A 3D model of human skeletal muscle innervated with stem cell-derived motor neurons enables epsilon-subunit targeted myasthenic syndrome studies

Mohsen Afshar Bakooshli 1, 2 ; Ethan S Lippmann 3 ; Ben Mulcahy 4 ; Elena Pegoraro 6 ; Howard Ginsberg 5 ; Mei Zhen 4 ; Randolph S Ashton 3 ; Penney M Gilbert 1, 2;

 1.  Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada; 2.  Donnelly Centre for Cellular and Biomolecular Research, Toronto, ON, Canada ; 3.  Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA; 4.  Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada; 5.  Department of Surgery, University of Toronto, Toronto, ON, Canada; 6.  Department of Neuroscience, University of Padova, Padova, Italy

 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 numerous functional NMJ connections within two weeks. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium transient imaging and electrophysiological recordings. Notably, we find epsilon acetylcholine receptor subunit protein expression and activity in our 3D co-culture demonstrating that the system supports a developmental shift from the embryonic to adult form of the receptor not previously reported in 2D culture. Further, co-culture treatment with myasthenia gravis (MG) patient sera demonstrates the ease of human disease studies using the system. This work provides the first method to model and evaluate adult NMJ development and disease in culture. In the next step we aim to evaluate the effect of complement inhibitors to rescue the phenotype observed in muscle-neuron co-cultures treated with MG patient sera.