Fig. 5.

Medium conditioned by native cerebellar cells induces terminal differentiation of markers for cerebellar granule neurons, Purkinje cells, and astroglia. ES cells differentiated into cerebellar neurons in medium supplemented with FGF8 and medium conditioned by purified cerebellar granule neurons or astroglia at two concentrations (0.25 mg/ml or 0.5 mg/ml) (see Materials and Methods). ES cells cultured in the low concentration of granule cell-conditioned medium expressed MATH1 (green, A). (B–E) At the high concentration of granule neuron-conditioned medium, ES cells expressed EN1 (B), a specific marker for the cerebellar territory cells. ES cells cultured in the low concentration of glial cell-conditioned medium expressed the cerebellar specific marker ZIC2 (C), the granule cell marker NEUROD (D), and the glial cell marker BLBP (E). (F) ES cells cultured in the higher concentration of glial-conditioned medium expressed the Purkinje cell marker. The neuronal marker class III β-tubulin (TUJ1) was expressed under all culture conditions (red). (Scale bar: 20 μm.)