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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.

迄今为止，在将成年干细胞分化为心肌细胞亚型方面，仅取得了有限的成果。在本研究中，我们成功地将大鼠毛囊相关多能干细胞（HAP）诱导分化为具有搏动能力的房性和室性心肌细胞，这些HAP干细胞位于膨大部区域。在异丙肾上腺素、活化素A、骨形态发生蛋白4（BMP4）、碱性成纤维细胞生长因子（bFGF）和环孢素A（CSA）的联合作用下，HAP干细胞在培养过程中分化为房性心肌细胞。在活化素A、BMP4、bFGF、Wnt生产抑制剂-4（IWP4）和血管内皮生长因子（VEGF）的联合作用下，HAP干细胞分化为室性心肌细胞。分化的房性心肌细胞经抗肌球蛋白轻链2a（MLC2a）抗体特异性染色。室性心肌细胞经抗肌球蛋白轻链2v（MLC2v）抗体特异性染色。定量聚合酶链反应（qPCR）结果显示，房性心肌细胞中MLC2a表达显著，室性心肌细胞中MLC2v表达显著。分化的房性和室性心肌细胞在钙离子成像中显示出典型的波形。分化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。本研究结果证明了从HAP干细胞中诱导心肌细胞亚型，即房性和室性心肌细胞，是可行的。