MED64 Abstracts

Cardiac myocytes derived from Nkx2.5-GFP knock-in murine embryonic stem cell: Electrophysiological differentiation and feasibility of transplantation

Jong-Kook Lee 1 , Kyoko Hidaka 2 , Keiko Miwa 1 , Rong-qian Shi 1 , Gen Itoh 3 , Takayuki Morisaki 2 , Itsuo Kodama 1

Circulation. 2002:106.II-68
(American Heart Association 75th Annual Scientific Meeting)

1 Dept. of Circulation, Res Inst of Environ Med, Nagoya University , 2 Dept. of Bioscience, National Cardiovascular Center Research Institute, 3 Dept. of Pathology, Aichi Medical University

Background: Electrophysiological differentiation of embryonic stem (ES) cell-derived cardiac myocytes (ESCMs) has not been clarified. We investigated the stage-dependent changes of pacemaking activity and action potential (AP) configuration during a long-term (3W) culture of myocytes derived from Nkx2.5-GFP knock-in ES cells, and tested their feasibility for in-vitro and in-vivo transplantation. Methods: Nkx2.5-GFP knock-in murine ES cells were established by inserting GFP into Nkx2.5 allele. Embryoid bodies obtained with "hanging-dropEmethod 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 electrodes (MED64) to examine their electrical coupling. In-vivo transplant was conducted by injecting 5´104 ESCMs into left ventricular wall of SCID mice, and ECGs were recorded after 7 days. Results: 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 ± 45 bpm, n=20). APs at this stage had prominent pacemaker depolarization, slow upstroke phase, and long duration (max diastolic potential -48 ± 2 mV; APD90 95 ± 15 ms, 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 ± 6 mV and 38 ± 2 ms, n=5) like atrial or ventricular myocytes. ESCMs co-cultured with neonatal ventricular myocytes on MED64 for 7 days showed complete synchronization of spontaneous excitation. In-vivo transplantation of ESCMs in 3 mice did not induce any arrhythmias or other abnormalities in ECGs. Conclusion: Cardiac myocytes derived from Nkx2.5-GFP knock-in ES cells can differentiate into matured atrial cells, ventricular cells or SA node cells. They are able to form functional syncitium with preexisting cardiac tissues.