Functional Connectivity under Optogenetic Control Allows Modeling of Human Neuromuscular Disease

Abstract

Capturing the full potential of human pluripotent stem cell (PSC)-derived neurons in disease modeling and regenerative medicine requires analysis in complex functional systems. Here we establish optogenetic control in human PSC-derived spinal motorneurons and show that co-culture of these cells with human myoblast-derived skeletal muscle builds a functional all-human neuromuscular junction that can be triggered to twitch upon light stimulation. To model neuromuscular disease we incubated these co-cultures with IgG from myasthenia gravis patients and active complement. Myasthenia gravis is an autoimmune disorder that selectively targets neuromuscular junctions. We saw a reversible reduction in the amplitude of muscle contractions, representing a surrogate marker for the characteristic loss of muscle strength seen in this disease. The ability to recapitulate key aspects of disease pathology and its symptomatic treatment suggests that this neuromuscular junction assay has significant potential for modeling of neuromuscular disease and regeneration.

Figure 1. Optogenetic control in hPSC derived spinal motorneurons (MNs)

(A) Clonal hESC line carrying the hSyn-ChR2-EYFP transgene staining for OCT4 (POU5F1) and DAPI. (B) At day 20 (D20) MN clusters express ChR2-EYFP, bright field (BF). (C) After purification MN clusters are enriched. (D) qRT-PCR, after purification sMN markers are up-regulated. (E) qRT-PCR, after purification non-neuronal markers are down-regulated. (F) At day 30 spinal MNs express ChR2-EYFP and stain for HB9 and ISL1. (G) At day 30 spinal MNs co-stain for ChAT and SMI32. (H) Alternative protocol ChR2-EYFP+ MNs. (I) At day 30 spinal MNs (alternative protocol) express ChR2-EYFP, HB9 and ISL1. (J) At day 60 spinal MNs (alternative protocol) express ChR2-EYFP, ChAT and SMI32. (K) Neuron in bright field and EYFP channel chosen for electrophysiology. (L) Beyond day 60 (D60+) hESC-derived MNs fire action potentials in response to depolarizing current injection. (M, N) Mature ChR2+ hESC-derived MNs faithfully fire action potentials in response to optogenetic stimulation. (O) Clonal hESC line carrying the hSyn-EYFP transgene staining for OCT4 and DAPI. (P) At day 30 purified spinal hESC-derived MNs express EYFP, HB9 and ISL1. (Q) Mature EYFP+ hESC-derived MN fires action potentials in response to current injection. (R) Mature EYFP+ hESC-derived MNs do not respond to light stimulation. Scale bars 100 µM. Error bars represent SEM.

Figure 2. Generation of functional human myofibers

(A) Human myoblasts derived from an adult donor (hMA, upper panel) and a fetal donor (hMF, lower panel). (B) Human myofibers at day 17 of differentiation. (C) Calcium imaging in human myofibers on day 35. Acetylcholine (ACh) induces a robust calcium transient. Each trace resembles a distinct fiber. Scale bars 100 µM.

Figure 3. Characterization of neuromuscular co-cultures

(A, E) Co-cultures of spinal hESC-derived MNs with adult (hMA) and fetal (hMF) derived myofibers 1 week (1W) after initiation, EYFP and bright field channels. (B, F) Co-cultures of spinal hESC-derived MNs with adult (hMA) and fetal (hMF) derived myofibers 6–8 weeks after initiation. (Online version of Fig. 3 containing videos). (C, G) Quantification of muscle twitches in co-cultures in response to optogenetic stimulation for 50s (upper panel) and 500s (lower panel). Each trace resembles a distinct fiber. (D, H) Vecuronium (2µM) blocks light-evoked contractility in adult (D) and fetal (H) myofibers. (I) EYFP and bright field picture of calcium imaging experiment shown in J. (J) Ratiometric analysis of calcium transients in myofibers in response to optogenetic stimulation for 2 min (upper panel) and 40 min (lower panel). Each trace resembles a distinct fiber. (K) Sharp microelectrode recording from a single myofiber. Generation of vecuronium-sensitive action potentials in response to optogenetic stimulation at 0.2 and 2 Hz. (L) Long-term stability of neuromuscular connectivity. Movement in individual regions was quantified on day 5, 15 and 25 and compared to movement on day 0, normalized at 100%. (M) Co-cultures contain a layer of vimentin+ and GFAP+ stroma. (N) Co-cultures show dense network of EYFP+ axons and desmin+ muscle fibers. (O) Multinucleated and striated myofiber in close contact with EYFP+ neuronal processes in contractile region. (P) High-power confocal imaging of clustered acetylcholine receptor (BTX) in close association with EYFP+ neuronal process and synaptophysin labeling. (Q, R) Contracting regions (CONTR, left) and non-contracting regions (NO CONTR, right) were compared for AChR clustering. Quantification of BTX+ dots revealed a significant increase in contracting / innervated regions. * p < 0.05. In C, D and G, H one pixel corresponds to 0.5 µm. Scale bars 100µm, except I, K 50 µm and P, Q 25 µm.

Figure 4. Modeling of neuromuscular transmission failure, typical of Myasthenia gravis

(A, D) Kinetogram of mature, contracting co-cultures of spinal MNs with adult myofibers (hMA) before the addition of myasthenia gravis (MG) IgG (patient 2) and complement (A) or control IgG and complement (D). (B, E) Same co-cultures as in A and D, on day 3 after the addition of myasthenia gravis IgG and complement (B) or control IgG and complement (E). (C, F) Same co-cultures as in B and E after the addition of pyridostigmine (PYR, 10 µM) on day 3. (G) Quantification of movement in cultures treated with MG IgG (patient #1 and 2) and complement or control IgG and complement on day 3 in % as compared to day 0. (H) Quantification of movement in cultures treated with MG IgG (patient #1 & 2 combined) and complement before and after the addition of pyridostigmine on day 3. (I) Recovery of movement on day 4 and day 6 after wash out of MG IgG (patient #1 & 2 combined) and complement on day 3. (J) Quantification of movement in cultures treated with MG IgG (patient #1), control IgG and in untreated cultures, all without complement. (K, L) Bright field and EYFP images of functional MN co-cultures with adult muscle (hMA) treated with MG IgG (patient #1) and complement or control IgG and complement at 48h in regions selected for calcium imaging. (M) Quantification of the calcium increase in response to optogenetic stimulation in MG and control cultures and after the addition of PYR. (N) Percentage of reactive fibers in response to optogenetic stimulation in MG and control cultures and after the addition of PYR. (O, P, Q) Immunocytochemistry and quantification (Q) for the deposition of the human complement component C3c (blue) onto the neuromuscular junction co-labeled for EYFP (green) and BTX (red) 24h after the addition of MG IgG (patient #1) or control IgG and complement. Area in small boxes with dotted line are magnified in boxes with solid line. Scale bars 100 µm in K, L; 10 µm in O, P. In A–F one pixel corresponds to 0.5 µm. n.s. = not significant, * p < 0.05, ** p< 0.01, *** p < 0.001. All error bars represent SEM.