A Stem-Cell Based Bioassay to Critically Assess the Pathology of Dysfunctional Neuromuscular Junctions

Chipman, Peter H.; Zhang, Ying; Rafuse, Victor F.

doi:10.1371/journal.pone.0091643

Cited by 32 publications

(32 citation statements)

References 52 publications

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“…This, together with the recent development of biotechnologies to use induced human pluripotent stem cells from healthy human subjects and patients, will enable the study of NMJ disease mechanisms, in vitro drug screening on human cells and the development of personalized medical procedures1516171819206667.…”

Section: Discussionmentioning

confidence: 99%

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

Rabieh

Ojovan

Shmoel

et al. 2016

Sci Rep

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In contrast to the extensive use of microelectrode array (MEA) technology in electrophysiological studies of cultured neurons and cardiac muscles, the vast field of skeletal muscle research has yet to adopt the technology. Here we demonstrate an empowering MEA technology for high quality, multisite, long-term electrophysiological recordings from cultured skeletal myotubes. Individual rat skeletal myotubes cultured on micrometer sized gold mushroom-shaped microelectrode (gMμE) based MEA tightly engulf the gMμEs, forming a high seal resistance between the myotubes and the gMμEs. As a consequence, spontaneous action potentials generated by the contracting myotubes are recorded as extracellular field potentials with amplitudes of up to 10 mV for over 14 days. Application of a 10 ms, 0.5–0.9 V voltage pulse through the gMμEs electroporated the myotube membrane, and transiently converted the extracellular to intracellular recording mode for 10–30 min. In a fraction of the cultures stable attenuated intracellular recordings were spontaneously produced. In these cases or after electroporation, subthreshold spontaneous potentials were also recorded. The introduction of the gMμE-MEA as a simple-to-use, high-quality electrophysiological tool together with the progress made in the use of cultured human myotubes opens up new venues for basic and clinical skeletal muscle research, preclinical drug screening, and personalized medicine.

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Section: Discussionmentioning

confidence: 99%

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

Rabieh

Ojovan

Shmoel

et al. 2016

Sci Rep

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“…Advanced states of NMJ maturation have been shown in conventional 2D hybrid cultures [Chipman et al, 2014]; however, no such structures have yet been presented in 3D constructs. Methods for maturing constructs in situ must be investigated in order to advance the development of current models toward more biomimetic tissues.…”

Section: Discussionmentioning

confidence: 99%

“…These structural and molecular motifs are well-established hallmarks of mature NMJs [Sanes and Lichtman, 1999, 2001] and are necessary for accurate modelling of these synapses for drug screening, disease modelling, and fundamental mechanistic studies. Recently, the development of more structurally mature NMJs in conventional co-culture has been reported [Chipman et al, 2014]. Mouse ESC-derived motor neurons encased in embryoid bodies and co-cultured with embryonic chick myotubes were shown to develop advanced end-plate morphologies reminiscent of NMJs in vivo, demonstrating that the achievement of such structures is possible in vitro.…”

Section: Introductionmentioning

confidence: 99%

Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System

Smith

Passey

Martin

et al. 2016

Cells Tissues Organs

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Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.

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“…However, we were particularly interested in characterizing the differentiation of muscle fibers in a neuronal context, replicating in vivo conditions. Much attention has been given to the development and improvement of the neuronal compartment with the emergence of embryonic stem cells (Chipman et al, 2014;Das et al, 2007;Soundararajan et al, 2007) and induced pluripotent stem cells (Bohl et al, 2015;Burkhardt et al, 2013;Egawa et al, 2012); however, the muscle counterpart has been underexploited and its potential underestimated. Previous studies failed to produce convincing data on muscle differentiation either because muscle cell lines, such as C2C12, incapable of producing highly differentiated myofibers, were used or because the muscle compartment was overlooked (Arnold et al, 2012;Chipman et al, 2014;Das et al, 2009Das et al, , 2010Umbach et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

“…This can only be achieved in the presence of all required cell types. We believe that although new models derived from ESC-MNs and iPSC-MNs allow for homologous co-culture systems and also hold promise for personal-targeted therapies, these systems might lack some of the required cues that allow for the precise formation of the NMJ structures, such as junctional folds in the muscle fiber (Chipman et al, 2014;Son et al, 2011). Moreover, maintenance of the coculture over time in these systems is fragile and still needs to be improved.…”

Section: Discussionmentioning

confidence: 99%

A system for studying mechanisms of neuromuscular junction development and maintenance

Vilmont¹,

Cadot²,

Ouanounou³

et al. 2016

Journal of Cell Science

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The neuromuscular junction (NMJ), a cellular synapse between a motor neuron and a skeletal muscle fiber, enables the translation of chemical cues into physical activity. The development of this special structure has been subject to numerous investigations, but its complexity renders in vivo studies particularly difficult to perform. In vitro modeling of the neuromuscular junction represents a powerful tool to delineate fully the fine tuning of events that lead to subcellular specialization at the pre-synaptic and post-synaptic sites. Here, we describe a novel heterologous co-culture in vitro method using rat spinal cord explants with dorsal root ganglia and murine primary myoblasts to study neuromuscular junctions. This system allows the formation and long-term survival of highly differentiated myofibers, motor neurons, supporting glial cells and functional neuromuscular junctions with post-synaptic specialization. Therefore, fundamental aspects of NMJ formation and maintenance can be studied using the described system, which can be adapted to model multiple NMJ-associated disorders.

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A Stem-Cell Based Bioassay to Critically Assess the Pathology of Dysfunctional Neuromuscular Junctions

Cited by 32 publications

References 52 publications

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

On-chip, multisite extracellular and intracellular recordings from primary cultured skeletal myotubes

Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System

A system for studying mechanisms of neuromuscular junction development and maintenance

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