As shown in Figure 3, activated satellite cells will turn into proliferative myoblasts through a gene regulation shift which involves down regulation of PAX7 and up-regulation of MYF5 and MYOD, both important transcription factors for muscle identity. During this transition, these transcription factors alter gene expression to transform the cell from a myoblast into a myocyte which will align and fuse with other neighboring myocytes to form multinucleated myotubes, which when performed in vitro is usually upon serum starvation (discussed later).  The cells here can be identified based on staining of important molecular markers, where up-regulation of Myogenin, Mrf4, and MHC mark terminally differentiated myotubes (Figure 4).   Concurrent with these genetic changes, internally the cell undergoes structural changes to form characteristics of sarcomeric organization to achieve contractile functionality through the coordinated use of actin, myosin, calcium, ATP hydrolysis, and other proteins (Figure 5).  Indeed, iPSC-derived human skeletal myotubes will spontaneously contract in culture which may be mediated by gap junctions which are present on skeletal muscle during development [Supplementary Video 1 & 20].  This is in contrast to many primary muscle cell lines, where spontaneous contractions are not as frequent and therefore contractions must be elicited exogenously. Additionally, skeletal muscle contraction promotes protein synthesis and myotube hypertrophy and is a vital component of the in vitro growing process [2122]. In an animal, single myotubes will align and form myofibrils, muscle fibers, fiber bundles, and finally muscle. However, re-creating this in a dish is not as trivial.