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.
Highlights
The skeletal muscle neuromuscular junction (NMJ) is a highly organized synapse formed between a motor neuron (MN) axon and a muscle fiber
We performed a side-by-side comparison of human skeletal muscle fiber populations derived in standard 2D culture versus 3D culture and uncovered differences in fiber maturation and acetylcholine receptors (AChRs) clustering (Figure 1—figure supplement 1A)
We report a simple method to co-culture 3D human skeletal muscle fiber tissues together with human pluripotent stem cells (PSCs)-derived MNs, while performing comparative studies to uncover biological inquiries and processes that are enabled by the availability of a 3D human neuromuscular culture system
Summary
The skeletal muscle neuromuscular junction (NMJ) is a highly organized synapse formed between a motor neuron (MN) axon and a muscle fiber. Despite significant progress toward directing human pluripotent stem cells (PSCs) to the motor neuron lineage (Ashton et al, 2015; Hu and Zhang, 2010; Lippmann et al, 2014; Maury et al, 2015; Shimojo et al, 2015; Zhang et al, 2001) and establishing electrically and chemically responsive human muscle fibers in vitro (Madden et al, 2015), the first reports of human NMJ models – 2D (Guo et al, 2011; Santhanam et al, 2018; Steinbeck et al, 2016) or 3D (Maffioletti et al, 2018; Osaki et al, 2018) human muscle fiber and motor neuron co-cultures – do not demonstrate synapse maturation via the gamma to epsilon AChR subunit switch. There are no reports of epsilon AChR protein expression or function in culture in the absence of enforced gene expression
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