaki T, Okano H, Nakahata T.
  • Journal Title: J.Neurosci.Res. 80 Pages: 456-466
• [Journal Article] Prospective characterization of neural stem cells by flow cytometry analysis using combination of the surface markers. (2005)
  • Author(s): Nagato M, Heike T, Kato T, Yamanaka Y, Yoshimoto M, Shimazaki T, Okano H, Nakahata T.
  • Journal Title: J Neurosci Res (In Press)
• [Journal Article] Comparative functional genomics revealed conservation and diversification of three enhancers of the isl1 gene for motor and sensory neuron-specific expression. (2005)
  • Author(s): Uemura O, Okada Y, Ando H, Guedj M, Higashijima S, Shimazaki T, Chino N, Okano H, Okamoto H.
  • Journal Title: Dev Biol 278 Pages: 587-606
• [Journal Article] A method for gene transfer, single isolation and in vitro assay for neural stem cells. (2005)
  • Author(s): Sakaguchi M, Sawamoto K, Shimazaki T, Kitamura T, Shibuya A, Okano H
  • Journal Title: Inflammation and Regeneration 25 Pages: 50-54
  • NAID: 10026498954
• [Journal Article] Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. (2004)
  • Author(s): Okada Y, Shimazaki T, Sobue G, Okano H.
  • Journal Title: Dev Biol 275 Pages: 124-142
• [Journal Article] Human neural stem/progenitor cells, expanded in long-term neurosphere culture, promote functional recovery after focal ischemia in Mongolian gerbils. (2004)
  • Author(s): Ishibashi S, Sakaguchi M, Kuroiwa T, Yamasaki M, Kanemura Y, Shizuko I, Shimazaki T, Onodera M, Okano H, Mizusawa H.
  • Journal Title: J Neurosci Res 78 Pages: 215-223
• [Journal Article] Isolation and transplantation of dopaminergic neurons generated from mouse embryonic stem cells. (2004)
  • Author(s): Yoshizaki T, Inaji M, Kouike H, Shimazaki T, Sawamoto K, Ando K, Date I, Kobayashi K, Suhara T, Uchiyama Y, Okano H
  • Journal Title: Neurosci Lett 363 Pages: 33-37
• [Publications] Kokuzawa J., Yoshimura S., Kitajima H., Shinoda J., Kaku Y., Iwama T., Morishita R., Shimazaki T., Okano H., Kunisada T., Sakai N.: "Hepatocyte growth factor promotes proliferation and neuronal differentiation of neural stem cells from mouse embryos."Mol Cell Neurosci. 24. 190-197 (2003)
• [Publications] Shimazaki T.: "Biology and clinical application of neural stem cells."Horm Res. 60. 1-9 (2003)
• [Publications] Mikami Y., Okano H., Sakaguchi M., Nakamura M., Shimazaki T., Okano H.J., Kawakami Y., Toyama Y., Toda M.: "Implantation of dendritic cells in the injured adult spinal coed results in activation of the endogenous neural/progenitor cells for den novo neurogenesis and axonal regeneration, leading to functional recovery."J Neurosci Res. (in press). (2004)
• [Publications] Ozawa Y., Nakao K., Shimazaki T., Takeda J., Akira, Gruss P., Hirano T., Oguchi Y., Okano H.: "Down-regulation of STAT3 activation is required as an intrinsic factor to complete rod photoreceptor differentiation."Mol Cell Neurosci. (in press). (2004)
• [Publications] Murayama, A., Matsuzaki, Y., Kawaguchi, A., Shimazaki, T., Okano, H.: "Flow cytometric analysis of neural stem cells in the developing and adult mouse brain"J. Neursci. Res.. 68. 837-847 (2002)
• [Publications] Okano, H., Yoshizaki, T., Shimazaki, T., Sawamoto, K: "Isolation and transplantation of dopaminergic neurons and neural stem cells"Parkinsonism Relat Disord. 1. 23 (2002)
• [Publications] Chojnacki, A., Shimazaki T., Gregg, C., Weinmaster, G., Weiss, S.: "Glycoprotein 130 signaling regulates Notch1 expression and activation in the self-renewal of mammalian forebrain neural stem cells"J. Neursci.. 23. 1730-1741 (2003)