Temporal transcriptomic profiling of human three-dimensional neuromuscular co-cultures

The principal organization of mammalian neuromuscular junctions (NMJs) shares essential features across species. However, human NMJs (hNMJs) exhibit distinct structural and physiological properties. While recent advances in stem-cell-based systems have significantly improved in vitro modeling of hNMJs, the extent to which these models recapitulate in vivo development remains unclear. Here, we performed temporal transcriptomic analysis of human three-dimensional (3D) neuromuscular co-cultures, composed of iPSC-derived motoneurons and skeletal muscle engineered from primary myoblasts. We found that the expression pattern follows a temporally coordinated gene expression program underlying NMJ maturation. The model recapitulates transcriptional features of NMJ development, including early myoblast fusion and presynaptic development, followed by a late-stage upregulation of postsynaptic markers and embryonic AChR subunits. Importantly, comparable transcriptional dynamics across two independent hiPSC lines confirm the reproducibility and robustness of this system. This study confirms on a transcriptional level that human 3D neuromuscular co-cultures are a robust and physiologically relevant model for investigating hNMJ development and function.