Table 2.

Recipes of different media based on time post-seeding

	BUTYRATE	R-SPONDIN 1	NOGGIN	hEGF	Y-27632	CHIR99021
First 48 h	YES (1 mM)	NO	NO	YES (25 ng/mL)	YES (10 μM)	NO
After 48 h	NO	YES (500 ng/mL)	YES (100 ng/mL)	YES (50 ng/mL)	NO	YES (3 μM)

After the first 48 h, the medium change was performed every other day. These supplements were added to the basal medium. R-spondin1 (Cat.# SRP3292 - Sigma-Aldrich, St. Louis, Missouri, USA), Noggin (Cat.# 21–7075-U020 - Tonbobio, San Diego, California, USA), CHIR99021 (Cat.# SML1046 - Sigma-Aldrich, St. Louis, Missouri, USA), Y-27632 (Cat.# Y0503 - Sigma-Aldrich, St. Louis, Missouri, USA)