Table 1.
Medium compositions.
| Component | PM | DM1 | DM2 | DM3 | DM4 | Manufacturer; # Catalog Number |
|---|---|---|---|---|---|---|
| Basal medium (BM)* | + | + | + | + | + | |
| B-27™ supplement | 1 × | 1 × | 1 × | 1 × | 1 × | Thermo Fisher Scientific, Waltham, MA, USA; # 17504001 |
| N-2 | 1 × | 1 × | 1 × | 1 × | 1 × | Thermo Fisher Scientific, Waltham, MA, USA; # 17502001 |
| Nicotinamide | 10 mM | − | − | − | − | Merck, Vienna, Austria; # 72 340 |
| n-Acetyl-l-Cysteine | 1 mM | − | − | − | − | Merck, Vienna, Austria; # A9165 |
| A 83–01 | 500 nM | 500 nM | 500 nM | − | − | Hycultec GmbH, Beutelsbach, Germany; # HY-10432 |
| SB 202190 | 10 µM | 10 µM | 10 µM | − | − | Hycultec GmbH, Beutelsbach, Germany; # HY-10295 |
| hGastrin I | 10 nM | − | − | − | − | Hycultec GmbH, Beutelsbach, Germany; # HY-P1097 |
| hHGF | 50 ng/mL | − | − | − | − | Thermo Fisher Scientific, Waltham, MA, USA; # 100-39H |
| mEGF | 50 ng/mL | 50 ng/mL | 50 ng/mL | 50 ng/mL | 50 ng/mL | Thermo Fisher Scientific, Waltham, MA, USA; # 315–09 |
| CHIR-99021 | − | − | 2.5 µM | − | − | BioGems; Westlake Village, CA, USA; # 2 520 691 |
| LY2157299 | − | − | 500 nM | − | − | TargetMol®, Wellesley Hills, MA, USA; # T2510 |
| hNoggin | 100 ng/mL | 100 ng/mL | 100 ng/mL | 100 ng/mL | − | Thermo Fisher Scientific, Waltham, MA, USA; # 120-10C |
| Wnt3A conditioned medium# | 43% v/v | 43% v/v | 43% v/v | − | − | In house manufacturing (Division of Small Animal Internal Medicine, University of Veterinary Medicine Vienna) |
| Rspo1 conditioned medium# | 10% v/v | 10% v/v | 10% v/v | 10% v/v | − | In house manufacturing (Division of Small Animal Internal Medicine, University of Veterinary Medicine Vienna) |
| Gentamicin | 20 µg/mL | 20 µg/mL | 20 µg/mL | 20 µg/mL | 20 µg/mL | Merck, Vienna, Austria; # G1272 |
| DAPT | − | − | − | − | 5 µM | Hycultec GmbH, Beutelsbach, Germany; # HY-13027 |
| Y-27632 dihydrochloride | 10 µM | 10 µM | 10 µM | 10 µM | − | TargetMol®, Wellesley Hills, MA, USA; # T1725 |
*BM consists of Advanced DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA), 10 mM HEPES (Merck, Vienna, Austria), 2 mM GlutaMAX™ (Thermo Fisher Scientific, Waltham, MA, USA), 100 U/mL penicillin/streptomycin (Merck, Vienna, Austria). Final concentrations are given.
#Cells for conditioned medium production were provided by Bio-Techne®, Abingdon, UK (Cultrex® HA-R-Spondin1-Fc 293 T cells) and as a generous gift from Prof. Dr. Clevers (L-Wnt-3A cells).