Confluent hPSC cultures were treated with Acutase (STEMCELL Technologies) to resuspend as single cells in mTeSR1 and Y-27632 (10 μM, Tocris) and plated on Matrigel. On the following day, differentiation into definitive endoderm was carried out as previously described (McCracken et al., 2014 (link)). Briefly, cells were treated with Activin A (100 ng mL-1, R&D systems, Minneapolis, MN) and BMP4 (50 ng mL-1, R&D systems) on the first day in RPMI 1640 media (Life Technologies). Cells in the following two days were treated with only Activin A (100 ng mL-1) in RPMI 1640 with increasing concentrations 0.2% and 2% of HyClone defined fetal bovine serum (dFBS, GE Healthcare Life Sciences).
For anterior foregut monolayer cultures, cells were treated for 3 days in Noggin (200 ng mL-1) in RPMI 1640 with 2% dFBS, with all-trans retinoic acid (2 μM, Sigma, St. Louis, MO) the 3rd day.
Alternately, for the generation of anterior foregut spheroids, from definitive endoderm, cells were treated with FGF4 (500 ng mL-1, R&D systems), Noggin (200 ng mL-1) for 3 days in RPMI 1640 with 2% dFBS. Additional factors were tested during this time (described in results), such as CHIR99021 (“chiron” or “chr”, 2 μM, Tocris), Wnt3a (500 ng mL-1, R&D systems), SB431542 (10 μM, Tocris), DEAB (10 μM, Sigma), and retinoic acid (2 μM).
For anterior foregut monolayer cultures, cells were treated for 3 days in Noggin (200 ng mL-1) in RPMI 1640 with 2% dFBS, with all-trans retinoic acid (2 μM, Sigma, St. Louis, MO) the 3rd day.
Alternately, for the generation of anterior foregut spheroids, from definitive endoderm, cells were treated with FGF4 (500 ng mL-1, R&D systems), Noggin (200 ng mL-1) for 3 days in RPMI 1640 with 2% dFBS. Additional factors were tested during this time (described in results), such as CHIR99021 (“chiron” or “chr”, 2 μM, Tocris), Wnt3a (500 ng mL-1, R&D systems), SB431542 (10 μM, Tocris), DEAB (10 μM, Sigma), and retinoic acid (2 μM).