Table 2:

Complete media with growth factor (CMGF⁺)

S. No	Components	Volume (10 mL)
1	CMGF−	1.5 mL
2	Wnt 3 conditioned media (ATCC L-Wnt3a cell line Cat #CRL-2647)	5 mL
3	B27 (50X)	200 μL
4	N2 (100X)	100 μL
5	n-Acetylcysteine (500 mM)	20 μL
6	Rspo-1 conditioned medium (R-Spondin HEK 293 cells)	2 mL
7	Noggin (293-Noggin cells)	1 mL
8	EGF (1000X final conc. 50 ng/mL)	10 μL
9	Gastrin (1000X final conc. 10 nM)	10 μL
10	Nicotinamide (final conc. 10 mM)	100 μL
11	A83 (TGFb type I receptor inhibitor) (1000X final conc. 500 nM)	10 μL
12	SB202190 (P38 inhibitor) (1000X final conc. 10 μM)	10 μL

Store at 4°C and can be used up to 2 weeks. Discard after 2 weeks.

Differentiation media: CMGF⁺ without Wnt 3A, Nicotinamide and SB202190, reduce R-Spondin and Noggin conditioned medium to half of the concentration.