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.

<table><tbody><tr><td>&amp;alpha;-bungarotoxin (Btx) Alexa fluor 555</td><td>Thermo Fisher Scientific</td><td>B35451</td><td>Antibody (1:1000)</td></tr><tr><td>Acetic Acid</td><td>CHEM-Lab NV</td><td>CL00.0116.1000</td><td>Coating component. H226, H314. P280</td></tr><tr><td>Aclar 33C sheet (SEM sheet)</td><td>Electron Microscopy Sciences</td><td>50425-25</td><td>Thickness: 7.8 mil</td></tr><tr><td>Agrin (recombinant human protein)</td><td>R&amp;amp;D systems</td><td>6624-AG-050</td><td>Media supplement</td></tr><tr><td>Alexa fluor IgG (H+L) 488 donkey-anti rabbit</td><td>Thermo Fisher Scientific</td><td>A21206</td><td>Antibody (1:1000)</td></tr><tr><td>Alexa fluor IgG (H+L) 555 donkey-anti goat</td><td>Thermo Fisher Scientific</td><td>A21432</td><td>Antibody (1:1000)</td></tr><tr><td>Alexa fluor IgG (H+L) 555 donkey-anti mouse</td><td>Thermo Fisher Scientific</td><td>A31570</td><td>Antibody (1:1000)</td></tr><tr><td>Alexa fluor IgG (H+L) 647 donkey-anti mouse</td><td>Thermo Fisher Scientific</td><td>A31571</td><td>Antibody (1:1000)</td></tr><tr><td>Ascorbic acid</td><td>Sigma</td><td>A4403</td><td>Media component</td></tr><tr><td>&amp;beta;III-tubulin (Tubulin)</td><td>Abcam</td><td>ab7751</td><td>Antibody (1:500)</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Thermo Fisher Scientific</td><td>31350010</td><td>Media component. H317. P280.</td></tr><tr><td>B-27 without vitamin A</td><td>Thermo Fisher Scientific</td><td>12587-010</td><td>Media component</td></tr><tr><td>BDNF (brain-derived neurotrophic factor)</td><td>Peprotech</td><td>450-02B</td><td>Growth factor</td></tr><tr><td>bFGF (recombinant human basic fibroblast growth factor)</td><td>Peprotech</td><td>100-18B</td><td>Growth factor</td></tr><tr><td>Choline acetyltransferase (ChAT)</td><td>Millipore</td><td>ab144P</td><td>Antibody (1:500)</td></tr><tr><td>Collagen from calfskin</td><td>Thermo Fisher Scientific</td><td>17104019</td><td>Coating component</td></tr><tr><td>CNTF (ciliary neurotrophic factor)</td><td>Peprotech</td><td>450-13B</td><td>Growth factor</td></tr><tr><td>DAPI Nucblue Live Cell Stain ReadyProbes reagent</td><td>Thermo Fisher Scientific</td><td>R37605</td><td>Immunocytochemistry component</td></tr><tr><td>DAPT</td><td>Tocris Bioscience</td><td>2634</td><td>Media supplement</td></tr><tr><td>Desmin</td><td>Abcam</td><td>Ab15200</td><td>Antibody (1:200)</td></tr><tr><td>DMEM/F12</td><td>Thermo Fisher Scientific</td><td>11330032</td><td>Media component</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650-100ML</td><td>Cryopreservation component. H315, H319, H335. P280.</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Thermo Fisher Scientific</td><td>14190250</td><td>no calcium, no magnesium</td></tr><tr><td>Ethanol</td><td>VWR</td><td>20.821.296</td><td>Sterilization. H225. P280</td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>10270106</td><td>Media component</td></tr><tr><td>Fluo-4 AM live cell dye</td><td>Thermo Fisher Scientific</td><td>F14201</td><td>Calcium imaging dye</td></tr><tr><td>Fluorescence Mounting Medium</td><td>Dako</td><td>S3023</td><td>Immunocytochemistry component</td></tr><tr><td>GDNF (glial cell line-derived neurotrophic factor)</td><td>Peprotech</td><td>450-10B</td><td>Growth factor</td></tr><tr><td>Glutaraldehyde</td><td>Agar Scientific</td><td>R1020</td><td>Fixation component. EUH071, H301, H314, H317, H330, H334, H410. P280.</td></tr><tr><td>Horse serum</td><td>Thermo Fisher Scientific</td><td>16050122</td><td>Media component</td></tr><tr><td>Human alkaline phosphatase</td><td>R&amp;amp;D systems</td><td>MAB1448</td><td>Antibody</td></tr><tr><td>ImageJ software</td><td>NIH</td><td></td><td>ICC analysis</td></tr><tr><td>IMDM</td><td>Thermo Fisher Scientific</td><td>12440053</td><td>Media component</td></tr><tr><td>Insulin transferrin selenium</td><td>Thermo Fisher Scientific</td><td>41400045</td><td>Media component</td></tr><tr><td>Islet-1</td><td>Millipore</td><td>ab4326</td><td>Antibody (1:400)</td></tr><tr><td>Knockout serum replacement</td><td>Thermo Fisher Scientific</td><td>10828-028</td><td>Cryopreservation component</td></tr><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></tr><tr><td>Leica SP8 DMI8 confocal microscope</td><td>Leica</td><td></td><td>ICC confocal microscopy</td></tr><tr><td>L-glutamine</td><td>Thermo Fisher Scientific</td><td>25030-024</td><td>Media component</td></tr><tr><td>Myogenin (MyoG)</td><td>Abcam</td><td>Ab124800</td><td>Antibody (1:500)</td></tr><tr><td>Myosin heavy chain (MyHC)</td><td>In-house, SCIL</td><td></td><td>Antibody (1:20)</td></tr><tr><td>N-2 supplement</td><td>Thermo Fisher Scientific</td><td>17502-048</td><td>Media component</td></tr><tr><td>Neurobasal medium</td><td>Thermo Fisher Scientific</td><td>21103049</td><td>Coating and media component</td></tr><tr><td>Neurofilament heavy chain (NEFH)</td><td>Abcam</td><td>AB8135</td><td>Antibody (1:1000)</td></tr><tr><td>Nikon A1R confocal microscope</td><td>Nikon</td><td></td><td>Live-cell calcium imaging microscopy</td></tr><tr><td>NIS-Elements AR 4.30.02 software</td><td>Nikon</td><td></td><td>Live-cell calcium imaging analysis</td></tr><tr><td>Non-essential amino acids</td><td>Thermo Fisher Scientific</td><td>11140050</td><td>Media component</td></tr><tr><td>Normal donkey serum</td><td>Sigma</td><td>D9663-10ML</td><td>Immunocytochemistry component</td></tr><tr><td>Olig2</td><td>IBL</td><td>18953</td><td>Antibody (1:1000)</td></tr><tr><td>Parafilm M</td><td>Sigma</td><td>P7793-1EA</td><td>Storing equipment</td></tr><tr><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></tr><tr><td>Penicillin/Streptomycin (5000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15070063</td><td>Media component</td></tr><tr><td>Petri dish (3 cm)</td><td>nunc</td><td>153066</td><td>Diameter: 3 cm</td></tr><tr><td>Petri dish (10 cm)</td><td>Sarstedt</td><td>833.902</td><td>Diameter: 10 cm</td></tr><tr><td>Plate (6-well)</td><td>Cellstar Greiner bio-one</td><td>657160</td><td>Culture plate</td></tr><tr><td>Pluronic F-127</td><td>Thermo Fisher Scientific</td><td>P3000MP</td><td>Fluo-4 dye solvent</td></tr><tr><td>Poly-L-ornithine (PLO)</td><td>Sigma</td><td>P3655-100MG</td><td>Coating component</td></tr><tr><td>Potassium chloride</td><td>CHEM-Lab NV</td><td>CL00.1133.1000</td><td>Calcium imaging reagent</td></tr><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></tr><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 &amp;mu;m</td></tr><tr><td>Xona Silicone microfluidics device</td><td>Xona Microfluidics</td><td>SND75</td><td>Microgroove length: 75 &amp;mu;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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Neuroscience

4.6K Views.  KU Leuven - University of Leuven. Deze relatief eenvoudige methode kan helpen bij onderzoek gericht op motorneuronen en neuromusculaire juncties in gezondheid en ziekte. Dit protocol maakt gebruik van standaard stamceltechnologie en in de handel verkrijgbare microfluïdische apparaten die de reproduceerbaarheid verhogen. De ontkoppeling van motorneuronen en spiercellen is een vroeg fenomeen bij verschillende neuromusculaire ziekten.Een eenvoudig gehumaniseerd model zou kunnen helpen bij het ontwikkelen van strategieën...

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

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical ...

Developmental Biology

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Bioengineering

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro&#160;models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as &#39;MyoTACTIC&#39;, and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform&#39;s ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

We describe a reproducible, automated, and unbiased imaging system for characterizing neuromuscular junction function using human engineered skeletal muscle tissue and optogenetic motoneurons. This system allows for the functional quantification of neuromuscular connectivity over time and detects diminished neuromuscular function caused by neurotoxins and myasthenia gravis patient serum.

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Three-Dimensional Motor Nerve Organoid Generation

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and neurodegenerative diseases. Although numerous studies of axons have been conducted, our understanding of formation and dysfunction of axon fascicles is still limited due to the lack of robust three-dimensional in vitro models. Here, we describe a step-by-step protocol for the rapid generation of a motor nerve organoid (MNO) from human induced pluripotent stem (iPS) cells in a microfluidic-based tissue culture chip. First, fabrication of chips used for the method is described. From human iPS cells, a motor neuron spheroid (MNS) is formed. Next, the differentiated MNS is transferred into the chip. Thereafter, axons spontaneously grow out of the spheroid and assemble into a fascicle within a microchannel equipped in the chip, which generates an MNO tissue carrying a bundle of axons extended from the spheroid. For the downstream analysis, MNOs can be taken out of the chip to be fixed for morphological analyses or dissected for biochemical analyses, as well as calcium imaging and multi-electrode array recordings. MNOs generated with this protocol can facilitate drug testing and screening and can contribute to understanding of mechanisms underlying development and diseases of axon fascicles.

This protocol provides a comprehensive procedure to fabricate human iPS cell-derived motor nerve organoid through spontaneous assembly of a robust bundle of axons extended from a spheroid in a tissue culture chip.

A fascicle of axons is one of the major structural motifs observed in the nervous system. Disruption of axon fascicles could cause developmental and ...

Generating Neuromuscular Junctions Using a Microfluidic Device

Source: Stoklund Dittlau, K. et al., <a href="https://app.jove.com/v/62959">Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.</a>&#160;J. Vis. Exp. (2021)
The video demonstrates the formation of neuromuscular junctions using a microfluidic device. Neural precursor cells and muscle progenitor cells are seeded into different wells within the device and left to mature into motor neurons and myotubes, respectively. The creation of a volume and chemical gradient leads to the development of active neuromuscular junctions.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
All reagents and equipment used in ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Developing Human iPSC-Derived Motor Nerve Organoids Using a Microfluidic Chip

Source: Osaki, T., et al. <a href="https://app.jove.com/v/61544">Three-Dimensional Motor Nerve Organoid Generation.</a> J. Vis. Exp. (2020).
This video demonstrates the method of creating motor nerve organoids by first culturing human induced pluripotent stem cells in a special medium to form spheroids, then differentiating them into motor neurons using specific inhibitors, and finally cultivating them in a microfluidic chip to extend axons and form motor nerve organoids.

1. SU-8 mold fabrication by photolithography
NOTE: This procedure involves hazardous chemicals. Use fume hood and PPE throughout.

	Clean the silicon ...

In Vitro Induction of Neuromuscular Junctions from Engineered Human-Induced Pluripotent Stem Cells

Source:Lin, C. et al., <a href="https://app.jove.com/v/61396">In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells.</a> J. Vis. Exp.(2020).
This experiment details a method for generating neuromuscular junctions from human-induced pluripotent stem cells. It uses a staged differentiation process to promote myotube and motor neuron development and synapse formation, yielding functional neuromuscular junctions.

1. Preparation of extracellular matrix (ECM)-coated plates

	Dilute ECM (see Table of Materials) with ice cold 1x PBS to a final concentration of 2%
	Add ...

Assessing a Functional Neuromuscular Junction Via Simultaneous Optical Stimulation and Video Recording

Prepare a 4-to-1 mix of 3 milligrams per milliliter collagen I and solubilized basement membrane matrix. Then, resuspend the myoblasts in this freshly prepared collagen mixture.
Next, add 15 microliters of this cell-collagen suspension to each muscle chamber of the bioreactor, making sure to spread the suspension across both pillars using the pipette tip. Allow the cell gel mixture to polymerize at 37 degrees Celsius for 30 minutes. Then, fill each bioreactor well with 450 microliters of myotonic growth media.
On day 11 of motor neuron differentiation, transfer the cells and media to a 50-milliliter tube, and allow the neurospheres to settle to the bottom for 5 minutes. Aspirate the supernatant and resuspend the cells in 15 milliliters of motor neuron suspension culture medium supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor and 10 nanograms per milliliter of glial-cell-derived neurotrophic factor.
On day 14 of the differentiation protocols, seed the motor neurons into the platform. Prepare a 4-to-1 gel mixture of 2 milligrams per milliliter collagen I and solubilized basement membrane matrix. Use a 400-nanometer cell strainer to select large neurospheres, and resuspend them in the prepared gel mixture.
Carefully, aspirate media from the reservoir and the neurosphere well. Then, add 15 microliters of the gel mixture into the neurosphere channel. Load a 10-microliter pipette with 10 microliters of gel, and pick one neurosphere. Deposit the neurosphere into the neurosphere channel, ensuring that it is in the chamber. Then, slowly raise the pipette while releasing the remaining gel once the neurosphere is deposited.
Allow the gel to polymerize for 30 minutes at 37 degrees Celsius. Then, add 450 microliters of NbActiv4 supplemented with 20 nanograms per milliliter of brain-derived neurotrophic factor and 10 nanograms per milliliter of glial-cell-derived neurotrophic factor to the reservoirs.
For imaging, use an inverted fluorescent microscope with a scientific complementary metal oxide semiconductor camera software. Set the binning to 2-by-2, exposure to 20 milliseconds, rolling shutter on, readout rate to 540 megahertz, dynamic range to 12-bit and gain 1, and sensor mode to overlap. Use a 2x objective on the microscope to image the microtissues, and attach a live-cell chamber to the microscope stage.
Select the region of interest that contains the innervated skeletal tissues to minimize the file size and processing time. Then, place a 594-nanometer long-pass emission filter between the sample and the imaging objective to filter out blue light pulses from the camera. Next, place a rectangular four-well plate containing four bioreactors into the live-cell cell chamber. Then, click Live View, and center, and focus the image with the desired ROI.
Download the custom macrocode from the GitHub folder to control the stage position, the Arduino board, and video acquisition. Then, set the output movie as day_tissuegroup_tissuename_experiment.ND2. Run the macrocode with the desired x- and y-coordinates set on the stage, and acquire a fast time-lapse lapse with 1,700 frames at 50 frames per second.
After imaging, replace the media and return the samples to the incubator. Allow at least 24 hours between image acquisition sessions to avoid tissue fatigue.

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

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