Key Findings:
- Spheroid culture of motor neurons generates mature cells with axons of 1000-1600 µm in length
- The motor neurons in this model form functional mature neuromuscular junctions with muscle fibers causing them to twitch and contract when stimulated
- This new human-specific model is a platform for drug development against motor neuron disease
In landmark work published in Scientific Reports Rimington et al. describe how they have succeeded in recreating the mature adult NMJ using Axol’s motor neuron progenitor cells (ax0078).[1] The team hope their 3D human NMJ model will replace animal models and monolayer cellular models, improving the translation of therapies to the clinic.
The team built the 3D NMJ by encouraging ax0078 motor neurons to synapse with primary muscle tissue cells obtained from healthy male adult volunteers and cultured in a 3D matrix. To do this, the team adopted a 3D spheroid culture approach that had previously been successful when culturing motor neurons with vascular cells.[2]
Spheroid Culture Generates more Mature Cells faster
Despite not using vascular cells in their study, the spheroid culture method generated more mature motor neurons in 14 days of culture than had previously been achieved in mono-layer culture. Further analysis with qPCR showed augmented transcription of motor neuron markers OLIG2, ISLET1 and SMI-32 mRNAs, ~400, ~150 and ~200-fold, respectively. Importantly, the spheroid culture also generated motor neurons with extensive axon growth of 1000-1600 µm in length, of which 90% were positive for the mature motor neuron marker SMI-32. This finding is in contrast to cells grown in monolayer culture where axons of 400 µm in length are more common.
Motor Neurons form Functional NMJs with Muscle Fibers
The group then seeded their motor neuron spheroids onto plates with the muscle tissue and found that within two weeks of co-culture they could observe muscle fibers twitching – consistent with innervation by motor neurons. To confirm, they blocked communication between the motor neurons and the muscle cells with the acetylcholine receptor blocker tubocurarine, which drastically reduced the intensity and frequency of the skeletal muscle fiber twitches.
Deeper analysis revealed the presence of pre- and post-synaptic markers juxtaposed between the motor neurons and the muscle tissue further supporting the formation of functional neuromuscular junctions.
The Path to Better Translation
Taken together, the improved growth and maturity of motor neurons and their successful co-culture with muscle fibers provide a functional human model of the neuromuscular junction, which has exciting implications for drug discovery and R&D efforts to understand motor neuron disease.
By monitoring twitch and contraction activity, scientists now have a functional readout of the activity of the NMJ as a whole system. That insight means they can derive motor neurons from patient tissue and use them in the model to understand how functionality at the NMJ level is affected. They can then develop drugs to reverse or mitigate the effect of the disease.
To date, many therapies for motor neuron diseases such as ALS have failed to make it out of the clinical trial phase.[3,4] One reason for this could be the suitability of the experimental models used in the pre-clinical research and drug development phases.[5] Dr Rimington and his colleagues are hopeful a human model of the NMJ will lead to therapeutics that better-translate into the clinic.
References:
- Rimington, R., Fleming, J., Capel, A., Wheeler, P., & Lewis, M. (2021). Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions. Scientific Reports, 11(1). doi: 10.1038/s41598-021-91203-5
- Osaki, T., Sivathanu, V., & Kamm, R. (2018). Engineered 3D vascular and neuronal networks in a microfluidic platform. Scientific Reports, 8(1). doi: 10.1038/s41598-018-23512-1
- Kiernan, M., Vucic, S., Talbot, K., McDermott, C., Hardiman, O., & Shefner, J. et al. (2020). Improving clinical trial outcomes in amyotrophic lateral sclerosis. Nature Reviews Neurology, 17(2), 104-118. doi: 10.1038/s41582-020-00434-z
- Petrov, D., Mansfield, C., Moussy, A., & Hermine, O. (2017). ALS Clinical Trials Review: 20 Years of Failure. Are We Any Closer to Registering a New Treatment?. Frontiers In Aging Neuroscience, 9. doi: 10.3389/fnagi.2017.00068
