Fig. 2. Neural differentiation of ES cells is regulated by cell density. ES cells were induced to differentiate by the 4-/4+ protocol at increasing seeding densities. After the induction, cells were plated onto PDL/laminin-coated coverslips and fixed 3-4 days post-plating. Coverslips were stained with anti-ß-tubulin 3 (red) and anti-nestin (green), and counterstained with Hoechst dye (blue) to visualize nuclei. (A) 10⁵ cells/ml seeding density; (B) 10⁶ cells/ml seeding density; (C) quantification of neuronal differentiation at different seeding densities (ND, none detected; error bars are s.d.). (D) Quantification of nestin-positive cells at low (10⁵ cells/ml) and high (10⁶ cells/ml) densities with or without RA treatment. Seeding at low density allows significant neural differentiation. Higher seeding density inhibits both neural and neuronal differentiation (LD, low density; HD, high density).