The neuromuscular junction (NMJ) is crucial in the neuromuscular system and relevant to motor neuron diseases, neuropathies, junction disorders, and myopathies. In amyotrophic lateral sclerosis (ALS), NMJ degeneration is an early pathological feature. Around 90% of ALS cases are sporadic, while 10% are familial, resulting from mutations in genes like SOD1, FUS, TARDBP, PFN1, and C9orf72. These genes affect proteostasis, RNA binding, and axonal transport. The high percentage of sporadic cases, numerous ALS genes, and the importance of human-specific genetic backgrounds necessitate iPSC-based modeling. Modeling the NMJ using human iPSCs has been challenging, with co-culture strategies not consistently producing NMJs from ALS iPSCs. Most previous models used primary human or mouse muscle, losing genotype-specific disease contributions. The robustness and reproducibility of these models across multiple iPSC lines remain unclear. This study aims to establish a cultured human sensorimotor organoid model to investigate distinct ALS variants and characterize NMJ impairment in ALS.

Existing research highlights the importance of the NMJ in ALS pathogenesis and the challenges in modeling this synapse using human iPSCs. Studies have successfully differentiated spinal motor neurons and skeletal muscle from iPSCs, but co-culture strategies have yielded inconsistent results in generating NMJs, particularly from ALS iPSCs. While some groups have generated NMJs using microfluidic devices and co-culturing ALS iPSC-derived motor neurons with control myoblasts, most studies rely on primary human or mouse muscle, limiting the investigation of genotype-specific contributions from the muscle itself. The lack of robust and reproducible models across multiple iPSC lines has hindered comprehensive disease modeling. This paper addresses these limitations by developing a novel sensorimotor organoid model capable of forming functional NMJs and examining their dysfunction in various ALS contexts.

The study used a novel approach to generate sensorimotor organoids from human iPSCs. Initially, five iPSC lines were used: two control lines and three ALS lines (two familial and one sporadic). iPSCs were differentiated in suspension to form spheres, patterned for a week, and then plated at a fixed density on matrigel-coated plates. The cultures were maintained for up to 15 weeks. Single-cell RNA sequencing (scRNA-seq) was used to confirm the development of multiple cell types, including motor and sensory neurons, astrocytes, microglia, and skeletal muscle. Immunocytochemistry and qPCR were used to validate the presence and identity of these cell types. Functional NMJs were assessed through pharmacological inhibition of contractions using curare and botulinum toxin, optogenetic stimulation of neurons expressing hSYN::ChR2, and calcium imaging. To reduce the variability associated with different genetic backgrounds, isogenic pairs of iPSC lines with familial ALS mutations in TARDBP, SOD1, and PFN1 were generated and compared to controls. Electron microscopy was used for high-resolution imaging of the NMJs and skeletal muscle. Statistical analyses included ANOVA, Kruskal-Wallis, Mann-Whitney, and F-tests for variance.

The study successfully generated sensorimotor organoids containing physiologically functional NMJs. ScRNA-seq confirmed the presence of diverse cell types, including motor and sensory neurons, astrocytes, microglia, and skeletal muscle. Organoids derived from all three ALS lines showed impaired NMJ function, as evidenced by reduced muscle contractions. Gene editing of iPSC lines to introduce familial ALS mutations (TARDBP, SOD1, PFN1) confirmed the NMJ abnormalities and significantly reduced both within-line and among-line variability in organoid cultures. Longitudinal analysis of neurite outgrowth in isogenic lines showed no difference between ALS and control lines, suggesting that NMJ dysfunction, not neuronal outgrowth impairment, is the primary defect. The study further demonstrated that different ALS gene mutations yielded distinct NMJ phenotypes: reduced innervation in SOD1 and PFN1 mutants, and reduced innervated NMJ area in TARDBP mutants.

The findings demonstrate that the developed sensorimotor organoid model successfully recapitulates key features of human ALS, including the formation of functional NMJs and their dysfunction in various ALS genetic backgrounds. The reduced variability in isogenic lines strengthens the model's reproducibility and reliability for disease modeling. The observation of distinct NMJ phenotypes in different ALS genetic variants highlights the heterogeneity of the disease and suggests potential for personalized therapeutic approaches. The model's ability to capture non-cell autonomous effects of different cell types (astrocytes, microglia, etc.) further enhances its utility in investigating the complex pathogenesis of ALS. The limitations regarding developmental versus degenerative contributions and the potential impact of organoid culture stress need further investigation through longitudinal experiments.

This study presents a novel and robust human sensorimotor organoid model for studying ALS and other neuromuscular diseases. The model's ability to generate functional NMJs and accurately recapitulate disease-specific phenotypes, along with the reduction in variability achieved through isogenic lines, establishes its utility for both basic research and drug discovery efforts. Future research should focus on longitudinal studies to disentangle developmental and degenerative contributions to the observed phenotypes, further explore the role of non-cell autonomous effects, and expand the model's application to a broader range of sensory and motor neuron diseases.

The study acknowledges the limitations of its ability to fully distinguish between developmental and degenerative contributions to the NMJ phenotypes observed. Longitudinal experiments are needed to address this. The potential impact of cellular stress exerted by organoid culture on the observed phenotypes also needs further investigation. Finally, the study's scope limited the exploration of the integration of various cell types beyond motor neurons and muscle, such as the role of astrocytes, microglia and vascular cells in the overall NMJ dysfunction observed.