Stimulation assays were performed on confluent hiPSC cultures maintained in mTeSR1 medium. HiPSCs were seeded at 1.5 × 105 cells/cm² on Matrigel-coated coverslips in mTeSR1 medium supplemented with Ri. The next day, medium was replaced by fresh mTeSR1 medium. The day after hiPSCs cultures were stimulated for 1 h with BMP4 (R&D, ref. 314-BP, 50 ng/mL) or ACTIVIN A (100 ng/mL), and 6 h with WNT3A (R&D, ref. 5036-WNT, ranging from 5 ng/mL to 50 ng/mL) in mTeSR1 medium. Cells were then fixed and signaling pathways activation was assessed through immunocytochemical analyses. Medium replacement protocols are summarized in Supplementary Table 5 .
For experiments of rescue and chemical induction of epithelial integrity, hiPSCs were seeded on Matrigel-coated coverslips at a density of 0.75 × 105 cells/cm² in mTeSR1 medium supplemented with Ri. The next day, medium was replaced by fresh mTeSR1 medium. The day after, medium was replaced for 1 day by mTeSR1 medium supplemented with 5 µM of LPA or 10 µM of Blebbistatin. Finally, confluent cultures were stimulated for 1 h with BMP4 (50 ng/mL) in mTeSR1 medium, and then fixed and analyzed by immunocytochemistry.
For long-term stimulation experiments of BMP and ACTIVIN pathways, hiPSCs were seeded on Matrigel-coated coverslips at a density of 1.1 × 105 cells/cm² in mTeSR1 medium supplemented with Ri. The next day, medium was replaced by fresh mTeSR1 medium. The day after, hiPSCs cultures were stimulated with BMP4 (50 ng/mL) or ACTIVIN A (100 ng/mL) in mTeSR1 medium. hiPSCs cultures were then fixed and analyzed by immunocytochemistry at 0, 2, 6, 9, 12, and 18 h following stimulation.
For experiments of rescue and chemical induction of epithelial integrity, hiPSCs were seeded on Matrigel-coated coverslips at a density of 0.75 × 105 cells/cm² in mTeSR1 medium supplemented with Ri. The next day, medium was replaced by fresh mTeSR1 medium. The day after, medium was replaced for 1 day by mTeSR1 medium supplemented with 5 µM of LPA or 10 µM of Blebbistatin. Finally, confluent cultures were stimulated for 1 h with BMP4 (50 ng/mL) in mTeSR1 medium, and then fixed and analyzed by immunocytochemistry.
For long-term stimulation experiments of BMP and ACTIVIN pathways, hiPSCs were seeded on Matrigel-coated coverslips at a density of 1.1 × 105 cells/cm² in mTeSR1 medium supplemented with Ri. The next day, medium was replaced by fresh mTeSR1 medium. The day after, hiPSCs cultures were stimulated with BMP4 (50 ng/mL) or ACTIVIN A (100 ng/mL) in mTeSR1 medium. hiPSCs cultures were then fixed and analyzed by immunocytochemistry at 0, 2, 6, 9, 12, and 18 h following stimulation.
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