Regeneration of kidney and urinary tract using human and cynomolgus monkey pluripotent stem cells

• Project/Area Number: 16K20131
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Urology
• Research Institution: Shiga University of Medical Science
• Principal Investigator: Kenichi Kobayashi 滋賀医科大学, 医学部, 非常勤講師 (40727434)
• Project Period (FY): 2016-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000); Fiscal Year 2019: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2018: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2017: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2016: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
• Keywords: 腎臓 / 多能性幹細胞 / カニクイザル / ES細胞 / ノックイン / 腎再生 / ADPKD / 腎分化 / 再生医学 / 遺伝学 / 移植 / 細胞発現制御

Outline of Final Research Achievements

Cynomolgus monkey ES (Cyn ES) cells can be generated in a similar manner as human ES cells. However, Cyn ES cells are difficult to maintain in an undifferentiated state by untrained researchers. For easier culture, we generated an OCT3/4-P2A tdTomato IRES ZeocinR Cyn ES cell line using CRISPR/Cas9 genome editing technology. The stop codon of the endogenous OCT3/4 locus was replaced with the P2A tdTomato IRES ZeocinR pA cassette by homologous recombination. This cell line enables us to isolate pluripotent stem cells and exclude differentiated cells by addition of zeocin, especially for culture without feeder cells.

Academic Significance and Societal Importance of the Research Achievements

ヒトの多能性幹細胞を用いた臓器再生の研究は日進月歩で、近い将来に腎機能を持つ腎臓オルガノイドが開発されることが期待されているが、臨床応用のためには、作製された腎オルガノイドに交通を持つ尿路、血管をいかに構築し、どこにどのように移植するかという課題を解決しなければならない。カニクイザルはヒトに最も近い実験動物モデルであり、臨床前試験に理想的な実験モデルとなりうる。
我々の研究成果は多能性幹細胞を用いた腎再生の過程において臨床前試験という重要な段階で大きな役割を果たすと考えられる。

Research Products

• [Journal Article] Generation of an OCT3/4 reporter cynomolgus monkey ES cell line using CRISPR/Cas9. (2019)
  • Author(s): Kobayashi K, Tsukiyama T, Nakaya M, Kageyama S, Tomita K, Murai R, Yoshida T, Narita M, Kawauchi A, Ema M.
  • Journal Title: Stem cell research Volume: Epub Pages: 101439-101439
  • DOI: 10.1016/j.scr.2019.101439
  • Peer Reviewed / Open Access
• [Journal Article] Monkeys mutant for PKD1 recapitulate human autosomal dominant polycystic kidney disease (2019)
  • Author(s): Tsukiyama T, Kobayashi K, Nakaya M, Iwatani C, Seita Y, Tsuchiya H, Matsushita J, Kitajima K, Kawamoto I, Nakagawa T, Fukuda K, Iwakiri T, Izumi H, Itagaki I, Kume S, Maegawa H, Nishinakamura R, Nishio, Nakamura S, Kawauchi A, Ema M
  • Journal Title: Nature Communications Volume: 10 (1) Issue: 1 Pages: 5517-5517
  • DOI: 10.1038/s41467-019-13398-6
  • NAID: 120006773159
  • Peer Reviewed / Open Access
• [Journal Article] Comprehensive evaluation of ubiquitous promoters suitable for the generation of transgenic cynomolgus monkeys† (2019)
  • Author(s): Seita Yasunari、Tsukiyama Tomoyuki、Azami Takuya、Kobayashi Kenichi、Iwatani Chizuru、Tsuchiya Hideaki、Nakaya Masataka、Tanabe Hideyuki、Hitoshi Seiji、Miyoshi Hiroyuki、Nakamura Shinichiro、Kawauchi Akihiro、Ema Masatsugu
  • Journal Title: Biology of Reproduction Volume: 印刷中 Issue: 6 Pages: 1440-1452
  • DOI: 10.1093/biolre/ioz040
  • NAID: 120006594188
  • Peer Reviewed / Open Access / Int'l Joint Research