Generation of β cells from hPSCs

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

In Brief

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.

https://resources.tocris.com/images/protocols/pscs-to-beta-cells.jpg

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    
https://resources.tocris.com/images/protocols/pscs-to-beta-cells-timeline.jpg?v=1

Reference

Pagliuca et al. (2014) Generation of functional human pancreatic β cells in vitro. Cell 159 428. PMID: 25303535

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