B
Unlabeled ESCs
*
Embryoid Bodies
*
10
ESCs
*
5
0
ES:End2 Control 10:1
Ratio
2:1
1:1
GFP-labeled ESCs
with End2 Cells
D
End2
ESC
500000
60
40
20
EBs at day 7
% Bry-GFP+ Cells
C
Day 3
40
*
20
100000
M
ed
nd
ia
iti
10
(
x 10 on
Co um ed
nd )
M
ed
iti
(3 on
ia
0u e
m dM
)
ed
ia
ES
+
En
d2
0
Co
10
x
Co
nd
iti
on
ed
5
0
Days of Differentiation
ES
Number of Cells
GFP-labeled ESCs
Actc1
Hand2
ES ES
+
En
d2
Relative
mRNA levels
15
End2 Cells
Beating foci (%)
A
Fig. S1. Endoderm-like (End2) cells promote the emergence of mesoderm in ES cells through a short-range signal. (A) Relative
expression of cardiac genes Actc1 and Hand2 in embryoid bodies (EBs) formed with increasing ratios of End2 cells relative to mES
cells (ESCs). (B) Percentage of beating foci observed in a mixed culture of EBs formed with (green/red) or without (yellow) End2
cells. (C) Number of mES cells and End2 cells at day 0 and after 5 days of co-culture. (D) Percentage of Bry-GFP+ mesodermal
cells in EBs differentiated with End2 cells, End2-conditioned medium, or conditioned medium concentrated via a 10 or 30 μm filter.
*P<0.05, error bars indicate s.e.m.
Cheng, Supp. Fig. 1
*
c
1
Fn
2
ar
Sp
C
ol
4A
1
1
4A
1A
ol
C
ol
C
on
tr
E
*
0.0
Control KD
Fn1
Control KD
Fn1/GFP
1
*
DAPI
0.5
Fn1 DAPI
0
G
Ig
1
Fn
ti-
An
Fn1
50% End2
30% End2
DAPI
H
Fn1 KD
50 um
F
Day 4
10% End2
Fn1
Fn1 KD
10
DAPI
5
0
K
I
Day 3
Control KD
15
50 um
Fn1 KD
Fn1 DAPI
Ki-67
200 uM
2
J
Day 3
Fn1 KD
1
0
PH3
Control KD
Relative Expression
*
0.2
C
siRNA
*
0% End2
% Positive Cells
0.4
*
Relative Number
of Bry-GFP+ Cells
0.6
ol
Relative mRNA Levels
0.8
D
C
B
% Positive Cells
A
PI
2
1
L
1.E+03
Day 2
Fn1 KD
0
Annexin V
Control KD
Oct 4
Nanog
ntl
90% Epiboly
1.E+02
1.E+01
Control
Cadherin α PKC DAPI
Control KD
1.E-01
Fn1
1.E-02
Fn1 KD
90% Epiboly
G
50 um
Fn1 KD
1.E+00
50 um
1.E-03
MO-fn1/fn1b
1.E-04
1.E-05
Cadherin α PKC DAPI
1.E+03
Day 3
1.E+02
50 um
1.E+01
Control Sample
1.E+00
1.E-01
1.E-02
1.E-03
1.E-04
1.E-05
1.E-06
1.E-06
n>50
50 um
Day 3
Cheng, Supp. Fig. 2
Fig. S2. Fibronectin promotes End2-mediated induction of mesoderm and precardiac mesoderm in vitro and in vivo. (A)
Knockdown efficiency of the indicated siRNAs assayed by qPCR. (B) Percentage of Bry-GFP+ cells in day-4 EBs cultured with End2
cells with control (IgG) or anti-Fn1 antibody. (C) EBs with increasing ratio of End2 cells stained with DAPI and Fn1 antibody. (D)
Adherent culture of mESBry-GFP EBs plated with End2 cells and stained for Fn1 (red) and GFP (green). (E) End2 cells 48 hours after
transfection with scrambled siRNA or siRNA against Fn1, stained for Fn1 (green) and β-catenin (red). (F) End2 cells 48 hours after
transfection with scrambled siRNA or siRNA against Fn1, stained for pan-cadherin (green) and aPKC (red). (G) EBs containing End2
cells transfected with scrambled siRNA or siRNA against Fn1, stained for pan-cadherin (green) and aPKC (red). (H) FACS analysis of
cell proliferation as assayed by Ki-67 or phosphohistone H3 (PH3) staining among Bry-GFP+ cells in EBs aggregated with control or
fibronectin-deficient End2 cells. (I) FACS analysis of cell death as assayed by propidium iodide (PI)+ and annexin V staining among
Bry-GFP+ populations in EBs aggregated with control or fibronectin-deficient End2 cells. (J) Expression of the pluripotent genes Oct4
and Nanog in day-2 EBs with control or Fn1-deficient End2 cells. (K) Scatter plot of ECM mRNA array from EBs co-cultured with
control or Fn1 knockdown End2 cells. (L) Whole-mount in situ hybridization of ntl in control or Fn1/Fn3 knockdown (MO-Fn1/3)
zebrafish embryos at 90% epiboly (9 hpf). *P<0.05, error bars indicate s.e.m.
B
Control KD
Control KD
Fn1 KD
*
8
6
4
*
2
3.0
2.0
1.0
0.0
AFP
Foxf1
Day 10
GATA4
*
*
*
Actc1
Flk1
*
CD34
*
*
CD45 MyoD
*
*
120
90
*
60
30
0
SM22 Osterix Alp1
C
M
VL
0
Fn1 KD
Day 3
ac
Z
10
Day 5
Relative Expression
Relative Expression
12
C
Relative Number of Bry-GFP+ Cells (%)
A
F
E
100
0.4
0.2
Fn1
Fn1
Control
Control
Col4a2
0
on
tr
ol
Control
C
0
Relative Fn1 Expression
0.6
0.5
*
20
*
10
EF
M
E
2
nd
C
ol
tr
on
EF
M
d2
En
Co-culture with Bry-GFP
0.2
-L
ys
-D
Culture
Substrate
in
e
0
(p
[Wnt3a] (ng/ml)
50
0.4
ly
25
0.6
Po
10
0.8
1
0
1.0
Fn
20
*
1.2
)
Bry-GFP+ Cells (%)
*
40
*
1.4
Relative TOPFlash Activity
Day 3
60
0
30
0
C
ES
*
Day 3
I
la Co
st nt
ic ro
su l
rfa
ce
H
*
Cell Type
SiRNA
40
*
10
0
D
*
0.8
K
1.0
*
IL
1.2
1.0
-K
Relative Number of Bry-GFP+ Cells
Relative number of Bry-GFP+ Cells
Day 3
*
G
Bry-GFP+ Cells (%)
D
Fig. S3. Fibronectin augments mesoderm induction through integrin-dependent activation of Wnt/β-catenin signaling.
(A) Expression of various endodermal markers in cells differentiated from Bry-GFP+ cells isolated from EBs with control or Fn1
knockdown (KD). (B) Expression of various mesodermal markers in cells differentiated from Bry-GFP+ cells isolated from EBs with
control or Fn1-deficient End2 cells. (C) Relative number of Bry-GFP+ cells induced by End2 cells in differentiating mESBry-GFP cells
Supp. Fig. 3
with overexpression of the integrin signaling mutant Tacβ1 or control (lacZ). (D) Relative number of Bry-GFP+Cheng,
cells induced
by End2
+
cells transfected with control and/or Fn1/Col4a2 siRNA. (E) Relative number of Bry-GFP cells in End2 co-cultures with mESBry-GFP
cells transfected with control or Ilk siRNA. (F) Relative Fn1 expression in ESCs, MEFs or End2 cells. (G) Percentage Bry-GFP+
cells observed in differentiation with ESCs alone, or co-culture with MEFs or End2 cells. (H) Percentage Bry-GFP+ cells induced by
increasing concentrations of Wnt3a. (I) Relative TOP-flash activity in differentiated ESCs cultured on Fn1 or poly-d-lysine. *P<0.05,
n=3 or greater. Error bars indicate s.e.m.
Movie 1. Spontaneous contraction of cardiomyocytes differentiated from End2-induced precardiac mesoderm.
Table S1. Expression of ECM genes in End2 cells versus ES and ectodermal cells
Symbol
Fn1
Lama3
Col4a1
Col4a2
Timp3
Itgav
Sparc
Cdh2
Sgce
Ctnnb1
Timp2
Col1a1
Cntn1
Thbs3
Vcam1
Adamts5
Mmp2
Ctnna2
Thbs2
Col3a1
Col5a1
Itgam
Tnc
Itgb1
Hapln1
Cdh3
Itgb3
Mmp13
Cd44
Pecam1
Thbs1
Postn
Adamts1
Spp1
Ncam1
Lamb2
Itgb4
Mmp14
Col6a1
Itgal
Selp
Mmp8
Mmp9
Lamb3
Sele
Log2 (FC)
2.36832369
–3.69437791
5.39362669
2.25451884
4.78813179
1.81114939
2.88647209
2.00801929
2.51501439
–1.62105666
4.80258464
6.11512519
–1.52143081
–1.55693326
6.87848319
2.66148604
2.41154834
2.24932944
–3.59531056
6.66594809
1.55671699
–2.18766771
3.28507064
0.90042484
–2.41886931
1.52066139
4.04664894
3.58515104
4.93159714
–5.15819076
4.53810729
2.96244189
2.73405889
–5.03487621
0.81139574
0.73336334
–1.00554206
1.39571854
7.11230019
–2.57583136
1.47631989
2.99186449
–3.36491961
–2.97790276
1.88041984
P-value
0.000482225
0.004787302
0.006466226
0.009827216
0.010284841
0.011167394
0.013530666
0.013980868
0.017630714
0.01829935
0.020218724
0.020504982
0.021217884
0.024294494
0.028849818
0.029912537
0.03266755
0.036734739
0.041485655
0.04231551
0.047171288
0.052602835
0.05851667
0.058915566
0.063244531
0.064073183
0.071629966
0.072185608
0.081185984
0.082377403
0.084706461
0.090221262
0.099148625
0.113469299
0.121124338
0.135373586
0.150664561
0.155233801
0.165616311
0.167756088
0.173485089
0.182355545
0.187441341
0.191517903
0.193782813
Expressed around
egg-cylinder and
gastrulation stage
+
+
+
–
+
–
–
–
+
Adamts2
Col4a3
Col2a1
Cdh1
Spock1
Mmp15
Icam1
Adamts8
Mmp11
Ctgf
Syt1
Emilin1
Mmp1a
Vcan
Itga3
Ncam2
Ecm1
Lama2
Mmp7
Itga2
Sell
Tgfbi
Timp1
Lama1
Hc
Mmp12
Ctnna1
Mmp3
Itgax
Itga5
Lamc1
Itga4
Itgb2
Cdh4
Fbln1
Itgae
Mmp10
Entpd1
Vtn
1.15955854
1.39159194
–4.03470811
–4.95819166
–3.01631216
–1.47104581
–0.11250206
–1.85968281
–0.56176436
0.28752319
1.51337214
–2.35108266
1.29737074
1.09386404
1.11160969
–1.48765866
0.62412304
0.28008739
1.40926059
0.90122809
0.80391174
–2.19130066
–1.43928676
–4.47066411
0.09429884
0.27556214
–0.05077756
0.61166869
0.04080364
0.24840284
0.01268754
–0.62558746
–0.84664986
–4.15708061
–0.77668296
–0.10477356
0.91885374
–0.15355406
0.00488484
0.203361496
0.209170667
0.209645434
0.223356991
0.240265978
0.240576219
0.243198795
0.25723376
0.259450606
0.269014355
0.270920915
0.292417197
0.300237402
0.319549642
0.321351939
0.343283912
0.360233824
0.367725047
0.371188088
0.376339274
0.384569169
0.394459931
0.40153966
0.408702404
0.426772266
0.435886078
0.53954357
0.543160669
0.549823798
0.561258646
0.605250367
0.638477331
0.652115056
0.744720352
0.748958051
0.773917062
0.794369608
0.831367709
0.89528792
Cut-offs were set at >4-fold increase, P>0.025.