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 3
rd 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).
Trisno S.L., Philo K.E., McCracken K.W., Cata E.M., RuizTorres S., Rankin S.A., Han L., Nasr T., Chatuvedi P., Rothenberg M.E., Mandegar M.A., Wells S.I., Zorn A.M, & Wells J.M. (2018). Esophageal organoids from human pluripotent stem cells delineate Sox2 functions during esophageal specification. Cell stem cell, 23(4), 501-515.e7.