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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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Time course analysis revealed highly efficient myogenic differentiation of Puro-bulk MYOD1-hiPSCs. (a) Brightfield images showing the time course of myogenic differentiation of G418-bulk and Puro-bulk MYOD1-hiPSCs as well as control 409B2-MYOD1-hiPSCs. After day 7, G418-bulk MYOD1-hiPSCs featured nonmuscle cells with flattened morphology and showed fewer myotubes and larger spaces among myotubes than Puro-bulk and clonal MYOD1-hiPSCs. Scale bar, 100 μm. (b) Time course gene expression analysis of G418-bulk, Puro-bulk, and control 409B2-MYOD1-hiPSCs along with myogenic differentiation. The amount of cDNA was normalized to that of human-specific β-ACTIN and is presented as the expression relative to that in undifferentiated hiPSCs (NANOG and OCT3/4), that in the human myoblast cell line Hu5/KD3 differentiated for 3 days (CD56, total and endogenous MYOD1, MYOG, MYF6, MEF2C, MYH2, MYH7, and TMEM8C), or that in EKN3-MYOD1-hiPSCs differentiated for 5 days (Tg MYOD1). The data are presented as the mean ± SEM, n = 3. *, p < 0.05, **, p < 0.01 vs. 409B2-MYOD1-hiPSCs. ANOVA followed by post hoc Bonferroni test. (c) Time course ICC analysis of Puro-bulk MYOD1-hiPSCs for MyoD, MyoG, and MHC along with myogenic differentiation. The number of MyoD⁺ cells gradually decreased after day 7, while MHC⁺ cells appeared around day 5 and formed aligned myotubes from day 7. Scale bar, 100 μm.