Identification, Isolation and Characterization of HCN4-Positive Pacemaking Cells Derived from Murine Embryonic Stem Cells during Cardiac Differentiation
Identification, Isolation and Characterization of HCN4-Positive Pacemaking Cells Derived from Murine Embryonic Stem Cells during Cardiac Differentiation
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DOI：
10.1111/j.1540-8159.2009.02614.x
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发表时间：
2010-03-01
影响因子：
1.8
通讯作者：
Hisatome, Ichiro
中科院分区：
文献类型：
--
作者：
Morikawa, Kumi;Bahrudin, Udin;Hisatome, Ichiro
背景：生物起搏器的发展是治疗缓慢型心律失常的一种潜在方法。基于其特异性细胞标记物——超极化激活环核苷酸门控（hyperpolarization activated cyclic nucleotide-gated， HCN）4，可以从分化的胚胎干细胞（ES）中提取起搏器细胞。本研究的目的是建立一种鉴定、分离和表征由HCN4启动子驱动的GFP分化ES细胞的起搏细胞的方法。方法与结果：聚合酶链反应（PCR）筛选和southern blot分析显示，HCN4p-EGFP转基因稳定整合到小鼠AB1 ES细胞的染色体中。RT-PCR和免疫染色结果显示，HCN4p-EGFP ES细胞及其亲本AB1 ES细胞系的特异性心脏起搏器标志物表达相似。虽然HCN4p-EGFP转基因可能对一般中胚层分化有轻微影响，但对胚胎干细胞的多能性、心脏特异性因子和心脏收缩蛋白的转录以及胚胎干细胞向起搏器细胞分化的能力没有影响。电生理研究表明，hcn4p - gfp阳性细胞在2 mM Cs+处理下表现出自发动作电位，表达HCN4基因编码的超极化激活阳离子电流If。结论：利用HCN4p-EGFP基因稳定转染的方法，可以对ES细胞源性起搏细胞进行鉴定、分离和鉴定。（pace 2010; 33:29 -303）
Background: Development of biological pacemaker is a potential treatment for bradyarrhythmias. Pacemaker cells could be extracted from differentiated embryonic stem (ES) cells based on their specific cell marker hyperpolarization-activated cyclic nucleotide-gated (HCN)4. The goal of this study was to develop a method of identification, isolation, and characterization of pacemaking cells derived from differentiated ES cells with GFP driven by HCN4 promoter.Methods and Results: Polymerase chain reaction (PCR) screening and southern blot analysis revealed that HCN4p-EGFP trans-gene was stably integrated into the chromosome of mouse AB1 ES cells. RT-PCR and immunostaining results showed similar expression of the specific cardiac pacemaker markers of the HCN4p-EGFP ES cells and its parental AB1 ES cell lines. Although HCN4p-EGFP trans-gene may have slight effect on the general mesodermal differentiation, it had no effect on the pluripotency of ES cells, on the transcription of cardiac specific factors and cardiac contractile proteins, and on the capability of ES cells to differentiate into pacemaker cells. Electrophysiological study indicated that HCN4p-GFP-positive cells revealed the spontaneous action potential, which was slowed by the treatment with 2 mM Cs+, and expressed the hyperpolarization-activeted cation current If encoded by HCN4 gene.Conclusion: By the approach of using stable transfectant of HCN4p-EGFP gene, the identification, isolation, and characterization of ES cell-derived pacemaking cells could be carried out. (PACE 2010; 33:290-303)