test

1
Supplementary Figure 1
2
A
B
SHEF5
a
b
Activin A (100 ng/ml), FGF2 (10 ng/ml)
a
b
0 mM
1 mM
c
d
c
d
5 mM
10 mM
e
f
e
f
20 mM
50 mM
ES
0 1 5 10 20
ES
0 1 5 10 20
U0126 Activin A (100ng/ml) FGF2 (10 ng/ml)
U0126 Activin A (100ng/ml) FGF2 (10 ng/ml)
U0126
0.5 mm
C
DMSO
U0126
D
DMSO
U0126
a
b
a
b
OCT4
SHEF5
SIX3
0.5 mm
Supplementary Figure 2
3
A
B
SHEF5
a
b
Activin A (100 ng/ml), FGF2 (10 ng/ml)
a
b
0 mM
1 mM
c
d
c
d
5 mM
10 mM
e
f
e
f
20 mM
50 mM
ES
0 1 5 10 20
ES
0 1 5 10 20
U0126 Activin A (100ng/ml) FGF2 (10 ng/ml)
U0126 Activin A (100ng/ml) FGF2 (10 ng/ml)
U0126
0.5 mm
C
DMSO
U0126
D
DMSO
U0126
a
b
a
b
OCT4
SHEF5
SIX3
0.5 mm
Supplementary Figure 2
4
BMP4
hESF9A
BMP4 FGF2 U0126
BMP4 FGF2
Supplementary Figure 3
5
BMP4
hESF9A
BMP4 FGF2 U0126
BMP4 FGF2
Supplementary Figure 3
6
Culture
U0126 - -
- - U0126
B
SHEF6
A
Seeding
-
None
Number of cells/cm2
Y27632
Y27632
Culture
U0126 - -
- - U0126
Seeding
U0126 - -
- - U0126
U0126 - -
Culture
C
D
Seeding
Day 5-10
Number of AP positive colonies/well
None
-
Y27632
Y27632
U0126 - -
- - U0126
U0126 - -
Culture
Seeding
Day 5-10
Supplementary Figure 4
7
Culture
U0126 - -
- - U0126
B
SHEF6
A
Seeding
-
None
Number of cells/cm2
Y27632
Y27632
Culture
U0126 - -
- - U0126
Seeding
U0126 - -
- - U0126
U0126 - -
Culture
C
D
Seeding
Day 5-10
Number of AP positive colonies/well
None
-
Y27632
Y27632
U0126 - -
- - U0126
U0126 - -
Culture
Seeding
Day 5-10
Supplementary Figure 4
8
A
SHEF4 passage 10
SHEF6 passage 10
a
b
HUES1 passage 11
c
BF
TUJ1
OCT4
B
a
b
c
Neural Differentiation
BF
FOXA2
OCT4
d
e
f
Mesendodermal Differentiation
BF
CDX2
OCT4
g
h
i
Extraembryonic Differentiation
0.5 mm
0.1 mm
Supplementary Figure 5
9
A
SHEF4 passage 10
SHEF6 passage 10
a
b
HUES1 passage 11
c
BF
TUJ1
OCT4
B
a
b
c
Neural Differentiation
BF
FOXA2
OCT4
d
e
f
Mesendodermal Differentiation
BF
CDX2
OCT4
g
h
i
Extraembryonic Differentiation
0.5 mm
0.1 mm
Supplementary Figure 5
10
A
SHEF4 passage 10
SHEF6 passage 10
a
b
HUES1 passage 11
c
BF
TUJ1
OCT4
B
a
b
c
Neural Differentiation
BF
FOXA2
OCT4
d
e
f
Mesendodermal Differentiation
BF
CDX2
OCT4
g
h
i
Extraembryonic Differentiation
0.5 mm
0.1 mm
Supplementary Figure 5
11
A
SHEF4 passage 10
SHEF6 passage 10
a
b
HUES1 passage 11
c
BF
TUJ1
OCT4
B
a
b
c
Neural Differentiation
BF
FOXA2
OCT4
d
e
f
Mesendodermal Differentiation
BF
CDX2
OCT4
g
h
i
Extraembryonic Differentiation
0.5 mm
0.1 mm
Supplementary Figure 5
12
A
SHEF4 passage 10
SHEF6 passage 10
a
b
HUES1 passage 11
c
BF
TUJ1
OCT4
B
a
b
c
Neural Differentiation
BF
FOXA2
OCT4
d
e
f
Mesendodermal Differentiation
BF
CDX2
OCT4
g
h
i
Extraembryonic Differentiation
0.5 mm
0.1 mm
Supplementary Figure 5
13
A
SHEF4 passage 10
SHEF6 passage 10
a
b
HUES1 passage 11
c
BF
TUJ1
OCT4
B
a
b
c
Neural Differentiation
BF
FOXA2
OCT4
d
e
f
Mesendodermal Differentiation
BF
CDX2
OCT4
g
h
i
Extraembryonic Differentiation
0.5 mm
0.1 mm
Supplementary Figure 5
14
A
SHEF4 passage 10
SHEF6 passage 10
a
b
HUES1 passage 11
c
BF
TUJ1
OCT4
B
a
b
c
Neural Differentiation
BF
FOXA2
OCT4
d
e
f
Mesendodermal Differentiation
BF
CDX2
OCT4
g
h
i
Extraembryonic Differentiation
0.5 mm
0.1 mm
Supplementary Figure 5
15
A
SHEF4 passage 10
SHEF6 passage 10
a
b
HUES1 passage 11
c
BF
TUJ1
OCT4
B
a
b
c
Neural Differentiation
BF
FOXA2
OCT4
d
e
f
Mesendodermal Differentiation
BF
CDX2
OCT4
g
h
i
Extraembryonic Differentiation
0.5 mm
0.1 mm
Supplementary Figure 5
16
A
SHEF4 passage 10
SHEF6 passage 10
a
b
HUES1 passage 11
c
BF
TUJ1
OCT4
B
a
b
c
Neural Differentiation
BF
FOXA2
OCT4
d
e
f
Mesendodermal Differentiation
BF
CDX2
OCT4
g
h
i
Extraembryonic Differentiation
0.5 mm
0.1 mm
Supplementary Figure 5
17
B
SHEF4
SHEF6
A
b
a
PD0325901 0.3 mM)
DMSO
P-ERK1/2
PD0325901 (0.3 uM)
Total ERK1/2
0.5 mm
C
OCT4
NANOG
SOX2
a
b
c
a
SSEA1
SHEF4 PD0325901
CD9
SSEA4
d
e
f
0.1 mm
TUJ1
FORSE1
BRACHYURY
D
a
b
c
SHEF4 Differentiation
FOXA2
SOX17
CDX2
d
e
f
0.1 mm
Supplementary Figure 6
18
B
SHEF4
SHEF6
A
b
a
PD0325901 0.3 mM)
DMSO
P-ERK1/2
PD0325901 (0.3 uM)
Total ERK1/2
0.5 mm
C
OCT4
NANOG
SOX2
a
b
c
a
SSEA1
SHEF4 PD0325901
CD9
SSEA4
d
e
f
0.1 mm
TUJ1
FORSE1
BRACHYURY
D
a
b
c
SHEF4 Differentiation
FOXA2
SOX17
CDX2
d
e
f
0.1 mm
Supplementary Figure 6
19
B
SHEF4
SHEF6
A
b
a
PD0325901 0.3 mM)
DMSO
P-ERK1/2
PD0325901 (0.3 uM)
Total ERK1/2
0.5 mm
C
OCT4
NANOG
SOX2
a
b
c
a
SSEA1
SHEF4 PD0325901
CD9
SSEA4
d
e
f
0.1 mm
TUJ1
FORSE1
BRACHYURY
D
a
b
c
SHEF4 Differentiation
FOXA2
SOX17
CDX2
d
e
f
0.1 mm
Supplementary Figure 6
20
B
SHEF4
SHEF6
A
b
a
PD0325901 0.3 mM)
DMSO
P-ERK1/2
PD0325901 (0.3 uM)
Total ERK1/2
0.5 mm
C
OCT4
NANOG
SOX2
a
b
c
a
SSEA1
SHEF4 PD0325901
CD9
SSEA4
d
e
f
0.1 mm
TUJ1
FORSE1
BRACHYURY
D
a
b
c
SHEF4 Differentiation
FOXA2
SOX17
CDX2
d
e
f
0.1 mm
Supplementary Figure 6