3D human induced pluripotent stem cell-derived bioengineered skeletal muscles for tissue, disease and therapy modeling.

Skeletal muscle is a complex tissue composed of multinucleated myofibers responsible for force generation that are supported by multiple cell types. Many severe and lethal disorders affect skeletal muscle; therefore, engineering models to reproduce such cellular complexity and function are 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 myofiber 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 d, 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.

Extracellular matrix hydrogel derived from decellularized tissues enables endodermal organoid culture.

Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically.

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering.

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development.