A novel approach to treat arrhythmias using embryonic stem-derived cardiac pacemaker cells

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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.