Native-extracellular matrix drives human neuromuscular organoid morphogenesis and functionality

ABSTRACT: Human neuromuscular organoids (NMOs) hold a great potential to study the pathophysiology of functional human skeletal muscle in vitro due to the presence of both muscular and neuronal compartments organized in a their three-dimensional (3D) context and to be derived from induced pluripotent stem cells (hiPSCs). An open question in the field remains how the morphology of NMOs can influence the maturation and functionality of the neuronal-coupled skeletal muscle. NMOs self-assemble into spheroids, thus not fully recapitulating the morphological organization of the innervated skeletal muscle. Proper development, maturation and function of the innervated skeletal muscle require defined 3D organization of the cells in a context of tissue-specific extracellular matrix (ECM). We hypothesized that extracellular structural imprinting along hiPSC differentiation could provide self-assembly guidance cues able to impact on NMO morphogenesis and on neuronal-coupled skeletal muscle maturation and functionality. Therefore, here we used a well characterized skeletal muscle ECM to guide the morphogenesis of differentiating hiPSC toward tissue-like structured NMOs (t-NMOs). T-NMOs resemble the morphological organization of the neuronal-coupled skeletal muscle, with elongated bundles of myofibers reached by long neural projections (up to 1mm length). In presence of the instructive native ECM, high degree of skeletal muscle maturation together with functional neuromuscular junctions were reached in t-NMOs in only 30 days. Finally, we used t-NMO to understand whether neuronal activation could lead to altered myogenic response in a pathologic context. To do so, we used hiPSCs derived from patients affected by Duchenne Muscular Dystrophy (DMD), a neuromuscular disease characterized by myofiber weakness and membrane fragility, as well as altered calcium handling. Upon neuronal stimulation, DMD t-NMO displayed key muscular phenotypes of the disease, such as altered functionality and calcium dynamics, as well as myofiber rupture.

ORGANISM(S): Homo sapiens

PROVIDER: GSE226477 | GEO | 2025/05/30

REPOSITORIES: GEO