In vitro and In vivo Analysis of a Rat Bipotential O – 2A Progenitor Cell Line Containing the Temperature‐sensitive Mutant Gene of the SV40 Large T Antigen

Abstract

Primary cultures from neonatal optic nerve contain pluripotential O-2A progenitor cells that are capable of differentiating into oligodendrocytes, type-2 astrocytes or adult O-2A progenitors (O-2Aadult). Since primary optic nerve cultures contain a mixture of glial cell types of which only a small number are O-2A progenitors, experiments on cell lineage and differentiation carried out using these cultures are both intrinsically limited and difficult to interpret. Ideally, cells from a clonal cell population would provide the optimal starting material for biological studies. In this paper we describe the creation of an O-2A progenitor cell line using a retrovirus carrying a temperature-sensitive mutant SV40 large T antigen gene. This cell line has provided sufficient numbers of cells to allow analysis of their in vitro properties and their behaviour following transplantation into an in vivo environment. At the non-permissive temperature (39 degrees C), these cells differentiate into oligodendrocytes and type-2 astrocytes in a similar fashion to O-2A progenitor cells from primary cultures (O-2Aprim). When grown in media containing platelet-derived growth factor and basic fibroblast growth factor, the cell numbers can be expanded in culture without differentiating, consistent with the behaviour of O-2Aprim progenitor cells. By exploiting this property, it has been possible to culture large numbers of O-2A progenitors for in vivo analysis. In this study we have shown that transplantation of this O-2A cell line into glia-free areas in adult rat spinal cord results in differentiation of a proportion of cells into oligodendrocytes which are capable of myelinating axons. Furthermore, differentiation of O-2A cells into astrocytes was also observed, indicating that the bipotentiality of these cells in vitro can also be demonstrated in vivo.