脊髄損傷に対する人工多能性幹細胞由来神経幹細胞移植の確立

2011 Fiscal Year Annual Research Report

• Project/Area Number: 21390426
• Research Institution: Keio University
• Principal Investigator: 中村 雅也 慶應義塾大学, 医学部, 講師 (30217898)
• Co-Investigator(Kenkyū-buntansha): 戸山 芳昭 慶應義塾大学, 医学部, 教授 (40129549) ; 石井 賢 慶應義塾大学, 医学部, 講師 (00276289)
• Keywords: 脊髄損傷 / 人工多能性幹細胞 / 移植 / 再生医療

Research Abstract

【目的】これまで我々は入工多能性幹細胞(iPS細胞)に着目し、マウス脊髄損傷に対するマウスiPS細胞由来神経幹細胞、さらにヒトiPS-NSCの有効性を報告してきた。ヒトiPS細胞を用いた脊髄損傷治療の臨床応用にむけた、前臨床研究の第一歩として霊長類であるサル脊髄損傷に対するヒトiPS-NSC移植の有効性と安全性を検証した。
【方法】1)ヒトiPS-NSCの培養と分化誘導:京都大学山中研で樹立されたiPS細胞株のうち、既にこれまでの研究で最も安全性が高いと評価された201B7株を使用し、独自に開発した方法でNSCへと分化誘導した。2)コモンマーモセット脊髄損傷モデル作製:成体コモンマーモセット9匹を使用した。頚髄圧挫損傷モデルは既に報告した方法で作製した(Iwanami et al,JNR2005)。3)損傷脊髄に対するiPS-NSC移植:移植群では損傷後9日に、損傷中心部にヒトiPS-NSCを1x10^6個/5μl移植した。対照群ではリン酸緩衝生理食塩水を注入した。免疫抑制剤を連日筋肉内注射した。4)運動機能評価:損傷後12週まで、original open fieid scoring scale(MIKY score),bar grip test,cage climbing testによる機能評価を行った。5)組織学的評価:機能評価後、脊髄を切り出しHE、LFB、Eriochrome Cyanine染色、各種免疫染染色を行い定量的に評価した。
【結果】ヒトiPS-NSCは長期間にわたり継代が可能であり、また複数回の継代後もニューロンへの神経分化能を維持することを確認した。LFB及びEriochrome Cyanine染色では、移植群の髄鞘面積は対照群に比べ有意に増加していた。また、移植細胞は損傷中心部の空洞周囲に生着し、NeuN陽性のニューロン、GFAP陽性のアストロサイト、Olig1陽性のオリゴデンドロサイトに分化していた。各種運動機能評価でも移植群は対照群に比べ,有意な運動機能の改善を認めた。
【考察】ヒトiPS-NSCを用いた脊髄再生医療の実現に向けて、安全性を担保することが何よりも重要であることは論を待たない。現時点において最も安全性が高いと考えられるヒトiPS-NSC細胞株を用いて本実験を行い、その有効性と安全性が確認できた。

Research Products

• [Journal Article] Beneficial compaction of spinal cord lesion by migrating astrocytes through glycogen synthase kinase-3 inhibition (2011)
  • Author(s): Renault-Mihara F, Toyama Y, Nakamura M, Okano H
  • Journal Title: EMBO Molecular Medicine Volume: 3(11) Pages: 682-69
  • Peer Reviewed
• [Journal Article] Significance of Re-myelination by Neural Stem/Progenitor Cells Transplanted Into the Injured Spinal Cord (2011)
  • Author(s): Yasuda A, Tsuji O, Nakamura M (co-correspondence), Okano H
  • Journal Title: Stem Cells Volume: 29(12) Pages: 1983-1994
  • Peer Reviewed
• [Journal Article] The dual origin of the peripheral olfactory system : placode and neural crest (2011)
  • Author(s): Katoh H, Toyama Y, Nakamura M, Okano H
  • Journal Title: Molecular Brain Volume: 23 Pages: 34
  • Peer Reviewed
• [Journal Article] Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice (2011)
  • Author(s): Nori S, Toyama Y, Yamanaka S, Nakamura M, Okano H
  • Journal Title: PNAS Volume: 108 Pages: 16825-16830
  • Peer Reviewed
• [Journal Article] Transplantation of human neural stem/progenitor cells overexpressing Galectin-1 improves functional recovery from focal brain ischemia in the Mongolian gerbil (2011)
  • Author(s): Yamane J, Kanemura Y, Nakamura M, Toyama Y, Mizusawa H, Okano H
  • Journal Title: Molecular Brain Volume: 27 Pages: 35
  • Peer Reviewed
• [Journal Article] Human Hepatocyte Growth Factor Promotes Functional Recovery in Primates after Spinal Cord Injury (2011)
  • Author(s): Kitamura K, Aoki M, Toyama Y, Okano H, Nakamura M
  • Journal Title: PLos One Volume: 6(11) Pages: e27706
  • Peer Reviewed
• [Journal Article] Toward using iPS cells to treat spinal cord injury : their safety and ther apeutic efficacy (2011)
  • Author(s): Miura K, Tsuji O, Nakamura M, Okano H
  • Journal Title: Inflammation and Regeneration Volume: 31 Pages: 1-9
  • Peer Reviewed
• [Journal Article] Ce ll therapy for spinal cord injury by neural stem/progenitor cells derived from iPS/ES cells (2011)
  • Author(s): Tsuji O, Miura K, Fujiyoshi K, Nakamura M, Okano H
  • Journal Title: Neurotherapeutics Volume: 8(4) Pages: 668-676
  • Peer Reviewed
• [Presentation] Therapeutic potential of appropriately evaluated 'safe' induced pluripotent stem cells for spinal cord injury (2011)
  • Author(s): Tsuji O, Fujiyoshi K, Miura K, Matsumoto M, Chiba K, Toyama Y, Yamanaka S, Okano H, Nakamura M
  • Organizer: CSRS 39^<th> Annual meeting
  • Place of Presentation: Arizona, USA
  • Year and Date: 20111208-20111210
• [Presentation] Transplantation of human iPSC-derived neurospheres for the treatment of spinal cord injury in NOD-scid mice (2011)
  • Author(s): Nori S, Okada Y, Yasuda A, Tsuji O, Takahashi Y, Kobayashi Y, Fujiyoshi K, Toyama Y, Yamanaka S, Nakamura M, Okano H
  • Organizer: CSRS 39^<th> Annual meeting
  • Place of Presentation: Arizona, USA
  • Year and Date: 20111208-20111210
• [Presentation] Pre-evaluated safe human iPS clone derived neural stem cells promoted functional recovery after spinal cord injury without tumorigenicity in adult common marmosets (2011)
  • Author(s): Kobayashi Y, Okada Y, Iwai H, Nishimura S, Nori S, Konomi T, Fujiyoshi K, Tsuji O, Toyama Y, Yamanaka S, Okano H, Nakamura M
  • Organizer: CSRS 39^<th> Annual meeting
  • Place of Presentation: Arizona, USA
  • Year and Date: 20111208-20111210
• [Presentation] Transplantation of human iPSC-derived neurospheres promote motor functional recovery after spinal cord injury in NOD-scid mice (2011)
  • Author(s): Nori S, Okada Y, Yasuda A, Tsuji O, Takahashi Y, Kobayashi Y, Fujiyoshi K, Toyama Y, Yamanaka S, Nakamura M, Okano H
  • Organizer: 41^<st> Annual meeting Neuroscience 2011
  • Place of Presentation: Washington DC, USA
  • Year and Date: 20111112-20111116
• [Presentation] Efficient derivation of neural stem cells from common marmoset ES cells and iPS cells (2011)
  • Author(s): Shimada H, Okada Y, Tomioka I, Sasaki E, Nakamura M, Okano H
  • Organizer: 41^<st> Annual meeting Neuroscience 2011
  • Place of Presentation: Washington DC, USA
  • Year and Date: 20111112-20111116
• [Presentation] Pre-evaluated safe human iPSC-derived neurospheres promote functional recovery after spinal cord injury without tumorigenecity in adult common marmosets (2011)
  • Author(s): Kobayashi Y, Okada Y, Iwai H, Nishimura S, Nori S, Konomi T, Fujiyoshi K, Tsuji O, Toyama Y, Yamanaka S, Nakamura M, Okano H.
  • Organizer: 41^<st> Annual meeting Neuroscience 2011
  • Place of Presentation: Washington DC, USA
  • Year and Date: 20111112-16
• [Presentation] Pre-evaluated safe human iPS clone derived neural stem cells promoted functional recovery after spinal cord injury without tumorigenecity in adult common marmosets (2011)
  • Author(s): Kobayashi Y, Okada Y, Iwai H, Nishimura S, Nori S, Konomi T, Fujiyoshi K, Tsuji O, Toyama Y, Yamanaka S, Nakamura M, Okano H
  • Organizer: ISSCR 2011
  • Place of Presentation: Toronto, Canada
  • Year and Date: 20110615-18
• [Presentation] Transplantation of human iPS cell-derived neurospheres for the treatment of spinal cord injury in nod-scid mice (2011)
  • Author(s): Nori S, Okada Y, Yasuda A, Tsuji O, Takahashi Y, Kobayashi Y, Fujiyoshi K, Toyama Y, Yamanaka S, Nakamura M, Okano H
  • Organizer: ISSCR 2011
  • Place of Presentation: Toronto, Canada
  • Year and Date: 20110615-18