INCREASED SURVIVAL OF RAT EGF-GENERATED CNS PRECURSOR CELLS USING B27SUPPLEMENTED MEDIUM

Previous studies suggest that a population of precursor cells from the developing and adult mouse striatum can be expanded in culture using serum-free, N2-supplemented medium and mitogenic factors such as epide rmal growth factor (EGF). Here we show that EGF-responsive precursor c ells from embryonic rat striatum and mesencephalon can also be expande d in culture, incorporate bromodeoxy uridine (BrDU) and develop into s pheres that either adhere to the surface of the culture dish or float freely in the medium. Addition of B27, a medium supplement that increa ses neuronal survival in primary CNS cultures, resulted in a tenfold i ncrease in the number of proliferating cells in vitro over the first w eek. The effects of B27-supplemented medium on precursor cell survival were only seen when primary cultures were used, such that dividing ce lls grown in B27 for 1 week could then be transferred to either B27 or N2 medium and show similar survival and division rates in response to EGF. After 1, 2 or 4 weeks of growth in B27-supplemented medium, diss ociated precursor cells from either striatal or mesencephalic cultures could be differentiated when exposed to a poly-1-lysine-coated substr ate in serum and EGF-free medium supplemented with B27. These cells th en matured into a mixed culture containing neurons (approximately 35% of cells), astrocytes (approximately 44% of cells), and oligodendrocyt es (approximately 10% of cells), based on immunocytochemical staining with microtuble-associated protein (MAP2), glial fibriallary acidic pr otein and galactocerebrosidase. When whole spheres of precursor cells were allowed to differentiate, every one examined was found to generat e neurons, astrocytes and oligodendrocytes in similar proportions. Our findings suggest that B27-supplemented medium provides an enhanced en vironment for dividing and differentiating multi-potential precursor c ells over the first week in vitro. This culture system gives in expand able source of well-characterised, multi-potential CNS precursors that can be labelled with BrdU and, as such, may prove useful for either d ifferentiation experiments in vitro or as a source of tissue for graft ing into the damaged CNS.