Neuromuscular Organoid Modeling Service

The link between nerve and muscle – the neuromuscular junction (NMJ) – is fundamental to movement. Understanding how motor neurons communicate with muscle fibers is key to tackling conditions like ALS and SMA where this connection falters. Studying the human NMJ directly is difficult, as traditional methods often miss the crucial 3D interactions and human-specific factors involved.

Creative Biolabs provides advanced human Neuromuscular Organoids (NMOs) to address this need. We create 3D models using human pluripotent stem cells (hPSCs), including patient-derived lines, that bring together motor neurons and skeletal muscle fibers. These components interact within the organoid, forming functional neuromuscular junctions in vitro, offering a unique system to study this critical interface.

Our Neuromuscular Organoid Platform: Key Features

We utilize cutting-edge techniques to create NMOs that recapitulate key aspects of the human motor unit:

• Integrated Cell Types: Our models successfully co-develop essential components, including spinal motor neurons (expressing markers like ISL1, CHAT) and functional skeletal muscle fibers (expressing markers like Desmin, Titin).
• Functional NMJ Formation: Motor neurons within the organoids extend axons to connect with muscle cells, forming structures that resemble neuromuscular junctions, identifiable by markers like α-bungarotoxin binding.
• Electrophysiological Activity & Contraction: These aren't static models. The motor neurons show electrical activity, and crucially, the NMJs formed are functional, capable of transmitting signals that lead to observable muscle cell contraction within the organoid system.
• Disease Relevance: We can generate NMOs using patient-derived iPSCs (e.g., from ALS or SMA patients) or employ gene editing (like CRISPR) to model specific disease mutations, allowing for the direct study of disease-related NMJ pathology.

Our Specialized Neuromuscular Organoid Services

We offer a dedicated suite of services focused on modeling the motor unit:

1. Custom NMOrg Generation:

• NMOs Generation: We create NMOs starting from hPSCs, including patient or gene-edited lines. Using protocols that guide cells through neuromesodermal progenitor (NMP) stages, we enable the simultaneous development and self-organization of both spinal cord neurons and skeletal muscle within a single 3D structure.

Fig.1 NMO generation from iPSCs of ALS patients and healthy controls.^1,3

• NMSOs Generation: For studies needing skeletal context, we also generate advanced tri-tissue neuromusculoskeletal organoids (NMSOs) where neural, muscular, and skeletal tissues co-develop and self-organize, offering a unique model of their interactions.

Fig.2 Deriving NMSOs from hPSCs.^2,3

• Disease & Patient Specificity: We utilize patient-derived iPSCs or perform CRISPR gene editing to build NMOs reflecting specific neuromuscular disease states (e.g., ALS, SMA), complete with matched isogenic controls.
• Advanced Systems: Construction of more complex models like cortico-spinal-muscle assembloids is possible for pathway studies.

2. Targeted Characterization:

• Component Validation: Detailed confirmation of both motor neuron (e.g., ISL1, CHAT) and muscle fiber (e.g., Desmin, Titin) presence and maturity using multi-label immunofluorescence.
• NMJ Structural Analysis: Specific staining (e.g., α-bungarotoxin for acetylcholine receptors) and high-resolution imaging to visualize the structure and integrity of neuromuscular junctions formed within the organoid.
• Molecular Confirmation: Gene expression analysis focused on markers relevant to motor neurons, muscle development, and synaptic function.

3. Neuromuscular Functional Assessment:

• Synaptic Transmission: Assays designed to measure the functional connection across the NMJ, potentially involving stimulated activity and recording downstream muscle responses.
• Muscle Contraction Analysis: Direct visualization or quantitative measurement of skeletal muscle fiber contraction within the organoid, confirming functional output.
• Electrophysiological Recordings: Measuring activity specifically from the motor neuron component using appropriate techniques (e.g., MEAs adapted for 3D cultures).

4. Neuromuscular Disease Modeling:

• Focused Model Creation: Developing NMOrg systems that specifically replicate key pathological features observed at the NMJ in human diseases like ALS or SMA (e.g., synapse withdrawal, axon degeneration markers, muscle fiber atrophy signals).
• Phenotypic Quantification: Measuring disease-relevant changes, such as the number and stability of NMJs, motor neuron health within the co-culture, or functional decline in muscle contraction.

5. Drug Discovery for Neuromuscular Targets:

• NMJ-Focused Screening: Testing compounds specifically for their ability to protect the NMJ, improve synaptic transmission, or enhance muscle function in the context of the organoid.
• Efficacy in Disease Models: Validating potential therapeutics by assessing their ability to rescue NMJ defects or improve outcomes in our ALS or SMA organoid models.

Our Workflow

References

1. Gao, Chong et al. "Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis." Cell reports vol. 43,3 (2024): 113892. doi:10.1016/j.celrep.2024.113892
2. Yin, Yao et al. "Generation of self-organized neuromusculoskeletal tri-tissue organoids from human pluripotent stem cells." Cell stem cell vol. 32,1 (2025): 157-171.e8. doi:10.1016/j.stem.2024.11.005
3. Distributed under Open Access License CC BY 4.0. The original image was modified.