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

我们研究了胚胎干（ES）细胞来源的心肌细胞（ESCMs）在长期培养过程中的电生理分化以及与天然心肌细胞的电耦合。通过将GFP插入Nkx2.5等位基因中建立Nkx2.5-GFP敲入小鼠ES细胞。第10天将悬滴法获得的拟胚体分散，通过流式细胞仪分离GFP阳性细胞。分选后的ESCMs培养21天，用膜片钳技术记录AP。ESCMs还与新生小鼠心室肌细胞在具有64个单极电极（MED 64）的培养皿上汇合培养，以检查它们的电耦合。分选为GFP阳性细胞的ESCMs占总群体的3-5%。大多数ESCMs（>95%）对心肌肌钙蛋白I呈免疫阳性。术后7天，约85%的ESCMs出现持续的自发搏动（148± 45 bpm，n=20）。此期AP起搏除极显著，上升相缓慢，持续时间长（最大舒张期电位-48 2 mV; APD_（<90>95± 15）ms，n=5），与SA结细胞相似。第21天，只有20%的ESCMs显示自发搏动，其余80%静止。静息细胞的AP与心房肌或心室肌细胞一样，静息膜电位较负，持续时间较短（-68 ± 6 mV和38± 2 ms，n=5）。在MED 64上与新生心室肌细胞共培养7天的ESCMs显示自发兴奋的同步化。来源于Nkx2.5-GFP敲入ES细胞的心肌细胞可以分化成各种类型的心肌细胞，包括心房细胞、心室细胞或SA结细胞。它们能够与天然心肌细胞形成功能性电耦合。

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.