Objective: Congenital myotonic dystrophy (CDM) represents the most severe form of myotonic dystrophy type 1 (DM1), a multisystemic genetic disorder caused by the expansion of CTG trinucleotide repeats in the DMPK gene. CDM is characterized by hypotonia, myotonia, and progressive muscle weakness from birth, frequently associated with respiratory distress and developmental delay. While skeletal muscle pathology has been extensively studied, increasing evidence suggests that the broader neuromuscular system, including motor neurons and their connectivity with skeletal muscle, is also affected. However, the developmental origins and the cellular mechanisms underlying neuromuscular dysfunction in CDM remain poorly understood.

Methods: To model human neuromuscular development and assess the impact of CDM, we generated neuromuscular organoids (NMO) from 3 control and 3 CDM patient-derived human induced pluripotent stem cell (hiPSC) lines. NMOs were differentiated for up to 100 days. Immunofluorescence and flow cytometry were used to quantify key cell populations at multiple time points. Functional properties were assessed by calcium imaging of myotube activity and contractility analysis using phase-contrast video microscopy.

Results: By day 10 of differentiation, both control and CDM-derived NMOs exhibited robust specification of early myogenic progenitors and motor neurons precursors, indicating successful early lineage commitments. By day 50, NMOs developed polarized tissues organization, with distinct neural and muscular compartments. Using electron microscopy, we observed well organized sarcomeric structures in muscle fibers, as well as synaptic contacts between neurons and myotubes. Quantitative analysis of muscle progenitor dynamics revealed comparable levels of PAX3/PAX7-positive cells in control and CDM NMOs between days 30 and 50. However, at day 100, CDM organoids exhibited a marked reduction in PAX7-positive cells, suggestive of impaired maintenance of the muscle stem cell (satellite-like) pool. This was accompanied by a reduction in skeletal muscle area, indicating defective myogenesis over time.

Conclusion: Our data demonstrate that NMOs recapitulate key features of early human neuromuscular development and provide tractable system to study CDM pathogenesis. We identify a progressive defect in the maintenance of the muscle progenitor pool in CDM organoids, potentially contributing to impaired muscle development and regeneration.