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

Summary

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.

Abstract

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'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.

Introduction

The neuromuscular junction (NMJ) is the chemical synapse between motoneurons (MNs) and skeletal muscle cells (SkM) that allows for muscle contraction. Toxins, such as neurotoxin α-bungarotoxin (BTX), or neuromuscular diseases (NMD) like myasthenia gravis (MG) can lead to degeneration of the NMJ and reductions in muscle control¹. Bioengineered human tissue models better recapitulate the functional and physiological mechanisms of human NMJs and offer greater translational potential than animal models.

While animal models have advanced the understanding of the formation and function of the NMJ, there are significant differences between human and animal synapses that limit the translation of results to humans and make in vivo characterization of the NMJ challenging^2,3,4. Studies have shown distinct physiological differences between mouse and human NMJs. Mice have larger NMJs and smaller active zone densities when compared to human NMJs⁴. Additionally, drug studies conducted in animal models do not always reflect the effects found in human clinical trials. Engineered human tissue models provide the opportunity to study the healthy development of the NMJ and the pathology of neuromuscular diseases and allow for drug screenings. Human induced pluripotent stem cells (hiPSCs)⁵ can be differentiated into a variety of cell types, including skeletal muscle cells^6,7 and motoneurons^8,9. hiPSCs can be generated easily from patient cells, allowing for better disease modeling¹⁰ and drug screening^11,12 through patient-specific tissue models.

Two-dimensional (2D) monolayer co-cultures of SkMs and MNs lack the morphology, phenotype, organization, and functional behavior of physiological NMJs. NMJs randomly form in 2D culture, which inhibits the isolation of motor units for analysis, limits accurate functional measurements, and prevents their use for repeated, systematic experiments¹³. Three-dimensional (3D) tissue models of NMJs overcome many of these limitations, recapitulating the morphological and functional characteristics of physiological NMJs^{7,14,15,16,17}. Using this model, the two tissue types are developed separately and then integrated by directing axonal growth, allowing for more organized NMJs to develop compared to 2D culture systems.

Our previous study demonstrated that combining optogenetics with tissue engineering can allow for accurate non-invasive stimulation and evaluation of NMJ function^18,19. Through genetic engineering, light-sensitive proteins can be integrated into the genome of hiPSCs. Integrating channelrhodopsin-2 (ChR2), an ion channel that opens in response to blue light, into the membrane of excitable cells such as neurons allows for non-contact spatiotemporal control over cell activation^20,21,22. hiPSCs carrying ChR2 can be differentiated into optogenetic motoneurons sensitive to blue light, removing the need for typical invasive electrodes that stimulate neurons and avoiding unwanted stimulation of the muscle cells by electrodes²³. This system uses optogenetic motoneurons to stimulate contractions in non-optogenetic skeletal muscle cells. Combining video acquisition and controlled blue light illumination allows for the co-cultured tissues to be simultaneously stimulated and recorded for NMJ function.

MG is caused by autoantibodies targeting nicotinic acetylcholine receptors (AChR), which results in decreased NMJ function and muscle weakness²⁴. It is diagnosed based on presented symptoms, electrodiagnosis, and detection of autoantibodies via serological blood tests. However, not all autoantibodies involved in MG have been identified, and some seronegative patients are diagnosed with MG but with no recognized antibodies^25,26. Our system allows for repeated functional assessment of the NMJ before and after the addition of serum from MG patients, providing invaluable insight into the functional and biochemical changes caused by the MG antibodies¹⁸. Our protocol illustrates how to produce 3D in vitro models of functional human NMJ that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. We demonstrate the versatility of the system in two platforms, a microfluidic device, and a larger open-well bioreactor platform.

Protocol

All cell lines for this work were created and used in compliance with the institutional guidelines of Columbia University, NY, USA.

1. Bioreactor preparation

1. Make bioreactor molds
   1. Download a bioreactor CAD file from the Supplementary CAD File or create a custom own design.
   2. Generate a CNC toolpath from the 3D model using CAM software.
   3. Machine acetal molds using a CNC milling machine.
2. Fabrication of bioreactors
   1. Mix a 10:1 base to curing agent mixture of polydimethylsiloxane (PDMS, 77 g of mixture per 4 platforms/molds).
   2. Place the mixture into a vacuum chamber, close all the valves, turn on the vacuum, and de-gas the mixture for at least 30 min until no air bubbles remain. Pour the mixture into molds and de-gas the molds in the vacuum chamber for 1 h.
   3. Close the molds with the top half of the mold in the correct orientation. Place a steel hexagonal rod over the center and clamp on both sides.
   4. Refill the top with PDMS.
   5. Cure the molds in a 65 °C oven for at least 4 h.
   6. Remove the platforms from the molds after cooling down to room temperature.
   7. Clean the devices in an ultrasonic bath using 1 h cycles of dish soap, 400 mL of 100% isopropanol, and distilled water.
   8. Dry overnight at 65 °C.
   9. Soak glass coverslips in 1% nonionic surfactant polyol for 30 min, and ensure that the coverslips do not stack so that they are all properly coated.
   10. Rinse well with distilled water and dry overnight at 65 °C.
   11. Use a plasma cleaner on high with 6 L/min of oxygen for 1-2 min to the treat glass coverslips and PDMS platform. Once treated, bond the two together by pressing the PDMS down onto the coverslip for at least 30 s.
   12. Autoclave the bonded devices before adding cells.

2. Building an optical stimulation setup

1. Using a 30 mm cage cube system, attach a 573 nm dichroic mirror in the center. Couple a red 627 nm LED with a 594 nm long-pass excitation filter and attach it to the top side of the cube, with the LED facing the mirror. Then attach a blue 488 nm LED with a 546 nm short-pass excitation filter to the adjacent side of the cube, with the LED facing the mirror as shown in the schematic in Figure 3A.
2. Attach a ring-actuated iris diaphragm to the bottom of the cage cube to control the size of the illuminated area.
3. Power each LED by a T-Cube LED driver and control via an Arduino Uno Rev3 board. Connect the side input of the LED driver to a power source plug, connect the middle output to the Arduino, and connect the side output directly to the LEDs.
4. Connect the Arduino Uno to a PC by a USB cable.
5. Download files from GitHub link (https://github.com/ofvila/NMJ-function-analysis).
6. Open the "Optical_Stimulation_Ramp_Arduino.ino" file from GitHub folder.
7. Set starting parameters: Steps = 30; startFrequency = 0.5; endFrequency = 3; pulseLength = 100.
8. Ensure that pin output 4 is assigned to the Blue LED driver and pin output 2 is assigned to the Red LED driver. Check the middle wires of the LED driver are connected to the ground and to the respective pin output.
9. Compile and load the "Optical_Stimulation_Ramp_Arduino.ino" program to Arduino.
10. Connect each LED driver to its corresponding channel.

3. Cell culture setup (day -21-0)

1. Primary skeletal muscle cells
   1. Thaw and expand primary skeletal myoblasts (obtained from Cook Myosite) for a maximum of six passages using Myotonic growth medium (+ supplement). Maintain cells in an incubator set to 37 °C and 5% CO₂.
   2. Change the media every 2 days.
   3. Once cells are about 60% confluent, passage them using 0.05% Trypsin-EDTA (1x, for 5 min at 37 °C). Collect the dissociated cells and add fresh media to neutralize the trypsin.
   4. Spin down the cells at 300 x g and aspirate the supernatant above the cell pellet.
   5. Resuspend the cells with MGM and seed 1:3 with 60 mL per triple-layer flask.
2. ChR2-hiPSC
   NOTE: All cell lines for this work were created and used in compliance with the institutional guidelines of Columbia University, NY, USA. In this protocol, ChR2-expressing hiPSCs were generated via CRISPR-Cas9 genome editing using previously described methods¹⁸, but any stable optogenetic cell lines (lentiviral, piggyBac, etc.) can be used in the same way. Constitutive promoters expressed in both iPSCs and motoneurons were chosen (CAG). The cells were found to have healthy karyotype, as noted in previous publications^{18, 19}.
   1. Coat 6-well plates with 1 mL/well of solubilized basement membrane matrix diluted in DMEM/F12 (1:80) and incubate the plates at room temperature for 1 h.
   2. Seed iPSCs on coated 6-well plates with 2 mL of feeder-free cell culture medium (iPSC media), exchanging 2 mL of media every other day and passaging every 5-7 days. Maintain cells in an incubator set to 37^{°C and 5% CO}₂.
   3. Passaging
      1. Dissociate the stem cells by incubating with 1 mL of enzyme-free stem cell releasing reagent for 4 min and mechanically shearing with a wide tip P1000 pipette.
      2. Seed cells at a ratio of 1:24 or 1:48 in iPSC media with 2 μM of Y-27632 dihydrochloride in 2 mL/well onto coated 6-well plates.

4. Skeletal muscle tissue seeding (day -3)

1. Isolate and count 30 x 10⁶ myoblasts using an automated cell counter.
2. Resuspend the myoblasts in a 4:1 mix of 3 mg/mL collagen I and solubilized basement membrane matrix (800 μL of collagen mixture + 200 μL of basement membrane matrix to reach a final volume of 1 mL). For the collagen component, add the accompanying neutralizing agent (9:1 mix of collagen to neutralizing agent) and dilute the mixture with 1x PBS to achieve a volume of 800 μL.
3. Add 15 μL of cell-collagen suspension to each muscle chamber of the bioreactor, being sure to spread the suspension across both pillars using the pipette tip (Figure 2).
4. Let the cell-gel mixture polymerize at 37 °C for 30 min and then fill each bioreactor well with 450 μL of Myotonic growth media.
5. After 3 days (once the gel has compacted), begin myotube differentiation.

5. Myotube differentiation (days 0-14)

1. Begin myotube infusion by switching tissues to 450 μL of fusion inducing media (FS, Table 1) for 7 days, changing the media every other day.
   NOTE: Try to begin myotube differentiation on the same day as motoneuron differentiation from hiPSCs in order to seed motoneurons at the end of both cell types' differentiation schedules.
2. On day 7 change the media to 450 μL of maturation media Ia (MMIa, Table 1).
3. On day 9, change to 450 μL of maturation media Ib (MMIb, Table 1).
4. On day 11, change the media to 450 μL of NbActiv4. Keep changing the media every 2 days until the motoneurons are seeded (Step 7).

6. Motoneuron differentiation (days 0-14)

NOTE: Our motoneuron differentiation protocol was adapted from Maury et al⁸.

1. On day 0, transfer 4 x 10⁶ ChR2- hiPSCs to an ultra-low attachment Petri dish with 15 mL of motoneuron suspension culture medium (MSCM, Table 1). Supplement MSCM with 3 μM CHIR99021, 0.2 μM LDN193189, 40 μM SB431542 hydrate and 5 μM Y-27632 dihydrochloride.
2. On day 2, isolate the neurospheres (NS) with a 37 μM reversible strainer and replate in 15 mL of MSCM with 3 μM CHIR99021, 0.2 μM LDN193189, 40 μM SB431542 hydrate, and 0.1 μM retinoic acid. NS should be visible in the Petri dish without using a microscope after day 2.
3. On day 4, transfer the cells and media to a 50 mL tube and allow the neurospheres to settle to the bottom (5 min). Aspirate the supernatant and resuspend the cells in 15 mL of MSCM supplemented with 0.5 μM SAG, 0.2 μM LDN193189, 40 μM SB431541, and 0.1 μM retinoic acid.
4. On day 7, repeat Step 6.3. but resuspend the cells in 15 mL of MSCM supplemented with 0.5 μM SAG and 0.1μM retinoic acid.
5. On day 9, repeat Step 6.3. but resuspend cells in 15 mL of MSCM supplemented with 10 μM DAPT.
6. On day 11, repeat Step 6.3. but resuspend the cells in 15 mL of MSCM supplemented with 20 ng/mL BDNF and 10 ng/mL GDNF.
7. On day 14, seed the motoneurons into the platform.

7. Seeding motoneurons aggregates in bioreactor (day 14)

1. Prepare a 4:1 gel mixture of 2 mg/mL collagen I and Matrigel (800 μL of collagen mixture + 200 μL of Matrigel to reach a final volume of 1 mL). For the collagen component, add the accompanying neutralizing agent (9:1 mix of collagen to neutralizing agent) and dilute the mixture with 1x PBS to achieve a final volume of 800 μL.
2. Use a 400 nm cell strainer to select large neurospheres and resuspend them in the gel mixture.
3. Aspirate media from the reservoir and carefully from the neurosphere well (Figure 2).
4. Add 15 μL of gel mixture into the neurosphere channel.
5. Load a 10 μL pipette with 10 μL of gel and then pick one neurosphere.
6. Deposit the NS into the neurosphere channel and ensure that the NS is in the chamber. Slowly raise the pipette while releasing the remaining gel once the NS is deposited. If unsure that the NS was correctly deposited, check its location using a microscope.
7. Allow the gel to polymerize for 30 min at 37 °C.
8. Add 450 μL of NbActiv4 supplemented with 20 ng/mL BDNF and 10 ng/mL GDNF to the reservoirs.
9. Change the media every other day to allow for axonal growth from NS to muscle tissue.

8. Simultaneous optical stimulation and video recording of NMJ function (day 24+)

1. For imaging, use an inverted fluorescent microscope with a scientific complementary metal-oxide-semiconductor (sCMOS) camera.
2. Set the camera software binning to 2x2, exposure to 20 ms, rolling shutter ON, readout rate to 540 MHz, dynamic range: 12-bit & gain 1, and sensor mode: overlap.
3. Use 2x objective on the microscope to image the microtissues.
4. Attach a live-cell chamber (37 °C, 5% CO₂) to the microscope stage.
5. Select the region of interest (ROI) that contains the innervated skeletal tissues tissue to minimize the file size and processing time.
6. Place a 594 nm long-pass emission filter between the sample and the imaging objective to filter out blue light pulses from the camera.
7. Place a rectangular 4-well plate containing 4 bioreactors (24 tissues) into the live-cell chamber.
8. Click Live View. Center and focus the image with the desired ROI.
9. Upload the custom macro code from the GitHub folder (https://github.com/ofvila/NMJ-function-analysis) to control the stage position, the Arduino board, and video acquisition.
10. Set output movie as: day_tissue group_tissue name_experiment.nd2.
11. Run the macro code with the desired X,Y coordinates set on the stage and acquire a fast time lapse with 1700 frames at 50 frames/s.
12. Replace the media after imaging and return the samples to the incubator. Allow at least 24 h between image acquisition sessions to avoid tissue fatigue.

9. Batch processing and analysis (day 24+)

1. Movie processing
   1. Use the custom MATLAB code to process the videos in batch analysis. The files can be downloaded from the GitHub folder (https://github.com/ofvila/NMJ-function-analysis). The functions are listed and explained in Table 2.
      NOTE: The code is compatible with .nd2 and .czi formats. It requires parallel processing to be activated in MATLAB and needs the bioformats package.
   2. Run the recursiveOSAnalysis script to analyze the movies through parallel processing. Have all of the acquired videos in the same folder and select that folder when running the code.
   3. Adjust the post-analysis parameters as needed.
      1. Change baselineTime (option 1) if spontaneous contraction is picked up at beginning of recording. This will show an initial reading way above 0 and will need the frame to be shifted to compensate.
      2. Change peakThreshold (option 2) if the contractions are not being registered. The code will detect contractions that are above 25% of the highest peak by default so that this value can be changed.
      3. Change minMinProminence and minMinWidth to adjust the sensitivity when detecting the start of each peak.
   4. Generate video and graph output.
      1. Run recursiveOSMovie to generate a video file for each tissue with its respective contractility trace.

10. Perturbation of NMJ function (day 24+)

1. Prepare treatment media by adding external effector to NbActiv4 basal media supplemented with 20 ng/mL BDNF and 10 ng/mL GDNF.
   1. For the MG sera experiment, use 20% MG patient sera in NbActiv4 + BDNF + GDNF.
   2. For the BTX experiment, use 5 μg/mL BTX in NbActiv4 + BDNF + GDNF.
2. Stimulate/image using Step 3.2. to record the baseline.
3. Replace the media in tissues with treatment media (450 μL/tissue).
4. Incubate for the desired time (48 h for patient sera, 20 min for BTX).
5. Stimulate/image again using Step 3.2. to record function after treatment.
6. Remove the treatment media and allow the tissues to rest for the desired time (48 h after sera removal)
   NOTE: Allow at least 24 h between stimulation and imaging to avoid tissue fatigue

Results

Neuromuscular junctions were generated by co-culturing optogenetic hiPSC-derived motoneurons with non-optogenetic skeletal muscle tissue. Human primary skeletal myoblasts (SkM) were seeded into the platforms and differentiated into multinucleated myotubes using the 2-week protocol. The optogenetic motoneurons were differentiated separately, but in parallel with the myotube differentiation, and then seeded into the platform (Figure 1). The tissues began contracting in response to blue light s...

Discussion

This system is an engineered 3D human tissue model that combines optogenetics and video processing to enable automated and unbiased evaluation of NMJ function. Using a standardized protocol, we have demonstrated the ability to measure changes in NMJ function during physiological development and characterize the damaging effects of pathologies such as neurotoxin exposure and myasthenia gravis patient sera.

Previous studies have reported the ability to model MG with optogenetic hPSC-derived moto...

Materials

Name	Company	Catalog Number	Comments
Cells
SkMDC	Cook Myosite	P01059-14M
Media and Supplements
Advanced DMEM/F12	ThermoFisher Scientific	12634-020
Bovine Serum Albumin solution	Millipore Sigma	A9576-50ML
G-5 Supplement (100X)	ThermoFisher Scientific	17503-012
Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)	ThermoFisher Scientific	10131-035
Insulin, Recombinant Human	Millipore Sigma	91077C-100MG
Matrigel	Corning	354277
mTeSR Plus	Stem Cell Technologies	100-0276
MyoTonic Growth Media Kit	Cook Myosite	MK-4444
N-2 Supplement	ThermoFisher Scientific	17502-048
NBactiv4 500 mL	BrainBits LLC	Nb4-500
Neurobasal Medium	ThermoFisher Scientific	21103-049
Neurobasal-A Medium	ThermoFisher Scientific	A13710-01
Pluronic F-127	Sigma Aldrich	P2443
ReLeSR	Stem Cell Technologies	5872
Plasticware
30 mm cage cube system	ThorLabs	CM1-DCH, CP33, ER1-P4 and ER2-P4
37 µm Reversible Strainer, large	Stem Cell Technologies	27250
546 nm short-pass excitation filter	Semrock	FF01-546/SP-25
573 nm dichroic mirror	Semrock	FF573-Di01–25x36
594 nm long- pass emission filter	Semrock	BLP01-594R-25
594 nm long-pass excitation filter	Semrock	BLP01-594R-25
Blue (470nm) Rebel LED on a SinkPAD-II 10mm Square Base - 65 lm @ 700mA	LuxeonStarLEDs	SP-05-B4
Carclo 29.8° Frosted 10 mm Circular Beam Optic - Integrated Legs	LuxeonStarLEDs	10413
Corning 60 mm Ultra-Low Attachment Culture Dish	Corning	3261
Heat sink	LuxeonStarLEDs	LPD-19-10B
Optics
pluriStrainer 400 µm, 25 pack, sterile	PluriSelect	43-50400-03
pluriStrainer 500 µm, 25 pack, sterile	PluriSelect	43-50500-03
Red (627nm) Rebel LED on a SinkPAD-II 10mm Square Base - 65 lm @ 700mA	LuxeonStarLEDs	SP-05-R5
ring-actuated iris diaphragm	ThorLabs	SM1D12D
T-Cube LED drivers	ThorLabs	LEDD1B, KPS101
Molds
Female Threaded Hex Standoffs,  3 1/2" 10-32, Partially Threaded 1/2"	McMaster	91920A046
Low-Profile C-Clamp	McMaster	1705A12
Growth Factors
Adenosine 3′,5′-cyclic monophosphate	Millipore Sigma	A9501-1G
CHIR 99021, 10 mg	Tocris	4423/10
DAPT 10 mg	R&D Systems	2634/10
Human CNTF, research grade, 5 µg	Miltenyl Biotec	130-096-336
Human Vitronectin Protein, CF	R&D Systems	2349-VN-100
Human Vitronectin Protein, CF	R&D Systems	2349-VN-100
IGF1 Recombinant Human Protein	ThermoFisher Scientific	PHG0078
Laminin mouse protein, natural	ThermoFisher Scientific	23017015
Recombinant Human Agrin Protein	R&D Systems	6624-AG-050
Recombinant Human GDNF Protein, CF 50ug	R&D Systems	212-GD-050/CF
Recombinant Human Neurotrophin 3 100 ug	Cell Sciences	CRN500D
Recombinant Human Neurotrophin-4	Cell Sciences	CRN501B
Recombinant Human Sonic Hedgehog/Shh (C24II) N-Terminus	R&D Systems	1845-SH-100
Recombinant Human/Murine/Rat BDNF 50 ug	Peprotech	450-02
Retinoic Acid, 50 mg	Millipore Sigma	R2625-50
SAG Smoothened Agonist	Millipore Sigma	566660
SB431542 10 mg	Stem Cell Technologies	72234
StemMACS LDN-193189	Miltenyl Biotec	130-103-925
Vitronectin from human plasma	Millipore Sigma	V8379-50UG
Y-27632 dihydrochloride	Tocris	1254
Antibodies
α-actinin mAb (Mouse IgG1)	Abcam	ab9465
Choline Acetyltransferase (ChAT) (Goat)	Millipore	AB144P
Desmin mAb (Mouse IgG1)	Dako	M076029-2
Myosin Heavy Chain (MHC) (Mouse IgG2b)	DSHB	MF20
Equipment
Arduino Uno R3	Arduino	A000066
Automated stage	Applied scientific instrumentation	MS- 2000 XYZ
Expanded plasma cleaner	Harrick Plasma	PDC-001 (115V)
Invitrogen Countess Automated Cell Counter	Marshal Scientific	I-CACC
IX-81 Inverted fluorescence microscope	Olympus	IX-ILL100LH
Series Stage Top Incubator System	Tokai Hit STX	TOKAI-HIT-STXG
Zyla 4.2 sCOMS Camera	Andor Technology	ZYLA-4.2P-CL10
Software
Arduino Software (IDE)	Arduino	IDE 1.8.19
Mastercam	Mastercam	Mastercam for Solidworks
Matlab	Matlab	R2021b
NIS elements	Nikon	Basic Research
Solidworks 3D CAD	Solidworks	Solidworks Standard