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. 2023 Nov 14;12:RP87081. doi: 10.7554/eLife.87081

Figure 1. Skeletal muscle organoid protocol and correlation to fetal development.

(A) Brightfield images of myogenic development stages, with corresponding cytokines/growth factors protocol applications. (B) Graph depicting organoid development in size (day 0, n = 51; day 3, n = 46; day 5, n = 48; day 7, n = 50; day 11, n = 50; day 17, n = 33; for each timepoint organoids from three independent derivations were measured). (C) Representative mesodermal T+, neuromesodermal T+, SOX2+, and paraxial mesodermal TBX6+ organoid expression (in live DAPI- cells) on day 5. (D) Representative organoid PAX3+, mesodermal SOX2-, and neural SOX2+ expression on day 7; PAX3/PAX7 coexpression on day 11. (E) Graph depicting qPCR values for anterior PAX3/UNCX/MEOX2/TBX18 and posterior TBX6/HES7 somitic mesodermal markers. (F) Graph depicting qPCR values for epaxial and hypaxial dermomyotomal PAX7/EN1/SIM1/LBX1 and neural crest TFAP2A/SOX10 markers. (G) Representative organoid neural plate border epithelial PAX3+/PAX7+/SOX2+/TFAP2A+, paraxial mesodermal PAX3+/SOX2-, and delaminating specified neural crest progenitor PAX3+/SOX10+ expression on day 17. (H) Representative organoid myogenic FastMyHC+, PAX7+, and neural SOX2+/TFAP2A+/SOX10+ expression on day 23. Dashed line represents the location of embryoid body embedded into Matrigel. Statistics: values at each timepoint represent the difference in mean relative expression for each gene (D7 = day 5–day 7, D11 = day 7–day 11, D17 = day 11–day 17, D23 = day 17–day 23) as derived by performing ordinary one-way ANOVA and Tukey’´s multiple-comparison tests (E, F). Scale bars: 200 μm (G), 100 μm (A, D, H), 50 μm.

Figure 1.

Figure 1—figure supplement 1. Lineage representation and organoid culture progression at early stages of differentiation protocol.

Figure 1—figure supplement 1.

(A) qPCR analysis depicts relative expression of pluripotent (OCT4, SOX2, NANOG), mesodermal/posterior somitic (BRACHYURY, MESOGENIN, TBX6, HES7), anterior somitic (PAX3, UNCX, TBX18, MEOX2), dermomyotomal (PAX7, SIM1, EN1, LBX1), and neural tube/crest markers (SOX2, PAX6, TFAP2A, SOX10). (B) Gating strategies and FACS intracellular quantification of PAX3 and PAX7 markers on days 10 and 16. (C) Tile scan overview on day 23 shows neural crest SOX10+ populations close to neural plate border epithelium (D) and SOX10-/PAX3+ populations at more outer locations (E). For each replicate, six organoids were pooled; gene expression was normalized to housekeeping gene. Statistics: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns: not significant. FACS plots, yellow: unstained population; red: isotype control; gray: PAX7+ or PAX3+ population. Scale bars, 500 uM (C), 200 uM (D, E).