Human muscle cultured in monolayer and cocultured with fetal rat spinal cord: importance of dorsal root ganglia for achieving successful functional innervation

Abstract

Adult human muscle cultured in monolayer was cocultured with explants of 13-14-d-old rat embryo using (a) ventral spinal cord (VSC), (b) transverse section of whole spinal cord (WSC), and (c) WSC with dorsal root ganglia (DRG) attached (WSC + DRG). AChR clusters and AChE- positive patches, both at the nerve-muscle contacts, were studied at 5, 12, and 21 d of coculture with each of the 3 spinal cord preparations. In addition, AChE-positive patches were studied after 31-64 d of coculture with WSC + DRG to evaluate further organization of those patches. Compared to VSC and WSC cocultures, WSC + DRG induced significantly more AChR clusters per muscle fiber at the nerve-muscle contacts at 5 d of coculture, and the percentage of muscle fibers containing AChR clusters was higher at all 3 time points quantitated. The number of AChE-positive sites was the same with all 3 spinal cord preparations in early (day 5) cocultures. Between 12 and 21 d of coculture, the number of muscle fibers containing AChE patches increased significantly only with WSC + DRG, correlating with the increased number of contracting muscle fibers in that coculture system. Only in human muscle cocultured with WSC + DRG was successful innervation of the cultured muscle fibers achieved, as manifested by (1) contractions in a continuous rhythm of large groups of muscle fibers that were reversibly blocked by 1 mM d-tubocurarine (aneurally cultured human muscle does not spontaneously contract); (2) well- developed cross-striations throughout the fiber; (3) well-organized AChE-positive sites; and (4) a trend from multifocal toward unifocal innervation of those muscle fibers. Our studies demonstrate that adult human muscle cultured in monolayer can be innervated by fetal rat spinal cord and that, in our system, DRG are essential for achieving functional innervation.

A system for studying mechanisms of neuromuscular junction development and maintenance

“…5G). We tested co-cultures with transverse sections of ventral root stripped of meninges and DRGs under the same conditions with growth factors and found, in accordance with other reports, that the number of NMJs was drastically reduced (Guettier-Sigrist et al, 1998;Kobayashi et al, 1987) (Fig. 5H).…”

In VivoNMDA Receptor Activation Accelerates Motor Unit Maturation, Protects Spinal Motor Neurons, and Enhances SMN2 Gene Expression in Severe Spinal Muscular Atrophy Mice

“…Cocultures of spinal cord explants (1 mm 3 ) and muscle cells were performed as described by Kobayashi et al (1987) with the following modifications. Spinal cord explants were obtained from control and severe SMA embryonic mice.…”

“…Explants from the whole transverse slices of 10.5-d-old mice embryo spinal cords including dorsal root ganglia (DRGs) were placed on the muscle monolayer. DRGs are essential to ensure a good innervation ratio (Kobayashi et al, 1987). The muscle culture was established through the differentiation of the wild-type muscle cell line made MBI (Cartaud et al, 2004).…”

Calcium currents and transients in co‐cultured contracting normal and Duchenne muscular dystrophy human myotubes

“…In contrast to aneurally cultured human skeletal cells, cocultured cells display spontaneous contractile activity (see Delaporte et al 1986;Kobayashi et al 1987;Saito et al 1990;Imbert et al 1995) a few days after the initiation of the fusion of myoblasts into myotubes. In the present experiments, this contractile activity (visually observed from 6 days after initiation of the fusion process) was accompanied by electrical activity (Fig.…”