Identification and separation of living cells based on synthetic RNA circuits detecting intracellular information

• Project/Area Number: 25870355
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: General medical chemistry; System genome science
• Research Institution: The University of Tokyo (2014-2015); Kyoto University (2013)
• Principal Investigator: ENDO Kei 東京大学, 新領域創成科学研究科, 助教 (40626074)
• Co-Investigator(Renkei-kenkyūsha): SAITO Hirohide 京都大学, iPS細胞研究所, 教授 (20423014)
• Research Collaborator: HAYASHI Karin
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2013: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
• Keywords: 細胞分画法 / mRNA / マイクロRNA / 人口遺伝子回路 / 合成生物学 / 再生医療 / miRNA / 遺伝子スイッチ / 細胞分画 / 遺伝子回路 / 細胞識別 / microRNA

Outline of Final Research Achievements

Methods for identifying the cell types are critical not only to analysis of specific tissues and cells, but also to preparation of particular cells for therapeutic purposes. Marker proteins on the surface of living cells have been investigated to identify target cells. However, it is still hard to find a specific set of antibodies that determines a variety of cell types. In addition, possible combinations of binary classification are quite limited.
In this project, we developed a method that enables to distinguish living cells based on intracellular markers in a quantitative manner. We focused on the activity of microRNAs, which are short, non-coding RNAs expressed in a cell. We designed and synthesized microRNA-responsive mRNAs that produce fluorescent proteins as a reporter, and transfected them directly to target cells. With the synthetic mRNAs, we succeeded in isolating multiple cell types based on subtle (less than 2-fold) difference in the microRNA activity.

Research Products

• [Int'l Joint Research] マサチューセッツ工科大学(米国)
• [Journal Article] High-resolution identification of cell types by microRNA-responsive mRNAs. (2016)
  • Author(s): Endo, K., Hayashi, K., and Saito, H.
  • Journal Title: Scientific Reports Volume: 6 Issue: 1 Pages: 21991-21991
  • DOI: 10.1038/srep21991
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Efficient Detection and Purification of Cell Populations Using Synthetic MicroRNA Switches. (2015)
  • Author(s): Miki K, Endo K, Takahashi S, Funakoshi S, Takei I, Katayama S, Toyoda T, Kotaka M, Takaki T, Umeda M, Okubo C, Nishikawa M, Oishi A, Narita M, Miyashita I, Asano K, Hayashi K, Osafune K, Yamanaka S, Saito H, Yoshida Y.
  • Journal Title: Cell Stem Cell. Volume: 16(6) Issue: 6 Pages: 699-711
  • DOI: 10.1016/j.stem.2015.04.005
  • Peer Reviewed / Open Access
• [Journal Article] Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. (2015)
  • Author(s): Wroblewska L, Kitada T, Endo K, Siciliano V, Stillo B, Saito H, Weiss R
  • Journal Title: Nature biotechnology Volume: 33 Issue: 8 Pages: 839-841
  • DOI: 10.1038/nbt.3301
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Efficient detection and purification of cell populations by synthetic microRNA switches. (2015)
  • Author(s): Miki, K.†, Endo, K.†, Takahahshi, S., Funakoshi, S., Takei, I., Katayama, S., Toyoda, T., Kotaka, M., Takaki, T., Umeda, M., Okubo, C., Nishikawa, M, Oishi, A., Narita, M, Miyashita, I., Asano, K., Hayashi, K., Osafune, K., Yamanaka, S., Saito, H., Yoshida, Y.
  • Journal Title: Cell Stem Cell Volume: 印刷中
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. (2015)
  • Author(s): Wroblewska, L., Kitada, T.†, Endo, K.†, Siciliano, V., Elizabeth, B., Saito, H. and Weiss, R.
  • Journal Title: Nature Biotechnology Volume: 印刷中
  • Peer Reviewed
• [Presentation] 合成 mRNA により miRNA 活性プロファイルに従って生細胞を精密に分画する (2015)
  • Author(s): 遠藤 慧
  • Organizer: 情報計算化学生物学会 2015年大会
  • Place of Presentation: タワーホール船堀（東京都江戸川区）
  • Year and Date: 2015-10-27
  • Invited
• [Presentation] High-resolution identification of cell types by microRNA-responsive mRNAs. (2014)
  • Author(s): Endo, K., and Saito, H.
  • Organizer: The 7th Takeda Science Foundation Symposium on Pharmasciences
  • Place of Presentation: Osaka, Japan
• [Presentation] 細胞内の情報にもとづいて細胞を生きたまま精製したい (2014)
  • Author(s): 遠藤 慧
  • Organizer: 日本再生医療学会（サテライトミーティング）
  • Place of Presentation: 京都府京都市
  • Invited
• [Presentation] mRNA のエンジニアリングに基づく動物細胞の遺伝子発現制御系 (2014)
  • Author(s): 遠藤 慧、齊藤 博英
  • Organizer: 日本農芸化学会
  • Place of Presentation: 神奈川県川崎市
  • Invited
• [Presentation] mRNA エンジニアリングによる外来遺伝子の発現制御 (2013)
  • Author(s): 遠藤 慧、井上 丹、齊藤 博英
  • Organizer: 日本 RNA 学会
  • Place of Presentation: 愛媛県松山市
• [Remarks] マイクロRNAをつかった細胞の選別方法の開発：高純度な心筋細胞の作製に成功
  • URL: https://www.cira.kyoto-u.ac.jp/j/research/finding/150522-085314.html
• [Remarks] 細胞の機能を精密に制御する人工回路をRNAで構築：ヒトの細胞で成功
  • URL: https://www.cira.kyoto-u.ac.jp/j/research/finding/150804-110938.html
• [Remarks] 合成RNAを利用した生細胞の高精度な同定と分離
  • URL: https://www.cira.kyoto-u.ac.jp/j/research/finding/160224-100000.html
• [Patent(Industrial Property Rights)] mRNAの設計方法 (2015)
  • Inventor(s): 齊藤 博英、遠藤 慧
  • Industrial Property Rights Holder: 齊藤 博英、遠藤 慧
  • Industrial Property Rights Type: 特許
  • Filing Date: 2015-10-28
• [Patent(Industrial Property Rights)] RNA-based logic circuits with RNA binding proteins, aptamers and small molecules. (2015)
  • Inventor(s): Weiss, R., Endo, K. 他７名
  • Industrial Property Rights Holder: Weiss, R., Endo, K. 他７名
  • Industrial Property Rights Type: 特許
  • Filing Date: 2015-09-08
  • Overseas
• [Patent(Industrial Property Rights)] miRNAの発現を指標として所望の細胞種を判別する方法 (2014)
  • Inventor(s): 齊藤 博英、遠藤 慧
  • Industrial Property Rights Holder: 齊藤 博英、遠藤 慧
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2014-003726
  • Filing Date: 2014-01-10
• [Patent(Industrial Property Rights)] 心筋細胞の選別方法 (2014)
  • Inventor(s): 齊藤 博英、吉田 善紀、三木 健嗣、遠藤 慧、高橋 誠弥
  • Industrial Property Rights Holder: 齊藤 博英、吉田 善紀、三木 健嗣、遠藤 慧、高橋 誠弥
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2014-058926
  • Filing Date: 2014-03-20