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Fig. 4. Differentiation potential of Gsc⁺ mesendoderm cells. (A) Fate of Gsc⁺ECD^- cells. Potential of Gsc⁺ECD^- cells to give rise to osteocytes (left, Alizarin Red staining) and endothelial cells (right). Osteoblastic markers such as Bglap1, Bglap2 and osteopontin (Opn) are expressed in the case of osteogenic induction. (Right) Endothelial colonies were visualized by immunostaining of Vecad and Pecam1, which are specific markers for endothelial cells. Arrowheads indicate colonies of endothelial cells. (B) Multicolour immunofluorescent images of day 6.5 culture. There are three different types of cells in this picture. Gsc^-ECD⁺Foxa2⁺ mature endoderm cells are larger than Gsc⁺ECD⁺Foxa2⁺ cells, but both form a continuous epithelial sheet. Gsc⁺ECD^-Foxa2^- cells outside the epithelial sheet correspond to mesoderm cells. (C) Fate analysis of day 4 Gsc⁺ECD⁺ cells at single cell level. Two-colour immunostaining of wells containing both Foxa2⁺ (dark purple, arrowheads) epithelial cells and ßR⁺/Vegfr2⁺ (red, arrows) mesoderm cells. All Foxa2⁺ cells form a single cluster, whereas all ßR⁺/Vegfr2⁺ cells are dispersed.

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