Organoid Differentiation and Imaging

Organoids were seeded in 3D in Matrigel (Corning, NY, USA) plugs as for passaging organoids. Following seeding, organoids were given hW-CMGF+ medium^{42 (link)} supplemented with 10 µmol Y-27632 Rock inhibitor, and for J2-NGN3 organoids, 200 µg/ml Geneticin for 2 days. After two days, media were changed according to organoid treatment group: undifferentiated: hW-CMGF+ medium, 10 µmol Y-27632 Rock inhibitor, and for J2-NGN3 organoids 200 µg/ml Geneticin; differentiated and uninduced: differentiation medium^{42 (link)} with 10 µmol Y-27632 Rock inhibitor; differentiated and induced (J2-NGN3 organoids only): differentiation medium, 10 µmol Y-27632 Rock inhibitor, and 1.0 µg/ml doxycycline (as doxycycline monohydrate, Sigma-Aldrich, St. Louis, MO, USA, dissolved in DMSO). These media conditions (at 500 µl) were continued for 4 days with daily media changes. After 4 days, 3D organoids were prepared for imaging using established methods.^{79 (link)} Epithelial cell boundaries were imaged by staining with Alexa 647 conjugated E-cadherin, and nuclei were stained with 0.07× NucBlue Fixed Cell Stain ReadyProbes (Invitrogen, USA) with secondary antibody application. Primary and secondary antibodies and their dilutions are listed in Supplemental Table S8.