Bioengineered optogenetic model of human neuromuscular junction

Abstract

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function.

Keywords: Disease modeling; Human tissue models; Myasthenia gravis; Neuromuscular junction; Optogenetics; iPS cells.

Figure 1.. Generation of myotubes and motoneurons (MN) from human primary skeletal myoblasts (hSkM).

(A) Overall strategy (B) Myotube differentiation protocol (MM = Maturation Media) (C) Immunofluorescence (IF) images of skeletal markers in hSkM-derived myotubes. (D) human induced pluripotent stem cells (hiPSC) MN differentiation protocol. (E) Membrane expression of YFP-Channelrhodopsin-2 (ChR2) in optogenetic iPSCs and (F) MNs. (G) Co-expression of ChR2 (green) and choline acetyltransferase (ChAT, red) in opto-hMNs. (H) Spontaneous electrical depolarization vs light-induced electrical depolarization in opto-hMNs as confirmed by multi-electrode array (MEA) recordings, in which each row shows the electrical activity of one electrode, blue dots represent spikes and pink boxes indicate the presence of burst (synchronous activity in the electrodes), while blue ticks signal the light stimulation pulses. Scale bars 100 um.

Figure 2.. Transcription-factor mediated differentiation of myotubes and motoneurons from iPSCs.

(A)Overall strategy. (B) PiggyBac plasmid for the expression of MYOD1 and knock down of OCT4 in hiPSCs. (C) hiPSC-derived skeletal myoblast (hiSkM) differentiation protocol (D) Bright field and (E) skeletal muscle marker IF images (F) hNIL- MN differentiation protocol. (G) Membrane expression of ChR2-YFP in optogenetic hNIL iPSCs and (H) MNs. (I) IF images for β-tubulin (green) and ChAT (red) (J) MEA recording showing spontaneous and light-stimulated electrical activity in optogenetic hNIL-MNs. Each row shows the electrical activity of one electrode, blue dots represent spikes,pink boxes indicate the presence of burst (synchronous activity in the electrodes), and blue ticks indicate light stimulation pulses. Scale bars 100 μm.

Figure 3.. Advanced culture systems for the formation of 3D NMJs.

A) Microfluidic platform. Right panel depicts the microfluidic chambers inside the platform, located between each pair of media reservoirs. B) Open-well system C) Timeline showing differentiation and seeding strategies. D)Innervated primary myoblast-derived skeletal muscle tissue formed in the microfluidic platform. E) Innervated iPSC-derived skeletal muscle tissue cultured in the open-well bioreactor. Scale bar 0.5 mm.

Figure 4.. Optical set-up for simultaneous stimulation and recording of engineered NMJs.

(A) Light path (B) Implementation of the custom optical set-up in a traditional inverted microscope provided with a live cell chamber, an automated stage and a CMOS camera.

Figure 5.. Automated analysis of NMJ function

(A) Video-processing algorithm for the batch analysis of movies taken during optical stimulation of NMJ cultures (B) Representative frame of the output generated by the analysis software showing the video frame and time stamp on the top, and the contractility trace graph in the bottom. The blue blinker indicates when the blue stimulation is happening, shown in the graph by the vertical lines. Light-pulses followed by a contraction (x) are counted as triggered and marked in green.

Figure 6.. Myasthenia Gravis effect on NMJ function.

(A-C) Contractility traces for tissue engineered NMJs using myotubes and MNs generated from hSkM in a microfluidic platform (as depicted in Fig 1) (A) before and (B) after 48 hours of treatment with 20% of MG sera and (C) 48 hours after its removal. (D) Quantification of NMJ function of treated and control groups before and after treatment and post-recovery, (n = 12; after treatment post-hoc ANOVA F = 0.0002; *indicates p = 0.0015 **indicates p = 3.5·10⁻⁶) (MG = Myasthenia Gravis; NHS = normal human serum). (E) Effect of sera from different patients (A, B, C) on tissue function (n = 15; after treatment post-hoc ANOVA F = 2·10⁻⁵; * indicates p< 0.01 vs control group). Treatment was started on day 14 with sequential dose increments at days 16, 18 and 20 (F) Effect of IgG isolated from seronegative patients on tissue function (SN = seronegative, SP = seropositive), (n=8, *indicates p = 0.02). Boxes= 25–75 percentiles; brackets = 1.5 standard deviations. n indicates the number of biological replicates.