Name: generation of human motor units with functional neuromuscular junctions in microfluidic devices
Uploaded: 2021-09-07T15:00:00
Duration: 10 min 48 s
Description: Watch this Scientific Journal Video about Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices at JoVE.com

The authors thank Nikky Corthout and Sebastian Munck from LiMoNe, Research Group Molecular Neurobiology (VIB-KU Leuven) for their advice on live-cell calcium transient fluorescence recordings. This research was supported by the Fulbright Commission to Belgium and Luxembourg, KU Leuven (C1 and &#34;Opening the Future&#34; Fund), the VIB, the Agency for Innovation by Science and Technology (IWT; SBO-iPSCAF), the &#34;Fund for Scientific Research Flanders&#34; (FWO-Vlaanderen), Target ALS, the ALS Liga Belgi&#235; (A Cure for ALS), the Belgian Government (Interuniversity Attraction Poles Program P7/16 initiated by the Belgian Federal Science Policy Office), the Thierry Latran Foundation and the &#34;Association Belge contre les Maladies neuro-Musculaires&#34; (ABMM). T.V. and J.B. are supported by Ph.D. fellowships awarded by FWO-Vlaanderen.

L.V.D.B. has a patent on the use of HDAC inhibitors in Charcot-Marie-Tooth disease (US-2013227717-A1), is a scientific co-founder of Augustine Therapeutics, and a member of its scientific advisory board. The other authors declare no competing interests.

The protocol describes a relatively easy-to-use method, which generates human motor units with functional NMJs in commercially available microfluidic devices in less than 30 days. It is described how the NMJs can be assessed morphologically through standard techniques such as ICC and SEM and functionally through live-cell calcium recordings.
A large advantage of this protocol is the use of stem cell technology. This allows for full adaptability in which NMJs can be evaluated in both health and disease, independently of the donor profile. The model has proven already successful and beneficial in ALS research, where we identified impairments in neurite outgrowth, regrowth, and NMJ numbers as novel phenotypes due to mutations in the FUS gene18. With this model, it is possible to expand the research to include sporadic forms of ALS, where the etiology is unknown, by using iPSCs from sporadic ALS patients. This provides an advantage over traditional animal models, which rely on transgenic overexpression of mutated genes to recapitulate human disease23,24. In addition, our fully human system allows for potential recapitulation of human-specific physiology and disease. Previous studies demonstrated the differences between rodent and human NMJ morphology25, which suggests that caution must be implemented when using rodents to address human NMJ pathology. Although this system is a relative simple in vitro setup, which lacks the complexity of an in vivo model, it was possible to demonstrate that the NMJ morphology displayed in the microfluidic devices resembled NMJs of human amputates25. Furthermore, this model allows for NMJ evaluation during NMJ formation and maturation, potentially revealing early disease phenotypes, which are absent, unidentifiable, or overlooked in human post-mortem samples.
MABs provide a valid option to generate myotubes, although their limited survival of 10 days is a disadvantage of the system. The myotube survival relies on their attachment to the surface, which is likely compromised by spontaneous contractions of the myofibers. After more than 10 days, most myotubes will have detached, rendering the NMJ culture unusable. Ideally, the myotubes would be generated from iPSCs as well. However, current protocols have proven difficult to reproduce26 due to variability in fusion index27,28,29,30.
By using commercially available microfluidic devices, we generated a standardized system, which is fully accessible. Other NMJ models exist31,32,33,34,35,36,37,38,39,40,41,42. However, they typically rely on single compartments, which lack the compartmentalization and fluidic isolation between cell types, or on custom-made culture vessels, which lowers the availability and potentially also the reproducibility. The microfluidic devices used for this protocol can be purchased with microgrooves of various lengths, which allows for further analysis such as axonal transport43,44 or axotomy18,45,46 investigations. The fluidic isolation between compartments further enables compartmentalized drug treatment of either motor neurons or myotubes, which can be favorable in therapy development. More companies specializing in microfluidics have emerged, which has opened up for a large selection of device design and features, further promoting the accessibility for in vitro research.
In conclusion, we have developed a protocol providing a reliable, versatile and easy method to culture human motor units with functional NMJs.

NMJs facilitate the communication between lower spinal motor neurons and skeletal muscle fibers through the release of neurotransmitters1. In motor neuron disorders like ALS and SMA, the NMJs degenerate, which causes a disruption in the communication with the muscles2,3,4,5,6,7. This results in patients gradually losing their muscle function, which causes them to be wheelchair-bound and eventually dependent on respiratory life-support due to progressive atrophy of vital muscle groups like the diaphragm. The exact underlying mechanisms responsible for this profound loss of NMJs in these disorders are unknown. Many studies have been done on transgenic animal models, which has given us some insights into the pathogenesis of NMJ degeneration5,6,8,9,10,11. However, to fully understand the pathology and counteract the denervation, it is important to have a human system, which allows full accessibility.
Here, the protocol describes a relatively simple way to generate human NMJs through the co-culturing of hiPSC-derived motor neurons and human primary MAB-derived myotubes using commercially available microfluidic devices. The use of microfluidics to polarize and fluidically isolate the somas and axons of neurons has been known since the first description of the &#39;Campenot&#39; chambers12 in the late 1970s. Since then, more microfluidic designs have been fabricated, including commercial options. The devices used in this protocol contain two compartments, and each compartment consists of two wells connected with a channel13. The two compartments are mirrored and connected with several microgrooves. These microgrooves have a size that facilitates neurite growth while maintaining fluidic isolation between the two compartments through a capillary hydrostatic pressure13,14. Using this system, it is possible to culture motor neurons in one compartment and muscle cells in the other one, each in their specific culture medium, while still facilitating a physical connection through neurites passing through the microgrooves and engaging with the muscle cells. This model provides a fully accessible and adaptable in vitro system of a human motor unit, which can be used to study early NMJ pathology in diseases like ALS and SMA.

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			<td>Antibody (1:1000)</td>
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			<td>Acetic Acid</td>
			<td>CHEM-Lab NV</td>
			<td>CL00.0116.1000</td>
			<td>Coating component. H226, H314. P280</td>
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			<td>Aclar 33C sheet (SEM sheet)</td>
			<td>Electron Microscopy Sciences</td>
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			<td>Thickness: 7.8 mil</td>
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			<td>Agrin (recombinant human protein)</td>
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			<td>Media supplement</td>
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			<td>Alexa fluor IgG (H+L) 488 donkey-anti rabbit</td>
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			<td>Antibody (1:1000)</td>
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			<td>Alexa fluor IgG (H+L) 555 donkey-anti goat</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21432</td>
			<td>Antibody (1:1000)</td>
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		<tr>
			<td>Alexa fluor IgG (H+L) 555 donkey-anti mouse</td>
			<td>Thermo Fisher Scientific</td>
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			<td>Antibody (1:1000)</td>
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			<td>Alexa fluor IgG (H+L) 647 donkey-anti mouse</td>
			<td>Thermo Fisher Scientific</td>
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			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A4403</td>
			<td>Media component</td>
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			<td>&#946;III-tubulin (Tubulin)</td>
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			<td>&#946;-mercaptoethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>31350010</td>
			<td>Media component. H317. P280.</td>
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			<td>B-27 without vitamin A</td>
			<td>Thermo Fisher Scientific</td>
			<td>12587-010</td>
			<td>Media component</td>
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		<tr>
			<td>BDNF (brain-derived neurotrophic factor)</td>
			<td>Peprotech</td>
			<td>450-02B</td>
			<td>Growth factor</td>
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			<td>bFGF (recombinant human basic fibroblast growth factor)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td>Growth factor</td>
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			<td>Choline acetyltransferase (ChAT)</td>
			<td>Millipore</td>
			<td>ab144P</td>
			<td>Antibody (1:500)</td>
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			<td>Collagen from calfskin</td>
			<td>Thermo Fisher Scientific</td>
			<td>17104019</td>
			<td>Coating component</td>
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			<td>CNTF (ciliary neurotrophic factor)</td>
			<td>Peprotech</td>
			<td>450-13B</td>
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			<td>DAPI Nucblue Live Cell Stain ReadyProbes reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37605</td>
			<td>Immunocytochemistry component</td>
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			<td>DAPT</td>
			<td>Tocris Bioscience</td>
			<td>2634</td>
			<td>Media supplement</td>
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			<td>Desmin</td>
			<td>Abcam</td>
			<td>Ab15200</td>
			<td>Antibody (1:200)</td>
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		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11330032</td>
			<td>Media component</td>
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			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650-100ML</td>
			<td>Cryopreservation component. H315, H319, H335. P280.</td>
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			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190250</td>
			<td>no calcium, no magnesium</td>
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			<td>Ethanol</td>
			<td>VWR</td>
			<td>20.821.296</td>
			<td>Sterilization. H225. P280</td>
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			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270106</td>
			<td>Media component</td>
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		<tr>
			<td>Fluo-4 AM live cell dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>F14201</td>
			<td>Calcium imaging dye</td>
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		<tr>
			<td>Fluorescence Mounting Medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td>Immunocytochemistry component</td>
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		<tr>
			<td>GDNF (glial cell line-derived neurotrophic factor)</td>
			<td>Peprotech</td>
			<td>450-10B</td>
			<td>Growth factor</td>
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		<tr>
			<td>Glutaraldehyde</td>
			<td>Agar Scientific</td>
			<td>R1020</td>
			<td>Fixation component. EUH071, H301, H314, H317, H330, H334, H410. P280.</td>
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			<td>Horse serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16050122</td>
			<td>Media component</td>
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			<td>Human alkaline phosphatase</td>
			<td>R&#38;D systems</td>
			<td>MAB1448</td>
			<td>Antibody</td>
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		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td></td>
			<td>ICC analysis</td>
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			<td>IMDM</td>
			<td>Thermo Fisher Scientific</td>
			<td>12440053</td>
			<td>Media component</td>
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		<tr>
			<td>Insulin transferrin selenium</td>
			<td>Thermo Fisher Scientific</td>
			<td>41400045</td>
			<td>Media component</td>
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			<td>Islet-1</td>
			<td>Millipore</td>
			<td>ab4326</td>
			<td>Antibody (1:400)</td>
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			<td>Knockout serum replacement</td>
			<td>Thermo Fisher Scientific</td>
			<td>10828-028</td>
			<td>Cryopreservation component</td>
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		<tr>
			<td>Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane</td>
			<td>Sigma</td>
			<td>L2020-1MG</td>
			<td>Coating component and media supplement</td>
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		<tr>
			<td>Leica SP8 DMI8 confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td>ICC confocal microscopy</td>
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		<tr>
			<td>L-glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024</td>
			<td>Media component</td>
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			<td>Myogenin (MyoG)</td>
			<td>Abcam</td>
			<td>Ab124800</td>
			<td>Antibody (1:500)</td>
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			<td>Myosin heavy chain (MyHC)</td>
			<td>In-house, SCIL</td>
			<td></td>
			<td>Antibody (1:20)</td>
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		<tr>
			<td>N-2 supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td>Media component</td>
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		<tr>
			<td>Neurobasal medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td>Coating and media component</td>
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		<tr>
			<td>Neurofilament heavy chain (NEFH)</td>
			<td>Abcam</td>
			<td>AB8135</td>
			<td>Antibody (1:1000)</td>
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		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon</td>
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		<tr>
			<td>NIS-Elements AR 4.30.02 software</td>
			<td>Nikon</td>
			<td></td>
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			<td>Thermo Fisher Scientific</td>
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			<td>Media component</td>
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		<tr>
			<td>Normal donkey serum</td>
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			<td>Antibody (1:1000)</td>
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			<td>Sigma</td>
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			<td>Paraformaldehyde</td>
			<td>Thermo Fisher Scientific</td>
			<td>28908</td>
			<td>Fixation component. H302, H312, H315, H317, H319, H332, H335, H341, H350. P280.</td>
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			<td>Penicillin/Streptomycin (5000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070063</td>
			<td>Media component</td>
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		<tr>
			<td>Petri dish (3 cm)</td>
			<td>nunc</td>
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			<td>Diameter: 3 cm</td>
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			<td>Petri dish (10 cm)</td>
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			<td>Diameter: 10 cm</td>
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			<td>Plate (6-well)</td>
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			<td>Pluronic F-127</td>
			<td>Thermo Fisher Scientific</td>
			<td>P3000MP</td>
			<td>Fluo-4 dye solvent</td>
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		<tr>
			<td>Poly-L-ornithine (PLO)</td>
			<td>Sigma</td>
			<td>P3655-100MG</td>
			<td>Coating component</td>
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		<tr>
			<td>Potassium chloride</td>
			<td>CHEM-Lab NV</td>
			<td>CL00.1133.1000</td>
			<td>Calcium imaging reagent</td>
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		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td>Media supplement. H302, H315, H360FD, H410. P280.</td>
		</tr>
		<tr>
			<td>RevitaCell supplement</td>
			<td>Thermo Fisher Scientific</td>
			<td>A2644501</td>
			<td>ROCK inhibitor solution</td>
		</tr>
		<tr>
			<td>Smoothened agonist</td>
			<td>Merch Millipore</td>
			<td>566660</td>
			<td>Media supplement</td>
		</tr>
		<tr>
			<td>Sodium cacodylate buffer</td>
			<td>Sigma</td>
			<td>C0250</td>
			<td>Fixation component. H301, H331, H350, H410. P280.</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-070</td>
			<td>Media component</td>
		</tr>
		<tr>
			<td>Synaptophysin (SYP)</td>
			<td>Cell Signaling</td>
			<td>5461S</td>
			<td>Antibody (1:1000)</td>
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		<tr>
			<td>T75 flask</td>
			<td>Sigma</td>
			<td>CLS3276</td>
			<td>Culture plate</td>
		</tr>
		<tr>
			<td>Titin</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>9D10</td>
			<td>Antibody (1:300)</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250ML</td>
			<td>Immunocytochemistry component. H302, H315, H318, H319, H410, H411. P280</td>
		</tr>
		<tr>
			<td>TrypLE express</td>
			<td>Thermo Fisher Scientific</td>
			<td>12605010</td>
			<td>MAB dissociation solution</td>
		</tr>
		<tr>
			<td>Tubocyrarine hydrochloride pentahydrate</td>
			<td>Sigma</td>
			<td>T2379-100G</td>
			<td>Acetylcholine receptor blocker. H301. P280.</td>
		</tr>
		<tr>
			<td>XonaChips pre-assembled microfluidic device</td>
			<td>Xona Microfluidics</td>
			<td>XC150</td>
			<td>Microgroove length: 150 &#956;m</td>
		</tr>
		<tr>
			<td>Xona Silicone microfluidics device</td>
			<td>Xona Microfluidics</td>
			<td>SND75</td>
			<td>Microgroove length: 75 &#956;m</td>
		</tr>
	</tbody>
</table>

generation of human motor units with functional neuromuscular junctions in microfluidic devices

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 &#945;-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.

Watch this Scientific Journal Video about Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices at JoVE.com

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices

We describe a method to generate human motor units in commercially available microfluidic devices by co-culturing human induced pluripotent stem cell-derived motor neurons with human primary mesoangioblast-derived myotubes resulting in the formation of functionally active neuromuscular junctions.

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle ...

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