Single-cell RNA sequencing of neuromuscular organoids reveals novel roles for the lncRNA HSCHARME in the neuromuscular axis

Many neuromuscular disorders arise from defects in early embryogenesis, underscoring the critical need to understand the mechanisms governing neuromuscular co-development. While traditional 2D cultures fail to capture the spatial and cellular complexity of this cross-talk, advanced 3D human Neuromuscular Organoids (hNMOs) have emerged as an invaluable platform for modeling physiological co-development [1]. To map ncRNA dynamics across distinct developmental stages, we first generated a longitudinal single-cell RNA sequencing (scRNA-seq) atlas of wild-type NMOs. This comprehensive transcriptomic profiling highlighted HSCHARME, a long non-coding RNA [2,3], as a dynamically expressed transcript tightly associated with the muscular developmental trajectory. To validate its biological relevance and mechanistically dissect its role, we generated HSCHARME Knock-Out (KO) hiPSC lines and derived mutant NMOs. The impact of HSCHARME depletion was then comprehensively evaluated at the transcriptomic, anatomical, and functional levels. We integrated scRNA-seq and immunofluorescence analyses on KO organoids to characterize the alterations in early lineage commitment. Furthermore, the physiological consequences of HSCHARME loss were assessed through macroscopic muscle contraction analyses and Microelectrode Array (MEA) recordings to monitor network electrical activity. Together, these multidisciplinary approaches demonstrate the essential role of HSCHARME in the proper specification and functional maturation of the neuromuscular axis, highlighting the utility of NMOs as a powerful platform for exploring the non-coding genome in early human embryogenesis.