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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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Transcriptome analyses revealed that Puro-bulk MYOD1-hiPSCs were capable of differentiating into more mature skeletal muscles. (a) Hierarchical clustering based on 2,794 genes identified as having significantly altered expression between undifferentiated MYOD1-hiPSCs (day 0) and skeletal muscles derived from control hiPSCs (409B2-MYOD1) (day 9) or human myoblast cell lines (Hu5/KD3). 2d, 3d, and 6d: 2 days, 3 days, and 6 days in myotube differentiation medium, respectively; Undiff.: undifferentiated iPSCs. The expression data were grouped using a hierarchical clustering algorithm (ward. D2) by average linkage with the Pearson distance and visualized by ComplexHeatmap ver. 2.13.1 (https://github.com/jokergoo/ComplexHeatmap)⁶⁵ and dendsort ver. 0.3.4 (https://github.com/evanbiederstedt/dendsort)⁶⁶. (b) Quantitative analysis of the number of skeletal muscle-related GO terms significantly enriched in each sample indicated (Puro-bulk vs. G418-bulk and Puro-bulk vs. Puro-clones) (FDR q-value < 0.01). (c) Quantitative analysis of the number of skeletal muscle-related pathways significantly enriched in each sample indicated (Puro-bulk vs. G418-bulk and Puro-bulk vs. Puro-clones) (FDR q-value < 0.01). (d) GO enrichment analysis. The top 10 upregulated gene sets in Puro- and G418-bulk with FDR q-values < 0.01 are shown. The red bar indicates muscle-related gene sets, and the blue bar indicates non-muscle-related gene sets. (e) Pathway gene set enrichment analysis. The top 10 pathways significantly enriched in Puro- and G418-bulk with FDR q-values < 0.01 are shown. The red bar indicates muscle-related pathways, and the blue bar indicates non-muscle-related pathways. (f) 3D image of the PCA based on 10,000 genes among undifferentiated MYOD1-hiPSCs (409B2_Undiff, Puro-bulk_Undiff), skeletal muscles derived from control iPSCs (409B2-MYOD1), 201B7-MYOD1-hiPSCs (Puro-bulk and 5 Puro-clones) (2 days in myotube differentiation medium), various Puro-bulk MYOD1-hiPSCs (TIGE9-, EKN3-, and YFE16) (3 and 6 days in myotube differentiation medium), and human myoblast cell line (Hu5/KD3)-derived skeletal muscles. The distinct distributions of undifferentiated cells and differentiated skeletal muscles are indicated. The gray arrow indicates the ‘virtual myogenic timeline’. 2d, 3d, and 6d: 2 days, 3 days, and 6 days in myotube differentiation medium, respectively; Undiff. : undifferentiated iPSCs; C1-C6: clones 1 to 6 of Puro-clones.