Wobus A M, Rohwedel J, Maltsev V, Hescheler J
Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany.
Toxicol In Vitro. 1995 Aug;9(4):477-88. doi: 10.1016/0887-2333(95)00023-2.
Permanent cultures of cardiac cells described so far have limited value for studying cell biology and pharmacology of the developing heart because of the loss of proliferative capacity and cardiac-specific properties of cardiomyocytes during long-term cultivation. Pluripotent embryonic carcinoma (EC) and embryonic stem (ES) cells cultivated as permanent lines offer a new approach for studying cardiogenic differentiation in vitro. We describe cardiogenesis in vitro by differentiating EC and ES cells by way of embryo-like aggregates (embryoid bodies) into spontaneously beating cardiomyocytes. During cardiomyocyte differentiation three distinct developmental stages were defined by expression of specific action potentials and ionic currents measured by the whole-cell patch-clamp technique. Whereas early differentiated cardiomyocytes are characterized by action potentials and ionic currents typical for early pacemaker cells, terminally differentiated cardiomyocytes show action potentials and ionic currents inherent to ventricular-, atrial- or sinus nodal-like cells. These functional characteristics are in accordance with the expression of alpha- and beta-cardiac myosin heavy chain at early differentiation stages and the additional expression of ventricular-specific MLC-2V and atrial-specific ANF genes at terminal stages demonstrated by reverse transcription polymerase chain reaction (RT-PCR) analysis. Pharmacological studies performed by measuring chronotropic responses and by analysing the Ca(2+) channel activity correspond to data obtained with cardiac cells from living organisms. For testing the influence of exogenous compounds on cardiac differentiation the teratogenic compound retinoic acid (RA) was applied during distinct stages of embryoid body development. A temporally controlled influence of RA on cardiac differentiation and expression of cardiac-specific genes was found. We conclude that ES cell-derived cardiomyocytes provide an excellent cellular model to study early cardiac development and to perform pharmacological and embryotoxicological investigations.
迄今为止所描述的心脏细胞永久培养物，对于研究发育中心脏的细胞生物学和药理学价值有限，因为在长期培养过程中，心肌细胞会丧失增殖能力和心脏特异性特性。作为永久细胞系培养的多能胚胎癌（EC）细胞和胚胎干细胞（ES），为体外研究心脏发生分化提供了一种新方法。我们描述了通过将EC细胞和ES细胞分化为类胚胎聚集体（胚状体），进而分化为自发搏动的心肌细胞的体外心脏发生过程。在心肌细胞分化过程中，通过全细胞膜片钳技术测量特定动作电位和离子电流的表达，确定了三个不同的发育阶段。早期分化的心肌细胞具有早期起搏细胞典型的动作电位和离子电流特征，而终末分化的心肌细胞则表现出心室、心房或窦房结样细胞固有的动作电位和离子电流。这些功能特征与逆转录聚合酶链反应（RT-PCR）分析所显示的，在早期分化阶段α-和β-心肌肌球蛋白重链的表达，以及在终末阶段心室特异性MLC-2V和心房特异性ANF基因的额外表达相一致。通过测量变时反应和分析Ca(2+)通道活性进行的药理学研究，与从生物体心脏细胞获得的数据相符。为了测试外源性化合物对心脏分化的影响，在胚状体发育的不同阶段应用了致畸化合物视黄酸（RA）。发现RA对心脏分化和心脏特异性基因表达具有时间控制的影响。我们得出结论，ES细胞来源的心肌细胞为研究早期心脏发育以及进行药理学和胚胎毒理学研究提供了一个优秀的细胞模型。
