of 12
Developmental Cell, Volume
59
Supplemental information
Basal delamination during mouse gastrulation
primes pluripotent cells for differentiation
Nanami Sato, Viviane S. Rosa, Aly Makhlouf, Helene Kretzmer, Abhishek Sampath
Kumar, Stefanie Grosswendt, Alexandra L. Mattei, Olivia Courbot, Steffen Wolf, Jerome
Boulanger, Frederic Langevin, Michal Wiacek, Daniel Karpinski, Alberto Elosegui-
Artola, Alexander Meissner, Magdalena Zernicka-Goetz, and Marta N. Shahbazi
Figure S1
A
0
1
2
3
Relative levels
Pou5f1
Nanog
Sox2
EpiLCs EpiSCs FAX FAXN
Fgf5
Otx2
0
5
10
15
20
Relative levels
EpiLCs EpiSCs FAX FAXN
ns
ns
B
Brachyury/DAPI/F-actin
Sox17/Sox1/DAPI/F-actin
E
Formative
N=83
35%
39%
26%
Brachyury
Sox17
Sox1
1.0
0.8
0.6
0.4
0.2
0.0
G
F
Single-layer/lumen (SL)
Multi-layer/lumen (ML)
Disorganized (D)
Differentiated
cells / spheroid
Podxl/aPKC/DAPI/F-actin
Integrin
β
1/DAPI/F-actin
C
D
Figure S1:
In vitro
capture and characterization of 3D Epiblast Stem Cells. (Related to Figure 1)
(A, B) Relative expression levels of
Pou5f1
(Oct4),
Nanog
,
Sox2
(A),
Fgf5
, and
Otx2
(B) in cells cultured in different
conditions. Data are shown as mean ± SEM. n = 6 samples, 3 independent experiments. Kruskal-Wallis test. ns:
non-significant.
(C, D) Immunostaining of 3D EpiSCs. Arrows indicate polarized localization of apical polarity markers. Scale bars:
20 μm.
(E) Immunostaining of cells cultured in 3D Matrigel using formative conditions. Scale bars: 20 μm.
(F) Morphological characterization of spheroids from (E). Data are shown as a pie chart, and the total number of
spheroids analyzed is indicated.
(G) Ratio of differentiated cells in spheroids from (E). Data are shown as mean ± SEM. Each dot represents an
individual spheroid. n = 42 (Brachyury), 41 (Sox17) and 41 (Sox1) spheroids. 3 independent experiments.
Figure S2
B
0
1
2
0
2
4
6
8
10
Days
Cell growth rate
3D
FAXN
2D
FAXN
2D
FA
ns
ns
H
0.0
0.5
1.0
1.5
2.0
2.5
Pou5f1
relative levels
3D F
AXN
2D F
AXN
2D F
A
3D F
AXN
2D F
AXN
2D F
A
Early
Late
ns
0.0
0.5
1.0
1.5
Fgf5
Relative levels
3D F
AXN
2D F
AXN
2D F
A
3D F
AXN
2D F
AXN
2D F
A
Early
Late
*
C
Normal
Abnormal
G
0
20
40
60
80
100
% Cells
3D F
AXN
2D F
AXN
2D F
A
3D F
AXN
2D F
AXN
2D F
A
Early
Late
71
18
70
31
73
35
66
42
77
30
51
44
**
Day 1
Early passage
P6-P8
Late passage
P18-P19
2D FA
2D FAXN
3D FAXN
A
D
3D F
AXN
2D F
AXN
2D F
A
3D F
AXN
2D F
AXN
2D F
A
0.00
0.02
0.04
0.06
0.08
Early
Late
****
****
****
Sox1
0.0
0.2
0.4
0.6
0.8
1.0
3D F
AXN
2D F
AXN
2D F
A
3D F
AXN
2D F
AXN
2D F
A
Early
Late
****
****
Brachyury
0.0
0.2
0.4
0.6
0.8
3D F
AXN
2D F
AXN
2D F
A
3D F
AXN
2D F
AXN
2D F
A
Early
Late
Sox17
****
****
E
F
Differentiated cells /
spheroid
Differentiated cells /
spheroid
Differentiated cells /
spheroid
% spheroids
100
80
60
40
20
0
ESCs
EpiSCs
ns
70
54
12
17
23
25
I
Brachyury
Sox17
Sox1
1.0
0.8
0.6
0.4
0.2
0.0
ns
ns
ns
J
Differentiated
cells / spheroid
ESCs
EpiSCs
Figure S2
Figure S2: Characterization of epiblast-derived 3D EpiSCs. (Related to Figure 1)
(A) Brightfield images of epiblast-derived cells at early (P6-P8) and late (P18-19) passages, cultured in different
conditions. Cells were either embedded in Matrigel (3D) or cultured in monolayer (2D). FAXN: bFgf2, Activin-A,
XAV939 and Noggin, FA: bFgf2 and Activin-A. Scale bars: 100 μm.
(B, C) Relative expression levels of
Pou5f1
(B) and
Fgf5
(C) in cells cultured under different conditions. Data are
shown as mean ± SEM. n = 6 samples per condition, 3 independent experiments. Kruskal-Wallis test. *p = 0.0109,
ns: non-significant.
(D-F) The ratio of differentiated cells in cells from Figure 1I at early and late passages. Data are shown as mean ±
SEM. Each dot represents an individual spheroid. In (D), n = 99, 40, and 41 (early passage) and 109, 41, and 41
(late passage). In (E), and (F) n = 104, 40, and 38 (early passage) and 102, 40, and 40 (late passage). 5 independ-
ent experiments. Kruskal-Wallis test, ****p < 0.0001.
(G) Proliferation rates for cells in (A). Data are shown as mean ± SEM. n = 8 samples per condition and time point.
4 independent experiments. Kruskal-Wallis test, ns = non-significant.
(H) Karyotype analysis based on chromosome spreads in cells from (A), at early and late passages. Cells that did
not have 40 chromosomes were considered abnormal. Data are shown as a contingency bar graph, and the number
of cells analyzed per category is indicated. 4 independent experiments. X
2
test, **p = 0.0039.
(I) Morphological characterization of 3D spheroids derived from ESCs or EpiSCs. Data are shown as a contingency
bar graph, and the number of spheroids per category is indicated. 3 independent experiments. X
2
test. ns: non-sig-
nificant.
(J) Ratio of differentiated cells in spheroids derived from ESCs or EpiSCs. Data are shown as mean ± SEM. Each
dot represents an individual spheroid. For ESCs n = 55 (Brachyury), and 50 (Sox17/Sox1) spheroids. For EpiSCs n
= 48 (Brachyury), and 50 (Sox17/Sox1) spheroids. 3 independent experiments. Mann-Whitney U test. ns: non-sig-
nificant.