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. 2023 May 25;13:8146. doi: 10.1038/s41598-023-34445-9

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Bulk MYOD1-hiPSCs established with puromycin selection achieved efficient myogenic differentiation and myotube maturation similar to that achieved by clonally established MYOD1-hiPSCs. (a) Schematic presentation of skeletal muscle differentiation from MYOD1-hiPSCs. Samples were collected at the times indicated with the closed triangles. (b) ICC analysis of myotubes derived from bulk and clonal MYOD1-hiPSCs established with G418 or puromycin selection and control 409B2-MYOD1-hiPSCs for the expression of MyoG and MHC at day 9 of differentiation. The nuclei were stained with Hoechst 33258. Scale bar, 200 μm. (c–e) Quantitative analysis of the parameters for skeletal muscle differentiation, including the proportions of MyoG⁺ cells among total cells (c), the proportions of MHC⁺ nuclei among total nuclei (d), and the proportion of MHC⁺ area in the total area (e). Puro-bulk MYOD1-hiPSCs exhibited differentiation potential similar to that of clonal MYOD1-hiPSCs and control 409B2-MYOD1-hiPSCs, whereas G418-bulk MYOD1-hiPSCs exhibited lower differentiation potential than the other MYOD1-hiPSCs. (f–h) Quantitative analysis of the parameters for myotube maturation, including the myotube thickness (f), number of nuclei per myotube (g), and MHC⁺ area per myotube (h). Puro-bulk MYOD1-hiPSCs exhibited myotube maturation potential similar to that of clonal MYOD1-hiPSCs and control 409B2-MYOD1-hiPSCs, whereas G418-bulk MYOD1-hiPSCs exhibited lower myotube maturation potential than the other MYOD1-hiPSCs. The data are presented as the mean ± SEM, n = 3. *p < 0.05, **p < 0.01. ANOVA followed by post hoc Bonferroni test.