Optimized Cell Culture Conditions

All media were prepared as reported previously^{57 (link)}. Advanced Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium supplemented with 1× GlutaMAX (Gibco), 10 mM HEPES (Sigma-Aldrich), and 100 Units/ml penicillin-streptomycin (Sigma-Aldrich) was used as the basal medium. Expansion medium (EM) is basal medium supplemented with 50% (vol/vol) Wnt3A-conditioned medium, 20% (vol/vol) R-spondin-1-conditioned medium, 10% (vol/vol) Noggin-conditioned medium, 1× B27 supplement (Gibco), 1 mM N-acetylcysteine (Sigma), 1× Primocin (InvivoGen), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), 500 nM A83-01 (Tocris), and 10 μM SB202190 (Tocris). Differentiation medium is comprised of basal medium (no penicillin-streptomycin added), 10% (vol/vol) Noggin-conditioned medium, 1 mM N-acetylcysteine (Sigma), 50 ng/ml human epidermal growth factor (R&D Systems), 10 nM [Leu-15]-gastrin (AnaSpec), 500 nM A83-01 (Tocris), and 10% fetal bovine serum (Sigma Aldrich). Wnt3A (American Type Culture Collection, Manassas, VA), R-spondin1 (kindly provided by Dr. Calvin Kuo, Stanford University, Stanford, CA), and Noggin^{58 (link)} (kindly provided by Dr. Marcel Bijvelds, Erasmus University, Rotterdam, the Netherlands) cell lines were maintained to produce conditioned media.