IMMORTALIZATION OF IMMATURE AND MATURE MOUSE ASTROCYTES WITH SV40 T-ANTIGEN

The ability of neonatal astrocytes to promote neurite outgrowth in vit ro and in vivo diminishes as astrocytes mature. This property correlat es with the developmental loss of the central nervous system's ability to regenerate after injury. Cell lines representative of immature and mature astrocytes would be useful for studies to determine difference s between these two populations. Previous work on immortalization of b ipotential neural/glial precursors and fully differentiated glial cell s suggests that immortalization of astrocytes at timed intervals of cu lture may yield cell lines trapped in different maturation states. To test this, neonatal mouse cortical astrocytes were immortalized by ret rovirus-mediated transfer of the SV40 T antigen (Tag) gene at 2, 6 and 17 days of culture. The clonal cell lines express Tag and are contact -inhibited. Three phenotypes that change as a function of astrocyte ma turation were examined to determine the fidelity with which the cell l ines represent immature and mature astrocytes. These were: (1) cell mo rphology, growth pattern and size, (2) level of glial fibrillary acidi c protein (GFAP) expression, and (3) neurite outgrowth promotion. Firs t, immature and mature lines resemble mortal type 1 astrocytes of corr esponding ages with respect to morphology and growth pattern, and reta in a quantitative difference in cell size (mature cells are larger). S econd, the pattern of GFAP expression is preserved, with immature line s expressing lower levels than mature cell lines, but the overall GFAP levels are significantly lower in immortalized cell lines compared to mortal cells. Finally, promotion of neurite outgrowth from embryonic chick retinal ganglion cells on monolayers of the cell lines was exami ned. While all neurite outgrowth measures are significantly greater fo r the immortalized lines than for control 3T3 cells, they are attenuat ed relative to mortal astrocytes. The age-related pattern of stronger outgrowth support on immature astrocytes is retained for neurite initi ation, but not retained for mean neurite length. Thus, SV40 Tag-immort alized astrocytes have a complex phenotype characterized by retention of age-related differences in morphology, growth pattern and cell size , and by a marked attenuation of some astrocyte-specific characteristi cs but retention of age-related differences in the expression level of these same characteristics, and finally, loss of the ability to suppo rt neurite extension at levels characteristic of immature astrocytes. (C) 1994 Wiley-Liss, Inc.