Establishment of ES cell-derived biological pacemaker using ion channel and its application to bradycardia arrhythmias

点击翻译按钮获取中文摘要

We are attempting to establish the cardiac pacemaker cells derived from embryonic stem (ES) cells. To visualize cardiac pacemaker cells in differentiating embryoid bodies (EBs), EGFP gene was knocked in at HCN4 locus, which encodes ion channels responsible for pacemaker activity. In the present study, we conducted to establish EGFP-knock in ES cell line at HCN4 locus. PCR screening revealed two positive clones as possible knock in ES cell line. Southern blot analysis showed that one of them, H7 clone, is correctly targeted at HCN4 locus. After induction to cardiac differentiation through the formation of EBs in H7 clone, the expression of GFP was specifically localized at their contracting region in the outgrowth of EBs. Analysis of cell sorting revealed that 0.1～0.3% of differentiated H7 cells in EBs were GFP positive and that 70～90% of the sorted GFP positive cells were morphologically homogenous and beat spontaneously. Patch clamp analysis showed that spontaneously beating cells revealed the comparable electrophysiological properties with cardiac pacemaker cells, such as automaticity and If current. These results assured that H7 cell line was suitable to investigate and purify cardiac pacemaker cells.