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.