Primers for qPCR.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells.

在现有研究中，研究者们成功地将成年干细胞中的毛囊相关多能干细胞（HAP）诱导为具有搏动功能的房室心肌细胞亚型。这些HAP干细胞位于毛囊隆突区域，是成年干细胞的一种。通过在培养过程中结合异丙肾上腺素、激活素A、骨形态发生蛋白4（BMP4）、基本成纤维细胞生长因子（bFGF）和环孢素A（CSA），HAP干细胞分化为房性心肌细胞。在培养过程中，结合激活素A、BMP4、bFGF、Wnt生产抑制剂-4（IWP4）和血管内皮生长因子（VEGF）时，HAP干细胞分化为室性心肌细胞。通过特异性染色抗肌球蛋白轻链2a（MLC2a）抗体，对分化后的房性心肌细胞进行染色；而室性心肌细胞则通过特异性染色抗肌球蛋白轻链2v（MLC2v）抗体。定量聚合酶链反应（qPCR）结果显示，在房性心肌细胞中显著表达MLC2a，在室性心肌细胞中显著表达MLC2v。在钙离子成像中，分化后的房性和室性心肌细胞均显示出特征性的波形。分化后的房性和室性心肌细胞形成了长心肌纤维，并以功能性合胞体的形式搏动，其结构与成年心肌细胞相似。本研究结果证实，从HAP干细胞中诱导产生心肌细胞亚型，即房性和室性心肌细胞，是可行的。