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

我们研究了胚胎干（ES）细胞衍生的心肌细胞（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偶联。通过在Nkx2.5等位基因中插入GFP，建立了Nkx2.5-GFP敲入小鼠ES细胞。悬滴法获得的胚状体于第10天分散，FACS分离gfp阳性细胞。分选后的escm培养21 d，膜片钳法记录APs。将escm与新生小鼠心室肌细胞融合培养在带有64个单极电极（MED64）的培养皿中，观察其电偶联。gfp阳性细胞的escm占总数的3-5%。大多数escm（约95%）心肌肌钙蛋白i免疫阳性，在FACS后7天，约85%的escm出现持续自发跳动（148±45bpm, n=20）。该阶段APs与SA结细胞相似，起搏器去极化明显，上搏期缓慢，持续时间长（最大舒张电位-48±2mV； APD_<90 bb0 95±15ms, n=5）。在第21天，只有20%的escm出现自发跳动，其余80%处于静止状态。与心房肌细胞和心室肌细胞相比，静止细胞的静息膜电位为负，持续时间更短（-68±6mV和38±2ms n=5）。escm与新生儿心室肌细胞在MED64上共培养7天，出现同步自发兴奋。Nkx2.5-GFP敲入ES细胞衍生的心肌细胞可分化为心房细胞、心室细胞或窦房结细胞等多种类型的心肌细胞。它们能够与天然心肌细胞形成功能性电偶联。

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.