| Project/Area Number |
14570653
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Circulatory organs internal medicine
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| Research Institution | Nagoya University |
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Principal Investigator |
LEE Jong-kook Nagoya University, Research Institute of Environmental Medicine, Research Associate, 環境医学研究所, 助手 (60303608)
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| Co-Investigator(Kenkyū-buntansha) |
MORISAKI Takayuki National Cardiovascular Center, Department of Bioscience, Director, バイオサイエンス部, 部長 (30174410)
HIDAKA Kyoko National Cardiovascular Center, Department of Bioscience, Laboratory Chief, バイオサイエンス部, 室長 (00216681)
KODAMA Itsuo Nagoya University, Research Institute of Environmental Medicine, Professor, 環境医学研究所, 教授 (30124720)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | Regenerative medicine / Embryonic stem cell / Arrhythmia / Bio-pacemaker / ペースメーカー細胞 |
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| Research Abstract |
We investigated the electrophysiological differentiation of embryonic stem (ES) cell-derived cardiac myocytes (ESCMs) during a long-term culture and electrical coupling with native myocytes. Nkx2.5-GFP knock-in murine ES cells were established by inserting GFP into Nkx2.5 allele. Embryoid bodies obtained with "hanging-drop" method were dispersed on day 10,and GFP-positive cells were isolated by FACS. ESCMs after the sorting were cultured for 21 days to record APs using patch-clamp technique. ESCMs were also cultured with neonatal mouse ventricular myocytes confluently on a dish with 64 unipolar electrodes (MED64) to examine their electrical coupling. ESCMs sorted as GFP-positive cells were 3-5% of total population. Most of the ESCMs (>95%) were immunopositive for cardiac troponin I. On 7-day after FACS,〜85% ESCMs showed continuous spontaneous beating (148±45bpm, n=20). APs at this stage had prominent pacemaker depolarization, slow upstroke phase, and long duration (max diastolic potential -48±2mV ; APD_<90> 95±15ms, n=5) like SA node cells. On 21-day, only 20% ESCMs showed spontaneous beating, and remaining 80% quiescent. APs of the quiescent cells had more negative resting membrane potential and shorter duration (-68±6mV and 38±2ms n=5) like atrial or ventricular myocytes. ESCMs co-cultured with neonatal ventricular myocytes on MED64 for 7 days showed synchronization of spontaneous excitation. Cardiac myocytes derived from Nkx2.5-GFP knock-in ES cells can differentiate into various types of cardiac cells including atrial cells, ventricular cells or SA node cells. They are able to form functional electrical coupling with native cardiac myocytes.
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