Abstract: Sarcopenia, the progressive loss of skeletal muscle mass and function with aging, affects millions worldwide and poses a growing burden on aging societies. Despite its prevalence, effective treatments remain elusive. Notably, the failure of clinical trials targeting myostatin highlights the limitation of animal models for drug development, underscoring the urgent need for human-based models. Neuromuscular organoids (NMOs) have emerged as promising human in vitro platform that recapitulate the motor unit, comprising both neural and muscle compartments. Here, we present an optimized NMO model with enhanced motor neuron differentiation and myofiber maturation, demonstrated by increased expression of markers including ChAT, OLIG2, MYH2, and MYH7. Our protocol also resulted in larger muscle tissue size, increased number of neuromuscular junctions, and critically, synchronous contractions of skeletal muscle tissue, distinct from the sporadic and localized twitching commonly observed in previous models. To investigate sarcopenia, we successfully induced age-associated phenotypes by long-term culture of the optimized NMOs. At day 200, NMOs exhibited hallmark features of sarcopenia, such as centralized nuclei in muscle fibers, reduced neurite density within the muscle compartment, and degradation of laminin surrounding muscle fibers. Gene expression analysis revealed a significant upregulation of inflammatory cytokines and a downregulation of oxidative phosphorylation (OXPHOS) genes, indicative of mitochondrial dysfunction. Additionally, increased DNA damage, elevated expression of senescence marker P16, and senescence-associated β-galactosidase activity were observed. These findings establish our optimized NMO system as a robust human model for sarcopenia, offering a valuable platform for studying disease mechanisms and identifying potential therapeutic targets.
