Elucidation of the pathogenesis of the pediatric intractable epilepsy using human cerebral and midbrain organoids

• Project/Area Number: 19K23952
• Research Category: Grant-in-Aid for Research Activity Start-up
• Allocation Type: Multi-year Fund
• Review Section: 0902:General internal medicine and related fields
• Research Institution: Nara Medical University
• Principal Investigator: Kaoru Kinugawa 奈良県立医科大学, 医学部附属病院, 研究員 (90846677)
• Project Period (FY): 2019-08-30 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: 脳幹オルガノイド / 中脳オルガノイド / 1細胞遺伝子発現解析 / 小児難治性てんかん / ZEB2遺伝子 / Mowat-Wilson症候群 / 脳オルガノイド / ゼブラフィッシュ

Outline of Research at the Start

小児難治性てんかんは多くが胎生期の中枢神経系の発生異常によって生じる。その多くは発生メカニズムが十分に解明されておらず、故に根本的な治療法も存在せず、病態解明が早急に必要とされている。本研究では、小児難治性てんかんの一つであるMowat-Wilson症候群の病態を、ヒト大脳・中脳オルガノイド及びゼブラフィッシュを用いて、ヒト中枢神経発生の面から分析・解明することを試みる。

Outline of Final Research Achievements

Recently, three-dimensional in vitro human brain models derived from human pluripotent stem cells, brain organoids, have been developed. Brain organoids technologies are thought to be useful for research on pediatric neurological disorders, such as epilepsy. The aim of this study was to elucidate the pathogenesis of Mowat-Wilson Syndrome using human brain organoids of cerebrum and brainstem. MWS is one of the pediatric intractable epilepsy due to deficits of ZEB2 (zinc finger E-box-binding homeobox 2) gene. We established a method to induce brainstem organoids. Various methods, such as immunohistochemistry and single-cell RNA-sequencing, suggested that brainstem organoids mimic the structure of human brainstem. We developed the ZEB2 knock-out human brain organoids of cerebrum and brainstem, and performed morphological analysis and quantitative PCR. These results suggested that brain organoids might be useful model to elucidate the pathogenesis of the pediatric neurological disorders.

Academic Significance and Societal Importance of the Research Achievements

小児難治性てんかんなどの小児神経疾患は、胎生期の中枢神経系の異常によって生じる。その多くは発生メカニズムが十分に解明されておらず、故に根本的な治療法がなく、病態解明が早急に必要とされていた。ヒト脳疾患の研究には、げっ歯類が用いられてきたが、ヒト脳との構造の違いから、疾患の病態解明を行うのは困難であった。本研究ではヒト多能性幹細胞、CRISPER/Cas9によるゲノム編集で得たZEB2欠損ヒト多能性幹細胞から、ヒト脳幹オルガノイド及びZEB2欠損ヒト脳幹オルガノイドを樹立した。これら脳オルガノイドは、小児の中枢神経疾患の発生過程の再現や病態解明に有用なツールとなりうると考えられた。

Research Products

• [Journal Article] Gene expression profiles of human cerebral organoids identify PPAR pathway and PKM2 as key markers for oxygen-glucose deprivation and reoxygenation (2021)
  • Author(s): Naoki Iwasa, Takeshi K. Matsui, Naohiko Iguchi, Kaoru Kinugawa, Naritaka Morikawa, Yoshihiko M. Sakaguchi, Tomo Shiota, Shinko Kobashigawa et. al.
  • Journal Title: Frontiers in Cellular Neuroscience Volume: － Pages: 605030-605030
  • DOI: 10.3389/fncel.2021.605030
  • Peer Reviewed / Open Access
• [Journal Article] Brainstem organoids from human pluripotent stem cells (2020)
  • Author(s): Nobuyuki Eura, Takeshi K. Matsui, Joachim Luginbuhl, Masaya Matsubayashi, Hitoki Nanaura, Tomo Shiota, Kaoru Kinugawa, Naohiko Iguchi, Takao Kiriyama, Canbin Zheng, Tsukasa Kouno, Yan Jun Lan, Pornparn Kongpracha, Pattama Wiriyasermkul, Yoshihiko M. Sakaguchi, Riko Nagata, Tomoya Komeda, Naritaka Morikawa, et al.
  • Journal Title: Frontiers in Neuroscience Volume: - Pages: 00538-00538
  • DOI: 10.3389/fnins.2020.00538
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Single-cell transcriptomics reveals multiple neuronal cell types in human midbrain-specific organoids (2020)
  • Author(s): Smits Lisa M.、Magni Stefano、Kinugawa Kaoru、Grzyb Kamil、Luginbuhl Joachim、Sabate-Soler Sonia、Bolognin Silvia、Shin Jay W.、Mori Eiichiro、Skupin Alexander、Schwamborn Jens C.
  • Journal Title: Cell and Tissue Research Volume: 382 Issue: 3 Pages: 463-476
  • DOI: 10.1007/s00441-020-03249-y
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Comparative analysis of human brain organoids of brainstem and midbrain at single-cell resolution (2020)
  • Author(s): Kinugawa Kaoru、Luginbuhl Joachim、Matsui Takeshi K.、Eura Nobuyuki、Sakaguchi Yoshihiko M.、Shin Jay W、Sugie Kazuma、Schwamborn Jens C.、Mori Eiichiro
  • Journal Title: bioRxiv Volume: -
  • DOI: 10.1101/2020.09.02.279380
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Oxygen-glucose deprivation and reoxygenation on human cerebral organoids alters expression related to lipid metabolism (2020)
  • Author(s): Iwasa Naoki、Matsui Takeshi K.、Morikawa Naritaka、Sakaguchi Yoshihiko M.、Shiota Tomo、Iguchi Naohiko、Nishimura Yuhei、Mori Eiichiro、Sugie Kazuma
  • Journal Title: bioRxiv Volume: -
  • DOI: 10.1101/2020.03.31.017335
  • Open Access
• [Presentation] Comparative analysis of human brain organoids of brainstem and midbrain at single-cell resolution (2021)
  • Author(s): Kaoru Kinugawa, Nobuyuki Eura, Eiichiro mori, Kazuma Sugie
  • Organizer: 62th Annual Meating of the Japanese Society of Neurology
  • Int'l Joint Research
• [Presentation] Single-cell RNA-sequencing analysis on human brainstem organoids (2020)
  • Author(s): Kaoru Kinugawa, Kazuma Sugie, Eiichiro Mori
  • Organizer: The 1st International Symposium on Human InformatiX
  • Int'l Joint Research
• [Presentation] Single-cell RNA-sequencing analysis on human brainstem organoids (2020)
  • Author(s): Kaoru Kinugawa, Takeshi K. Matsui, Nobuyuki Eura, Hitoki Nanaura, Tomo Shiota, Naohiko Iguchi, Takao Kiriyama, Eiichiro Mori, Kazuma Sugie
  • Organizer: 61th Annual Meating of the Japanese Society of Neurology
  • Int'l Joint Research
• [Presentation] Single cell RNA sequencing analysis on human brainstem organoids (2020)
  • Author(s): Kaoru Kinugawa, Kazuma Sugie, Eiichiro Mori
  • Organizer: The 1st International Symposium on Human InformatiX
• [Presentation] A human cerebral organoid model for ZEB2-deficient Mowat-Wilson syndrome (2019)
  • Author(s): Nobuyuki Eura, Takeshi K. Matsui, Hitoki Nanaura, Tomo Shiota, Eiichiro Mori, Kazuma Sugie
  • Organizer: 60th Annual Meating of the Japanese Society of Neurology
  • Int'l Joint Research