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Fig. 6. Nodal-expressing ES cells promote the differentiation of definitive endoderm from genetically unmanipulated ES cells. (A) 1x10⁴ Nodal-expressing ES cells were plated on an upper insert and 5x10³ genetically unmanipulated ES cells were plated on a gelatin-coated lower chamber and cultured for 7 days. (B) The phosphorylation of Smad2, an effector of Nodal signaling, and the phosphorylation of Smad1/5/8 in each culture condition. CM; conditional medium of Nodal expressing ES cells. (C) The gene expression of Nodal signaling in genetically unmanipulated ES cells. Co-culture with Nodal-expressing ES cells or CM upregulated the gene expression of downstream transcriptional targets of Nodal in genetically unmanipulated ES cells. (D) Mature definitive endoderm markers were induced in the cells co-cultured with Nodal-expressing ES cells or CM. This effect is blocked by Cripto-neutralizing antibody. (E) Flow cytometric analysis of the expression of the definitive endoderm progenitor, CXCR4, in the cells co-cultured with Nodal-expressing ES cells or CM. FSC, forward scattered light. (F) Expression of definitive endoderm genes (Foxa2 and TTF-1) in genetically unmanipulated ES cells co-cultured with Nodal-expressing ES cells. The original magnification was 400x. (G) A flow cytometric analysis of the expression of GFP in the Pdx1-GFP knock-in ES cells co-cultured with Nodal-expressing ES cells.

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