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 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 α-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.
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 'Campenot' 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.
Written informed consent was obtained from all subjects, who provided their samples for iPSC generation and MAB harvesting. The procedure was approved by the medical ethics committee of the University Hospital Leuven (n° S5732-ML11268) and by the UK's main research ethics committee as part of the StemBANCC project. All reagents and equipment used in this protocol are listed in the Table of Materials and should be used sterile. Media should be heated to room temperature (RT) before use unless otherwise specified. For an overview of the co-culture protocol, please see Figure 1.
1. Differentiation of motor neuron progenitors from iPSCs
2. Derivation and maintenance of human MABs
NOTE: MABs are vessel-associated mesenchymal stem cells, which in this case have been harvested from biopsies obtained from a 58-year-old healthy donor. Alternative commercial sources are available. The protocol to obtain MABs is briefly explained. For further information, refer to the detailed protocol17. All MAB media should be heated to 37 °C before use.
3. Preparation of pre-assembled microfluidic devices – Day 9
NOTE: The protocol is adapted from the microfluidic device manufacturer's neuron device protocol and has been adjusted for the use of both pre-assembled and silicone devices. Here, pre-assembled devices are used for immunocytochemistry (ICC) and live-cell calcium transient recordings, while silicone devices are used for SEM. The timeline of the protocol follows the timeline for the motor neuron differentiation protocol.
4. Preparation of silicone microfluidic devices – Day 9
5. Plating of NPCs in microfluidic devices – Day 10
NOTE: According to the motor neuron differentiation protocol15, plating of day 10 NPCs occurs on a Thursday.
6. Plating of MAB in microfluidic devices – Day 17
7. Implementation of a volumetric and chemotactic gradient to promote the growth of motor neuron neurites towards the MAB compartment
Figure 1: Schematic overview of the motor unit protocol in microfluidic devices. Differentiation timeline and co-culture overview from day 0 to day 28 according to the timeline of the motor neuron differentiation protocol22. Motor neuron differentiation from iPSCs is initiated at day 0 and performed as stated previously for the following 10 days15. On day 9, the device is sterilized and coated with PLO-laminin. MABs are thawed for expansion in T75 flasks. On day 10, the motor neuron-NPCs are plated in both wells and the channel of one compartment (light grey) of the device, where their differentiation into motor neurons is continued for a week. MABs are plated in both wells and the channel of the opposite compartment (dark grey) on day 17. On day 18, MABs differentiation into myotubes is begun. On day 21, a volumetric and chemotactic gradient is established to promote motor neuron-neurite polarization through the microgrooves of the device. The motor neuron compartment received 100 µL/well of motor neuron basal medium without growth factors (light green compartment), while the myotube compartment received 200 µL/well of motor neuron basal medium with 30 ng/mL of growth factors (dark green compartment) (Table 2 and Table 3). The culture is continued with the volumetric and chemotactic gradient for an additional 7 days until analysis at day 28. Bright-field images show cell morphology at day 0, day 11, day 18, and day 28 cultured in pre-assembled microfluidic devices. Scale bar, 100 µm. This figure has been modified from Stoklund Dittlau, K. et al.18. Cell illustrations have been modified from Smart Server medical Art22. Please click here to view a larger version of this figure.
8. Fixation and ICC
NOTE: All steps should be done carefully to prevent detachment of the neuronal cultures. Do not remove liquid from the channels during the following steps.
9. Fixation and preparation of the device for SEM
NOTE: When changing liquids, always keep a small amount to cover the culture to avoid cell collapse. This protocol uses highly toxic substances, and it is required to work with personal protective equipment and in a fume hood during the entire process.
10. Assessment of NMJ functionality using live-cell calcium imaging
Generation of NMJs in microfluidic devices
To generate a human motor unit with functional NMJs in commercially available microfluidic devices, human iPSC-derived motor neurons and human MAB-derived myotubes were used. The quality of the starting cell material is important, and especially the fusion capability of the MABs into myotubes is crucial for a successful outcome of this protocol. MABs are easy to keep in culture. However, it is important to assess the fusion capability of each batch before applying them to the microfluidic devices (Supplemental Figure 1A,B)18. Any batches, which do not show myotube formation after 10 days of differentiation, should not be used. The fusion index in Supplemental Figure 1B was determined by calculating the percentage of nuclei within myotubes positive for each myotube marker of the total number of nuclei per image. We found that a fusion index of approximately 8% was sufficient for our co-culture in generating NMJs.
It is always important to commence a motor neuron differentiation from a pure culture of iPSCs. The purer the input – the purer the outcome. The motor neuron differentiation protocol generates motor neuron cultures, which are typically 85%-95% positive for motor neuron markers (Supplemental Figure 1C,D)18. The remaining cells will usually be undifferentiated precursor cells, which in some cases will undergo extensive proliferation and hereby have a negative impact on the quality of the culture. To get the best outcome of this protocol, the motor neuron differentiation efficiency should be evaluated before applying the day 10 motor neuron-NPCs into the device. In addition, a NPC quality check can be performed at day 11 to evaluate the expression of NPC marker Olig2 (Supplemental Figure 1E,F).
Initially, the motor neuron-NPCs and the MABs were plated at the same time point on day 10. Here, the MAB differentiation was initiated on day 11. The volume and growth factor gradient implemented on day 14 allowed us to evaluate the NMJ formation at day 21, thereby shortening the protocol by one week. Interestingly, we could observe characteristic NMJ formation by ICC (Supplemental Figure 2A). However, we were not able to acquire a functional output via the live-cell calcium recordings this early in the motor neuron differentiation (data not shown). We concluded that the motor neurons were not yet mature enough to form functional NMJ connections with the myotubes, even though the NMJ morphology looked promising. This is in line with our previous observations that spontaneous action potentials in motor neurons, recorded through patch-clamp electrophysiological analysis, only occur at day 35 of motor neuron differentiation15.
In addition, we attempted to prolong motor neuron maturation, as well as the co-culture sustainability, by maturing the motor neurons in the device for 2 weeks (day 24), before plating the MABs. Unfortunately, a large amount of spontaneous motor neuron-neurite crossing through microgrooves was observed, which resulted in the inhibition of MAB attachment (Supplemental Figure 2B). Due to the lack of myotube formation in the channel, we were unsuccessful in identifying NMJs at day 36 and therefore applied the 28-day protocol (Figure 1).
Identification, quantification, and morphological characterization of in vitro NMJs
After following the 28-day protocol (Figure 1), fully functional NMJs could be obtained. Both in vivo and in vitro, NMJs are characterized immunohisto- or immunocytochemically through the co-localization of a presynaptic marker and a postsynaptic marker. In this study, a combination of neurofilament heavy chain (NEFH) and SYP as a presynaptic marker combination was used, which allowed the following of a single neurite from the soma of the motor neuron towards the most distal process. On the muscle side, Btx is widely used as a postsynaptic marker for AChRs, and was likewise used in this study. The supplementation of agrin and laminin promotes the clustering of the AChRs at the sarcolemma19,20,21, making it easier to identify AChRs in vitro and likewise increases the number of AChRs and NMJs present18.
In order to locate and calculate the NMJs in an unbiased manner, each myotube is identified through myosin heavy chain (MyHC)-positivity and imaged in z-stacks at 40x magnification using an inverted confocal microscope. For very long myotubes, multiple z-stacks were acquired. For image analysis, the number of co-localizations between NEFH/SYP and Btx is counted manually through each z-stack, and the number of co-localizations is normalized to the number of myotubes present in the z-stack (Figure 2A-C)18. Not all myotubes will have NMJs, as seen in the quantification of innervated myotubes (Figure 2D). Consequently, it is important to perform an unbiased recording approach, where all myotubes are imaged, independent of Btx presence.
It is possible to identify two types of morphologies in this in vitro system. The NMJs either appear as single contact point NMJs, where a neurite touches upon a cluster of AChRs at one interaction point, or multiple contact point NMJs, where a neurite will fan out and engage with the AChR cluster over a larger surface. These two morphologies can be identified both immunocytochemically (Figure 2A)18 and with SEM (Figure 2B)18, and can likewise be quantified (Figure 2C)18. Overall, the multiple contact points facilitate a broader connection through a large muscle embedment, which points towards a more mature NMJ formation. In contrast, the single contact point NMJs are considered less mature due to the early developmental state of the culture.
Functional evaluation of in vitro NMJs
To evaluate the functionality of the NMJs, live-cell calcium transient recordings were used (Figure 3)18. Taking advantage of the fluidically isolated system of the microfluidic devices, the motor neuron soma side was stimulated with a high concentration (50 mM) of potassium chloride while simultaneously recording an influx in calcium in the myotubes, which were loaded with the calcium-sensitive Fluo-4 dye (Figure 3A). Almost immediately upon motor neuron activation, we could observe a calcium influx in the myotubes through a characteristic wave formation, which confirms a functional connection through the motor neuron-neurite and the myotube (Figure 3A-C)18. No spontaneous calcium waves nor spontaneous myotube contractions were observed, although myotube contraction upon direct stimulation with potassium chloride was observed. The specificity of the connection was further confirmed by adding the competitive AChR antagonist, tubocurarine hydrochloride pentahydrate (DTC) to the myotube compartment (Figure 3A), which resulted in an inhibition of calcium influx (Figure 3C). This effect confirmed that the connection between motor neurons and myotubes resulted in fully functional NMJs. To evaluate the number of active myotubes through NMJ stimulation, the myotube compartment was stimulated directly with potassium chloride to identify the total number of active myotubes in this compartment. Approximately 70% of the myotubes were active through motor neuron-stimulated activation with potassium chloride (Figure 3D)18.
These results confirm the optimal NMJ formation, number, morphology, and functionality through co-culturing of the iPSC-derived motor neurons and MAB-derived myotubes during a 28-day protocol.
Figure 2: NMJ formation in microfluidic devices. (A) Confocal micrographs of NMJ formation in pre-assembled microfluidic devices at day 28. NMJs are identified through the co-localization (arrowheads) of presynaptic markers (NEFH and SYP) and postsynaptic AChR marker (Btx) on MyHC-stained myotubes. NMJs are identified morphologically through single or multiple contact point formation between neurites and AChR clusters. DAPI label nuclei. Scale bar, 25 µm. Inset shows a magnification of an NMJ. Inset scale bar, 10 µm. (B) SEM of NMJ morphology in silicone microfluidic devices at day 28. Arrowheads depict neurite embedment into the myotube. Scale bar, 2 µm. Inset shows a magnification of NMJ. Inset scale bar, 1 µm. (C) Quantification of total number of NMJs per myotube as well as the number of single and multiple contact point NMJs per myotube. Graph is shown as mean ± standard error of the mean from four biological replicates. Statistical significance is determined with Mann-Whitney test with * p < 0.05. (D) Quantification of the percentage of innervated myotubes. Graph is shown as mean ± standard error of the mean from four biological replicates. This figure has been modified from Stoklund Dittlau, K. et al.18. Please click here to view a larger version of this figure.
Figure 3: Confirmation of NMJ functionality. (A) Schematic illustration of live-cell transient calcium recordings of NMJ functionality in pre-assembled microfluidic devices at day 28 before and after NMJ blockage with tubocurarine (DTC)22. Motor neurons in the light green compartment are stimulated with 50 mM potassium chloride (KCl), which causes an intracellular motor neuron response through the neurites. This evokes an influx of calcium (Ca2+) in myotubes, which are labeled with calcium-sensitive Fluo-4 dye (dark green compartment). (B) Fluo-4 fluorescence micrographs of pre-stimulation, intensity peak and post-stimulation of a myotube depicting a wave of intracellular calcium increase upon motor neuron stimulation with KCl. Inset shows a magnification of an innervated active myotube. Scale bars, 100 µm. Inset scale bar, 200 µm. (C) Representative calcium influx curves in myotubes after motor neuron stimulation with KCl (arrow) confirming NMJ functionality. Myotube 1-3 show characteristic calcium curves through motor neuron-myotube innervation, while myotube A-C DTC depicts curves after NMJ blocking with DTC. (D) Ratio of motor neuron-stimulated active myotubes on the total number of active myotubes. This figure has been modified from Stoklund Dittlau, K. et al.18. Cell illustrations have been modified from Smart Server medical Art22. Please click here to view a larger version of this figure.
Supplemental Figure 1: Motor neuron verification, MAB fusion index, and NPC quality control. (A) Confocal images of MAB-derived myotubes 10 days after initiation of differentiation. Myotubes are labelled with myotube markers: desmin, MyHC, myogenin (MyoG) and titin. Nuclei are stained with DAPI. Scale bar, 100 µm. (B) Quantification of MAB fusion index 10 days after initiation of differentiation. Upon starvation, MABs fuse into multinucleated myotubes, which were quantified for myotube marker positivity (AB+). Graph depicts mean ± standard error of the mean from three biological replicates. (C) Confocal images of iPSC-derived motor neurons at day 28 of differentiation, which are labelled with motor neuron markers NEFH, choline acetyltransferase (ChAT) and Islet-1 in addition to pan-neuronal marker βIII-tubulin (Tubulin). Nuclei are stained with DAPI. Scale bars, 75 µm. (D) Quantification of the number of cells, which are positive for motor neuron and pan-neuronal markers (AB+). Graph depicts mean ± standard error of the mean from three biological replicates. (E) Confocal images of iPSC-derived NPCs at day 11 of motor neuron differentiation, which are labelled with NPC marker Olig2 and pan-neuronal marker βIII-tubulin (Tubulin). Nuclei are stained with DAPI. Scale bars, 50 µm. (F) Quantification of the number of NPCs, which are positive for Olig2 and βIII-tubulin (AB+). Graph depicts mean ± standard error of the mean from three biological replicates. This figure has been modified from Stoklund Dittlau, K. et al.18. Please click here to download this File.
Supplemental Figure 2: Optimization of co-culture protocol (A) Confocal images of NMJ formation at day 21 of motor neuron differentiation, when MABs are seeded at the same time point as NPCs at day 10. NMJs are identified through the co-localization (arrowheads) of presynaptic markers (NEFH and SYP) and postsynaptic AChR marker (Btx) on MyHC-stained myotubes. Scale bar (left), 10 µm. Scale bar (right), 5 µm. (B) Bright-field image of the myotube channel at day 24 depicting spontaneous motor neuron-neurite crossing inhibiting the attachment of MABs. Scale bar, 100 µm. Please click here to download this File.
Reagent | Stock concentration | Final concentration |
IMDM | 1x | 80% |
Fetal bovine serum | 15% | |
Penicillin/Streptomycin | 5000 U/mL | 0.5% |
L-glutamine | 50x | 1% |
Sodium pyruvate | 100 mM | 1% |
Non-essential amino acids | 100x | 1% |
Insulin transferrin selenium | 100x | 1% |
bFGF (added fresh) | 50 μg/mL | 5 ng/mL |
Table 1: MAB growth medium. Medium can last 2 weeks at 4 °C. bFGF is added fresh on the day of use.
Reagent | Stock concentration | Final concentration |
DMEM/F12 | 50% | |
Neurobasal medium | 50% | |
Penicillin/Streptomycin | 5000 U/mL | 1% |
L-glutamine | 50x | 0.5 % |
N-2 supplement | 100x | 1% |
B-27 without vitamin A | 50x | 2% |
β-mercaptoethanol | 50 mM | 0.1% |
Ascorbic acid | 200 μM | 0.5 μM |
Table 2: Motor neuron basal medium. Medium can last 4 weeks at 4 °C.
Day | Reagent | Stock concentration | Final concentration | Compartment |
Day 10/11 | Smoothened agonist | 10 mM | 500 nM | Both |
Retinoic acid | 1 mM | 0.1 μM | ||
DAPT | 100 mM | 10 μM | ||
BDNF | 0.1 mg/mL | 10 ng/mL | ||
GDNF | 0.1 mg/mL | 10 ng/mL | ||
Day 14 | DAPT | 100 mM | 20 μM | Both |
BDNF | 0.1 mg/mL | 10 ng/mL | ||
GDNF | 0.1 mg/mL | 10 ng/mL | ||
Day 16 | DAPT | 100 mM | 20 μM | Both |
BDNF | 0.1 mg/mL | 10 ng/mL | ||
GDNF | 0.1 mg/mL | 10 ng/mL | ||
CNTF | 0.1 mg/mL | 10 ng/mL | ||
Day 18 | BDNF | 0.1 mg/mL | 10 ng/mL | Motor neuron |
GDNF | 0.1 mg/mL | 10 ng/mL | ||
CNTF | 0.1 mg/mL | 10 ng/mL | ||
Day 21+ | BDNF | 0.1 mg/mL | 30 ng/mL | Myotube |
GDNF | 0.1 mg/mL | 30 ng/mL | ||
CNTF | 0.1 mg/mL | 30 ng/mL | ||
Agrin | 50 μg/mL | 0,01 μg/mL | ||
Laminin | 1 mg/mL | 20 μg/mL | ||
Day 21+ | No supplements | Motor neuron |
Table 3: Motor neuron medium supplements. Supplements are added fresh on the day of use to the motor neuron basal medium.
Day | Reagent | Stock concentration | Final concentration | Compartment |
Day 18 | DMEM/F12 | 97% | MAB | |
Sodium pyruvate | 100 mM | 1% | ||
Horse serum | 2% | |||
Agrin | 50 μg/mL | 0.01 μg/mL |
Table 4: MAB differentiation medium. Medium can last 2 weeks at 4 °C. Agrin is added fresh on the day of use.
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.
The authors have nothing to disclose.
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 "Opening the Future" Fund), the VIB, the Agency for Innovation by Science and Technology (IWT; SBO-iPSCAF), the "Fund for Scientific Research Flanders" (FWO-Vlaanderen), Target ALS, the ALS Liga België (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 "Association Belge contre les Maladies neuro-Musculaires" (ABMM). T.V. and J.B. are supported by Ph.D. fellowships awarded by FWO-Vlaanderen.
α-bungarotoxin (Btx) Alexa fluor 555 | Thermo Fisher Scientific | B35451 | Antibody (1:1000) |
Acetic Acid | CHEM-Lab NV | CL00.0116.1000 | Coating component. H226, H314. P280 |
Aclar 33C sheet (SEM sheet) | Electron Microscopy Sciences | 50425-25 | Thickness: 7.8 mil |
Agrin (recombinant human protein) | R&D systems | 6624-AG-050 | Media supplement |
Alexa fluor IgG (H+L) 488 donkey-anti rabbit | Thermo Fisher Scientific | A21206 | Antibody (1:1000) |
Alexa fluor IgG (H+L) 555 donkey-anti goat | Thermo Fisher Scientific | A21432 | Antibody (1:1000) |
Alexa fluor IgG (H+L) 555 donkey-anti mouse | Thermo Fisher Scientific | A31570 | Antibody (1:1000) |
Alexa fluor IgG (H+L) 647 donkey-anti mouse | Thermo Fisher Scientific | A31571 | Antibody (1:1000) |
Ascorbic acid | Sigma | A4403 | Media component |
βIII-tubulin (Tubulin) | Abcam | ab7751 | Antibody (1:500) |
β-mercaptoethanol | Thermo Fisher Scientific | 31350010 | Media component. H317. P280. |
B-27 without vitamin A | Thermo Fisher Scientific | 12587-010 | Media component |
BDNF (brain-derived neurotrophic factor) | Peprotech | 450-02B | Growth factor |
bFGF (recombinant human basic fibroblast growth factor) | Peprotech | 100-18B | Growth factor |
Choline acetyltransferase (ChAT) | Millipore | ab144P | Antibody (1:500) |
Collagen from calfskin | Thermo Fisher Scientific | 17104019 | Coating component |
CNTF (ciliary neurotrophic factor) | Peprotech | 450-13B | Growth factor |
DAPI Nucblue Live Cell Stain ReadyProbes reagent | Thermo Fisher Scientific | R37605 | Immunocytochemistry component |
DAPT | Tocris Bioscience | 2634 | Media supplement |
Desmin | Abcam | Ab15200 | Antibody (1:200) |
DMEM/F12 | Thermo Fisher Scientific | 11330032 | Media component |
DMSO | Sigma | D2650-100ML | Cryopreservation component. H315, H319, H335. P280. |
Dulbecco's phosphate-buffered saline (DPBS) | Thermo Fisher Scientific | 14190250 | no calcium, no magnesium |
Ethanol | VWR | 20.821.296 | Sterilization. H225. P280 |
Fetal bovine serum | Thermo Fisher Scientific | 10270106 | Media component |
Fluo-4 AM live cell dye | Thermo Fisher Scientific | F14201 | Calcium imaging dye |
Fluorescence Mounting Medium | Dako | S3023 | Immunocytochemistry component |
GDNF (glial cell line-derived neurotrophic factor) | Peprotech | 450-10B | Growth factor |
Glutaraldehyde | Agar Scientific | R1020 | Fixation component. EUH071, H301, H314, H317, H330, H334, H410. P280. |
Horse serum | Thermo Fisher Scientific | 16050122 | Media component |
Human alkaline phosphatase | R&D systems | MAB1448 | Antibody |
ImageJ software | NIH | ICC analysis | |
IMDM | Thermo Fisher Scientific | 12440053 | Media component |
Insulin transferrin selenium | Thermo Fisher Scientific | 41400045 | Media component |
Islet-1 | Millipore | ab4326 | Antibody (1:400) |
Knockout serum replacement | Thermo Fisher Scientific | 10828-028 | Cryopreservation component |
Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane | Sigma | L2020-1MG | Coating component and media supplement |
Leica SP8 DMI8 confocal microscope | Leica | ICC confocal microscopy | |
L-glutamine | Thermo Fisher Scientific | 25030-024 | Media component |
Myogenin (MyoG) | Abcam | Ab124800 | Antibody (1:500) |
Myosin heavy chain (MyHC) | In-house, SCIL | Antibody (1:20) | |
N-2 supplement | Thermo Fisher Scientific | 17502-048 | Media component |
Neurobasal medium | Thermo Fisher Scientific | 21103049 | Coating and media component |
Neurofilament heavy chain (NEFH) | Abcam | AB8135 | Antibody (1:1000) |
Nikon A1R confocal microscope | Nikon | Live-cell calcium imaging microscopy | |
NIS-Elements AR 4.30.02 software | Nikon | Live-cell calcium imaging analysis | |
Non-essential amino acids | Thermo Fisher Scientific | 11140050 | Media component |
Normal donkey serum | Sigma | D9663-10ML | Immunocytochemistry component |
Olig2 | IBL | 18953 | Antibody (1:1000) |
Parafilm M | Sigma | P7793-1EA | Storing equipment |
Paraformaldehyde | Thermo Fisher Scientific | 28908 | Fixation component. H302, H312, H315, H317, H319, H332, H335, H341, H350. P280. |
Penicillin/Streptomycin (5000 U/mL) | Thermo Fisher Scientific | 15070063 | Media component |
Petri dish (3 cm) | nunc | 153066 | Diameter: 3 cm |
Petri dish (10 cm) | Sarstedt | 833.902 | Diameter: 10 cm |
Plate (6-well) | Cellstar Greiner bio-one | 657160 | Culture plate |
Pluronic F-127 | Thermo Fisher Scientific | P3000MP | Fluo-4 dye solvent |
Poly-L-ornithine (PLO) | Sigma | P3655-100MG | Coating component |
Potassium chloride | CHEM-Lab NV | CL00.1133.1000 | Calcium imaging reagent |
Retinoic acid | Sigma | R2625 | Media supplement. H302, H315, H360FD, H410. P280. |
RevitaCell supplement | Thermo Fisher Scientific | A2644501 | ROCK inhibitor solution |
Smoothened agonist | Merch Millipore | 566660 | Media supplement |
Sodium cacodylate buffer | Sigma | C0250 | Fixation component. H301, H331, H350, H410. P280. |
Sodium pyruvate | Life Technologies | 11360-070 | Media component |
Synaptophysin (SYP) | Cell Signaling | 5461S | Antibody (1:1000) |
T75 flask | Sigma | CLS3276 | Culture plate |
Titin | Developmental Studies Hybridoma Bank | 9D10 | Antibody (1:300) |
Triton X-100 | Sigma | T8787-250ML | Immunocytochemistry component. H302, H315, H318, H319, H410, H411. P280 |
TrypLE express | Thermo Fisher Scientific | 12605010 | MAB dissociation solution |
Tubocyrarine hydrochloride pentahydrate | Sigma | T2379-100G | Acetylcholine receptor blocker. H301. P280. |
XonaChips pre-assembled microfluidic device | Xona Microfluidics | XC150 | Microgroove length: 150 μm |
Xona Silicone microfluidics device | Xona Microfluidics | SND75 | Microgroove length: 75 μm |