Basic research for regenerating kidney with functional structure by selective extraction of renal progenitor cells

• Project/Area Number: 19K09678
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 56030:Urology-related
• Research Institution: Nagoya City University
• Principal Investigator: Nakane Akihiro 名古屋市立大学, 医薬学総合研究院(医学), 准教授 (70464568)
• Co-Investigator(Kenkyū-buntansha): 西尾 英紀 名古屋市立大学, 医薬学総合研究院(医学), 助教 (10621063) ; 林 祐太郎 名古屋市立大学, 医薬学総合研究院(医学), 教授 (40238134) ; 安井 孝周 名古屋市立大学, 医薬学総合研究院(医学), 教授 (40326153) ; 丸山 哲史 名古屋市立大学, 医薬学総合研究院(医学), 教授 (50305546) ; 水野 健太郎 名古屋市立大学, 医薬学総合研究院(医学), 准教授 (70448710)
• Project Period (FY): 2019-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2021: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2020: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2019: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: 再生医療 / 腎 / ES細胞 / FACS / 腎臓 / 遺伝子導入

Outline of Research at the Start

これまで私たちは、ES細胞を用い、腎臓の再生を試みてきた。本研究は、これらの成果を発展させ、ES細胞を腎構成細胞や腎前駆細胞に分化させてセルソーティングで選択的に回収し、必要な細胞のみ生体に移植を行う、新しい技術の確立を目的とする。まずPax2をES細胞に遺伝子導入し、培養・分化させる。次にEBの培養各段階での発現遺伝子を確認する。さらにEBからの腎前駆細胞抽出するために、遺伝子発現が整った状態のEBに対しセルソーティングを行い、解析に必要な細胞を回収する。最後にセルソーティングで回収した細胞を免疫不全・腎不全マウスに移植し、腎再生医療へ応用できる実験系を確立する。

Outline of Final Research Achievements

Pax2 transgenic ES cells were differentiated under the condition of addition of activin A and retinoic acid. Expression of various genes was evaluated by PCR method, and expression of BMP 7, Ret, Pax 8, aquaporin-1, and Podocin was enhanced. Expression of aquaporin-1 was also confirmed in immunostaining. EBs expressing the Pax2 gene was double-labeled with Pax2 and aquaporin-1 and cell sorting was carried out.It was possible to confirm increase of positive cells of Pax2 and aquaporin-1. From the results of this study, It was considered that the constitution ratio of some renal constituent cells or progenitor cells increased, and differentiation from ES cells to renal cells was induced by expressing the Pax2 gene.

Academic Significance and Societal Importance of the Research Achievements

末期腎不全から血液透析を行う患者数は増加し、年間約 1 兆円以上の医療費や根本的治療の腎移植のドナー不足が問題視されている。解決策として臨床応用され始めている再生医療の開発が望まれている。発生や構造が複雑な腎臓では再生医療の研究が他の臓器に比べ遅れていたが、近年では腎構成細胞の再生が報告され始めている。そこで腎構成細胞の幹細胞をセルソーティングの技術を用い再生することはこの分野の研究の一つの方向性となると考えられた。目的となる幹細胞の選定にはさらなる研究を継続が必要であると考えられた。

Research Products

• [Journal Article] Low Serum Inhibin B/Follicle-Stimulating Hormones and Anti-Mullerian Hormone/Follicle-Stimulating Hormones Ratios as Markers of Decreased Germ Cells in Infants with Bilateral Cryptorchidism (2021)
  • Author(s): Kato T, Mizuno K, Matsumoto D, Nishio H, Nakane A, Kurokawa S, Kamisawa H, Maruyama T, Iwatsuki S, Umemoto Y, Yasui T, Hayashi Y
  • Journal Title: J Urology Volume: 207 Issue: 3 Pages: 701-709
  • DOI: 10.1097/ju.0000000000002344
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Disorganization of claudin-11 and dysfunction of the blood-testis barrier during puberty in a cryptorchid rat model (2020)
  • Author(s): Kato Taiki、Mizuno Kentaro、Nishio Hidenori、Moritoki Yoshinobu、Kamisawa Hideyuki、Kurokawa Satoshi、Nakane Akihiro、Maruyama Tetsuji、Ando Ryosuke、Hayashi Yutaro、Yasui Takahiro
  • Journal Title: Andrology Volume: - Issue: 5 Pages: 231-235
  • DOI: 10.1111/andr.12788
  • Peer Reviewed
• [Presentation] Potential role of EEF1A1 and TPT1 genes expressed in the spermatogonial stem cells as a predictive factor of subsequent testicular growth (2020)
  • Author(s): Mizuno Kentaro, Nishio Hidenori, Kato Taiki, Kamisawa Hideyuki, Moritoki Yoshinobu, Kurokawa Satoshi, Nakane Akihiro, Maruyama Tetsuji, Yasui Takahiro, Hayashi Yutaro
  • Organizer: American Urological Association Annual Meeting 2020
  • Int'l Joint Research
• [Presentation] Kdm5a activates the protein kinase A signaling pathway in mouse spermatogonial GC-1 cells (2020)
  • Author(s): Nishio Hidenori, Mizuno Kentaro, Kato Taiki, Kamisawa Hideyuki, Kurokawa Satoshi, Nakane Akihiro, Maruyama Tetsuji, Yasui Takahiro, Hayashi Yutaro
  • Organizer: American Urological Association Annual Meeting 2020
  • Int'l Joint Research
• [Presentation] Disorganization of claudin-11 and dysfunction of the blood-testis barrier during puberty in a cryptorchid rat model (2020)
  • Author(s): Kato Taiki, Mizuno Kentaro, Nishio Hidenori, Moritoki Yoshinobu, Kamisawa Hideyuki, Kurokawa Satoshi, Nakane Akihiro, Maruyama Tetsuji, Ando Ryosuke, Hayashi Yutaro, Yasui Takahiro
  • Organizer: American Urological Association Annual Meeting 2020
  • Int'l Joint Research