Epicult b media

Cells were plated in 2% Matrigel-containing maintenance media (DMEM F12 with horse serum, hydrocortisone C, Cholera toxin, Insulin, ciprofloxacin, B27 supplement, and EGF), or differentiation media (Epicult B media containing B supplement [STEMCELL Technologies], Heparin, penicillin/streptomycin, EGF, and FGF) in low-adhesion 96-well plates. Spheres with diameters measuring 20 μM or greater were counted 7 days later. The percent of SFCs was determined as the number of spheres formed per cell plated. At least three wells were plated for each experiment to obtain technical repeats that were averaged. Each experiment was performed at least three times to obtain biological replicates. Two-tailed Student’s t-tests on averages of biological replicates were performed to determine p-values. p-values smaller than .05 were determined to be significant. For FACS analysis and serial passaging, colonies were isolated by incubating in cell recovery solution (Corning) on ice for 1.5 h and dissociated with Dispase, DNase, and .25% Trypsin-EDTA before being passed through a .40-μM filter and resuspended in Hank’s balanced salt solution containing 2% FBS and DNase. Diptheria toxin (DTx, Sigma) added to maintenance media with 2% Matrigel prior to plating fMaSCs.

For organoid-forming assays, cells were grown at a clonal density of 2 × 10³ cells/cm² in 24-well ultra-low attachment plates that had been coated with 1.2% poly(2-hydroxyethyl methacrylate) to prevent adhesion and growth of the primary MECs. The cells were grown in media containing EPiCult-B media (STEMCELL Technologies, Vancouver, BC, Canada) supplemented with 5% Matrigel, 5% FBS, 10 ng/ml EGF, 20 ng/ml basic fibroblast growth factor, 4 mg/ml heparin, and 10 μM Y-27632. Cells were left for 10 days to form organoids, which were then counted. For activating Wnt signalling in organoid cultures, recombinant mouse Wnt3A (R&D Systems, Minneapolis, MN, USA) or glycogen synthase kinase 3 (GSK3) inhibitor (GSK3i, CHIR99021; Sigma-Aldrich) was added to the organoid cultures on day 0 at concentrations of 100 ng/ml or 50 nM, respectively. For gene expression analysis, RNA was collected from cells on day 2, and the RNA expression was measured using qRT-PCR. Note that addition of Rock inhibitor (Y-27632) is important for the expansion of pluripotent stem cells because it helps maintain the stem cells in their undifferentiated state, and they survive longer in culture, and note also that the Rock inhibitor increases the efficiency of colony formation.