Hi, I am working on iPSC to skeletal muscle differentiation using transgene-free small molecule protocol. The protocol was successful in many papers and has been used by many researchers. After completing the differentiation, I am trying to differentiate my cells further into a mature myotube state. This is the basic unit of muscle structures. The myocytes or muscle cells fuse together to form myotubes. They appear to have multiple nuclei and are long and thick in morphology. I have tried using higher concentrations of horse serum (5%, 10%) and tried to include insulin to accelerate the process. However, I do not see any multinucleation of the myocytes.

When characterizing the cells, the cells were found to express desmin, myod1, and myogenin which are muscle-related proteins.

Any and every suggestion from you will be helpful in my progress. Thank you in advance.

protocol used - obtained from Chal et al. (iPSC to skeletal muscle differentiation)

differentiation media - based on dmem f12, with insulin and horse serum.