Human three-dimensional engineered muscle tissue characterization and intramyocellular lipid modeling

Three-dimensional engineered muscle tissues (EMTs) are transformative tools for modeling skeletal muscle physiology and pathology in vitro. Here, we perform a comprehensive comparison of EMTs derived from primary human myoblasts (hP-Myo) and hiPS-derived myoblasts (hiPS-Myo) to evaluate their structural, functional, and transcriptional characteristics. Contractile performance was quantified using a magnetic force-sensing platform, revealing that hP-Myo EMTs generate ~2 fold higher twitch forces and enhanced tetanic responses compared to hiPS-Myo EMTs. Tissue architecture and maturation were assessed and demonstrated significant larger myofiber diameters in hP-Myo EMTs. Transcriptomic profiling highlighted that hP-Myo EMTs maintain a mature skeletal muscle-like signature, marked by enriched pathways linked to sarcomere organization and fast-/slow-twitch fiber specification. To model metabolic dysfunction, hiPS-Myo EMTs were subjected to lipid overload, recapitulating hallmarks of intracellular lipid (IMCL) accumulation, including impaired contractility, blunted force-frequency responses, and dysregulated lipid metabolism genes.