A trial for the clarification of the precise relationship between filaggrin gene mutation and atopic dermatitis by using human pluripotent stem cell

• Project/Area Number: 24591651
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Dermatology
• Research Institution: Tokyo Medical and Dental University
• Principal Investigator: IGAWA Ken 東京医科歯科大学, 医学部附属病院, 講師 (00372441)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2014: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2013: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2012: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: iPS細胞 / 人工ヌクレアーゼ / アトピー性皮膚炎 / フィラグリン / 角化細胞 / ヒト / Cas9/CRISPR / TALENs / ヒトiPS細胞 / ケラチン遺伝子 / フィラグリン遺伝子 / 人工多能性幹細胞 / ゲノム編集 / TALEN

Outline of Final Research Achievements

A filaggrin is one of important proteins which consist skin barrier and the mutations of this gene are reported to be essential precipitating factor of atopic dermatitis.In this study, we try to knock-out filaggrin gene (FLG) in human iPS cells and keratinocytes will be differentiated from FLG-KO hiPS cells and their parental hiPS cells. Then, finally, we will get isogenic (only FLG-KO or FLG-normal) keratinocytes. In this situation, we can evaluate the precise effect of FLG mutation (KO) to keratinocytes physiology.In order to KO FLG in hiPS cells, we decided to use engineered nucleases mediated gene targeting. First, we made FLG specific transcription activator-like effector nuclease (FLG-TALENs) , but we failed. Then, we switched to use CRISPR/Cas9 system and finally we succeeded and we got several clones of hiPS cells which possessed FLG-mutations (KO).

Research Products

• [Journal Article] Removal of reprogramming transgenes improves the tissue reconstitution potential of keratinocytes generated from human induced pluripotent stem cells (2014)
  • Author(s): Ken Igawaa, Chikara Kokubu, Kosuke Yusa, Kyoji Horie, Yasuhide Yoshimura, Kaori Yamauchi, Hirofumi Suemori, Hiroo Yokozeki, Masashi Toyoda, Nobutaka Kiyokawa, Hajime Okita, Yoshitaka Miyagawa, Hidenori Akutsu, Akihiro Umezawa, Ichiro Katayama, Junji Takeda.
  • Journal Title: Stem Cells Transl Med Volume: 3 Issue: 9 Pages: 992-1001
  • DOI: 10.5966/sctm.2013-0179
  • Peer Reviewed / Open Access
• [Presentation] Efficient keratinocytes differentiation from transgene-free human induced pluripotent stem cell line: Implication for therapeutic application. (2013)
  • Author(s): Igawa K
  • Organizer: International Investigative Dermatology
  • Place of Presentation: Edinburgh, Scotland
• [Presentation] Efficient keratinocytes differentiation from transgene-free human induced pluripotent stem cell line: Implication for therapeutic application. (2013)
  • Author(s): 井川 健
  • Organizer: 第8回 箱根カンファレンス
  • Place of Presentation: 淡路島
  • Invited
• [Presentation] A trial of in vitro reconstitution of human skin using transgene-free induced pluripotent stem cells. (2012)
  • Author(s): Ken IGAWA, Kyoji HORIE, Kosuke YUSA, Junji TAKEDA, Ichiro KATAYAMA
  • Organizer: 10th annual meeting of ISSCR
  • Place of Presentation: Yokohama
• [Presentation] induced keratinocytes from transgene-free but not transgene-residual human induced pluripotent stem cells phenotypically resemble primary human keratinocytes. (2012)
  • Author(s): Ken IGAWA, Kyoji HORIE, Hiroo YOKOZEKI, Ichiro KATAYAMA, Junji TAKEDA.
  • Organizer: The 42nd Annual Meeting of the European Society for Dermatological Research
  • Place of Presentation: Venice, Italy