Examination of 'true' meanings of filaggrin mutation in human epidermal keratinocytes.

• Project/Area Number: 15K09759
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Dermatology
• Research Institution: Dokkyo Medical University (2017); Tokyo Medical and Dental University (2015-2016)
• Principal Investigator: IGAWA KEN 獨協医科大学, 医学部, 教授 (00372441)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: iPS細胞 / アトピー性皮膚炎 / フィラグリン / ゲノム編集 / 表皮角化細胞 / 人工ヌクレアーゼ / K14 / 角化細胞

Outline of Final Research Achievements

With our established systems of obtaining iKCs from hiPSCs and new technology of programmable nucleases, especially CRISPR/Cas9 system, we tried to clarify the precise effects of filaggrin gene (FLG) mutations in keratinocytes. A guide RNA that targeted appropriate site of human FLG was designed by web-based tool and cloning into the backbone vector of CRIPSR/Cas9 (hFLG-CRISPR/Cas9). We transfected hFLG-CRISPR/Cas9 into hiPSCs and obtained the several clones of hiPSCs which possessed random mutations in FLG. Then, original hiPSC and FLG-mutated hiPSCs were differentiated into epidermal keratinocytes using our established protocols and we obtained the normal iKCs and FLG-mutated iKCs. Under this condition, we can compare the phenotypes of normal and FLG-mutaed iKCs of the same genetic background.
Thus, the results obtained from this system should be "true" meanings of FLG mutation in keratinocytes and should be important information for the understanding of AD pathogenesis.

Research Products

• [Presentation] iPS細胞の基礎と臨床 (2018)
  • Author(s): 井川 健
  • Organizer: 第117回日本皮膚科学会総会
  • Invited
• [Presentation] To clarify the effect of the filaggrin gene mutation in keratinocytes by using CRISPR/Cas9 system and human induced pluripotent stem cells. (2016)
  • Author(s): Ken Igawa
  • Organizer: The 41th Annual Meeting of the Japanese Society for Investigative Dermatology
  • Place of Presentation: Sendai, Japan
  • Year and Date: 2016-12-09
  • Int'l Joint Research
• [Presentation] A trial to clarify the effect of the filaggrin gene mutation to keratinocytes biology by using CRISPR/Cas9 system and human induced pluripotent stem cells. (2015)
  • Author(s): Igawa K
  • Organizer: European Society for Dermatological Research 2015
  • Place of Presentation: ロッテルダム、オランダ
  • Year and Date: 2015-09-09
  • Int'l Joint Research
• [Presentation] REMOVAL OF REPROGRAMMING TRANSGENES IMPROVES THE TISSUE RECONSTITUTION POTENTIAL OF KERATINOCYTES GENERATED FROM HUMAN INDUCED PLURIPOTENT STEM CELLS. (2015)
  • Author(s): Igawa K
  • Organizer: World Congress of Dermatology 2015
  • Place of Presentation: バンクーバー、カナダ
  • Year and Date: 2015-06-08
  • Int'l Joint Research