In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

In the present study, we demonstrate that the mitotic activity of O-2A lineage cells observed in vivo during demyelination has been maintained in vitro. This proliferation might be mediated by factors active in the culture milieu or by signals experienced in vivo which persist in vitro. The nature of the signal(s) triggering this proliferation is presently unknown. Reactive astrocytes may secrete mitogenic factors while microglia can synthesize lymphokines in response to trauma (Giulian, 1987; Giulian et al., 1988) . Since microglial cells are present in our cultures of adult spinal cord and are much more prevalent in cultures of demyelinated tissue, it is possible that O-2A lineage cells proliferate due to factors released into the culture milieu by these cells. In addition to soluble factors, denuded axons or axolemmal components, which are mitogenic for oligodendrocytes from developing CNS (Chen and DeVries, 1989), may act as mitogens for O-2A lineage cells during demyelination. Denuded axons in demyelinated lesions may stimulate proliferation directly, or may "prime" O-2A lineage cells to divide in response to specific growth factors. Previous studies have shown that oligodendrocytes isolated from adult spinal cord and expressing GC, but not myelin basic protein, can divide in the presence of neurons (Wood and Bunge, 1986) . Along with O-2A progenitors, oligodendrocytes expressing GC also proliferated in our cultures from remyelinating animals. Thus, proliferating oligodendrocytes could also be a major source of remyelinating cells. Type 2 astrocytes and cells with a mixed oligodendrocyte-astrocyte phenotype also proliferated in response to demyelination. Cells with characteristics of both o l i~ and astrocyte phenotypes have been described in several in vivo studies of demyelinating tissue (Bunge et al., 1961; Carrollet al., 1987; Godfralnd et al., 1989; Paine, 1989) . Additionally, ciliary neurotrophic factor, which induces transient GFAP expression in neonatal O-2A progenitors in vitro, is much more abundant in regenerating CNS tissue (Nieto-Sampedro et al., 1983) and might possibly induce a mixed phenotype in vivo. The presence of phenotypic characteristics of both oligodendrocytes and astrocytes in the same cell indicates that some degree of plasticity is possible between these two differentiation pathways. Mixed phenotype cells might be precursor cells at an intermediate bipotential stage which are responding to two differentiation signals simultaneously, or may be mature cells in a state of transdifferentiation between astrocyte and oligodendrocyte phenotypes. By either mechanism, mixed phenotype cells might provide additional ways to increase the number of oligodendrocytes available for remyelination. Now that we have established and characterized an in vitro system of glial cells derived from normal and demyelinating/remyelinating adult CNS, we can design future experiments to determine which of the proliferating cells provide new oligodendrocytes in remyelination and which factors enhance this pathway. Since we have recently developed a similar in vitro system from adult human CNS (Dorn et al., 1990) , such experiments could also be performed with human oligodendrocyte lineage cells. The study of the growth and differentiation properties of the glial cell types involved in remyelination may lead to the elaboration of strategies to promote remyelination in human demyelinating diseases.