Table 2 Seven-stage differentiation protocol

S0D0

Pluripotent stem cell

mTeSR^TM1

10 µM Y27632

S0D1

mTeSR^TM1

S1D1–D3

Definitive endoderm

MCDB131

2 mM GlutaMAX

100 ng/ml GDF8

1 µM MCX (S1D1 only)

0.5% FAF-BSA

0.1 µM MCX (S1D2 only)

10 mM Glucose (Final concentration)

2.7 g/L bicarbonate (Final concentration)

1:5000 ITS-X (3D differentiation only)

S2D1–D3

Primitive gut tube

MCDB131

2 mM GlutaMAX

50 ng/ml FGF7

1% FAF-BSA

10 mM Glucose (Final concentration)

2.7 g/L bicarbonate (Final concentration)

0.25 mM Vitamin C

S3D1–D2

Posterior foregut

BLAR

2 mM GlutaMAX

25 ng/ml FGF7

1 µM Retinoic Acid

2% FAF-BSA

0.25 µM SANT-1

10 mM Glucose (Final concentration)

300 nM TPPB

3.6 g/L bicarbonate (Final concentration)

250 nM LDN

1:200 ITS-X

0.25 mM Vitamin C

S4D1–D3

Pancreatic progenitor

BLAR

2 mM GlutaMAX

2 ng/ml FGF7

0.05 µM Retinoic Acid

2% FAF-BSA

0.25 µM SANT-1

10 mM Glucose (Final concentration)

200 nM TPPB

3.6 g/L bicarbonate (Final concentration)

250 nM LDN

1:200 ITS-X

0.25 mM Vitamin C

S4D4

Aggrewell aggregation

Pancreatic progenitor

BLAR

2 mM GlutaMAX

2 ng/ml FGF7

0.05 µM Retinoic Acid

2% FAF-BSA

0.25 µM SANT-1

10 mM Glucose (Final concentration)

200 nM TPPB

3.6 g/L bicarbonate (Final concentration)

250 nM LDN

1:200 ITS-X

1:50 Laminin 521 (100 µg/mL stock)

0.25 mM Vitamin C

10 µM Y27632

S5D1–D3

PBS-Mini: 60 rpm

Endocrine progenitor

BLAR

2 mM GlutaMAX

50 nM Retinoic Acid

2% FAF-BSA

0.25 µM SANT-1

20 mM Glucose (Final concentration)

10 µM ALK5i

2.7 g/L bicarbonate (Final concentration)

100 nM LDN

1:200 ITS-X

1 µM T3

10 µM ZnSO4

10 µg/ml heparin

10 µM Y27632 (S5D1 only)

8 U/mL DNAse (S5D1 only)

S6D1-D7

PBS-Mini: 60 rpm

Immature endocrine cells

BLAR

2 mM GlutaMAX

10 µM ALK5i

2% FAF-BSA

100 nM LDN

20 mM Glucose (Final concentration)

1 µM T3

2.7 g/L bicarbonate (Final concentration)

100 nM Gamma secretase inhibitor

1:200 ITS-X

10 µg/mL heparin

10 µM ZnSO4

S7 D1–D8+ 

PBS-Mini: 60 rpm

Maturing endocrine islet-like cells

BLAR

2 mM GlutaMAX

10 nM T3

2% FAF-BSA

1 mM N-Acetyl cysteine

20 mM Glucose (Final concentration)

0.5 µM ZM 447

1:200 ITS-X

10 µg/mL heparin

1:200 RPMI Vitamin supplement

5 µM AZT (S7D1-D4 only)

1:200 MEM Non-essential amino acid

1 µM DEZA (S7D1-D4 only)

1:2000 Lipid concentrate

1:200 Sodium pyruvate

1:2000 Trace elements A

1:2000 Trace elements B

2.7 g/L bicarbonate (Final concentration)