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This is intended as a guide only; for full experimental details please read the reference provided.
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.
|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|