Culture of embryonic stem cells in serum-free conditions. The Sox1-GFP cells gave rise to bright green fluorescence in spheres that remained unattached during culture on non-tissue culture-coated plastic. The floating spheres on a Petri dish at day 1 of culture did not fluoresce (A is the GFP channel; B is phase; and C is the merged image) but became uniformly bright green 4 d after the start of culture (D is the GFP channel; E is phase; and F is the merged image). Expression of stem cell markers was assessed by RT-PCR (G). Stem cell markers Oct4 and NANOG decreased after differentiation of ES cells to neural progenitors in floating spheres (FS). Otx2 a marker for early placodal and forebrain neurons was maintained. Sox1 expression was first apparent at the time that neural progenitors formed. Floating spheres (I, black arrow) were fluorescent while attached spheres (I, white arrow) were not (H is the phase image and I is the merged phase and GFP images). Scale bars are 50 microns.