Developing Single Cell Culture Platform for Human Pluripotent Stem Cells

• Project/Area Number: 26289065
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Intelligent mechanics/Mechanical systems
• Research Institution: Osaka University (2017); Kyoto University (2014-2016)
• Principal Investigator: Liu Li 大阪大学, 医学系研究科, 特任准教授(常勤) (50380093)
• Co-Investigator(Kenkyū-buntansha): 陳 勇 京都大学, 物質－細胞統合システム拠点, 特定拠点教授 (70512458)
• Co-Investigator(Renkei-kenkyūsha): NAKATSUJI Norio 京都大学, 物質細胞統合システム拠点, 教授 (80237312) ; KOTERA Hidetoshi 京都大学, 工学研究科, 教授 (20252471) ; WASHITSU Masao 東京大学, 工学研究科, 教授 (10201162)
• Project Period (FY): 2014-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥16,770,000 (Direct Cost: ¥12,900,000、Indirect Cost: ¥3,870,000); Fiscal Year 2016: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2015: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2014: ¥7,150,000 (Direct Cost: ¥5,500,000、Indirect Cost: ¥1,650,000)
• Keywords: 幹細胞 / ナノファイバー / 多能性能 / 接着力 / マイクロデバイス / マイクロ・ナノデバイス / 再生医学 / マイクロ流路 / マイクロ流体

Outline of Final Research Achievements

By using a micro-nano devices, we successfully isolated and culture single cell-derived two novel pluripotent cell subtypes co-existing in the conventional hiPSCs population. Both types can be stably maintained and keep their properties inherited during proliferation. One type holds higher pluripotency, and is more sensitive to the microenvironment. We discovered a serum response factor-based regulation loop, which explained the relevance among cell-matrix adhesion, cell-cell adhesion, pluripotent stem cell morphology and pluripotency. We also found the high cell-matrix adhesion repressed the expression of some pluripotency-related genes, implying that the low-adhesion substrate may be a more suitable substrate for high-pluripotency state cells culture. A mathematical model was constructed and experimentally verified. It helped to provide a mechanistic view on the relationship among adhesion, pluripotent stem cell morphology and pluripotency.

Research Products

• [Int'l Joint Research] Peking University, Beijing(China)
• [Int'l Joint Research] Ecole Normale Supertre, Paris(France)
• [Int'l Joint Research] Peking University, Beijing(China)
• [Int'l Joint Research] Ecole Normale Superteure, Paris(France)
• [Int'l Joint Research] Peking University/Beijing/China(中国)
• [Int'l Joint Research] Ecole Normale Superieure/Paris/France(フランス)
• [Journal Article] Culture Substrate Made of Elastomeric Micro-Tripod Arrays for Long-Term Expansion of Human Pluripotent Stem Cells (2017)
  • Author(s): Li, J., Zhang, F., Yu, L., Fujimoto, N., Yoshioka, M., Li, X., Shi, J., Kotera, H., Liu, L*, Chen, Y*
  • Journal Title: Journal of Materials Chemistry B. Volume: 5 Issue: 2 Pages: 236-244
  • DOI: 10.1039/c6tb02246d
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Human Pluripotent Stem Cell-Derived Cardiac Tissue-like Constructs for Repairing the Infarcted Myocardium (2017)
  • Author(s): Li Junjun、Minami Itsunari、Shiozaki Motoko、Yu Leqian、Yajima Shin、Miyagawa Shigeru、Shiba Yuji、Morone Nobuhiro、Fukushima Satsuki、Yoshioka Momoko、Li Sisi、Qiao Jing、Li Xin、Wang Lin、Kotera Hidetoshi、Nakatsuji Norio、Sawa Yoshiki、Chen Yong、Liu Li
  • Journal Title: Stem Cell Reports Volume: 9 Issue: 5 Pages: 1546-1559
  • DOI: 10.1016/j.stemcr.2017.09.007
  • NAID: 120006358786
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Nano-on-micro fibrous extracellular matrices for scalable expansion of human ES/iPS cells (2017)
  • Author(s): Li Liu, Ken-ichiro Kamei, Momoko Yoshioka, Minako Nakajima, Junjun Li, Nanae Fujimoto Shiho Terada, Yumie Tokunaga, Yoshie Koyama, Hideki Sato, Koichi Hasegawa, Norio Nakatsuji, Yong Chen
  • Journal Title: Biomaterial Volume: 124 Pages: 47-54
  • DOI: 10.1016/j.biomaterials.2017.01.039
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Extracellular Recordings of Patterned Human Pluripotent Stem Cell-Derived Cardiomyocytes on Aligned Fibers (2016)
  • Author(s): Li, J.; Minami, I.; Yu, L.; Tsuji, K.; Nakajima, M.; Qiao, J.; Suzuki, M.; Shimono, K.; Nakatsuji, N.; Kotera, H.; Liu, L*.; Chen, Y*.
  • Journal Title: Stem cells international Volume: 2634013 Issue: 1 Pages: 1-9
  • DOI: 10.1155/2016/2634013
  • NAID: 120005829439
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] human pluripotent stem cell clones are revealed by a single cell culture platform (2017)
  • Author(s): L. Yu et.al.
  • Organizer: Biomedical Engineering Society (BMES)
  • Int'l Joint Research
• [Presentation] A Single Human Pluripotent Stem Cell Culture Platform (2016)
  • Author(s): Leqian Yu, Junjun Li, Wei Yang, Fuchou Tang, Nanae Fujimoto, Minako Nakajima, Yong Chen, Li Liu*, Hidetoshi Kotera*
  • Organizer: 29th International Microprocesses and Nanotechnology Conference (MNC)
  • Place of Presentation: Kyoto, (Japan)
  • Year and Date: 2016-11-08
  • Int'l Joint Research
• [Presentation] Low cell-matrix adhesion microenvironment promote maintain of high-pluripotency state stem cells (2016)
  • Author(s): L. Yu, J. Li, J. Hong, Y. Takashima, K. Okita, F. Tang, Y. Chen, C. Tang, H. Kotera, L. Liu*
  • Organizer: 10 Years of iPSCs Cell Symposium
  • Place of Presentation: Berkeley (USA)
  • Year and Date: 2016-09-25
  • Int'l Joint Research
• [Presentation] Down-Regulated Cell-Matrix Adhesion Promote Maintain of Naive-like Human Pluripotent Stem Cells (2016)
  • Author(s): Yu, Leqian, Li Junjun, Liu, Li*, Fujimoto, Nanae, Nakajima, Minako, Chen, Yong, Kotera, Hidetoshi
  • Organizer: 13th International Society for Stem Cell Research (ISSCR) Annual Meeting
  • Place of Presentation: San Francisco (USA)
  • Year and Date: 2016-06-22
  • Int'l Joint Research
• [Presentation] Low Cell-Matrix Adhesion Regulate Pluripotent Stem Cells Shape and Naïve-Related Genes Expression via Serum Response Factor Based Signaling (2016)
  • Author(s): Leqian Yu
  • Organizer: CiRA/ISSCR 2016 International Symposia
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 2016-03-22
  • Int'l Joint Research
• [Presentation] Identification of a novel naïve state-like cell type in human pluripotent stem cells population (2016)
  • Author(s): Leqian Yu
  • Organizer: 第１５回日本再生医療学会
  • Place of Presentation: Osaka, Japan
  • Year and Date: 2016-03-17
• [Presentation] Developing Single Cell Culture Platform for Human Pluripotent Stem Cells (2015)
  • Author(s): Leqian Yu
  • Organizer: The 6th Japan-China-Korea MEMS/NEMS Conference
  • Place of Presentation: Xi'an, China
  • Year and Date: 2015-09-23
  • Int'l Joint Research
• [Presentation] ナノファイバーを用いた再生医療への応用 (2015)
  • Author(s): Li Liu
  • Organizer: 2015年度日本機械学会年次大会
  • Place of Presentation: Sapporo, Japan
  • Year and Date: 2015-09-13
  • Invited
• [Presentation] Single Cell Culture of Human Pluripotent Stem Cells (2015)
  • Author(s): Leqian Yu
  • Organizer: International Society for Stem Cell Research (ISSCR) 2015
  • Place of Presentation: Stockholm, Sweden
  • Year and Date: 2015-06-24
  • Int'l Joint Research
• [Patent(Industrial Property Rights)] 心筋細胞に分化させるための多能性幹細胞の製造方法 (2017)
  • Inventor(s): 劉莉、于楽謙、李俊君
  • Industrial Property Rights Holder: 劉莉、于楽謙、李俊君
  • Industrial Property Rights Type: 特許
  • Filing Date: 2017
• [Patent(Industrial Property Rights)] 環状心筋細胞塊 (2016)
  • Inventor(s): 劉莉、Junjun LI
  • Industrial Property Rights Holder: 劉莉、Junjun LI
  • Industrial Property Rights Type: 特許
  • Filing Date: 2016-12-28