Cell types required to efficiently innervate human muscle cells in vitro

Previous studies carried out in our laboratory have shown that myofibers fo rmed by fusion of muscle satellite cells from donors with spinal muscular a trophy (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 satell ite cells. In order to study the kinetics of nerve and muscle cell degenera tion in nerve-muscle cocultures implicating SMA muscle cells, we attempted to simplify the nervous component of the coculture and identify the nerve c ell types necessary for a successful innervation. We demonstrate here that motoneurons alone mere unable to innervate myotubes. However, when three ce ll types (motoneurons, sensory neurons, and Schwann cells) were added onto a reconstituted muscular component consisting of cloned muscle satellite ce lls and cloned muscular fibroblasts, myotubes contracted, indicating that f unctional 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 ner ve cells on innervation; we observed that physical contacts among sensory n eurons, motoneurons, and myotubes are required for a successful innervation ; in contrast Schwann cells can be replaced by a Schwann-cell-conditioned m edium, indicating that these cells produce a putative soluble "innervation- promoting factor." Obviously such a reconstituted system does not reflect t he in uivo situation but it allows the formation of functional motor synaps es and could therefore allow us to elucidate neuromuscular disease pathogen esis, especially that of spinal muscular atrophy. (C) 2000 Academic Press