Fluorescent reporter lines in human iPS cells offer useful platforms to study cardiac subtype specification, cell-based therapies of cardiac disease and drug development.

• Project/Area Number: 26501003
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Regenerative medicine
• Research Institution: Tottori University
• Principal Investigator: Shirayoshi Yasuaki 鳥取大学, 医学(系)研究科(研究院), 准教授 (90249946)
• Co-Investigator(Kenkyū-buntansha): 李 佩俐 鳥取大学, 医学(系)研究科(研究院), 助教 (40464292) ; 森川 久未 鳥取大学, 医学部附属病院, 助教 (90707217)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2014: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: ヒトES細胞 / ヒトiPS細胞 / イオンチャネル / 蛍光タンパク質 / 心筋分化 / 再生医療 / ペースメーカ細部 / ペースメーカ細胞 / iPS細胞 / 可視化 / 心筋分化誘導 / HCN4 / MLC2v / CRISPR/Cas9 / 心筋細胞 / HCN4イオンチャネル

Outline of Final Research Achievements

In order to use human ES/iPS cells in the fields of regenerative medicine and drug development, it is crucial to identify functionally distinct cardiac subtypes and to study their functional aspects in healthy and diseased conditions. To isolate nodal pacemaker cells and ventricular cardiomyocytes, we have established the dual cardiac fluorescent reporter human ES/iPS cells, in which HCN4 ion channel and MLC2v myosin light chain genes are knocked in with eGFP and mCherry, respectively.
Isolated HCN4-eGFP-positive (HCN4+) cells expressed endogenous HCN4 and exhibited action potential characteristic of a nodal-phenotype including If current. Using imaging techniques, we demonstrated that HCN4+ cells established electrical coupling with HL-1 atrial CMs. These results demonstrated the potential of human ES/iPS cells-derived HCN4+ cells to act as a rate-responsive biological pacemaker.

Research Products

• [Int'l Joint Research] コロンビア大(米国)
• [Journal Article] Molecular mechanisms underlying the pilsicainide-induced stabilization of hERG proteins in transfected mammalian cells (2017)
  • Author(s): Onohara, Takeshi; Hisatome, Ichiro; Kurata, Yasutaka; Li, Peili; Notsu, Tomomi; Morikawa, Kumi; Otani, Naoyuki; Yoshida, Akio; Iitsuka, Kazuhiko; Kato, Masaru; Miake, Junichiro; Ninomiya, Haruaki; Higaki, Katsumi; Shirayoshi, Yasuaki; Nishihara, Takashi; Itoh, Toshiyuki; Nakamura, Yoshinobu; Nishimura, Motonobu
  • Journal Title: J. Arrhythmia, Volume: 32 Issue: 3 Pages: 226-233
  • DOI: 10.1016/j.joa.2016.09.003
  • Peer Reviewed / Open Access
• [Journal Article] M3 Muscarinic Receptor Signaling Stabilizes a Novel Mutant Human Ether-a-Go-Go-Related Gene Channel Protein via Phosphorylation of Heat Shock Factor 1 in Transfected Cells (2016)
  • Author(s): Mahati E., Li P., Kurata Y., Maharani N., Nobuhito I., Sakata S., Ogura K., Miake J., Aiba T., Shimizu W., Nakasone N., Ninomiya H., Higaki K., Yamamoto K., Nakai A., Shirayoshi Y., Hisatome I.
  • Journal Title: Circulation Journal Volume: 80 Issue: 12 Pages: 2443-2452
  • DOI: 10.1253/circj.CJ-16-0712
  • NAID: 130005284978
  • ISSN: 1346-9843, 1347-4820
  • Peer Reviewed / Open Access
• [Journal Article] Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1. (2016)
  • Author(s): Kondo T., Hisatome I., Yoshimura S., Mahati E., Notsu T., Li P., Iitsuka K., Kato M., Ogura K., Miake J., Aiba T., Shimizu W., Kurata Y., Sakata S., Nakasone N., Ninomiya H., Nakai A., Higaki K., Kawata Y., Shirayoshi Y., Yoshida A., Yamamoto K.
  • Journal Title: Journal of Arrhythmia Volume: 32 Issue: 5 Pages: 433-440
  • DOI: 10.1016/j.joa.2015.10.005
  • NAID: 120007146982
  • Peer Reviewed / Open Access
• [Journal Article] Prion Protein and Stage Specific Embryo Antigen 1 as Selection Markers to Enrich the Fraction of Murine Embryonic Stem Cell-Derived Cardiomyocytes. (2016)
  • Author(s): Ikeda N, Nakayama Y, Nakazawa N, Yoshida A, Ninomiya H, Shirayoshi Y.
  • Journal Title: Yonago Acta Med. Volume: 59 Pages: 126-134
  • NAID: 120005775321
  • Peer Reviewed
• [Journal Article] Electrophysiological Properties of iPS Cell-Derived Cardiomyocytes from a Patient with Long QT Syndrome Type 1 Harboring the Novel Mutation M437V of KCNQ1 (2016)
  • Author(s): Tatsufumi Sogo; Kumi Morikawa; Yasutaka Kurata; Peili Li; Takafumi Ichinose; Shinsuke Yuasa; Daizou Nozaki; Junichiro Miake, Haruaki Ninomiya, Wataru Shimizu, Keiichi Fukuda, Kazuhiro Yamamoto, Yasuaki Shirayoshi and Ichiro Hisatome.
  • Journal Title: Regenerative Therapy Volume: 4 Pages: 9-17
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] 生物学的ペースメーカの展望 (2016)
  • Author(s): 森川久未、白吉安昭、久留一郎
  • Journal Title: 心電図 Volume: 35 Pages: 264-268
  • NAID: 130005139479
• [Journal Article] E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells. (2015)
  • Author(s): Li P, Kurata Y, Maharani N, Mahati E, Higaki K, Hasegawa A, Shirayoshi Y, Yoshida A, Kondo T, Kurozawa Y, Yamamoto K, Ninomiya H, Hisatome I.
  • Journal Title: J Mol Cell Cardiol. Volume: 86 Pages: 138-146
  • DOI: 10.1016/j.yjmcc.2015.07.018
  • Peer Reviewed / Open Access
• [Journal Article] Stabilization of Kv1.5 channel protein by the inotropic agent olprinone. (2015)
  • Author(s): Endo R, Kurata Y, Notsu T, Li P, Morikawa K, Kondo T, Ogura K, Miake J, Yoshida A, Shirayoshi Y, Ninomiya H, Higaki K, Kuwabara M, Yamamoto K, Inagaki Y, Hisatome I.
  • Journal Title: Eur J Pharmacol. Volume: 765 Pages: 488-494
  • DOI: 10.1016/j.ejphar.2015.09.013
  • Peer Reviewed / Open Access
• [Journal Article] Molecular Mechanisms Underlying Urate-Induced Enhancement of Kv1.5 Channel Expression in HL-1 Atrial Myocytes (2015)
  • Author(s): Maharani N, Ting YK, Cheng J, Hasegawa A, Kurata Y, Li P, Nakayama Y, Ninomiya H, Ikeda N, Morikawa K, Yamamoto K, Makita N, Yamashita T, Shirayoshi Y, Hisatome I.
  • Journal Title: Circulation Journal Volume: 79 Issue: 12 Pages: 2659-2668
  • DOI: 10.1253/circj.CJ-15-0416
  • NAID: 130005110934
  • ISSN: 1346-9843, 1347-4820
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Carvedilol Suppresses Apoptosis and Ion Channel Remodelling of HL-1 Cardiac Myocytes Expressing E334K cMyBPC. (2015)
  • Author(s): Endo R, Bahrudin U, Notsu T, Tanno S, Onohara T, Yamaguchi S, Ikeda N, Surastri B, Nakayama Y, Ninomiya H, Shirayoshi Y, Inagaki Y, Yamamoto K, Yoshida A, Hisatome I.
  • Journal Title: Drug Res (Stuttg). Volume: 66 Issue: 03 Pages: 126-129
  • DOI: 10.1055/s-0035-1555772
  • Peer Reviewed / Open Access
• [Journal Article] P19 cells overexpressing Lhx1 differentiate into the definitive endoderm by recapitulating an embryonic developmental pathway. (2015)
  • Author(s): Hasagawa A, Shirayoshi Y.
  • Journal Title: Yonago Acta medica Volume: 58 Pages: 15-22
  • NAID: 120005596734
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Apoptosis induced by an uromodulin mutant C112Y and its suppression by topiroxostat. (2014)
  • Author(s): Utami SB, Mahati E, Li P, Maharani N, Ikeda N, Bahrudin U, Munemura C, Hosoyamada M, Yamamoto Y, Yoshida A, Nakayama Y, Higaki K, Nanba E, Ninomiya H, Shirayoshi Y, Ichida K, Yamamoto K, Hosoya T, Hisatome I.
  • Journal Title: Clin Exp Nephrol. 2014 Sep 20. Volume: 00 Issue: 4 Pages: 000-0
  • DOI: 10.1007/s10157-014-1032-8
  • Peer Reviewed
• [Journal Article] Instability of KCNE1-D85N that Causes Long QT Syndrome: Stabilization by Verapamil (2014)
  • Author(s): Sakata S, Kurata Y, Li P, Notsu T, Morikawa K, Miake J, Higaki K, Yamamoto Y, Yoshida A, Shirayoshi Y, Yamamoto K, Horie M, Ninomiya H, Kanzaki S, Hisatome I.
  • Journal Title: Pacing Clinical Electrophysiology Volume: Epub ahead of print Issue: 7 Pages: 1-11
  • DOI: 10.1111/pace.12360
  • Peer Reviewed / Open Access
• [Presentation] Fluorescent human iPS reporter lines offer useful platforms to study cardiac subtype specification and cell-based therapies of cardiac disease. (2017)
  • Author(s): Yasuaki Shirayoshi, Kenta Fukumura, Kumi Morikawa, Ichiro Hisatome
  • Organizer: ISSCR 2017
  • Place of Presentation: Boston, USA
  • Year and Date: 2017-06-14
  • Int'l Joint Research
• [Presentation] Fluorescent Human iPS/ES Reporter Lines Offer the Opportunity to Track Cardiac Subtype Cells in Cell-based Therapies and Drug Development (2017)
  • Author(s): Yasuaki Shirayoshi, Kumi Morikawa, Kenta Fukumura, Ichiro Hisatome
  • Organizer: 第81回 日本循環器学会学術集会
  • Place of Presentation: 石川県立音楽堂他、金沢
  • Year and Date: 2017-03-17
• [Presentation] 虚血性心不全モデルに対する脂肪幹細胞シートと心不全薬の併用効果の検討 (2017)
  • Author(s): 山本堅士郎、丹野翔伍、足立真彩、森川久未、大月優樹、大谷直由、陶山淑子、八木俊路朗、荻野和秀、三明淳一朗、白吉安昭、山本一博、西村元延、久留一郎
  • Organizer: 第16回 日本再生医療学会総会
  • Place of Presentation: 仙台国際センター、仙台
  • Year and Date: 2017-03-07
• [Presentation] ヒトiPS細胞に由来するHCN4陽性心筋前駆細胞の解析 (2016)
  • Author(s): 福村健太、横井文香、森川久未、野崎大蔵、久留一郎、白吉安昭
  • Organizer: 第39回 日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜、横浜
  • Year and Date: 2016-11-30
• [Presentation] 選別純化したヒト多能性幹細胞由来分化誘導心筋に関する電気生理学的特性と薬剤応答性の評価 (2016)
  • Author(s): 白吉安昭、森川久未、山内香織、横井文香、福村健太、野崎大蔵、末盛博文、久留一郎
  • Organizer: 第43回 日本毒性学会
  • Place of Presentation: ウインクあいち、名古屋
  • Year and Date: 2016-06-29
• [Presentation] Generation of Biological Pacemaker Derived fromHuman Pluripotent Stem Cells (2016)
  • Author(s): 白吉安昭
  • Organizer: 第80回日本循環器学会学術集会
  • Place of Presentation: 仙台国際センター、仙台
  • Year and Date: 2016-03-18
  • Invited
• [Presentation] Electrophysiological properties of iPS cell-derived cardiomyocytes from patients with long QT syndrome type 1 harvoring a novel mutation KCNQ1. (2015)
  • Author(s): Yasutaka Kurata, Peili Li, Kumi Morikawa, Junichiro Miake, Kazuhiro Yamamoto, Yasuaki Shirayoshi, Ichiro Hisatome
  • Organizer: 第３０回日本不整脈学会学術集会・第３２回日本心電学会学術集会合同学術大会
  • Place of Presentation: 国立京都国際会館、京都
  • Year and Date: 2015-07-28
• [Presentation] Isolation and Characterrization of HCN4 positive cells derived from human embryonic stem cells. (2015)
  • Author(s): Morikawa K, Nozaki D, Shirayoshi Y, Hisatome I.
  • Organizer: International Society for Stem cell Rsearch 13th Annual Meeting
  • Place of Presentation: Stocjholm Sweden
  • Year and Date: 2015-06-24 – 2015-06-27
• [Presentation] Isolation and Characterization of HCN4 positive cells derived from human embryonic stem cells (2015)
  • Author(s): Kumi Morikawa, Nozaki Daizou, Yasuaki Shirayoshi, Ichiro Hisatome.
  • Organizer: ISSCR (International Society of Stem Cell Research) 13th Annual Meeting
  • Place of Presentation: STockholm, Sweden
  • Year and Date: 2015-06-24
  • Int'l Joint Research
• [Presentation] HCN4 positive cells derived from pluripotent stem cells show automaticity and pace making ability. (2015)
  • Author(s): Shirayoshi Y, Morikawa K, Miake J, Hisatome I.
  • Organizer: 第79回日本循環器学会学術集会
  • Place of Presentation: 大阪国際会議場等、大阪
  • Year and Date: 2015-04-24 – 2015-04-26
  • Invited
• [Presentation] Electrical properties of human iPS cell^derived cardiac myocytes from the patient with ling QT syndrome harboring a novel mutation of KCNQ1. (2014)
  • Author(s): Sogo T, Ichinose T, Ikeda N, Shirayoshi Y, Morikawa K, Kurata Y, Miake J, Yamamoto K, Yuasa S, Fukuda K, Hisatome I.
  • Organizer: 第29回日本不整脈学会、第31回日本心電図学会、合同学術大会
  • Place of Presentation: 東京プリンスホテル、東京
  • Year and Date: 2014-07-22 – 2014-07-25
• [Book] The 7th Takao International Symposium procceding Book (2015)
  • Author(s): Morikawa K, et al.
  • Publisher: Springer
• [Remarks] 鳥取大学大学院 医学系研究科 機能再生医科学専攻 遺伝子再生医療学講座 再生医療学
  • URL: http://www.tottori-u.ac.jp/remed/
• [Remarks] 生物学的ペースメーカの作製とその応用
  • URL: http://www.tottori-u.ac.jp/introduction/grad/297/430/563/1345.html
• [Remarks] 鳥取大学大学院 医学系研究科 機能再生医科学専攻 遺伝子再生医療学講座 再生医療学部門
  • URL: http://www.med.tottori-u.ac.jp/remed/
• [Remarks] 生物学的心臓ペースメーカー細胞の作製とその応用
  • URL: http://www.med.tottori-u.ac.jp/introduction/grad/297/430/563/1345.html
• [Remarks] 鳥取大学大学院 医学系研究科 機能再生医科学専攻 遺伝子再生医療学講座 再生医療学部門
• [Remarks] 生物学的心臓ペースメーカー細胞の作製とその応用