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. 2022 Jun 15;41(14):e110655. doi: 10.15252/embj.2022110655

Figure 1. Three‐component embryoids (TCEs) are permissive for myogenic specification.

Figure 1

  • A
    Schematic outlining embryonic and adult cell lines that were screened for their effects on the induction of the myogenic marker Pax7 in heterotypic human‐induced pluripotent stem cell (hiPSC) suspension embryoids.
  • B, C
    Expression of the human Pax7 mRNA in embryoids containing different ratios of hiPSCs and growth‐arrested embryonic fibroblasts (GAeFib) or hiPSCs and embryonic endothelial cells (eEC). Mixtures of the respective cell types were analyzed before (D0), and 7 and 13 days (D7 and D13) after aggregation.
  • D
    Human Pax7 mRNA expression in TCEs containing hiPSCs, GAeFib, and eEC.
  • E, F
    Size and human Pax7 mRNA expression in hiPSC mono‐aggregates or in TCEs when combined with biochemical induction of the Wnt and FGF pathways (iTCEs).
  • G
    Representative Pax7 immunostaining of sections of D7 iTCEs.
  • H–K
    mRNA expression of the mesoderm marker brachyury (T), the paraxial mesoderm markers mesogenin 1 (Msgn1) and T‐box 6 (Tbx6), and the dermomyotome marker Pax3 in hiPSCs and iTCEs at D7 and D13.
  • L
    Representative Pax7 immunostaining of a D13 iTCE.
  • M–P
    Representative stainings for actin, the extracellular matrix component laminin, the mesoderm marker homeobox 1 (Meox1), the endoderm marker alpha‐fetoprotein (AFP), and the mesoderm marker smooth muscle actin (SMA) in D7 or D13 iTCEs.
  • Q
    Flow cytometry quantification of the number of Pax7 positive cells in hiPSC mono‐aggregates, TCEs, and iTCEs.
  • R, S
    RNA fluorescence in situ hybridization for the myogenic commitment markers Myf5 and MyoD in cells derived from D13 iTCEs (R) and primary adult human skeletal muscle myogenic progenitors (hskMPs) (S) by flow cytometry and in representative post‐sort cytospins.

Data information: G, L, M–P, R, S. Scale bars = 25 μm. B–F, H–K, Q. Data are represented as means ± SEM from n ≥ 4 independent experiments. B–S. hiPSC donor 1. R, S. Representative sorts of n = 3 repeats are provided in Appendix Table S2. P‐values are *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using ANOVA followed by a Bonferroni post hoc test.