3D Human Induced Pluripotent Stem Cell-derived Bioengineered Skeletal Muscles for Tissue, Disease and Therapy Modelling

Abstract Skeletal muscle is a complex tissue composed of multinucleated myofibres responsible for force generation, supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function is instrumental for investigating muscle pathophysiology and developing therapies. Here, we detail the modular 3D bioengineering of multilineage skeletal muscles from human induced pluripotent stem cells, which are first differentiated into myogenic, neural and vascular progenitor cells, and then combined within 3D hydrogels under tension to generate an aligned myofibre scaffold containing vascular networks and motor neurons. 3D bioengineered muscles recapitulate morphological and functional features of human skeletal muscle, including establishment of a pool of cells expressing muscle stem cell markers. Importantly, bioengineered muscles provide a high-fidelity platform to study muscle pathology, such as emergence of dysmorphic nuclei in muscular dystrophies caused by mutant lamins. The protocol is easy to follow for operators with cell culture experience and takes between 9 and 30 days, depending on the number of cell lineages in the construct. We also provide examples of applications of this advanced platform for testing gene and cell therapies in vitro , as well as for in vivo studies, providing proof-of-principle of its potential as a tool to develop next-generation neuromuscular or musculoskeletal therapies.