Figure 4. Micropatterned soft substrates enhance sarcomere formation, differentiation, and maturation of control iPSC-derived myotubes.

Representative bright field images of plated myotubes; myotubes on unpatterned substrates were irregularly distributed at 1 week (A) and 4 weeks (E) of differentiation. In contrast, highly-aligned myotubes were maintained on micropatterned lanes with controlled stiffness (B and F). MHC immunocytochemistry revealed that micropatterned soft substrates induced more multi-nuclear fusion in iPSC-derived myotubes (D) than the unpatterned culture (C). Represented by striated patterns of MHC-positive signals, sarcomere structures were visible in the myotubes on micropatterned soft substrates at 4 weeks of differentiation (H), although such subcellular characteristic was not identified in the myotubes plating on unpatterned rigid substrates (G). (I) Representative image field analyzed by digital image correlation (DIC) in the myotube contraction videos. The heatmap is created using contracted and at rest video frames, with positive displacement (red) in the longitudinal (X-axis) direction. (J) The displacement change quantifies individual contractions. Representative changes of maximum displacement from the entire field of view are plotted for three contraction events. (K) The displacements in the transverse and longitudinal direction during the course of a single contraction. The unpatterned myotubes had similar displacements in two directions, while the myotubes plated on micropatterned lanes had a much larger displacement longitudinally along the lane axis, with minimal displacement in the transverse direction. (L) Immunostaining against alpha-bungarotoxin was used to label acetylcholine receptors (AChR) (arrows) on MHC⁺ myotubes.