A83 01

Except where specified, reset cells were ʻre-primed' before initiating differentiation. Cells were plated on Geltrex in t2iLGö and after 48 h the medium was changed to E8. Cultures were maintained in E8, passaging at confluence. Lineage-specific differentiation was initiated between 25 and 44 days.
Definitive endoderm was induced according to Loh et al. (2014) (link). Cells were cultured in CDM2 medium (in-house according to Loh et al., 2014) supplemented with 100 ng/ml activin A (produced in-house), 100 nM PI-103 (Bio-Techne, 2930), 3 µM CHIR99021, 10 ng/ml FGF2, 3 ng/ml BMP4 (Peprotech) for 1 day. For the next 2 days the following supplements were applied: 100 ng/ml activin A, 100 nM PI-103, 20 ng/ml FGF2, 250 nM LDN193189.
For lateral mesoderm induction (Loh et al., 2016 (link)), cells were treated with CDM2 supplemented with 30 ng/ml activin A, 40 ng/ml BMP4 (Miltenyi Biotech, 130-098-788), 6 µM CHIR99021, 20 ng/ml FGF2, 100 nM PI-103 for 1 day, then with 1 µM A8301, 30 ng/ml BMP4 and 10 µM XAV939 (Sigma-Aldrich).
For neural differentiation via dual SMAD inhibition (Chambers et al., 2009 (link)), cells were treated with N2B27 medium supplemented with 500 nM LDN193189 (Axon, 1509) and 1 μM A 83-01 (Bio-Techne, 2939) for 10 days, then passaged to plates coated with poly-L-ornithine and laminin and further cultured in N2B27 without supplements.

Between 2014 and 2017, tumor samples were collected from human pancreatic ductal adenocarcinoma cancer (PDAC) patients under IRB12-1108 and IRB13-1149. Samples were obtained from patients undergoing pancreatic resections at The University of Chicago Medicine (UCM) facilities. Tumor samples were digested and established into organoids according to established protocols previously published^{9 (link)}. Briefly, organoids were grown and cultured by embedding dissociated tumor cells in growth-factor-reduced (GFR) Matrigel (Corning, 356231) and cultured in complete media (Intesticult [Stemcell Technologies, 6005], A83-01 [0.5 µM, Sigma, SML0788], fibroblast growth-factor 10 [FGF10, 100 ng/ml, Gibco, PHG024], Gastrin I [10 nM, Sigma, 17105-041], N-acetyl-L-cysteine [10 mM, Sigma, A9165], Nicotinamide [10 mM, Sigma, N0636], B27 supplement [1x, Gibco, 17504-044], Primocin [1 mg/ml, InvivoGen, ant-pm-1], and Y-27632 [10.5 μM Tocris, 1254]). Organoids were passaged via mechanical dissociation and TrypLE Express (Fisher Scientific, 12605-010) to single cells before being loaded on to the platform.

The ExM induction protocol was modified based on previous reports [38 (link),39 (link)]. Prior to induction, all cell types were brought into the same hypoxic, feeder-free condition on 10 μg/mL Biolaminin 521. For this, nESC were depleted of feeder cells [24 (link)] and prESC were placed in the hypoxic incubator 24 h after plating. After reaching confluency, cells were briefly washed with PBS and incubated in N2B27 medium supplemented with 30 ng/mL ACTIVIN A, 40 ng/mL BMP4 (PeproTech, 120-05ET), 6 μM CHIR99021, 20 ng/mL FGF2 (PeproTech, 100-18B), 100 nM PIK90 (Axon MedChem, 1362), and 1:1000 Penicillin–Streptomycin. After 24 h, following a brief wash with PBS, cells were treated with N2B27 medium supplemented with 1 μM A8301 (Sigma, St. Louis, MO, USA, SML0788), 30 ng/mL BMP4, 1 μM C59 (Axon MedChem, 2287), and 1:1000 Penicillin–Streptomycin for 14 days, with a medium change of every 1–2 days.

For construction of organoids, 200–500 crypts per well were suspended in Matrigel (Corning) as described.^{58 (link)} Complete ENR medium (all components from Thermo Fisher Scientific unless noted) were comprised of advanced DMEM/F12 (Gibco), antibiotic-antimycotic (×100), 1 mM N-acetyl cysteine (Sigma-Aldrich), B27 supplement, N2 supplement, EGF, Noggin (R&D Systems), R-spondin-1-conditioned medium, and Y-27632 (Sigma). Y-27632 was added to the ENR medium for the first 48–72 h of culture only and then removed during the medium change. The ENR medium was replaced every 2 to 3 days. Colon organoids were cultured in the ENR medium supplemented with Wnt3 conditioned media (WENR). Human colon organoids were cultured in WENR medium supplemented with gastrin, nicotinamide, A83-01, and SB202190 (all from Sigma) as described.^{59 (link)} Isolated ISCs and Paneth cells were co-cultured in ENR medium supplemented with Jagged-1 (1 µM; Anaspec). Wnt-C59 (50 µM; Abcam) was used as a porcupine (PORCN) inhibitor. The surface areas of SI and colon organoids were measured microscopically by taking several random non-overlapping photos of organoids in a well using an inverted microscope (Carl Zeiss). Each photo was analyzed using ImageJ software (NIH) and the Zen image program (Carl Zeiss). Organoid perimeters for area measurements were defined manually using automated ImageJ software.