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Brightfield
FACSDiva Version 6.1.3
AHF-Mef2c-DsRed
Nkx2.5-eGFP
Merge
D6 ESC-EB
A
B
FACS isolation ESC-derived FHF and SHF cell populations
SHF
Mef2c
Mef2c
SHF
Nkx2.5eGFP
FHF
SHFMef2cDsRed
Ccccccccccc
Figure S1. Isolation of ESC-derived early FHF and SHF marked cells. (A) Brightfield and immunofluorescence images of Embryoid
body (EB) at day 6 (D6) of cardiac differentiation. The AHF-Mef2c-DsRed and Nkx2.5-eGFP reporter system mark fractions of FHF
and SHF ESC-derived cells. (B) Double labeled Nkx2.5-eGFP/AHF-Mef2c-DsRed ESC lines were differentiated by hanging droplet
formation and were dissociated into single cell suspension on EB day 6. FACS plots show isolation strategy for the 3 populations used in
this work; Nkx2.5-eGFP+/AHF-Mef2c-DsRed- (FHF), Nkx2.5-eGFP+/AHF-Mef2c-DsRed+ (SHF) and Nkx2.5-eGFP-/AHF-Mef2cDsRed- (NEG) cells.
B
A
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DNA
DNA / cTnT / Ki67
Channel
Exposure
Exposure
Printed on: Thu Feb 2, 2012 07:41:33 EST
(unstained) (stained)
cTnT
(488nm)
197.534ms
197.534ms
Ki67 (594nm)
733.441ms
733.441ms
DNA (350nm)
156.357ms
156.357ms
Figure S2: Immunofluorescence control of ESC-derived SHF ventricular CMs
(A) Images of unstained (left panel) and stained (right panel) ventricular myocytes. (B) Table indicating the exposure times for the
immunofluorescence images shown in A. No significant interference of the cardiac reporter system with immunofluorescence was
observed.
FHF +DMSO
A
!-SA/ Ki67 / DNA
MF20 / Ki67 / DNA
cTnT / MLC2v / Ki67 / DNA
C
Overlap of MLC2v+
and cTnT+ cells
B
("
FHF +BIO
!#'"
!#&"
!#%"
!#$"
!-SA/ Ki67 / DNA
MF20 / Ki67 / DNA
cTnT / MLC2v / Ki67 / DNA
!-SA/ Ki67 / DNA
MF20 / Ki67 / DNA
cTnT / MLC2v / Ki67 / DNA
!"
)*+,"
-.,"
F
E
Overlap of MLC2v+
and cTnT+ cells
SHF +DMSO
D
NS
("
SHF +BIO
!#'"
NS
!#&"
!#%"
!#$"
!-SA/ Ki67 / DNA
MF20 / Ki67 / DNA
cTnT / MLC2v / Ki67 / DNA
!"
)*+,"
-.,"
Figure S3: GSK-3 inhibition promotes expansion of early ventricular myocytes
Immunofluorescence images of (A) differentiating or (B) expanding early FHF marked ventricular cardiomyocytes (vCMs) stained
for Troponin T (cTnT), Myosin Light Chain 2v (MLC2v), Ki67 and DAPI. (C) Quantification of cTnT+/MLC2v+ cells represented
over total cTnT+ cell number. Idem for (D) differentiating and (E) expanding early SHF vCMs, and (F) quantification. (n = 3). NS=not
significant.. Errorbars indicate standard deviation. Scale bars represent 50µm.
B
SHF1 D6
FHF2 D6
SHF2 D6
FHF3 D6
SHF3 D6
FHF1 D6+6 BIO
SHF1 D6+6 BIO
FHF2 D6+6 BIO
SHF2 D6+6 BIO
FHF3 D6+6 BIO
SHF3 D6+6 BIO
FHF1 D6+6 DMSO
SHF1 D6+6 DMSO
FHF2 D6+6 DMSO
SHF2 D6+6 DMSO
FHF3 D6+6 DMSO
SHF3 D6+6 DMSO
FHF1 D6+6 IWR
SHF1 D6+6 IWR
FHF2 D6+6 IWR
SHF2 D6+6 IWR
FHF3 D6+6 IWR
SHF3 D6+6 IWR
SHF3 D6+6 IWR
SHF2 D6+6 IWR
SHF1 D6+6 IWR
SHF3 D6+6 DMSO
SHF2 D6+6 DMSO
SHF3 D6+6 BIO
SHF1 D6+6 DMSO
SHF2 D6+6 BIO
SHF1 D6+6 BIO
SHF1 D6
FHF1 D6
SHF3 D6
SHF ESC-vCMs
FHF3 D6+6 IWR
FHF2 D6+6 IWR
FHF1 D6+6 IWR
FHF3 D6+6 DMSO
FHF2 D6+6 DMSO
FHF1 D6+6 DMSO
FHF3 D6+6 BIO
FHF2 D6+6 BIO
FHF1 D6+6 BIO
FHF3 D6
FHF2 D6
FHF1 D6
FHF ESC-vCMs
SHF2 D6
A
D
0.1
300
600 250
500 200
150
Tnnt2 expression Tnnt2 expression
C
y = -50.46x + 202.72
R² = 0.60926
100
50
0
1
-50
Axin2 expression
10
0.01 400 300 200 y = -­‐97.886x + 404.7 R² = 0.70943 100 0 0.1 1 -­‐100 Axin2 expression 10 Figure S4: Unsupervised consensus clustering of ESC-derived FHF and SHF
Unsupervised consensus clustering of 3 experiments containing (A) FHF- and (B) SHF-marked cells at day 6 (D6), expanded in
6-bromoindirubin-3’-oxime (BIO) (D6+5 BIO), differentiated in carrier control media (D6+5 DMSO) and enhanced differentiation
with Wnt inhibition (D6+5 IWR). This reveals that FHF and SHF marked cells cluster individually at D6, D6+5 BIO and D6+5 DMSO
together with IWR. This clustering validates the experimental reproducibility of isolation, expansion and differentiation of FHF and
SHF-marked cells. FHF1 represents a pooled experiment. Linear regression plotted for gene expression values of Axin2 (x-axis) versus
Tnnt2 (y-axis) for (C) FHF and (D) SHF cells treated with BIO, DMSO or IWR.
2
0
FHF ESC-vCM SHF ESC-vCM
p<0.05
8
6
p<0.05
4
2
0
FHF ESC-vCM SHF ESC-vCM
C
p<0.01
15
10
p<0.01
5
0
140
120
100
80
60
40
20
0
LV P1
RV P1
p<0.01
p<0.01
LV P1
RV P1
15
D
p<0.05
10
5
0
4
EV 14.5
p<0.05
3
2
1
0
EV 14.5
Relative Axin2 expression
4
20
Relative Lef1 expression
6
B
Relative Axin2 expression
p<0.05
Relative Lef1 expression
p<0.05
Relative Axin2 expression
8
Relative Lef1 expression
Relative Lef1 expression
Relative Axin2 expression
A
7
6
5
4
3
2
1
0
5
p<0.01
hESC-CM
p<0.05
4
3
2
1
0
hESC-CM
Figure S5: Wnt target gene expression
Relative gene expression of direct Wnt target gene Axin2 and Lef1 in (A) FHF- and SHF-marked cells represented as 6-bromoindirubin3’-oxime (BIO) 6-bromoindirubin-3’-oxime (BIO) at D6+5 over DMSO control, (B) LV and RV Catnb/(ex3)Myl2 over littermate
controls at P1, (C) ex vivo (EV) E14.5 after 6 days of BIO over DMSO control and (D) human ESC-derived cardiomyocytes (CM) after
6 days in BIO over DMSO control. N=3. Errorbars indicate standard deviation.
Brightfield
AHF-Mef2c-DsRed
E9.5 left view
A
Nkx2.5-eGFP
Merge
LV
RV
RV
B
E9.5 left view
PM
RV
RV
C
E9.5 right view
PM
IFT
OFT
LV
OFT
RV
RV
LV
FACSDiva Version 6.1.3
D6 iPSC-EB
D
E
FACS isolation of iPSC-derived early FHF and SHF cell populations
Mef2c
SHF
Mef2c
SHF
Nkx2.
5eGFP
FHF
SHFMef2cDsRed
Figure S6: iPSC chimera formation reveals expression of dual cardiac reporter system
Chimera embryos were generated by injecting double-labeled iPSC into wildtype E3.5 blastocysts and were sub sequentially implanted
into pseudo pregnant females. Whole mount fluorescence microscopy was performed on chimeric embryos at E9.5. (A, B) Left lateral
and (C) right lateral views of E9.5 embryos demonstrate the double transgenic ESC contribution and accurate fluorescence marking of
FHF and SHF structures. Scale bar 50μm. (D) Brightfield and immunofluorescence images of Embryoid body (EB) at day 6 (D6) of
cardiac differentiation. The AHF-Mef2c-DsRed and Nkx2.5-eGFP reporter system mark fractions of FHF and SHF iPSC-derived cells.
Scale bar 50μm. (E) Double labeled Nkx2.5-eGFP/AHF-Mef2c-DsRed iPSC lines were differentiated by hanging droplet formation and
were dissociated into single cell suspension on EB day 6. FACS plots show isolation strategy for the 3 populations used in this work;
Nkx2.5-eGFP+/AHF-Mef2c-DsRed- (FHF), Nkx2.5-eGFP+/AHF-Mef2c-DsRed+ (SHF) and Nkx2.5-eGFP-/AHF-Mef2c-DsRed(NEG) cells.
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Printed on: Thu Jan 26, 2012 09:42:51 EST
-3 <
SHF D6+6 BIO +6 IWR
FHF D6+6 BIO +6 IWR
SHF D6+6 BIO +6 DMSO
FHF D6+6 BIO +6 DMSO
SHF D6+6 BIO
FHF D6+6 BIO
NEG D6
A
<3
Figure S7: Differentiation of expanded ventricular myocytes
(A) Heatmap displaying the relative RNA expression of ~20 selected genes in FHF and SHF-marked cells treated with BIO (D6+5
BIO), subsequently cultured with control media (D6+5 BIO +5 CTR) or with IWR (D6+5 BIO +5 IWR. Gene expression in all groups
was row normalized to the same not transgenic-marked (NEG D6) populations used in Figure 1E. (n = 3). Low expression in blue, high
expression in red.
LV
B
RV
5
Fold change
cTnT+/Ki67+ cells
Fold change cTnT+ cells
A
4
3
2
1
0
DMSO
BIO 2.0
IWR 16
LV
RV
10
8
6
4
2
0
DMSO
BIO 2.0
IWR 16
Figure S8: Wnt responsiveness of left and right ventricular cardiomyocytes
Quantification of (A) cardiac troponin T (cTnT) positive and (B) cTnT and Ki67+ fetal ventricular myocytes from the left ventricle (LV)
and right ventricle (RV) treated with BIO, IWR or DMSO. Error bars represent standard deviation. (n=3).
A
!-catenin
Ki67
Merge
Figure S9: Expression of active β-catenin in the fetal mouse heart
(A) Immunofluorescence for active β-catenin (green) and Ki67 (red) in E12.5 hearts.
Compact myocardium Trabecular myocardium
A
Control P30
Catnb/ex3/Myl2/cre P30
cTnT / Ki67 / DNA
cTnT / Ki67 / DNA
cTnT / Ki67 / DNA
cTnT / Ki67 / DNA
Figure S10: Increased proliferation in Catnb/(ex3)Myl2 animals is transient
(A) Immunofluorescence images of P30 Catnb/(ex3)Myl2 and littermate controls for trabecular and compact myocardium. Cardiomyocytes
were stained with Troponin T (cTnT) (green), cycling cells with Ki67 (red) and nuclei with DAPI (blue). Scale bars represent 50µm.
Movie 1: Differentiated mouse embryonic stem cells carrying the double fluorescent AHF-Mef2c-DsRed and Nkx2.5-GFP cardiac
reporter system.
Movie 2: Mouse ES cell-derived SHF early ventricular myocytes treated with a Gsk3 inhibitor for 6 days and re-plated on micropatterned surfaces.
Movie 3: Human ES cell-derived cardiomyocytes treated with a Gsk3 inhibitor for 6 days.
Gene
Tnnt2
Tnnc1
Tnni
Mybpc3
Synpo
Myl3
Myl2
Actn1
Actn2
Tbx20
Gata4
Gja1
Myh6
Myh7
Ttn
Gjc1
Myl7
Mef2c
Nkx2-­‐5
Smyd1
Tbx5
Myl1
Hand2
Axin2
Lef1
Bmp2
Bmp4
Bmp5
Wnt2
Wnt5a
Wnt11
Bmper
Nppa
Isl1
Forward primer sequence
CAGAGGAGGCCAACGTAGAAG
GCGGTAGAACAGTTGACAGAG
CTCTGCCAACTACCGAGCCTA
CAGGGAAGAAACCAGTGTCAG
GGAGGTGCAGGTCACATTAGC
TGGGGAAGCCAAAACAGGAAG
CATTCTCAACGCATTCAAGGTG
GACCATTATGATTCCCAGCAGAC
TGGCACCCAGATCGAGAAC
AAACCCCTGGAACAATTTGTGG
CCCTACCCAGCCTACATGG
ACAGCGGTTGAGTCAGCTTG
GCCCAGTACCTCCGAAAGTC
ACTGTCAACACTAAGAGGGTCA
TCAAGGAGGAAGCGTCCAAAG
CAGAGCCAACCAAAACCTAAGC
GGCACAACGTGGCTCTTCTAA
ATCCCGATGCAGACGATTCAG
GGTCTCAATGCCTATGGCTAC
TCAGTGACCAGAGAGGGCTAC
AATGGTCCGTAACTGGCAAAG
TGCAAGCTATCTCCAACAACAA
GCAGGACTCAGAGCATCAACA
ATGAGTAGCGCCGTGTTAGTG
GCCACCGATGAGATGATCCC
GGGACCCGCTGTCTTCTAGT
TTCCTGGTAACCGAATGCTGA
TTACTTAGGGGTATTGTGGGCT
CTCGGTGGAATCTGGCTCTG
CAACTGGCAGGACTTTCTCAA
ATGCGTCTACACAACAGTGAAG
GCCTGGGATTACCTGCTGC
GCTTCCAGGCCATATTGGAG
ATGATGGTGGTTTACAGGCTAAC
Table S1. Primer sequences
Table S2. Cell numbers
Download Table S2
Reverse primer sequence
CTCCATCGGGGATCTTGGGT
CCAGCTCCTTGGTGCTGAT
CTCTTCTGCCTCTCGTTCCAT
GCTGCCAAACCATACTTGTCATT
CAAGAAACCCCGTTGATCGC
AGCCATCAGTTTCTCTACCTCA
TGCGAACATCTGGTCGATCTC
CGGAAGTCCTCTTCGATGTTCTC
GTGGAACCGCATTTTTCCCC
CATCTCTTCGCTGGGGATGAT
ACATATCGAGATTGGGGTGTCT
GAGAGATGGGGAAGGACTTGT
GCCTTAACATACTCCTCCTTGTC
TTGGATGATTTGATCTTCCAGGG
GACTTCTTCGGATGCCTGTGA
CTGCACACATAAAATGGGTGGA
TGCAGATGATCCCATCCCTGT
AACAGCACACAATCTTTGCCT
GCCAAAGTTCACGAAGTTGCT
AGCTCAATCTTGCCATTGTTGAA
GGATAATGTGTCCAAACGGGTC
CCAGCAACGCTTCTACCTCT
AGGTAGGCGATGTATCTGGTG
GGGCATAGGTTTGGTGGACT
TTGATGTCGGCTAAGTCGCC
TCAACTCAAATTCGCTGAGGAC
CCTGAATCTCGGCGACTTTTT
CCGTCTCTCATGGTTCCGTAG
CACATTGTCACACATCACCCT
CATCTCCGATGCCGGAACT
GTAGCGGGTCTTGAGGTCAG
ACACATTATGCAAGGGTTGTCTG
GGGGGCATGACCTCATCTT
TCGATGCTACTTCACTGCCAG