#apaperaday: 3D human induced pluripotent stem cell–derived bioengineered skeletal muscles for tissue, disease and therapy modeling

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: 3D human induced pluripotent stem cell–derived bioengineered skeletal muscles for tissue, disease and therapy modeling

Today’s pick is from Nature Protocols by Pinton et al on 3D skeletal muscle cultures. Paper contains a detailed protocol (DOI: 10.1038/s41596-022-00790-8). I will here provide context and show some beautiful images, for details, read the paper.

Skeletal muscle is a complex tissue, containing multinucleated muscle fibers, muscle stem cells (satellite cells), but also connective tissue, motorneurons, blood vessels and immune cells. Cell cultures usually are a 2D layer of only muscle cells.

With IPSCs one now has the option to differentiate cells into fetal tissues. For skeletal muscles this can be done with only muscle, or with mixes of neurons and muscle, or mixes of endothelium and pericytes and muscle, or mixing everything. Authors here provide the how to.

The how to is not only how to get 3D muscle bundles (in hydrogel & between scaffolds, but also how to analyze them to ensure quality. 3D muscles can be used to study how myofibers are formed and whether there is pathology, e.g. misshapen nuclear envelopes for laminopathies.

Furthermore, the contractility and calcium handling can be studied in healthy systems and those carrying pathogenic variants. When there is a deficit for these aspects, also therapeutic approaches can be evaluated in this system.

Authors stress that FBS batches can influence procedures and that ideally batch tests are done before switching en masse to a new batch. Authors have both transgene induced and transgene free protocols to generate muscle bundles. Each has pros and cons:

The transgene (myoD overexpression) induces robust & controlled myogenic differentiation, but is not physiological so less suited to study fibre formation. The transgene free method (using a kit) is more physiological, but less robust & more variable, containing other cell types.

The muscle fiber bundles are about 8-10 mm long and 2 mm wide. mRNA and protein expression studies can be done for quality control. When neurospheres or neuronal precursor cells (also derived from IPSCs) are added, these can form motor neurons and neuromuscular junctions.

Authors are clear about the limitations (fetal muscle and of course not the same as a muscle in a body), but each model system has limitations. It is a matter of matching the questions to the model system. e.g. studying systemic delivery won’t work in this system.

But, studying pathology, fiber formation and screening therapeutic candidates will work. Authors stress that expertise is needed to work with the system – this is good advice and good to realize: you need time to invest into becoming an expert (generally through making mistakes).

I thank the authors for sharing their detailed protocol and like the modular format so people can mix and match as needed for their own work.