Stem cell derived phenotypic human neuromuscular junction model for dose response evaluation of therapeutics

Abstract

There are currently no functional neuromuscular junction (hNMJ) systems composed of human cells that could be used for drug evaluations or toxicity testing in vitro. These systems are needed to evaluate NMJs for diseases such as amyotrophic lateral sclerosis, spinal muscular atrophy or other neurodegenerative diseases or injury states. There are certainly no model systems, animal or human, that allows for isolated treatment of motoneurons or muscle capable of generating dose response curves to evaluate pharmacological activity of these highly specialized functional units. A system was developed in which human myotubes and motoneurons derived from stem cells were cultured in a serum-free medium in a BioMEMS construct. The system is composed of two chambers linked by microtunnels to enable axonal outgrowth to the muscle chamber that allows separate stimulation of each component and physiological NMJ function and MN stimulated tetanus. The muscle's contractions, induced by motoneuron activation or direct electrical stimulation, were monitored by image subtraction video recording for both frequency and amplitude. Bungarotoxin, BOTOX^® and curare dose response curves were generated to demonstrate pharmacological relevance of the phenotypic screening device. This quantifiable functional hNMJ system establishes a platform for generating patient-specific NMJ models by including patient-derived iPSCs.

Keywords: BioMEMS; Dose response; Drug discovery; Human; Neuromuscular junction.

Fig. 1. NMJ BioMEMS system

A) The overall system layout consisted of a co-culture chip with separate populations of motoneurons and skeletal muscle connected through micro-tunnels. A microscope was used to image the co-culture and detect skeletal muscle contractions upon electrical stimulation. Stimulation is provided by two silver electrodes dipped into the bath of either chamber. Electrical pulses on the motoneuron-side evoked action potentials that traveled down the axons to the neuromuscular junction. Electrical pulses on the muscle-side cause direct depolarization of the myotubes. B) A PDMS co-culture construct was made using a master of SU8 on silicon. The molded PDMS was peeled off and cut to create two open-air compartments for the cells. The cut PDMS was then plasma bonded to a silicon or silica substrate depending on whether it was viewed by an upright or inverted microscope. C) Motion capture was used to transform a contraction video into an amplitude plot. A single frame of video was taken when the myotubes were not engaged in active movement. Subsequent frames were normalized to this reference frame. When a pixel varied from normal, such as when a myotube moved, the motion capture produces a bright spot. Restricting the field of view to the myotube of interest and plotting the intensity of the normalized pixels gave a time trace of myotube movement.

Fig. 2. Morphological characterization of the co-cultures in NMJ platform

A) Phase images of co-cultures maintained in the dual chamber BioMEMs construct. (a) Barrier with tunnels visible between the motoneuron and muscle chambers. Scale bar: 200 μm. (b) Human MNs in the NMJ chamber. Scale bar: 50 μm. (c) Human SKMs in the NMJ chamber, demonstrating the differentiation of myotubes and well distribution of axons (yellow arrows) in the muscle chamber. Scale bar: 50 μm. (d) Higher magnification indicating axons from human MNs projected through the microtunnels and the emerging axons in the muscle chamber. Scale bar: 25 μm. (e) Phase images from the muscle chamber at high magnifications, displaying the frequent close contacts between axons and myotubes (green arrows). Scale bar 25 μm. B&C) Immunocytochemical characterization of co-cultures. Cultures stained with Myosin Heavy Chain (green), neurofilament (red) and Dapi (blue) indicated the formation of myotubes and neuritic extensions, as well as their physical interactions in the muscle chamber. Scale bars 50 μm.

Fig. 3. Immunocytochemical characterization of NMJ formation in the dual chamber system

Axons were stained with antibody against the synaptic vesicle protein synaptophysin while Acetyl choline receptors (AchRs) on the myotubes were immunostained with BTX-488. A) Low magnification images indicate the distribution of BTX-positive myotubes and synaptic vesicle-rich axons in the muscle chamber. B) & C) High magnification images demonstrate the close apposition of vesicle-rich axonal terminals with clusters of AchRs. Scale bars are 100 μm for row A and 50 μm for rows B & C.

Figure 4. Testing of NMJ function and its inhibition by gradual increase of Curare. Stimulations at 0.33 Hz is presented as an example

Myotube contracted reliably in response to each stimulation at MN side (D0=0). As the increasing doses of Curare was added to the muscle chamber, myotube contraction became attenuated, skipped and finally completely disappeared (D6=160 μM).

Fig. 5. Formation of functional NMJs and evidence of temporal summation of MN-stimulated muscle contractions

When electrical stimulation at various frequencies were applied to the MN chamber, myotube contractions at the correspondent frequencies were recorded, indicating the formation of functional NMJs in the chamber system. When stimulations were applied rapidly and repeatedly at increasing frequencies and threshold was reached when muscle relaxation was not possible between contractions, temporal summation was observed. At 1 Hz infused tetanus was evident and at 2 Hz fused tetanus was established. All traces were demonstrated at the same scale as indicated.

Fig. 6. Dose-response effects for curare

A) The dose response curves for curare followed a two-site binding model at all frequencies tested. The fitted parameters of biphasic dose-response curves for curare resulted in two concentrations for half-maximum inhibitory responses to curare, with the first IC_{50_1} = 82 nM (h_c1 = −9) and the second IC_{50_2} = 83 μM (h_c2 = −4). Asterisks (*) denote concentrations significantly different than control (p<0.05). B) For curare dose response curves, the plateau p between the two EC₅₀ values followed an inversely proportional, logarithmic dependency to the stimulation frequency f. C) The number of successfully completed muscle contractions showed multi-frequency effects in their dose response as well.

Fig. 7. Dose-response curve for α- bungarotoxin and BOTOX®

A) The muscle response to increasing bungarotoxin concentrations followed a hormetic, biphasic dose-response at all frequencies tested Low concentrations of bungarotoxin with EC₅₀ = 10 nM (h_c1 = 4.0) had a small excitatory effect, before the onset of inhibitory effects by higher concentrations IC₅₀ = 83 nM (h_c2 = 8.0). Asterisks (*) denote concentrations significantly different than control (p<0.05). B) BOTOX® caused a simple, monophasic dose-response in muscle contractions at all frequencies tested. The half-maximum inhibitory response was at IC₅₀ = 0.54 U (h_c = 1.2).