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In vitro production of insulin-responsive skeletal muscle tissue from mouse embryonic stem cells by spermine-induced differentiation method

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The treatment of an embryoid body with spermine for a short duration can trigger the generation of a 3-dimensional multilayer myotube sheet (MMTS) that shows pulsatile activity. MMTS was previously characterized as a model of skeletal muscle tissue. In the present work, the insulin responsiveness of MMTS was investigated because it is an essential function for a model of skeletal muscle. The glucose uptake activity of MMTS was analyzed by confocal microscopy using fluorescent glucose analogs, namely 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) and its l-glucose counterpart, 2-NBDLG. The specific uptake rate of glucose was estimated from the difference between the fluorescent signals of 2-NBDG and 2-NBDLG. It was enhanced by insulin stimulation to 3.6 times higher than the control without insulin, and this insulin responsiveness was maintained for 5 days. The advantages of the 3-dimensional structure of MMTS are discussed in the contexts of its potential in vivo and in vitro uses.

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We thank Prof. Emer. Hideaki Matsuoka of Tokyo University of Agriculture and Technology for his valuable advice in bioimaging. The work was supported in part by the Strategic Research Promotion Program, the Ministry of Education, Culture, Sports, Science, and Technology, on the research subject “Development of Next Generation Bioresources”.

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Correspondence to Mikako Saito.

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Saito, M., Ishida, A. & Nakagawa, S. In vitro production of insulin-responsive skeletal muscle tissue from mouse embryonic stem cells by spermine-induced differentiation method. Human Cell 30, 162–168 (2017).

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