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

ES细胞源离子通道生物起搏器的建立及其在心动过缓心律失常中的应用

• 批准号: 20590866
• 负责人: HISATOME Ichiro
• 金额: $ 3万
• 依托单位: Tottori University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2008
• 资助国家: 日本
• 起止时间: 2008 至 2010
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-20590866/
• 关键词: ペースメーカ; ES細胞; イオンチャネル; 生物学的ペースメーカ; HCN4; 自動能; 房室ブロックモデル; 細胞移植

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.

我们正在尝试从胚胎干细胞（ES细胞）中建立心脏起搏细胞。为了观察分化中的胚状体（EBs）中的心脏起搏细胞，在HCN 4位点敲入EGFP基因，其编码负责起搏活性的离子通道。在本研究中，我们进行了建立EGFP敲入ES细胞系HCN 4基因座。PCR筛选出2个阳性克隆，为ES细胞系中可能的敲除。Southern杂交分析表明，其中一个克隆H7正确定位于HCN 4位点。在H7克隆中通过EB的形成诱导心脏分化后，GFP的表达特异性地定位于EB生长的收缩区域。细胞分选分析显示，EB中0.1~0.3%的分化H7细胞为GFP阳性，70~90%的分选出的GFP阳性细胞形态均一，并有自发性搏动。膜片钳分析显示，自发搏动的细胞显示出与心脏起搏细胞相当的电生理特性，如自律性和If电流。这些结果表明，H7细胞系适合于研究和纯化心脏起搏细胞。

项目成果

特許

专利

• 发表时间: 2008

心血管疾患診療のエクセレンス

卓越的心血管疾病治疗

• 发表时间: 2008
• 作者: Mizuta E;et al.;古瀬祥之;久留一郎
• 通讯作者: 久留一郎

Long-term reliability of AAI mode pacing in patients with sinus node dysfunction and low Wenckebach block rate

• DOI: 10.1093/europace/eum290
• 发表时间: 2008-02-01
• 期刊: EUROPACE
• 影响因子: 6.1
• 作者: Adachi, Masamitsu;Igawa, Osamu;Hisatome, Ichiro
• 通讯作者: Hisatome, Ichiro

Regional difference in dynamical property of sinoatrial node pacemaking: Role of Na+ channel current

• DOI: 10.1529/biophysj.107.112854
• 发表时间: 2008-07-15
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Kurata, Yasutaka;Matsuda, Hiroyuki;Shibamoto, Toshishige
• 通讯作者: Shibamoto, Toshishige

ldentification, lsolation and Characterization of HCN4-Positive Pacemaking Cells Derived from Murine Embryonic Stem Cells during Cardiac Differentiation.

心脏分化过程中源自鼠胚胎干细胞的 HCN4 阳性起搏细胞的鉴定、分离和表征。

• 发表时间: 2010
• 期刊: Pacing Clin Electrophysiol. 33
• 作者: Morikawa K;et al.
• 通讯作者: et al.