Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system

Abstract

Functional in vitro models composed of human cells will constitute an important platform in the next generation of system biology and drug discovery. This study reports a novel human-based in vitro Neuromuscular Junction (NMJ) system developed in a defined serum-free medium and on a patternable non-biological surface. The motoneurons and skeletal muscles were derived from fetal spinal stem cells and skeletal muscle stem cells. The motoneurons and skeletal myotubes were completely differentiated in the co-culture based on morphological analysis and electrophysiology. NMJ formation was demonstrated by phase contrast microscopy, immunocytochemistry and the observation of motoneuron-induced muscle contractions utilizing time-lapse recordings and their subsequent quenching by d-Tubocurarine. Generally, functional human based systems would eliminate the issue of species variability during the drug development process and its derivation from stem cells bypasses the restrictions inherent with utilization of primary human tissue. This defined human-based NMJ system is one of the first steps in creating functional in vitro systems and will play an important role in understanding NMJ development, in developing high information content drug screens and as test beds in preclinical studies for spinal or muscular diseases/injuries such as muscular dystrophy, Amyotrophic lateral sclerosis and spinal cord repair.

Figure 1

Schematic diagram of the culture protocol and timeline.

Figure 2

Phase contrast micrographs A. Myocytes in the expansion phase before differentiation was induced. Scale bar: 100 μm. B. Multi-nuclei myotubes induced after differentiation. Scale bar: 25 μm. C. Myotubes and neurons exhibited healthy morphology after plating in the co-culture system. Scale bar: 50 μm. D. Physical connections were established between neurons and myotubes in the co-culture (indicated by arrows) Scale bar: 40 μm. E. Striated myotubes were present in the co-culture system as indicated by the yellow arrow. Scale bar: 25 μm. F. A micrograph of a neuron with MN morphology that extended an axon (red arrow) towards a striated myotube (yellow arrow). Scale bar: 25 μm.

Figure 3

A&B. Representative voltage clamp (A) and current clamp (B) trace recording from the myotubes in the co-culture. C&D. Representative voltage clamp (C) and current clamp (D) trace recording from the motoneurons in the co-culture. Insert pictures indicate the cells from which the recordings were obtained.

Figure 4

Synapse formation as indicated by immunocytochemical analysis. A. Immunocytochemical identification of each component of the co-culture. Co-immunostaining of MHC (myosin heavy chain) and β III Tubulin in a 19 day co-culture demonstrated that axonal terminals branched in the vicinity of the myotube and wrapped heavily around the myotube. Scale bar: 50 μm. B&C. Potential synaptic sites (yellow arrows in B and arrowheads in C) demonstrated by co-localization of nerve terminals (indicated by synaptophysin) and AchR (indicated by BTX-488) at day 15 in coculture. Scale bar: 100 μm in B and 50 μm in C.