Preparation of stage-specific differentiation media

Day	Additives to the media	Stock concentration	Final concentration	Dilution
BE1a
0–1 (d0)	Act A	100 μg/mL	100 ng/mL	1:1,000
	CHIR-99021	10 mM	2 μM	1:5,000
1–3 (d1,2)	Act A	100 μg/mL	100 ng/mL	1:1,000
	FGF2	50 μg/mL	5 ng/mL	1:10,000
BE1b
3–6 (d3,4,5)	FGF10	50 μg/mL	50 ng/mL	1:1,000
	DorsoM	5 mM	0.75 μM	1:6,666
	Wnt3a	25 μg/mL	3 ng/mL	1:8,333
BE3
d6–9 (d6,7,8)	FGF10	50 μg/mL	50 ng/mL	1:1,000
	LDN-193189	1 mM	200 nM	1:5,000
	SANT-1	1 mM	0.25 μM	1:4,000
	RA	10 mM	2 μM	1:5,000
	Glucose	1 M	16 mM∗	1:62.5
d9–13 (d9,10,11,12)	LDN-193189	1 mM	200 nM	1:5,000
	EGF	50 μg/mL	100 ng/mL	1:500
	Nicotinamide	1 M	10 mM	1:100
	Indolactam V	660 μM	330 nM	1:2,000
	Glucose	1 M	16 mM∗	1:62.5

Prepare differentiation media at the day of medium change; day of medium change is given in brackets; d: day.