Figure 2
MyoTACTIC supports formation of hMMTs with aligned myotubes exhibiting hypertrophy and adult myosin heavy chain expression. (a) Representative phase-contrast images of hMMTs depicting the remodeling of the extracellular matrix by human myoblasts over time. Day 0 marks the time for switching to differentiation media. Scale bar 500 µm. (b) Schematic diagram of the timeline for hMMT culture. hMMTs are cultured in growth media (GM) lacking bFGF for the first two days (day -2 to day 0) and then the media is switched to differentiation media (DM) on Day 0. (c) Representative confocal stitched image of a hMMT cultured for 2 weeks, immunostained for sarcomeric α-actinin (SAA, red) and exposed to DRAQ5 (blue) to counter stain the nuclei. Scale bar 500 µm. (d) Dot plot indicating the width of hMMTs over the course of culture time. (n = minimum of 16 hMMTs from 3 muscle patient donors per time point). (e) Representative confocal images of myotubes formed in hMMTs and immunostained for SAA (red) and nuclei (blue) after 7, 10, and 14 days in differentiation media. Scale bar 50 µm. (f) Quantification of hMMT myotube diameter over time. **p < 0.01, ***p < 0.001 (n = minimum of 9 hMMTs from 3 muscle patient donors per time point). (g) Representative western blot images of myosin heavy chain (MHC) isoforms (fast and slow), SAA, and β-tubulin over culture time (Day 7, 10, and 14). The blots were cropped and stained separately (see Methods for more detail). Full length blots are presented in Supplementary Fig. 8. (h–i) Bar graph quantification of relative (h) MHC-fast and (i) MHC-slow protein expression in hMMTs over culture time. *p < 0.05 (n = 3 blots from 3 muscle patient donors, where each blot was run in a set of single experiment using lysate of 4 hMMTs (per time point) from a single patient donor lysed together. Blots were then processed in parallel to generate the bar graphs shown in (h,i)). In (d,f) each symbol represents data from one patient donor. In (h,i) values are reported as mean ± SEM. In (f,h,i) significance was determined by one-way ANOVA followed by multiple comparisons to compare differences between groups using Tukey’s multiple comparisons test.
