Document S1. Figures S1–S6 and Tables S1 and S2

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Cell Stem Cell, Volume 22
Supplemental Information
YAP/TAZ-Dependent Reprogramming
of Colonic Epithelium Links ECM Remodeling
to Tissue Regeneration
Shiro Yui, Luca Azzolin, Martti Maimets, Marianne Terndrup Pedersen, Robert P.
Fordham, Stine L. Hansen, Hjalte L. Larsen, Jordi Guiu, Mariana R.P. Alves, Carsten F.
Rundsten, Jens V. Johansen, Yuan Li, Chris D. Madsen, Tetsuya Nakamura, Mamoru
Watanabe, Ole H. Nielsen, Pawel J. Schweiger, Stefano Piccolo, and Kim B. Jensen

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Figure S1: Phases of tissue regeneration following DSS induced colitis
A) Time course of regeneration following DSS induced colitis with representative
images of H&E staining of each phase. Scale bars, 100 µm.
B) Time course analysis for expression of Reg3b and Sca1 (both green) during
ulceration and regeneration. Tissue is counter stained with DAPI (blue). Scale bar
100µm.
C-D) Ki67 (C) and MUC2 (D) detected during homeostasis and the repair phase.
Tissue is counterstained with E-cadherin (red) and DAPI (blue). Scale bar 50µm.
E) Heat map analysis of differentially expressed probe sets (fold change > 2.0, FDR <
0.05) from comparisons of epithelial cells isolated from homeostatic tissue and
Sca1low as well as Sca1high cells isolated from mice that have been exposed to DSS.
F) GO term enrichment analysis illustrating biological processes and pathways that
are significantly (p<0.05) enriched (PANTHER GO slim database) in the Sca1high
populations when compared to homeostatic epithelium.

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Figure S2: Expression of fetal markers in intestinal epithelium and organoids
A) Time course analysis for expression of the two fetal markers Anxa1 and
Tacstd2/Trop2 (both green) during the course of treatment and recovery from
experimental colitis. Tissue is counter stained with DAPI (blue). Scale bar 100µm.
B-C) Trop2 (green) expression in organoids derived from the adult small intestine (B)
or in enterospheres derived from the fetal small intestine (C). Tissue is counter stained
with E-cadherin (red) and DAPI (blue). Scale bar 100µm.

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Figure S3: Characterization of the repairing epithelium
A) qPCR analysis for Col1a2 and Col1a1 in sorted homeostatic epithelial cells and
Sca1high epithelial cells. Data is presented as mean ± SEM (n=3; Col1a2: p=0.059;
Col1a1: p=0.019 based on two-sided Student’s t-test).
B) Quantification of confocal immunofluorescence images for β1 integrin, FAK, pSrc
and Phalloidin in homeostatic and repair phase. The Y-axis represents the average
gray scale value determined at the basolateral membrane for β1 integrin, FAK and
pSrc, and max intensity of gray scale value determined at apical side for Phalloidin.
Each dot represents single measurements. Signals were obtained for three animals per
group and the average for the biological triplicates were compared (p=0.033, 0.015,
0.049 and 0.019, respectively based on two-sided Student’s t-test).
C) Time course analysis for expression of YAP (green) during the course of treatment
and recovery from experimental colitis. Tissue is counterstained with DAPI (blue)
and E-cadherin (red). Scale bar 100µm.
D-F) Representative images from H&E stained sections of the distal part of the colon
at day 12 following the administration of DSS from animals treated daily with either
vehicles control (Ctrl; D) FAK inhibitor (E) or Src inhibitor (F) from day 8. Scale bar
250µm. Demarcated area is shown at higher magnification. Scale bar 100µm.
G) Length of denuded regions in the colon in the different animal groups. Each dot
represent independent animals, and data is presented as the mean ± SEM (Ctrl vs FAK
inhibitor p=0.016; Ctrl vs Src inhibitor p=0.037 based on two-sided Student’s t-test).
H) Expression of Ki67 (green) in samples from animals treated vehicles (Ctrl), FAK
or Src inhibitor. Scale bar 50µm.

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Figure S4: Recapitulating features of tissue repair in vitro
A) Representative images and quantification of organoid/spheroid seeding efficiency
using different culture matrices and conditions. Cells were seeded in either collagen
type 1, Matrigel or a 1:1 mix of collagen type 1/Matrigel cultured in either
EGF/Noggin/Rspondin (ENR) or ENR supplemented with Wnt3a (W), PGE2 or
Wnt3a and the Cox inhibitor Indomethacin (W – Indo). Bars represent mean ± SEM
(n=3 for all conditions). Scale bar 100µm.
B) Quantification of Phalloidin localization in both Matrigel organoids and Collagen
cultures. The Y-axis represents the max intensity of gray scale value determined at the
apical membrane in each independent sample. Each dot represents one measurement
(n=6 for Matrigel, n=8 for Collagen, p=7.7x10-6 based on two-sided Student’s t-test).
C) GSEA showing enrichment of an Lgr5 intestinal stem cell gene signature in
Matrigel relative to collagen cultures.
D) Phase contrast image of fetal colonic sphere derived from E16.5 murine colon.
Scale bar, 100 µm. qPCR analysis for cultured materials when compared to freshly
harvested colonic crypts from adult animals. Data is presented as mean ± SEM (n=3).
E) Seeding efficiency of collagen cultures treated with C3 toxin (0.2, 1 and 3µg/mL),
Mevastatin (0.3, 1 and 3µM), Cytochalasin D (0.6, 2 and 6µM), FAK-inhibitor
(PF573228; 3, 5 and 10µM) and Src inhibitor (Dasatinib; 3, 5 and 10µM), when
compared to samples treated with DMSO. The bars indicate the average ± SEM (n=3)
ND: no growth detected (C3-toxin p<0.0001; Mevastatin: 1µM p=0.02, 10µM
p<0.0001; Cyto D p<0.0001; FAKinhib and Srcinhib p<0.0001 based on an ordinary one-
way ANOVA test with Dunnett’s multiple comparisons test with a single pooled
variance).

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Figure S5: YAP/TAZ are required for in vitro growth and establishing the
repairing epithelium expressing fetal markers
A) Seeding efficiency of control and Vil-CreERT2;Yapfl/fl;Tazfl/fl (YAP/TAZ cDKO)
spheroids cultured in collagen type 1, when exposed to 4-hydroxy tamoxifen (4OHT)
following seeding. The bars indicate the average ± SEM (n=3; p=2.9x10-6 based on a
based on two-sided Student’s t-test).
B) Western Blot for YAP and TAZ from isolated small intestinal crypts 15 days after
administration of tamoxifen.
c) H&E images of colon from control (Yapfl/fl;Tazfl/fl) and cDKO (Vil-
CreERT2;Yapfl/fl;Tazfl/fl) animals 15 days after administration of tamoxifen. Scale bar
50µm.
d) Expression of Ki67, YAP and Sca1 (green) in different regions of the colon in
YAP/TAZ cDKO animals at day 13 following administration of DSS reveals two
distinct phenotypes either repairing epithelium or aberrant epithelial cysts. Scale bar
50µm.

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Figure S6: Tissue regeneration is recapitulated using transplantation
experiments as a reversible process
A) Whole mount imaging of engrafted patches in the distal colon of animals
transplanted with either wt (red) or Vil-CreERT2;Yapfl/fl;Tazfl/fl (green) small intestinal
organoids at day 8 and day 11 following initiation of DSS administration. Top panel
were analyzed at day 12 (before 4-hydroxy tamoxifen treatment) and bottom panel at
day 16 (3 days after 4-hydroxy tamoxifen treatment).
B-C) Detection of Mucin2 (MUC2, green, left), alkaline phosphatase (ALP, purple,
middle) and carbonic anhydrase II (CAII, green, right) in transplant derived from
small intestinal epithelial cells (red) cultured in Matrigel (B) or collagen type I (C).
The demarcated line in serial sections indicates engrafted regions. Scale bars, 50 µm.
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Supplemental Table 1. Related to Figure 1
PANTHER GO-Slim Biological Process
Total
#
Observed
#
Expected
Fold
Enrich
P value
Lipid metabolic process
559
57
19
3.0
2.8x10-10
Cellular amino acid metabolic process
224
30
8
3.9
1.8x10-7
Generation of precursor metabolites and
energy
227
28
8
3.6
3.6x10-6
Fatty acid beta-oxidation
28
10
1
10.3
1.9x10-5
Fatty acid metabolic process
195
24
7
3.6
4.0x10-5
Respiratory electron transport chain
153
21
5
4.0
4.1x10-5
Metabolic process
6472
282
224
1.3
8.3x10-4
Cellular amino acid catabolic process
54
11
2
5.9
1.0x10-3
Coenzyme metabolic process
94
14
3
4.3
1.9x10-3
Carbohydrate metabolic process
428
34
15
2.3
2.5x10-3
Primary metabolic process
5565
238
192
1.2
2.8x10-2
Homeostatic process
240
21
8
2.5
3.3x10-2
PANTHER Pathways
Total
#
Observed
#
Expected
Fold
Enrich
P value
Pyrimidine Metabolism
10
6
0
17.4
2.7x10-4
GO terms enriched among genes up-regulated by epithelial cell isolated from
homeostatic tissue (non-DSS), when compared to Sca1high epithelial cells isolated
during tissue repair. The table shows the number of genes in each GO category (Total
#), the number of genes up-regulated in the homeostatic sample (Observed #), the
relative enrichment, and the associated P-values using Bonferroni correction for
multiple testing.

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Supplemental Table 2. Related to Figure 1
PANTHER GO-Slim Biological Process
Total
#
Observed
#
Expected
Fold
Enrich
P value
Cellular component morphogenesis
433
25
8
3.2
1.4x10-4
Developmental process
1892
63
34
1.8
5.5x10-4
Cell death
387
20
7
2.9
9.0x10-3
Cytokine-mediated signaling pathway
122
10
2
4.5
2.4x10-2
Locomotion
129
10
2
4.3
3.7x10-2
Cell adhesion
369
18
7
2.7
4.4x10-2
PANTHER Pathways
Total
#
Observed
#
Expected
Fold
Enrich
P value
Integrin signalling pathway
191
16
3
4.6
1.1x10-4
Inflammation mediated by chemokine and
cytokine signaling pathway
262
18
5
3.8
3.5x10-4
p53 pathway
87
8
2
5.1
3.6x10-2
CCKR signaling map
163
11
3
3.7
3.8x10-2
GO terms enriched among genes up-regulated by Sca1high epithelial cells isolated
during tissue repair, when compared to epithelial cells from homeostatic conditions.
The table shows the number of genes in each GO category (Total #), the number of
genes up-regulated in Sca1high epithelial cells (Observed #), the relative enrichment,
and the associated P-values using Bonferroni correction for multiple testing.
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