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Primers for qPCR.

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posted on 2024-01-26, 18:31 authored by Nanako Takaoka, Michiko Yamane, Ayami Hasegawa, Koya Obara, Kyoumi Shirai, Ryoichi Aki, Hiroyasu Hatakeyama, Yuko Hamada, Nobuko Arakawa, Manabu Tanaka, Robert M. Hoffman, Yasuyuki Amoh

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.

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