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Elife. 2019 May 14;8. pii: e44530. doi: 10.7554/eLife.44530.

A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction.

Author information

1
Donnelly Centre, University of Toronto, Toronto, Canada.
2
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada.
3
Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, United States.
4
Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, United States.
5
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada.
6
Department of Cell and Systems Biology, University of Toronto, Toronto, Canada.
7
Department of Surgery, University of Toronto, Toronto, Canada.
8
Department of Biology, University of Toronto Mississauga, Mississauga, Canada.
9
Department of Pharmaceutics, Utrecht University, Utrecht, Netherlands.
10
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada.
11
INSERM, Association Institut de Myologie, Centre de Recherche en Myologie, Sorbonne Universite, Paris, France.
12
Department of Neuroscience, University of Padova, Padova, Italy.
13
Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Canada.
14
Department of Physiology, University of Toronto, Toronto, Canada.
15
Department of Molecular Genetics, University of Toronto, Toronto, Canada.
16
Department of Biochemistry, University of Toronto, Toronto, Canada.

Abstract

Two-dimensional (2D) human skeletal muscle fiber cultures are ill-equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-cultures. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. Hence, this work offers a simple method to model and evaluate adult human NMJ de novo development or disease in culture.

KEYWORDS:

3D co-culture; acetylcholine receptor subunit epsilon; developmental biology; human; motor neuron; myasthenia gravis; neuromuscular junction; regenerative medicine; skeletal muscle; stem cells

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