Elsevier

Experimental Cell Research

Volume 259, Issue 1, 25 August 2000, Pages 204-212
Experimental Cell Research

Regular Article
Cell Types Required to Efficiently Innervate Human Muscle Cells in Vitro

https://doi.org/10.1006/excr.2000.4968Get rights and content

Abstract

Previous studies carried out in our laboratory have shown that myofibers formed by fusion of muscle satellite cells from donors with spinal muscular atrophy (SMA) type I or II undergo a characteristic degeneration 1.5–3 weeks after innervation with rat embryonic spinal cord explants. The only cells responsible for degeneration of innervated cocultures are SMA muscle satellite cells. In order to study the kinetics of nerve and muscle cell degeneration in nerve–muscle cocultures implicating SMA muscle cells, we attempted to simplify the nervous component of the coculture and identify the nerve cell types necessary for a successful innervation. We demonstrate here that motoneurons alone were unable to innervate myotubes. However, when three cell types (motoneurons, sensory neurons, and Schwann cells) were added onto a reconstituted muscular component consisting of cloned muscle satellite cells and cloned muscular fibroblasts, myotubes contracted, indicating that functional neuromuscular junctions were formed. We concluded that the three cell types were required for a successful innervation. Moreover, we studied the effects of culture medium conditioned by different combinations of nerve cells on innervation; we observed that physical contacts among sensory neurons, motoneurons, and myotubes are required for a successful innervation; in contrast Schwann cells can be replaced by a Schwann-cell-conditioned medium, indicating that these cells produce a putative soluble “innervation-promoting factor.” Obviously such a reconstituted system does not reflect the in vivo situation but it allows the formation of functional motor synapses and could therefore allow us to elucidate neuromuscular disease pathogenesis, especially that of spinal muscular atrophy.

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      In addition, human ESC-derived MNs have been investigated for their capability of innervating C2C12 cells in a serum-containing system [30–34], and MNs derived from human fetal spinal cord stem cells were demonstrated to be able to form functional NMJs with rat myotubes derived from embryonic skeletal muscles in a defined serum-free system [27]. Separately, cloned human skeletal muscle satellite cells have been used extensively for the study of in vitro NMJ formation or related diseases in combination with rat spinal explants or dissociated MNs in serum-containing systems [30–34]. In this study, we endeavored to develop an entirely human-based in vitro neuromuscular junction system, in which both MNs and SKMs were derived from stem cells, in a defined, serum-free system.

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      This also is an essential requirement for drug discovery applications. Furthermore, the reported importance of culturing motoneurons, sensory neurons and Schwann cells together with muscle to form a significant number of neuromuscular junctions in vitro makes this basic medium even more critical [24,25]. Schwann cell interaction with axons in the periphery is essential for efficient myelin sheath formation.

    • Synaptogenetic mechanisms controlling postsynaptic differentiation of the neuromuscular junction are nerve-dependent in human and nerve-independent in mouse C2C12 muscle cultures

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      Stronger nerve-dependency of human muscle is not surprising. Our previous observations [17,19,20] as well as reports from other laboratories [16,24] agree that human myotubes never contract spontaneously, remain poorly differentiated and ultimately die if they are not innervated. On the other hand rodent muscle contract spontaneously and reach high level of differentiation without any presence of the nerve.

    • Expression and distribution of acetylcholinesterase among the cellular components of the neuromuscular junction formed in human myotube in vitro

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      The temporal coincidence between maturation of the NMJ and lineage progression of neurons and glial cells was found to be practically the same as observed in vivo [5]. Important insight into the intercellular relationships in this coculture was provided by Guettier-Sigrist et al. [6]. They found that rat motor neurons alone were unable to innervate human myotubes and that three cell types (motoneurons, sensory neurons and Schwann cells) are required for a successful functional innervation.

    • Schwann cells and astrocytes induce synapse formation by spinal motor neurons in culture

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