Establishment of genetically modified biological pacemaker cell based on the in-silico model

由于ES源性心脏起搏器细胞的特性和谱系尚不清楚，我们建立了体外培养体系，通过形成胚状体（EB）将小鼠ES细胞分化为心脏细胞，并纯化Nkx2.5（+）细胞系，以绿色荧光蛋白（GFP）为报告细胞，研究ES源性心脏细胞自律性电生理特性的变化。胚胎干细胞在EB形成后6 ~ 8天内分化为心肌细胞。胚胎样体的心肌细胞免疫组织化学表达肌原肌氨酸，其水平取决于它们的搏动频率。异丙肾上腺素、卡巴卡醇、腺苷、利多卡因、硝苯地平、Ni^<2+>、Ba^<2+>、Cs^+和E-4031对心脏搏动频率均有影响，表明多种心脏特异性受体（β1-、M2-、a1受体）和离子通道（Na^+通道、t型、l型Ca^<2+>通道、I_<Kl>通道、I_…More通道、I_<kr>通道）在药理学上的表达。从EB中纯化的Nkx2.5/GFP（+）心肌细胞表达心脏特异性收缩蛋白和受体，代表与心脏起搏器细胞、心房或心室类型细胞相对应的电生理表型。具有自动性的es源性心肌细胞V_<max>、最大舒张电位和V_<th>呈显著正相关，提示具有自动性的es源性心肌细胞由Na^+通道依赖细胞和Ca^<2+>通道依赖细胞组成，尽管其自动性在药理学上是由Na^+通道、t型和l型Ca^<2+>通道、I_<kl>通道、I_f通道和I_<kr>通道的表达介导的。此外，我们发现它们对Ni^<2+>的自动性响应以微分时间依赖的方式显着衰减，而它们对Cs^+的灵敏度不变。这些结果表明，t型Ca^<2+>通道可能在es源性起搏器细胞分化早期对起搏器活性起关键作用，而I_f通道在整个分化过程中持续归因于自动性。综上所述，1)具有自动性的Nkx2.5（+）系细胞具有分化为多种起搏器细胞类型的潜力；2)es源性具有自动性的心脏细胞中t型Ca通道和I_f通道的差异可能是收集起搏器细胞的关键。少

Since the characteristics and the lineage of ES-derived cardiac pacemaker cells are still unknown, we established in vitro culture system to differentiate mouse ES cells into cardiac cells via formation of embryoid bodies(EB) and to purify Nkx2.5(+) cell lineages, using green fluorescence protein(GFP) as a reporter and studied the changes in electrophysiological characteristics of the ES-derived cardiac cells with automaticity. The ES cells differentiated into cardiac cells within 6 to 8 days after formation of EB. Cardiac myocytes in embryoid bodies immunohistochemically expressed the sarcomeric tropomyosine, of which levels depends on their frequency of pulsation. The frequency of their pulsation was influenced by isoproterenol, carbachol, adenosine, lidocaine, nifedipine, Ni^<2+>,Ba^<2+>,Cs^+ and E-4031, indicating the expression of various cardiac-specific receptors (β1-, M2-, A1-receptors) as well as ion channels (Na^+ channels, T-type, L-type Ca^<2+> channels, I_<Kl> channels I_f … More channels, and I_<kr> channels) pharmacologically. Nkx2.5/GFP(+) cardiomyocytes purified from EB expressing cardiac-specific contractile proteins and receptors represented electrophysiological phenotypes corresponding to the pacemaker-cell, atrial or ventricular types cells of the heart. There were significant positive correlations between V_<max>, maximum diastolic potential and V_<th> of the ES-derived cardiac cells with automaticity, suggesting the ES-derived cardiac cells with automaticity were composed of Na^+ channel-dependent and Ca^<2+>-channel-dependent cells, although their automaticity was mediated by expression of Na^+ channels, T-type, and L-type Ca^<2+> channels, I_<kl>, channels I_f channels, and I_<kr> channels, pharmacologically. Furthermore, we found that the response of their automaticity to Ni^<2+> was significantly attenuated in a differentiation time-dependent manner with their constant sensitivities to Cs^+. These results suggested that T-type Ca^<2+> channels might play the pivotal role for the pacemaker activities in ES-derived pacemaker cells in an early stage of their differentiation, while I_f channels persistently attributing to the automaticity during entire differentiation period. In conclusion, 1)Nkx2.5(+) lineage cells with automaticity possess the potential to differentiate into various pacemaker cell types, 2)the differences of T-type Ca channel and I_f channel in ES-derived cardiac cells with automaticity might be the key for collecting the pacemaker cells. Less