Maltsev V A, Wobus A M, Rohwedel J, Bader M, Hescheler J
Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Freie Universität Berlin, Germany.
Circ Res. 1994 Aug;75(2):233-44. doi: 10.1161/01.res.75.2.233.
Cardiomyocytes differentiated in vitro from pluripotent embryonic stem (ES) cells of line D3 via embryo-like aggregates (embryoid bodies) were characterized by the whole-cell patch-clamp technique during the entire differentiation period. Spontaneously contracting cardiomyocytes were enzymatically isolated by collagenase from embryoid body outgrowths of early, intermediate, and terminal differentiation stages. The early differentiated cardiomyocytes exhibited an outwardly rectifying, transient K+ current sensitive to 4-aminopyridine and an inward Ca2+ current but no Na+ current. The Ca2+ current showed all features of L-type Ca2+ current, being highly sensitive to 1,4-dihydropyridines but not to omega-conotoxin. Cardiomyocytes of intermediate stage were characterized by the additional expression of cardiac-specific Na+ current, the delayed K+ current, and If current. Terminally differentiated cardiomyocytes expressed a Ca2+ channel density about three times higher than that of early stage. In addition, two types of inwardly rectifying K+ currents (IK1 and IK,Ach) and the ATP-modulated K+ current were found. During cardiomyocyte differentiation, several distinct cell populations could be distinguished by their sets of ionic channels and typical action potentials presumably representing cardiac tissues with properties of sinus node, atrium, and ventricle. Reverse transcription polymerase chain reaction revealed the transcription of alpha- and beta-cardiac myosin heavy chain (MHC) genes synchronously with the first spontaneous contractions. Transcription of embryonic skeletal MHC gene at intermediate and terminal differentiation stages correlated with the expression of Na+ channels. The selective expression of alpha-cardiac MHC gene in ES cell-derived cardiomyocytes was demonstrated after ES cell transfection of the LacZ construct driven by the alpha-cardiac MHC promoter region followed by ES cell differentiation and beta-galactosidase staining. In conclusion, our data demonstrate that ES cell-derived cardiomyocytes represent a unique model to investigate the early cardiac development and permit pharmacological/toxicological studies in vitro.
通过胚胎样聚集体（胚状体）从D3系多能胚胎干细胞体外分化而来的心肌细胞，在整个分化期间采用全细胞膜片钳技术进行表征。自发收缩的心肌细胞通过胶原酶从早期、中期和终末分化阶段的胚状体生长物中酶解分离出来。早期分化的心肌细胞表现出对4-氨基吡啶敏感的外向整流性瞬时钾电流和内向钙电流，但无钠电流。钙电流表现出L型钙电流的所有特征，对1,4-二氢吡啶高度敏感，但对ω-芋螺毒素不敏感。中期心肌细胞的特征是额外表达心脏特异性钠电流、延迟钾电流和If电流。终末分化的心肌细胞表达的钙通道密度比早期高约三倍。此外，还发现了两种内向整流钾电流（IK1和IK,Ach）以及ATP调节的钾电流。在心肌细胞分化过程中，几个不同的细胞群体可以通过它们的离子通道组和典型动作电位来区分，这些电位可能代表具有窦房结、心房和心室特性的心脏组织。逆转录聚合酶链反应显示，α和β心肌肌球蛋白重链（MHC）基因的转录与首次自发收缩同步。胚胎骨骼肌MHC基因在中期和终末分化阶段的转录与钠通道的表达相关。在由α-心脏MHC启动子区域驱动的LacZ构建体转染ES细胞后，进行ES细胞分化和β-半乳糖苷酶染色，证明了α-心脏MHC基因在ES细胞衍生的心肌细胞中的选择性表达。总之，我们的数据表明，ES细胞衍生的心肌细胞代表了一个研究早期心脏发育的独特模型，并允许进行体外药理学/毒理学研究。
