Retinoic acid

Ascorbic Acid or AA (Fisher Scientific, Cat No A61-25, CAS 5081-7) was prepared from a stock of 1 M, and used at 10 mM. A reduced form of glutathione or GSH (Alfa Aesar, Cat No AAJ6216606, CAS 70-18-8) was prepared from a stock of 0.5 M, and used at 10 mM. Pyrrolidinedithiocarbamate or PDTC (Cayman Chemicals, Cat No 20713, CAS 5108-96-3) was prepared from a stock of 10 mM, and used at 10 μM. Retinoic Acid or RA (Cayman Chemical, Cat No 11017, CAS 302-79-4) was prepared from a stock of 100 mM (dissolved in dimethyl sulfoxide (DMSO)), and used at 1 mM. The fluorescent dye for ROS assays, 4-Amino-5-methylamino-2′,7′-difluorofluorescein diacetate (H₂DCFDA) (Sigma, Cat No D6883, CAS 4091-99-0), was dissolved in DMSO at a stock concentration of 100 mM and used at 10 μM. Fluconazole (Cayman Chemical, Cat No 11594, CAS 86386-73-4) was dissolved in DMSO as a 50 mg/ml stock and used at 32 μg/ml. Hydrogen peroxide (Cat No H325-100) was obtained from Fisher Scientific.

For differentiation, iPSCs were accutase-treated and plated into a 6-well Matrigel-coated dish at a density of 1x10⁶ per well in E8 medium with ROCK-inhibitor Y27632 (10 μM; Stemgent). On the next day, iPSCs were differentiated into definitive endoderm by exposing them to Activin A (100 ng/ml; R&D) and Wnt3A (25 ng/mL only on the first day; Peprotech) in RPMI 1640 (Gibco) for 3 days. During these 3 days, the cells were exposed to increasing concentrations of 0, 0.2, and 2% defined FBS (dFBS, Hyclone). After definitive endoderm induction, the cells were directed to form foregut spheroids by culturing them for the next 3 days in Advanced DMEM/F12 medium (Gibco) containing 2% dFBS, 2 μM CHIR99021 (2 μM; Cayman), FGF4 (500 ng/mL; Peprotech), LDN193189 (2 μM; Cayman), and retinoic acid (2 μM; Cayman). This resulted in semi floating spheroids, which were then selectively picked and transferred on to Matrigel-coated experimental plates for further maturation and experimentation. For maturing the picked foregut spheroids, they were cultured in a medium containing Advanced DMEM/F12 with N2 (Invitrogen), B27 (Invitrogen), GlutaMax, Penicillin/streptomycin/Antimycotic, and EGF (100 ng/ml; Peprotech). Media was replaced every 2–3 days as necessary and the spheroids are allowed to develop into an epithelial monolayer until day 20.

For differentiation into iHTNs, iPSCs were accutase-treated and plated as single cells in 6-well Matrigel-coated plates at a density of approx. Overall, 1x10⁶ cells per well in E8 medium with ROCK-inhibitor Y27632 (10 μM; Stemgent). The next day iHTN differentiation was initiated by neuroectoderm differentiation by dual SMAD inhibition using LDN193189 (1 μM, Cayman) and SB431542 (10 μM, Cayman) and this treatment is carried on for 48 h. This was followed by Sonic hedgehog activation by Smoothened agonist SAG (1 μM, Tocris) and purmorphamine (PMN, 1 μM, Tocris) and Wnt signaling inhibition using IWR-endo (10 μM, Cayman) from day 3 to 8 to direct the cells towards ventral diencephalon with regular media change every 2 days. From day 9 to 13, the cells are slowly made to exit cell cycle using DAPT (10 μM, Cayman) in the presence of ventralizing agent retinoic acid (0.1 μM, Cayman). On day 14, the cells were treated with Accutase and re-plated onto laminin-coated plates in the presence of maturation medium containing brain-derived neurotrophic factor BDNF (10 ng/mL, Miltenyi) and maintained until day 40.