Extended Data Fig. 1. Primary epithelial stem cells possess differentiation potential and stemness, related to Fig. 1.
(A) For the cell expansion, dissociated cell aggregates were embedded in fresh matrigel then developed into new spheroids. Urothelial spheroids can be passaged every 3 days using 1:2–1:3 dilutions depending on cell density. (B-D) Primary USCs originated from 8 week old C3H/HeN mice were cultured in matrigel with 50% CM. After 3 days of culture in 50% CM, media were changed to fresh 50% CM or 5% CM at 3, 5, and 7 days, then RNAs were isolated at 1, 2, 3 (yellow), 5 (green), and 7 days (orange) (USC culture n = 12, 9, 13, 12, 12, 11, 11 respectively). (B) Gene expression of p63, (C) Axin2, a Wnt signaling marker, and (D) Upk3a, a urothelial cell differentiation marker, was measured by qRT-PCR and data is represented as mean ± SD. Significance was determined by an unpaired (two-tailed) t test. (E) To culture bladder organoids in matrigel, USCs were pre-cultured in 50% CM for 3 days, gently dissociated, then passaged into fresh matrigel for culture in 50% CM or 0% CM for 5 days, while media were changed every 2 days. After 5 day culture, USC spheroids were fixed with 10% neutral buffered formaldehyde (NBF) and prepared for paraffin embedding. The slide with paraffin sections were stained with hematoxylin and eosin (H&E) and immunostained for Upk3a (red), E-cadherin (yellow), and DAPI (blue) or K20 (red), K5 (yellow), and DAPI (blue).