Table 1 Overview of recent protocols for stepwise differentiation of pancreatic β cells from human PSCs

(Rezania et al., 2014)

Stage 1: DE

GDF8 + MCX-928/CHIR

H1

Planar culture/air–liquid interface

No/

No

 ~ 60% PDX1⁺/NKX6-1⁺

 ~ 42% NKX6.1⁺/CHGA⁺

 ~ 50% insulin⁺/NKX6-1⁺

Stage 2: PGT

FGF7

Stage 3: PP1

FGF7 + RA + TPB + LDN + SANT1

Stage 4: PP2

FGF7 + RA + TPB + LDN + SANT1

Stage 5: EP

RA + SANT1 + ALK5iII + T3 + LDN

Stage 6: IB

ALK5iII + T3 + LDN + GSiXX

Stage 7: β cell

ALK5iII + T3 + N-Cys + R428

(Pagliuca et al., 2014)

Stage 1: DE

Activin A + CHIR

HUES8

Suspension culture

No/

No

 > 55% PDX1⁺/NKX6-1⁺

n.d

33% ± 2% C-peptide⁺/ NKX6-1⁺

Stage 2: PGT

KGF

Stage 3: PP1

KGF + RA + PdBU + LDN + SANT1

Stage 4: PP2

KGF + RA + SANT1

Stage 5: EP

RA + SANT1 + ALK5iII + T3 + XXI + Betacellulin

Stage 6: β cell

ALK5iII + T3

(Russ et al., 2015)

Stage 1: DE

Activin A + WNT3A

MEL1- INS^GFP/W

Low-adherence plates

No/

No

 ~ 90% PDX1⁺/NKX6-1⁺

n.d

17% ± 6%

C-peptide⁺/ NKX6-1⁺

Stage 2: PGT

KGF + TGF-βiIV

Stage 3: PP1

RA

Stage 4: PP2

EGF + KGF

Stage 5: EP

TPB + ALK5iII + Noggin + KGF

Stage 6: β cell

No factors

(Millman et al., 2016)

Stage 1: DE

Activin A + CHIR

ND (non-diabetic) iPSC;

T1D (Type 1 diabetic) iPSC

Suspension culture

No/

No

(ND) 52% -79% PDX1⁺/NKX6-1⁺

(T1D) 59% -88% PDX1⁺/NKX6-1⁺

n.d

(ND)27% ± 2% C-peptide⁺/ NKX6-1⁺

(T1D)24% ± 2% C-peptide⁺/ NKX6-1⁺

Stage 2: PGT

KGF

Stage 3: PP1

KGF + RA + PdBU + LDN + SANT1 + Y27

Stage 4: PP2

KGF + RA + SANT1 + Activin A + Y27

Stage 5: EP

RA + SANT1 + ALK5iII + T3 + XXI + Betacellulin

Stage 6: β cell

ALK5iII + T3

(Ghazizadeh et al., 2017)

Stage 1: DE

Activin A + CHIR

H1;

HUES8; HES3- INS^GFP/W

Planar culture/air–liquid interface

No/

No

n.d

n.d

34% C-peptide⁺

Stage 2: PGT

FGF7

Stage 3: PP1

FGF7 + RA + TPB + LDN + SANT1

Stage 4: PP2

FGF7 + RA + TPB + LDN + SANT1

Stage 5: EP

RA + SANT1 + ALK5iII + T3 + LDN

Stage 6: IB

ALK5iII + T3 + LDN + GSiXX

Stage 7: β cell

ALK5iII + T3 + LDN + H1152

(Velazco-Cruz et al., 2019)

Stage 1: DE

Activin A + CHIR

HUES8

Suspension culture

Stage 6/

No

n.d

96% ± 1% CHGA⁺

52% C-peptide⁺/ NKX6-1⁺

Stage 2: PGT

KGF

Stage 3: PP1

KGF + RA + PdBU + LDN + SANT1 + Y27

Stage 4: PP2

KGF + RA + SANT1 + Activin A + Y27

Stage 5: EP

RA + SANT1 + ALK5iII + T3 + XXI + Betacellulin

Stage 6: β cell

ESFM

(Veres et al., 2019)

Stage 1: DE

Activin A + CHIR

HUES8

Suspension culture

Stage 6/

CD49a⁺ at Stage 6

n.d

 ~ 95% CHGA⁺

80% C-peptide⁺/ NKX6-1⁺

Stage 2: PGT

KGF

Stage 3: PP1

KGF + RA + PdBU + LDN + SANT1 + Y27

Stage 4: PP2

KGF + RA + SANT1 + Activin A + Y27

Stage 5: EP

RA + SANT1 + ALK5iII + T3 + XXI + Betacellulin

Stage 6: β cell

No factors

(Rosado-Olivieri et al., 2019)

Stage 1: DE

Activin A + CHIR

HUES8

Suspension culture

No/

No

 ~ 43.6% PDX1⁺/NKX6-1⁺

12.1 ± 2% NGN3⁺

38.6 ± 3.9% C-peptide⁺/ NKX6-1⁺

Stage 2: PGT

KGF

Stage 3: PP1

KGF + RA + PdBU + LDN + SANT1

Stage 4: PP2

KGF + RA + SANT1 + Activin A + Y27

Stage 5: EP

RA + SANT1 + ALK5iII + T3 + XXI + Betacellulin + verteporfin

Stage 6: β cell

verteporfin

(Nair et al., 2019)

Stage 1: DE

Activin A + WNT3A

MEL1- INS^GFP/W

Suspension culture

Stage 6/

INS: GFP; at Stage 6

 > 70% PDX1⁺/NKX6-1⁺

99% CHGA⁺

85% C-peptide⁺/ NKX6-1⁺

Stage 2: PGT

KGF + TGF-βiIV

Stage 3: PP1

TTNPB

Stage 4: PP2

TTNPB + EGF + KGF

Stage 5: EP

ALK5iII + T3 + LDN + XXI

Stage 6: β cell

ALK5iII + T3

(Mahaddalkar et al., 2020)

Stage 1: DE

Activin A + WNT3A

H1; H9;

HUES8;

MEL1-NKX6.1^GFP

Suspension culture

Stage 3/

CD177⁺ at Stage 1

 ~ 60% PDX1⁺/NKX6-1⁺

n.d

 ~ 62% insulin⁺/NKX6-1⁺

Stage 2: PGT

FGF7 + IWP2

Stage 3: PP1

FGF7 + RA + TPB + LDN + SANT1

Stage 4: PP2

FGF7 + RA + TPB + LDN + SANT1

Stage 5: EP

RA + SANT1 + ALK5iII + T3 + LDN

Stage 6: β cell

ALK5iII + T3 + LDN + XXI

(Hogrebe et al., 2020)

Stage 1: DE

Activin A + CHIR

HUES8

Planar culture

Stage 6/

No

n.d

 ~ 80% CHGA⁺;

 ~ 54% NKX6-1⁺/CHGA⁺

 ~ 40% C-peptide⁺/ NKX6-1⁺

Stage 2: PGT

KGF

Stage 3: PP1

KGF + RA + TPPB + LDN + SANT1

Stage 4: PP2

KGF + RA + TPPB + LDN + SANT1

Stage 5: EP

RA + SANT1 + ALK5iII + T3 + XXI + Betacellulin + Latrunculin A

Stage 6: β cell

ESFM

(Yoshihara et al., 2020)

Stage 1: DE

Activin A + CHIR

HUES8

Suspension culture

No/

No

n.d

n.d

50% ~ 60% insulin⁺/ NKX6-1⁺

Stage 2: PGT

FGF7

Stage 3: PP1

FGF7 + RA + TPB + LDN + SANT1 + ALK5iII

Stage 4: PP2

FGF7 + RA + SANT1 + LDN + ALK5iII

Stage 5: EP

SANT1 + ALK5iII + T3 + LDN + GSiXX

Stage 6: β cell

ALK5iII + T3 + N-Cys + R428 + rhWNT4

(Liu et al., 2021)

Stage 1: DE

Activin A (115–111-100 ng/ml) + CHIR

H1

Planar culture/air–liquid interface

Stage 5/

No

81 ± 4% PDX1⁺/NKX6-1⁺

n.d

60% ~ 82% insulin⁺/ NKX6-1⁺

Stage 2: PGT

KGF + Dorsomorphin

Stage 3: PP1

KGF + RA + Noggin + SANT1

Stage 4: PP2

EGF + Nicotinamide + Noggin

Stage 5: PP-10C

LDN + T3 + SANT1 + Repsox + RA + ZnSO₄ + TPB + EGF + Nicotinamide + GABA

Stage 6: EP

FSK + LDN + TBP + Repsox + KGF + SANT1 + RA + T3

Stage 7: IB

LDN + T3 + Repsox + ZnSO4 + GSiXX + RA + HGF + IGFI + PD

Stage 8: β cell

BTC + ISX-9 + G-1 + Deza + ZM447439 + H1152 + CI-1033