Factors that influence oligodendrocyte proliferation are poorly characterized or unidentified. Although, cell culture systems have great potential for identifying and characterizing these molecules, at the present time continued division of oligodendrocytes has not been achieved in vitro, and this provides a barrier to further study of this important developmental process. We believe that conventional culture techniques employing serum-supplemented medium are not permissive for oligodendrocyte cell division, since in vivo data suggests that the oligodendrocytes should be capable of some proliferation. We propose to first define the molecular requirements for growth of the CO-13-7 hybrid cell line, which expresses differentiated properties of oligodendrocytes, by replacing serum with hormones, growth factors, and other molecules. After optimization of a serum-free defined medium and substratum, these defined culture conditions will be tested for their ability to sustain the division of cell lines of other cell types and species. It will also be tested for ability to select for and to maintain the survival of dissociated new born rat cerebral hemisphere cells in vitro. Selection for oligodendrocytes will be assessed by immunocytochemical and biochemical criteria. The ability of these conditions to stimulate the proliferation of selected or isolated oligodendrocytes will be evaluated by the incorporation of tritiated thymidine into trichloroacetic acid-precipitable material as well as combination autoradiography and immunocytochemistry techniques for cultures of mixed phenotype. The defined medium and substratum bill be further modified, if required, to obtain substantial growth of oligodendrocytes and will be evaluated for species- and central nervous system locus-specificity. Developmental differences will be determined with rat cerebral hemisphere oligodendrocytes. The availability of proliferating oligodendrocytes will alleviate the problem of low yields obtained presently from tedious and expensive isolation procedures. The development of defined culture conditions will provide a model system for further investigation of the regulation of oligodendrocyte proliferation. In addition, there are many other aspects of development, differentiation, and pathology (e.g. multiple sclerosis) of this cell type that will benefit from the availability of an in vitro system of purified oligodendrocytes grown in serum-free defined culture conditions. We are interested in pursuing studies of myelinogensis with this model system.