Differentiation of human ES cell derived crater cells in suspension culture in the presence of MedII conditioned medium. Serum free EB/MedII and EB differentiated in suspension in the presence of FGF2/MedII (A) or FGF2 (B). Toluidine blue stained plastic sections of sfEB/MedII (C) and sfEB (D), a partial rosette in sfEB (arrowhead) is indicated. (E) sfEB/MedII rosette, higher magnification of boxed region shown in (C). Presumptive extracellular matrix in central cavity (red dots), and surrounding the rosette (green dot) are indicated. (F) DAPI stained sfEB/MedII rosette indicating the central core of proliferative cells. Condensed mitotic chromosomes are indicated by the arrowheads. (G) DAPI stained sfEB/MedII rosette indicating characteristic elongated radial nuclei and a centralized mitotic figure (arrowhead). (H) Percentage of DAPI stained rosette nuclei in sfEB (white column, 21+/-17%) and in sfEB/MedII (black column, 58.2+/-3.9%) (P < 0.02). Images and scale bars: (A,B) 10×, 100 μm. (C,D) 20×, 50 μm. (E) 40×, 25 μm. (F,G) 60×, 10 μm.