Extended Data Fig. 2: Optimization of PrE/ExEM-like cells priming protocol from human HENSM naïve ESCs.

a, representative immunofluorescence images of WT WIBR3 (W3) nESCs induced in RCL media for 6 days, showing expression of GATA4 (cyan), SOX17 (yellow), and BST2 (red); nuclei (DAPI, white). GATA4 marks both SOX17+ PrE-like and BST2+ ExEM-like populations (see also Fig. 1e). This panel is an extended version of Fig. 1e but showing staining patterns of more markers. Scale bar, 100 µm. b, representative immunofluorescence images of iGATA6 nESCs induced in RCL media for 6 days (with or without DOX), showing expression of SOX17 (yellow) and BST2 (red). In both set-ups, BST2⁺ (ExEM-like) and SOX17⁺ (PrE-like) cell populations have a mutually exclusive expression pattern. Scale bar, 200 µm. c, representative immunofluorescence images of WT nESCs in RCL media for 6 days. FOXF1 (yellow), BST2 (red), GATA4 (cyan), nuclei (DAPI, white). BST2⁺ cells are also GATA4⁺/FOXF1⁺, excluding the possibility that they represent residual pluripotent cells in the RCL induced cultures from HENSM ESCs. Scale bar, 100 µm. Bottom, 4x zoom, scale bar, 25 µm.