Generation of Functional Neuromuscular Junctions from Human Pluripotent Stem Cell Lines

Abstract

Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca(2+) concentration in sarcoplasm, while electrical stimulation evoked Ca(2+) transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.

Keywords: cell culture; embryonic; human; induced pluripotent stem cell; nerve cell engineering; skeletal muscle.

Figure 1

Cells from non-selected spheres mature into neurons and myotubes derived from ESCs (HS306). Cell populations contain neural progenitors and mature neurons with neurites (A, white arrows), and non-striated (A, black arrows) and striated (B) multinucleated myotubes. In these cultures, myotubes are spontaneously contracting (A, Video S1). Eventually, the contraction and movement may lead to the detachment and further degeneration of the myotube (A, black arrowhead). Time-lapse imaging of the same view reveals that myoprecursors and myotubes are highly mobile, and the fusion of precursor cells into myotubes is a dynamic process (C,D, Video S2). Scale bars: (A) 100 μm; (B) 50 μm; (C,D) 100 μm.

Figure 2

Immunostaining confirms that the cells from non-selected spheres mature into neurons and myotubes. Neurosphere-derived cells mature predominantly (>90%) into neuron specific, class III β-tubulin (Tuj1) positive neurons with small nuclei and long projections (A, white arrows). These cells do not express a skeletal muscle marker myosin heavy chain (MHC) (B). Non-selected (mixed) spheres produce both neurons (C) and thick MHC positive (D) myotubes (D, white arrows) that are also positive to phalloidin (E), labeling the actin filaments of myo-oriented cells. In some MHC positive myotubes, the striation is visible (F, white arrows). In (A–F) the nuclei were stained with Hoechst 333042 (blue). Scale bars: (A–D) (iPSC-derived), 100 μm; (E) (ESC-derived), 50 μm; (F) (iPSC-derived), 30 μm.

Figure 3

Neural genes (A,B) are strongly down-regulated and myogenic markers (C–E) up-regulated in the non-selected (mixed) sphere-derived cell populations when compared to neurospheres. Results are shown as the normalized expression of the target genes against the expression of the endogenous β-actin gene. Results are expressed as mean ± SD, n = 4, and analyzed with paired T-test, ^** <0.005, ^*** <0.001. Map-2, microtubule- associated protein; MyoD1, myogenic differentiation 1; Myog, myogenin; Pax-6, paired box protein; RYR1, ryanodine receptor 1. All cells were derived from iPSCs.

Figure 4

Neurons and myotubes that are in close proximity seem to connect with each other potentially via neuromuscular junction (NMJ). A representative phase contrast image (A; ESC-derived) shows an elongated muscle cell contacting with two neuronal cells (arrows). Immunocytochemistry shows that neuron specific III β-tubulin (Tuj1) positive neuronal cells (B; iPSC-derived, in red) grow along and surround the phalloidin positive myotubes (in green). A proportion of doublecortin (DCX) positive neurons (C; iPSC-derived, in red; also myotubes express DCX) express a synaptic protein, synaptic vesicles (SV2; C in green). In (D) (iPSC-derived), DCX positive neurons (in red) contacting with multinucleated myotubes co-express (in yellow) neurofilament H (SMI-32; in green), a motoneuronal protein. In (E) (ESC-derived), α-bungarotoxin (in green) specifically binding to the nicotinic acetylcholine receptors densely expressed at the postsynaptic site of NMJ is detected on the surface of myotubes (the phase contrast insert shows presumably a neuron contacting the myotube). In (F) (confocal image, iPSC-derived), a thick myotube (big white arrow) expressing post-synaptic membranes, visualized with red-labeled α-bungarotoxin, is contacting with a synaptophysin positive nerve ending (green; small white arrow). At the site of NMJ the colocalization is seen (in yellow color; yellow big arrow). In (B–D) the nuclei were stained with Hoechst 33042 (blue). Scale bars: (A–D), 50 μm; (E,F), 10 μm.

Figure 5

Functional characterization of iPSC-derived myotubes. (A) Membrane potential (Vm) recording of spontaneously active myotube showing repetitive firing and narrow spike-like APs. (B) In part of the myotubes current injection evokes a single relatively long AP. (C) Carbacholine application (red arrow) causes strong depolarization of the myotube membrane. Note the fast phase (inset) most likely induced by the current through nAChR. (D) Acetylcholine (100 μM) application (red arrow) induces a robust increase in cytosolic [Ca²⁺] of the myotubes, whereas (E) 0.5 Hz electrical stimulation (red arrows) evokes repetitive calcium transients in myotubes. All traces are representative examples out from at least 15 recordings.

Figure 6

Functional characterization of NMJs in iPSC-derived cells. (A) Activation of NMDARs in neurons by application of NMDA (500 μM, red arrow) induces AP firing of adjacent myotubes. (B) Membrane potential recording of miniature end-plate potentials (mEPPs) of myotubes (upper line), example of expanded mEPP trace (lower line, right part) and main characteristics of mEPP (lower line, left part, mEPP: amplitude, rise time (RT) and decay of mEPP). (C) Hyperpolarizing effect (H-effect) of nimbex (2 μM) a nicotinic receptor blocker on resting membrane potential (application—the first red arrow, wash—the second red arrow). All traces are representative examples out from at least 8 recordings.