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. 2019 Feb 20:12:1-9.
doi: 10.2147/SCCAA.S187655. eCollection 2019.

Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells

Jasdeep Saini  1 , Alessandro Faroni  2   3 , Marwah Abd Al Samid  1 , Adam J Reid  2   3 , Adam P Lightfoot  1 , Kamel Mamchaoui  4 , Vincent Mouly  4 , Gillian Butler-Browne  4 , Jamie S McPhee  5 , Hans Degens  1   6   7 , Nasser Al-Shanti  1
Affiliations

Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells

Jasdeep Saini et al. Stem Cells Cloning. .

Abstract

Background: Neuromuscular junctions (NMJs) consist of the presynaptic cholinergic motoneuron terminals and the corresponding postsynaptic motor endplates on skeletal muscle fibers. At the NMJ the action potential of the neuron leads, via release of acetylcholine, to muscle membrane depolarization that in turn is translated into muscle contraction and physical movement. Despite the fact that substantial NMJ research has been performed, the potential of in vivo NMJ investigations is inadequate and difficult to employ. A simple and reproducible in vitro NMJ model may provide a robust means to study the impact of neurotrophic factors, growth factors, and hormones on NMJ formation, structure, and function.

Methods: This report characterizes a novel in vitro NMJ model utilizing immortalized human skeletal muscle stem cells seeded on 35 mm glass-bottom dishes, cocultured and innervated with spinal cord explants from rat embryos at ED 13.5. The cocultures were fixed and stained on day 14 for analysis and assessment of NMJ formation and development.

Results: This unique serum- and trophic factor-free system permits the growth of cholinergic motoneurons, the formation of mature NMJs, and the development of highly differentiated contractile myotubes, which exhibit appropriate configuration of transversal triads, representative of in vivo conditions.

Conclusion: This coculture system provides a tool to study vital features of NMJ formation, regulation, maintenance, and repair, as well as a model platform to explore neuromuscular diseases and disorders affecting NMJs.

Keywords: NMJ; coculture; motoneuron; motor neuron; myoblast; myotube; neuromuscular junction.

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Conflict of interest statement

Disclosure The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Cholinergic motoneurons co-localize with myotubes at day 14. Notes: Representative images of human myotube cocultured with embryonic rat motoneuron stained for choline acetyltransferase (ChAT) (red), myosin heavy chain (MHC) (green), and DAPI (blue). Scale bars =7.5 µm.
Figure 2
Figure 2
Characterization of neuromuscular junction formation at day 14. Notes: Representative images of coculture stained for β-III-tubulin (green), α-bungarotoxin (α-BTX) (red), and DAPI (blue). Scale bars =5 µm.
Figure 3
Figure 3
Percentage of various neuromuscular junction (NMJ) morphologies at day 14. Notes: Cocultured and aneurally cultured myotubes. Results are expressed as mean ± SD, n=4, analyzed with unpaired t-test; ****P<0.0001.
Figure 4
Figure 4
Characterization of myotubes at day 14. Notes: Representative images of (A) a myotube cocultured with embryonic rat spinal cord and (B) an aneurally cultured myotube. Myotubes were stained for ryanodine receptor (RyR) (green) and dihydropyridine receptor (DHPR) (red); scale bars =7.5 µm. (C) The percentage of myotubes with triads from cocultured and aneurally cultured myotubes. Results are expressed as mean ± SD, n=10, analyzed with unpaired t-test; ****P<0.0001.
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References

    1. van der Worp HB, Howells DW, Sena ES, et al. Can animal models of disease reliably inform human studies? PLoS Med. 2010;7(3):e1000245. - PMC - PubMed
    1. Haase G. Motor neuron diseases: cellular and animal models. In: Meyers RA, editor. Reviews in Cell Biology and Molecular Medicine. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA; 2006.
    1. Prather RS, Lorson M, Ross JW, Whyte JJ, Walters E. Genetically engineered pig models for human diseases. Annu Rev Anim Biosci. 2013;1(1):203–219. - PMC - PubMed
    1. Suuronen EJ, Mclaughlin CR, Stys PK, Nakamura M, Munger R, Griffith M. Functional innervation in tissue engineered models for in vitro study and testing purposes. Toxicol Sci. 2004;82(2):525–533. - PubMed
    1. Demestre M, Orth M, Föhr KJ, et al. Formation and characterisation of neuromuscular junctions between hiPSC derived motoneurons and myotubes. Stem Cell Res. 2015;15(2):328–336. - PubMed