ヒト多能性幹細胞からの効率的な心筋細胞分化に向けた新規低分子化合物の開発

• Project/Area Number: 22K08164
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 53020:Cardiology-related
• Research Institution: Keio University
• Principal Investigator: 岸野 喜一 慶應義塾大学, 医学部(信濃町), 助教 (20594568)
• Project Period (FY): 2022-04-01 – 2025-03-31
• Project Status: Granted (Fiscal Year 2023)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2024: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2023: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2022: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: 再生医療 / ヒトiPS細胞由来心筋細胞 / BMPシグナル / 低分子化合物

Outline of Research at the Start

近年ヒトiPS細胞由来の心筋細胞が移植細胞ソースや疾患解析ツールとして注目されているが，高効率の心筋分化誘導方法の確立は十分とはいえない。心筋分化誘導においては，分化初期のBMPシグナルの活性化が重要であり，同シグナルを活性化する低分子化合物は未だ存在しない為，当研究では低分子化合物のスクリーニングによりヒト多能性幹細胞からの心筋分化に有効なBMPシグナル作動化合物を同定することを目的とする。

Outline of Annual Research Achievements

①in silicoパイロットスクリーニングで選出した候補化合物の有効性評価
約80万個の化合物データベースライブラリーから，標的因子の構造に基づいたpharmacophore及びドッキングスコアを用いて行ったパイロットスクリーニングにより選出された候補化合物の有効性評価を行った。評価系としては，ヒト細胞株HEK293とBMPシグナル下流遺伝子Id-1プロモーターを有する既知のBMP反応性ルシフェラーゼベクターを用いたルシフェラーゼアッセイ及びヒトiPS細胞の心筋分化誘導系を用いた。実際に選出した低分子化合物を添加し，FACS，PCR，免疫染色などを用いて定量的に評価した。ヒトiPS細胞に同定した化合物を添加した結果, BMPシグナル下流遺伝子のId-1の発現増加を認め, 心筋細胞への分化系においてはトロポニンT陽性細胞の増加を認めた。
②in silicoフルスクリーニングによる候補化合物の選出
パイロットスクリーニングで得られた候補化合物の有効性評価結果を踏まえて，さらに低濃度で効果の高い化合物を選出する為，大規模化合物データベースを用いたin silicoフルスクリーニングを行った。具体的には，化合物の化学構造式と①で得られたBMPシグナル相対活性値から，ソフトウェアAutoQSARを用いて，定量的構造活性相関式の算出を行い，約800万個の化合物を含むライブラリーから，さらに高い効果を示し，かつリード化合物の条件を備えた化合物を選出した。次に，パイロットスクリーニング時同様に，選出された候補化合物と標的因子ドメインとのドッキングスコアを算出し，GBVI/WSA_dGを用いた結合親和性の評価をin silicoで行い，50個の候補化合物を選出した。ヒト細胞株HEK293を用いたBMP反応性ルシフェラーゼアッセイで候補化合物の有効性を定量的評価し、絞り込みを実施している。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

当初の研究実施計画に基づいて研究が進行出来ている。

Strategy for Future Research Activity

今後は，標的因子の立体構造に基づいたin silicoフルスクリーニングで選出した候補化合物の有効性をヒト細胞株HEK293を用いたBMP反応性ルシフェラーゼアッセイやヒトiPS細胞の心筋分化誘導系を用いて，再度定量的に評価する。

Research Products

• [Journal Article] Coronary Artery Bypass Grafting for a Young Female Patient With Chronic Active Epstein-Barr Virus Infection. (2024)
  • Author(s): Iwata J, Kodaira M, Kishino Y, Anzai A, Kanazawa H, Hayashida K, Kato J, Yamazaki M, Shimizu H, Ieda M.
  • Journal Title: JACC Case Report Volume: 29 Issue: 5 Pages: 102221-102221
  • DOI: 10.1016/j.jaccas.2024.102221
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Pluripotent stem cell-based cardiac regenerative therapy for heart failure. (2024)
  • Author(s): Soma Y, Tani H, Morita-Umei Y, Kishino Y, Fukuda K, Tohyama S.
  • Journal Title: Journal of Molecular and Cellular Cardiology Volume: 187 Pages: 90-100
  • DOI: 10.1016/j.yjmcc.2023.12.001
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Cardiac Regenerative Therapy Using Human Pluripotent Stem Cells for Heart Failure: A State-of-the-Art Review. (2023)
  • Author(s): Kishino Y, Tohyama S, Morita Y, Soma Y, Tani H, Okada M, Kanazawa H, Fukuda K.
  • Journal Title: Journal of Cardiac Failure Volume: 29 Issue: 4 Pages: 503-513
  • DOI: 10.1016/j.cardfail.2022.10.433
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Scalable production of homogeneous cardiac organoids derived from human pluripotent stem cells (2023)
  • Author(s): Moriwaki Taijun、Tani Hidenori、Haga Kotaro、Morita-Umei Yuika、Soma Yusuke、Umei Tomohiko C.、Sekine Otoya、Takatsuna Kaworu、Kishino Yoshikazu、Kanazawa Hideaki、Fujita Jun、Fukuda Keiichi、Tohyama Shugo、Ieda Masaki
  • Journal Title: Cell Reports Methods Volume: 3 Issue: 12 Pages: 100666-100666
  • DOI: 10.1016/j.crmeth.2023.100666
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Seamless and non-destructive monitoring of extracellular microRNAs during cardiac differentiation from human pluripotent stem cells (2023)
  • Author(s): Sekine Otoya、Kanaami Sayaka、Masumoto Kanako、Aihara Yuki、Morita-Umei Yuika、Tani Hidenori、Soma Yusuke、Umei Tomohiko C.、Haga Kotaro、Moriwaki Taijun、Kawai Yujiro、Ohno Masatoshi、Kishino Yoshikazu、Kanazawa Hideaki、Fukuda Keiichi、Ieda Masaki、Tohyama Shugo
  • Journal Title: Stem Cell Reports Volume: 18 Issue: 10 Pages: 1925-1939
  • DOI: 10.1016/j.stemcr.2023.08.011
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Heart-derived collagen promotes maturation of engineered heart tissue (2023)
  • Author(s): Tani Hidenori、Kobayashi Eiji、Yagi Shinomi、Tanaka Keisuke、Kameda-Haga Kotaro、Shibata Shinsuke、Moritoki Nobuko、Takatsuna Kaworu、Moriwaki Taijun、Sekine Otoya、Umei Tomohiko C、Morita Yuika、Soma Yusuke、Kishino Yoshikazu、Kanazawa Hideaki、Fujita Jun、Hattori Shunji、Fukuda Keiichi、Tohyama Shugo
  • Journal Title: Biomaterials Volume: 299 Pages: 122174-122174
  • DOI: 10.1016/j.biomaterials.2023.122174
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Perspectives of hospitalized heart failure patients: preferred and perceived participation roles in treatment decisions (2023)
  • Author(s): Sekine Otoya、Kitakata Hiroki、Kohsaka Shun、Fujisawa Daisuke、Nakano Naomi、Shiraishi Yasuyuki、Kishino Yoshikazu、Katsumata Yoshinori、Yuasa Shinsuke、Fukuda Keiichi、Kohno Takashi
  • Journal Title: Heart and Vessels Volume: 38 Issue: 10 Pages: 1244-1255
  • DOI: 10.1007/s00380-023-02275-4
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Unlocking the Pragmatic Potential of Regenerative Therapies in Heart Failure with Next- Generation Treatments (2023)
  • Author(s): Yoshikazu Kishino, Keiichi Fukuda
  • Journal Title: Biomedicines Volume: 11 Issue: 3 Pages: 915-915
  • DOI: 10.3390/biomedicines11030915
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Effect of pulmonary artery pressure-guided therapy on heart failure readmission in a nationally representative cohort (2022)
  • Author(s): Yoshikazu Kishino, Toshiki Kuno, Aaqib H Malik, Gregg M Lanier, Daniel B Sims, Ernesto Ruiz Duque, Alexandros Briasoulis
  • Journal Title: ESC Heart Failure Volume: 9 Issue: 4 Pages: 2511-2517
  • DOI: 10.1002/ehf2.13956
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Recurrence of Myopericarditis Following mRNA COVID-19 Vaccination in a Male Adolescent (2022)
  • Author(s): Umei Tomohiko C.、Kishino Yoshikazu、Watanabe Keiko、Shiraishi Yasuyuki、Inohara Taku、Yuasa Shinsuke、Fukuda Keiichi
  • Journal Title: CJC Open Volume: 4 Issue: 3 Pages: 350-352
  • DOI: 10.1016/j.cjco.2021.12.002
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Scalable manufacturing of clinical-grade differentiated cardiomyocytes derived from humaninduced pluripotent stem cells for regenerative therapy (2022)
  • Author(s): Yuika Morita, Yoshikazu Kishino, Keiichi Fukuda, Shugo Tohyama
  • Journal Title: Cell Proliferation Volume: 55 Issue: 8
  • DOI: 10.1111/cpr.13248
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Recurrent pulmonary thrombosis after balloon pulmonary angioplasty in chronic thromboembolic pulmonary hypertension (2022)
  • Author(s): Tomohiko C Umei, Yoshikazu Kishino, Takashi Kawakami, Keiichi Fukuda
  • Journal Title: European Heart Journal Volume: 43 Issue: 37 Pages: 3593-3593
  • DOI: 10.1093/eurheartj/ehac419
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] The complement C3-complement factor D-C3a receptor signalling axis regulates cardiac remodelling in right ventricular failure (2022)
  • Author(s): Shogo Ito, Hisayuki Hashimoto, Yoshikazu Kishino,Yoshihiro Fukumoto, Keiichi Fukuda, Shinsuke Yuasa, et al.
  • Journal Title: Nature Communication Volume: 13 Issue: 1 Pages: 5409-5409
  • DOI: 10.1038/s41467-022-33152-9
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Social Isolation and Implementation of Advanced Care Planning Among Hospitalized Patients With Heart Failure (2022)
  • Author(s): Kitakata Hiroki、Kohno Takashi、Kohsaka Shun、Fujisawa Daisuke、Nakano Naomi、Sekine Otoya、Shiraishi Yasuyuki、Kishino Yoshikazu、Katsumata Yoshinori、Yuasa Shinsuke、Fukuda Keiichi
  • Journal Title: Journal of the American Heart Association Volume: 11 Issue: 21
  • DOI: 10.1161/jaha.122.026645
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Apixaban-Associated Diffuse Alveolar Hemorrhage in an Elderly Man with Multiple Complications (2022)
  • Author(s): Takuya Ozawa, Hideki Terai, Akiyoshi Kajino, Shiro Otake, Ayaka Saito, Miyuki Nishie, Yoshikazu Kishino, Hirofumi Kamata, Kentaro Hayashida, Makoto Ishii, Koichi Fukunaga
  • Journal Title: American Journal of Case Reports Volume: 23
  • DOI: 10.12659/ajcr.937809
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Production of functional cardiomyocytes and cardiac tissue from human induced pluripotent stem cells for regenerative therapy. (2021)
  • Author(s): Tani H, Tohyama S, Kishino Y, Kanazawa H, Fukuda K*.
  • Journal Title: J Mol Cell Cardiol. Volume: 164 Pages: 83-91
  • DOI: 10.1016/j.yjmcc.2021.11.008
  • Peer Reviewed / Int'l Joint Research