Controlled early differentiation of human ES cell colonies (A) Undifferentiated HESC colony on mouse embryonic fibroblasts. (B) Crater containing colony derived from passaging HESC onto freshly plated mouse embryonic fibroblasts. A central crater of monolayer cells (arrowhead) is surrounded by undifferentiated HESC (*), and differentiating cells in an outer ring adjacent to the feeders (arrow). (C,D) OCT-4 expression in undifferentiated HESC (C) and crater cells (D). (E,F) DAPI staining of the same fields as (C,D). (G,H) merged images of (C-F). Mitotic cells (arrowheads) and some of the apparently apoptotic cells (white dots) are indicated. (I) Anti-SSEA-4 immunostaining of a crater colony. Crater cells (arrowhead) do not stain for the SSEA-4 antigen, a marker of undifferentiated HESC, while the surrounding HESC are SSEA-4+ (arrow). Images and scale bars: (A,B) 10×, 100 μm. (C-H) 40×, 50 μm. (I) 10×, 100 μm.