Schwann cells proliferate but fail to differentiate in defined medium.

Abstract

Primary cultures of dorsal root ganglia cells from 18-to 21-day rodent embryos were studied for their ability to express Schwann cell function in a defined medium lacking serum and embryo extract. It was confirmed that Schwann cells, but not fibroblasts, are able to proliferate in response to contact with axons when cultured in this defined medium. We here report that in this medium, however, differentiation of Schwann cells was arrested before completion of ensheathment and before initiation of myelin formation. (13) to support proliferation of neuroblastoma cells; they observed neuronal growth and Schwann cell (but not fibroblast) proliferation in their dissociated cell cultures. In this paper, we report that, when this defined medium is used in our standard sensory ganglion preparations, Schwann cell proliferation occurs and differentiation is initiated but fails to progress to typical axonal ensheathment and myelination, even after many weeks in culture. Electron microscopic study of these cultures shows that secretory products of the neuron-related Schwann cells [basal lamina and collagen fibrils; (8)] are absent from the extracellular space. Ensheathment, appearance of basal lamina and fibrils, and myelination occur within several days, however, after serum and embryo extract are added to the defined medium. We will discuss the implication of these observations on the role of secretion in the expression of Schwann cell function. MATERIALS AND METHODSDRG were dissected from either rat or mouse 18-to 21-day embryos, and their connective tissue capsules were removed by using forceps (Dumont, modified) having exceptionally fine tips. Ganglia were used as explants or were dissociated by incubation at 370C for 30 min in 0.25% trypsin, followed by trituration with a pipette and filtration through a nylon membrane having 15-Mm pores. The ganglia or dissociated cells were plated on Aclar minidishes coated with reconstituted rat tail collagen and incubated as described (14). Explant cultures were established and maintained in either N2 medium alone or in N2 medium supplemented with serum, serum and embryo extract, or specific additions (Table 1). Dissociated cell cultures were initially plated in a starter (i.e., antimitotic) medium (AMM; see below) containing serum, embryo extract, and an antimitotic agent and designed to (i) promote vigorous initial axonal growth, (fi) permit optimal neuronal differentiation, and (ii) suppress the development of nonneuronal cells. Cell cultures were shifted to N2 medium 10-14 days later. Cultures were fed every 2-3 days with an appropriate medium. Compositions were as follows: the standard medium (SM) contained 65% Eagle's minimal essential medium (15), 25% human placental serum, 10% 9-day chicken embryo extract, glucose at 6 g/liter, and crude nerve growth factor (16) at :t50 biological units per ml; the AMM had the same basic composition as the SM, but also included 10 MtM uridine and fluorodeoxyuridine and had only 10% serum and 2% embryo extract; the N2 medium was pre...

Glial Cell Line-Derived Neurotrophic Factor Alters Axon Schwann Cell Units and Promotes Myelination in Unmyelinated Nerve Fibers

“…Similar to NGF, when Schwann cells were cocultured and then "triggered" by the addition of ascorbate, they promptly began myelination. A virtually identical sequence has been found with NGF (Moya et al, 1980). The present study demonstrates the ability of GDNF to support small sensory neurons in culture and promote myelination.…”

Glypican-1 and α4(V) Collagen Are Required for Schwann Cell Myelination

“…Cocultures incubated in serum-free medium without ascorbic acid lack any fibrillar or basal lamina matrix structures that can be detected by immunofluorescent staining with specific antibodies (Fig. 7) or by electron microscopy (Moya et al, 1980). One day after addition of ascorbic acid to initiate ECM assembly deposition of ␣(V) collagen into matrix is discernible by confocal microscopy (Fig.…”

“…In cocultures of Schwann cells and sensory neurons, Schwann cells fail to myelinate axons in the absence of basal lamina (Moya et al, 1980). Myelination is restored by providing exogenous basal lamina (Carey et al, 1986) or ascorbic acid, which stimulates endogenous extracellular matrix (ECM) assembly (Carey and Todd, 1987;Eldridge et al, 1987).…”

Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina

“…Cultures were maintained on N2 for 3 weeks to allow full Schwann cell repopulation. Some cultures were then switched to E-15 medium to induce myelination and basal lamina formation (Moya et al, 1980;Eldridge et al, 1986); others were retained on N2 to provide Schwann cells that were unable to organize ECM. Then, 2-3 d before placement of retinal explants, DRG neurons were removed from the central regions of all cultures, both those providing SC with ECM and those providing SC without ECM.…”