Differentiation of glial cells and motor neurons during the formation of neuromuscular junctions in cocultures of rat spinal cord explant and human muscle

Abstract

Motor axons extending from embryonic rat spinal cord explants form fully mature neuromuscular junctions with cocultured human muscle. This degree of maturation is not observed in muscle innervated by dissociated motor neurons. Glial cells present in the spinal cord explants seem to be, besides remaining interneurons, the major difference between the two culture systems. In light of this observation and the well documented role of glia in neuronal development, it can be hypothesized that differentiated and long-lived neuromuscular junctions form in vitro only if their formation is accompanied by codifferentiation of neuronal and glial cells and if this codifferentiation follows the spatial and temporal pattern observed in vivo. Investigation of this hypothesis necessitates the characterization of neuronal and glial cell development in spinal cord explant-muscle cocultures. No such study has been reported, although these cocultures have been used in numerous studies of neuromuscular junction formation. The aim of this work was therefore to investigate the temporal relationship between neuromuscular junction formation and the differentiation of neuronal and glial cells during the first 3 weeks of coculture, when formation and development of the neuromuscular junction occurs in vitro. The expression of stage-specific markers of neuronal and glial differentiation in these cocultures was characterized by immunocytochemical and biochemical analyses. Differentiation of astrocytes, Schwann cells, and oligodendrocytes proceeded in concert with the differentiation of motor neurons and neuromuscular junction formation. The temporal coincidence between maturation of the neuromuscular junction and lineage progression of neurons and glial cells was similar to that observed in vivo. These findings support the hypothesis that glial cells are a major contributor to maturity of the neuromuscular junction formed in vitro in spinal cord explant-muscle cocultures.

Schwann Cells Promote Synaptogenesis at the Neuromuscular Junction via Transforming Growth Factor-β1

“…A similar synaptogenic effect of mammalian Schwann cells has been reported in mammalian motoneuron cultures (Ullian et al, 2004), as well as in rat motoneuron and human muscle fiber cocultures (Guettier-Sigrist et al, 2000;Mars et al, 2001). It would be intriguing to test whether these synaptogenic effects of Schwann cells are also mediated by TGF-␤1.…”

Insulin-induced exocytosis in single, in vitro innervated human muscle fibres: a new approach

“…In the present study we used an in vitro system in which motor axons extending from embryonic rat spinal cord explants functionally innervate co-cultured human muscle. Under such conditions a high degree of differentiation of the human skeletal muscle fibres is obtained that is never observed in aneural human muscle [1,6].…”

A 3D-printed platform for modular neuromuscular motor units

“…Cells expressed green fluorescent protein (GFP) under the control of the post-mitotic MN-specific Hb9 promoter (Supplementary Figure S3a); thus, we could visually confirm differentiation of MNs without the addition of exogenous factors or antibodies. Fluorescent imaging revealed that EBs also contained proliferating glia (Figure 2c), which are known to contribute to the formation and maintenance of the NMJ and differentiation and survival of MNs 11 .…”

“…Early research on the formation of NMJs in two dimensions (2D) has primarily focused on either the co-culture of excised or isolated muscle and neural tissues in vitro 11,12 , or the differentiation of mouse [13][14][15][16] or human [17][18][19] embryonic stem cells into MNs (usually through the formation of embryoid bodies or EBs), which were then co-cultured with excised or engineered muscle tissues. Beyond applications in regenerative medicine and therapeutics 20 , however, only a few studies have produced 3D NMJ platforms or applied neuromuscular research to living cellular systems using embryonic or neural stem cells [21][22][23] .…”