Reagents
1 0.05% Trypsin/EDTA, 25300120, Gibco
2 10 cm2 tissue-culture dish, 353003, BD Falcon
3 15 ml tube, 352097, BD Falcon
4 24-well transwell insert, 8770, BD Falcon
5 50 ml tube, 352098, BD Falcon
6 7.5% Bovine serum albumin, 15260037, Gibco
7 Accutase/EDTA, AT104, Innovative Cell Technologies
8 Activin A, 338-AC, R&D System
9 All-trans Retinoic acid, 0695, R&D System
10 Ascorbic acid, A4544, Sigma
11 B27, 17504044, Gibco
12 β-mercaptoethanol, M6250, Sigma
13 BMP4, 214-BP, R&D System
14 CHIR 99021, 4423, R&D System
15 c-KIT-PE, 313204, Biolegend
16 CXCR4-APC, 306510, Biolegend
17 FGF10, 345-FG, R&D System
18 FGF2, 233-FB, R&D System
19 FGF7, 251-KG, R&D System
20 Fibronectin, 1918-FN, R&D System
21 Glutamax, 35050061, Gibco
22 Growth factor reduced matrigel, 354230, Corning
23 Ham's F12, 10-080-CV, Cellgro
24 Iscove's Modified Dulbecco's Medium (IMDM), 10-016-CV, Cellgro
25 IWP2, 3533, R&D System
26 knockout serum replacement, 10828028, Gibco
27 low-adherin plate, 3471, costar
28 MEM Non-Essential Amino Acids Solution, 11140050, Gibco
29 Monothioglycerol, M6145, Sigma
30 Mouse embryonic fibroblasts, GSC-6201G, GlobalStem
31 N2, 17502048, Gibco
32 Noggin, 6057-NG, R&D System
33 Non-tissue culture-treated plate, 351146, BD Falcon
34 Penicillin-streptomycin, 30-002-CI, Cellgro
35 Primocin, ant-pm-2, InvivoGen
36 SB 431542, 1614, R&D System
37 Y-27632, 1254, R&D System
Media
Stop media
IMDM 500 ml
FBS 25 ml
GultaMax 5 ml
Penicillin-streptomycin 5 ml
hPSC maintenance media
DMEM/F12 400 ml
Knockout serum 100 ml
β-mercaptoethanol 0.1 mM
Primocin 1 ml
FGF2 20 ng/ml
GlutaMax 5 ml
Serum-free differentiation (SFD) media
IMDM 750 ml
Ham’s F-12 250 ml
N2 5 ml
B27 10 ml
7.5% BSA 7.5 ml
Penicillin-streptomycin 1%
GultaMax 10 ml
Ascorbic acid 50 μg/ml
Monothioglycerol 0.4 μM
Embryoid bodies/primitive streak formation media
Y-27632 10 μM and BMP4 3 ng/ml in SFD media
Endoderm induction media
Y-27632 10 μM, BMP4 0.5 ng/ml, FGF2 2.5 ng/ml, and Activin A 100 ng/ml in SFD media
Anteriorization media-1
Noggin 100 ng/ml and SB431542 10 μM in SFD media
Anteriorization media-2
SB431542 10 μM and IWP2 1 μM in SFD media
Ventralization media/Branching media
CHIR99021 3 μM, FGF10 10 ng/ml, FGF7 10 ng/ml, BMP4 10 ng/ml, and all-trans Retinoic acid 50 nM in SFD media