Results The Tumor Matrix Extract Polymerizes In Vitro into a Basement Membrane-like Structure

Exposing rat Schwann cells co-cultured with nerve cells to a reconstituted basement membrane induced the formation of myelin segments by Schwann cells. This occurred in a serum-free culture medium in which, in the absence of this matrix, Schwann cells proliferate but fail to differentiate. This reconstituted basement membrane was prepared from solubilized extracellular matrix proteins synthesized by a basement membrane-producing murine tumor. The major constituents of this reconstituted matrix are collagen type IV, laminin, heparan sulfate proteoglycan, entactin, and nidogen. The matrix also elicited striking morphological changes in Schwann cells, inducing them to spread longitudinally along the nerve fibers (a necessary early step in the process of ensheathment of nerve fibers). Several observations indicated that the effect of the matrix was exerted directly on Schwann cells and not indirectly through an effect on nerve cells. First, the matrix-induced cell spreading occurred only in areas in which Schwann cells directly contacted the matrix; Schwann cells that were associated with the same nerve fibers but that did not themselves directly contact the matrix did not exhibit spreading. Second, the matrix-induced alteration in Schwann cell morphology was observed in cultures in which the nerve cells were removed. These results provide direct evidence that basement membrane contact induces normal Schwann cell differentiation, and support the idea that Schwann cell differentiation in vivo may be regulated by the appearance of the basement membrane, which normally envelops terminally differentiating Schwann cells. T HE extracellular matrix has been shown to influence the behavior of many cell types, including cells of neural origin, during embryonic development. Cellular functions affected by extracellular matrix include proliferation, migration, adhesion, morphology, and terminal differentiation (reviewed in references 14-16, 26, and 28). In most cases the molecular mechanisms responsible for these effects are not known. We have been studying the development in culture of Schwann cells and nerve cells obtained from sensory ganglia of rat embryos. Under appropriate culture conditions these Schwann cells produce an extracellular matrix consisting of a basement membrane and associated collagen fibrils that ultrastructurally and biochemically is similar to the extracellular matrix of the peripheral nerve endoneurium (7, 9, 11). Under certain conditions, however, the formation of this extracellular matrix by Schwann cells is blocked. This occurs, for example, in cultures treated with cis-hydroxyproline, an analogue of proline that inhibits collagen hydroxylation (10), and in cultures fed the serum-free medium N2 (8, 21). It has been shown that in these cultures the Schwann cells fail to differentiate normally, as demonstrated by their inability to provide ensheathment and myelination for sensory nerve fibers (10, 21). These observations, and others (4), have led to the suggestion that the presence of a suitable extracellular matrix is necessary for normal Schwann cell development (4, 21). Until recently, however, it was not possible to demonstrate a direct effect of exogenously added extracellular matrix on Schwann cell development in the absence of de novo matrix production. Thus, it could not be ruled out that the culture conditions that prevented extracellular matrix production by Schwann cells were also affecting other cellular functions important for Schwann cell differentiation. In this paper we present evidence that an exogenous basement membrane-like extracellular matrix, under conditions in which extracellular matrix production by Schwann cells does not occur, elicits normal Schwann cell development, as evidenced by the spreading of Schwann cells onto sensory nerve fibers and the formation of myelin segments around sensory nerve fibers. Materials and Methods