Generation of β cells from hPSCs
This is intended as a guide only; for full experimental details please read the reference provided.
In Brief Download the PDF of this protocol
Pagliuca et al. describe the generation of insulin-producing pancreatic β cells from human pluripotent stem cells (PSCs) in vitro in 4 - 5 weeks using a cocktail of small molecules and proteins.
Briefly, using a suspension-based culture system hESCs and hPSCs were seeded at 6 x 105 cells/ml. After 48 h and 72 h, respectively, differentiation was started by changing the media to Day media. Pancreatic β cells were generated as follows: Days 0-3: Definitive endoderm (DE) induction; Days 4-6; Generation of primitive gut tube (PGT); Days 7-13: Pancreatic specification; Days 14-27+ β cell maturation. The resulting β cells released insulin in response to glucose stimulation in vitro and following transplantation into mice.
Cocktails
| DE Induction | PGT Generation | Pancreatic Specification | Endoderm Specification | β-cell Differentiation | |||||
|---|---|---|---|---|---|---|---|---|---|
| Activin A | 100 ng/ml | KGF | 50 ng/ml | KGF | 50 ng/ml | SANT-1 (Cat.No. 1974) | 0.25 μM (Days 14-17 only) | RepSox (Cat.No. 3742) | 10 μM |
| CHIR 99021 (Cat.No. 4423) | 3 μM | Activin A | 100 ng/ml | Retinoic Acid (Cat.No. 0695) | 2 μM | Retinoic Acid (Cat.No. 0695) | 100 nM (25 nM from Day 18) | T3 (Cat.No. 6666) | 3 μM |
| SANT-1 (Cat.No. 1974) | 0.25 μM | Compound E (Cat.No. 6476) | 1 μM | ||||||
| LDN 193189 (Cat.No. 6053) | 200 nM (Day 7-8 only) | RepSox (Cat.No. 3742) | 10 μM | ||||||
| Phorbol 12,13-dibutyrate (Cat.No. 4153) | 10 μM (Day 7-8 only) | T3 (Cat.No. 6666) | 1 μM | ||||||
| Betacellulin | 20 ng/ml | ||||||||
