期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 175, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/62959
关键词
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资金
- Fulbright Commission
- KU Leuven
- VIB
- Agency for Innovation by Science and Technology (IWT
- SBO-iPSCAF)
- Fund for Scientific Research Flanders (FWO-Vlaanderen)
- Target ALS
- ALS Liga Belgie
- Belgian Government [P7/16]
- Thierry Latran Foundation
- Association Belge contre les Maladies neuro-Musculaires (ABMM)
- FWO-Vlaanderen
This study successfully created an in vitro model of a human motor unit with functional NMJs, identified and characterized the NMJs through immunocytochemistry and scanning electron microscopy, and confirmed the functionality of the NMJs.
Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker alpha-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.
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