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. 2021 Jun 4;2(6):100298. doi: 10.1016/j.xcrm.2021.100298

Figure 2.

Figure 2

Electrical field stimulation (EFS) with a modified replating method induced the maturation, sarcomere formation, and contractile activities of hiPSC skeletal myotubes

(A) A schematic diagram of EFS with the modified replating method. EFS began at day 10 at 2 V, 2 ms, and 0.5 Hz, and the voltage was increased to 5 V and 10 V on days 12 and 14, respectively.

(B) Fusion index analysis of 409B2, 409B2 ex45KO, DMDΔ44-ctrl, and DMDΔ44 myotubes was differentiated using modified replating methods with EFS and gel culture.

(C) Immunocytochemical analysis of α-actinin of matured 409B2, 409B2 ex45KO, DMDΔ44-ctrl, and DMDΔ44 myotubes differentiated by the modified replating method with EFS. Scale bar represents 20 μM. The right panel is a quantitative analysis of the percentage of cells possessing a sarcomere-like pattern of α-actinin staining.

(D) Transmission electron microscopy analysis of mature 409B2, 409B2 ex45KO, DMDΔ44-ctrl, and DMDΔ44 myotubes differentiated by the modified replating method with EFS. Rectangles denote magnified areas. Scale bar represents 3 μm.

(E) Heatmap analyses using a SI8000 motion imaging system of motion pixels of matured 409B2, 409B2 ex45KO, DMDΔ44-ctrl, and DMDΔ44 myotubes differentiated using the previous and modified replating methods with EFS. The right panel is a quantitative analysis of the contracting area. Scale bar represents 100 μm.

Data represent the mean ± SD and were analyzed by an unpaired t test from six biological replicates (B and C) and five biological replicates (E). ∗p < 0.05.