Mina Gouti

Scalable human models that capture interactions between distinct tissues remain limited, constraining mechanistic insight and therapeutic prediction. Here, we established a scalable, automation-compatible human neuromuscular organoid (NMO) platform that enables integrated analysis of neuronal and muscle lineages in spinal muscular atrophy (SMA). Patient-derived NMOs reproducibly self-organise into spinal cord and skeletal muscle compartments and form functional neuromuscular circuits. SMA NMOs recapitulate early disease features, including reduced survival motor neuron (SMN) protein levels and impaired neuromuscular junction (NMJ) maturation. Single-nucleus RNA sequencing identifies lineage-specific transcriptional changes across neuronal and muscle compartments preceding functional deficits. Using this platform, we compared two clinically relevant SMN2 splicing modulators and observed distinct, cell-type-dependent responses. While both compounds increased SMN levels and NMJ number, only one enhanced myofiber growth and improved contractile function. These findings highlight muscle maturation, rather than NMJ number alone, as a key determinant of functional recovery and establish NMOs as a scalable system for studying cell-type-specific therapeutic responses.

Organoids derived from human pluripotent stem cells (hPSCs) are emerging as powerful models for studying development and disease. Despite their physiological relevance, the predictive power of organoids remains limited by the immature state of the constituent cells, posing a major challenge for mechanistic studies of adult physiology and late‐onset disorders. Here, we establish a strategy for enhancing the maturation status of human neuromuscular organoids (NMOs) through chronic Electrical Pulse Stimulation (EPS). We demonstrate that low‐frequency EPS, applied during the early stages of NMO development and maintained over several weeks, enhances neuromuscular maturation and functional output. Independent of stimulation waveform dynamics, EPS‐trained NMOs (EPS‐NMOs) display stronger and more frequent spontaneous contractions that persist long after stimulation has ceased. Quantitative imaging and transcriptomic analyses reveal a robust improvement in EPS‐NMO skeletal muscle and neural tissue morphology, coordinated regulation of lineage‐specific biomarkers, and upregulation of gene programs associated with neuromuscular maturation. Mechanobiological measurements further demonstrate increased tissue stiffness and faster relaxation dynamics in EPS‐NMOs, consistent with enhanced excitation‐contraction coupling (ECC) and force generation. Collectively, these findings establish EPS as a powerful, non‐invasive, and on‐demand modality for promoting the morphological and functional maturation of complex organoid systems.

Organoids derived from human pluripotent stem cells (hPSCs) are emerging as powerful models for studying development and disease. Despite their physiological relevance, the predictive power of organoids remains limited by the immature state of the constituent cells, posing a major challenge for mechanistic studies of adult physiology and late-onset diseases and disorders. Here, we establish a strategy for enhancing the maturation status of human neuromuscular organoids (NMOs) through chronic Electrical Pulse Stimulation (EPS). We demonstrate that low-frequency EPS, applied early on during NMO development and maintained over several weeks, promotes structural and functional maturation of neuromuscular junctions (NMJs). Independent of stimulation waveform dynamics, EPS-trained NMOs (EPS-NMOs) displayed stronger and more frequent spontaneous contractions that persisted long after stimulation ceased. Quantitative imaging and transcriptomic analyses revealed a robust improvement in EPS-NMO skeletal muscle and neural tissue morphology, coordinated regulation of lineage-specific biomarkers, and upregulation of gene programmes associated with mature neuromuscular function. Mechanobiological measurements further demonstrated increased EPS-NMO tissue stiffness and faster relaxation dynamics, consistent with advanced excitation–contraction coupling and force generation. Collectively, these findings establish EPS as a powerful, non-invasive, and on-demand modality for driving the morphological and functional maturation of complex organoid systems.

The complex neuromuscular network that controls body movements is the target of severe diseases that result in paralysis and death. Here, we report the development of a robust and efficient self-organizing neuromuscular junction (soNMJ) model from human pluripotent stem cells that can be maintained long-term in simple adherent conditions. The timely application of specific patterning signals instructs the simultaneous development and differentiation of position-specific brachial spinal neurons, skeletal muscles, and terminal Schwann cells. High-content imaging reveals self-organized bundles of aligned muscle fibers surrounded by innervating motor neurons that form functional neuromuscular junctions. Optogenetic activation and pharmacological interventions show that the spinal neurons actively instruct the synchronous skeletal muscle contraction. The generation of a soNMJ model from spinal muscular atrophy patient-specific iPSCs reveals that the number of NMJs and muscle contraction is severely affected, resembling the patient’s pathology. In the future, the soNMJ model could be used for high-throughput studies in disease modeling and drug development. Thus, this model will allow us to address unmet needs in the neuromuscular disease field.

In June 2022, the second meeting on ‘Engineering Multicellular Systems’, organized by the European Molecular Biology Laboratory and the Institute of Bioengineering of Catalonia, took place in Barcelona. Stem cell and systems biologists, physicists and engineers from all over the world gathered to discuss how recent breakthroughs in organoid technologies, engineering and mechanobiology are boosting our understanding of early morphogenesis, organogenesis and organ function with applications in tissue engineering, disease modeling and drug screening. The meeting was organized with sustainability in mind, and included an ethics session and an outreach public activity.

Organoids are miniature, organ-like structures derived from stem cells. While techniques for developing organoids for different tissues have advanced in the past decade, it has remained a challenge to simultaneously grow two different tissues into a single functional organoid. We have recently developed a 3D neuromuscular organoid that allows the simultaneous generation of spinal neurons and skeletal muscles. Here we discuss their future applications in modeling neuromuscular disorders.