Clonal analysis of astrocyte diversity in neonatal rat spinal cord cultures

Abstract

Within the mammalian CNS, astrocytes appear to be a heterogeneous class of cells. To assay the number of distinct types of astrocytes in the rat spinal cord, cell lineage and phenotypic analyses were carried out on cultures from newborn rat spinal cord and five distinct types of astrocytes were observed. Proliferating precursors for each class of astrocyte were isolated by low density culture and shown to give rise to 5 distinct and morphologically homogeneous clusters of GFAP + astrocytes. Immunocytochemical analysis with antibodies A2B5 and Ran-2, which identify different glial lineages in optic nerve cultures, demonstrated that many clusters included both A2B5+ and A2B5- cells. Similarly, many clusters also possessed a mixture of Ran-2+ and Ran-2-cells, suggesting that in spinal cord cultures, in contrast to optic nerve cultures, expression of these antigens is regulated by individual cells rather than by cell lineage. Single-cell cloning studies, revealed that the abundance and proliferative capacity of individual astrocyte precursors differed depending on the type of astrocyte. To assay the effects of a complex cellular environment on the composition of astrocyte clones, lineage analysis was performed in complete spinal cord cultures using a replication deficient retrovirus. Although similar morphologically homogeneous clones of cells to those seen with single-cell clones were observed, the proliferative capacity and relative abundance of the distinct astrocyte precursors differed from that seen in single-cell cloning studies. Together these observations suggest that in spinal cord, gliogenesis is considerably more complex than in the optic nerve and that cultures of newborn rat spinal cord contain multiple, distinct populations of astrocytes.