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....

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).
   <....

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