Stem Cell Reports, Volume 9
Supplemental Information
Downregulation of LGR5 Expression Inhibits Cardiomyocyte Differen-
tiation and Potentiates Endothelial Differentiation from Human Pluripo-
tent Stem Cells
Rajneesh Jha, Monalisa Singh, Qingling Wu, Cinsley Gentillon, Marcela K.Preininger, and Chunhui Xu
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Supplemental Experimental Procedures Growth Curve of Undifferentiated hPSCs Cells from control shRNA and LGR5 shRNA cultures were dissociated and plated at 5x104 cells/well in Matrigel-coated 24-well plates. Cells were fed daily by replacing MEF-CM supplemented with bFGF and live cell counts in triplicate wells were recorded daily for 6 days using trypan blue and hemocytometer. Immunocytochemistry Cells were washed once with PBS and fixed at room temperature with 2% paraformaldehyde (vol/vol) for 15 min. Further, non-specific bindings were blocked by incubation in PBS containing 5% normal goat serum overnight at 4°C. The cells were then treated with optimum concentration of primary antibodies (Table S1) diluted in 1% NGS in PBS for 2 h at room temperature. After 3 times of cold PBS washing to remove unbound antibodies, cells were incubated with fluorescence-conjugated secondary antibody for 1 h at room temperature; and after further washing with PBS, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories) for the visualization of nuclei. Finally, stained cells were subjected to imaging under a microscope. Real-time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Levels of gene expression were determined by qRT-PCR using ABI 7500 instrument (Applied Biosystems, Foster City, CA) with SYBR green and primers listed in Table S2. Briefly, total RNA was isolated at indicated time points using an Aurum total RNA mini kit (Bio-Rad) following manufacturer’s protocol. Further, 1 µg of total RNA was reverse-transcribed to cDNA using Superscript III enzyme and random primers in 20 µl reaction volume per the manufacturer’s instruction in SuperScript® VILO™ cDNA Synthesis Kit
(Life Technologies). Reaction mixture was subjected to a cyclic temperature of 25C for
10 min, 42C for 2 h and 85C for 5 min. After completion of the RT step, reaction mixture was diluted 10 times and 1.5 µl of cDNA was used to perform real-time PCR in a total 15 µl reaction mixture containing 1X iTaq SyBr green and 200 nmol of each primers. PCR
was performed at initial activation temperature at 95C for 10 min; amplification for 40
cycles at 95C for 15 sec and 60oC for 1 min followed by dissociation step to check the specificity of reaction. The relative levels of gene expression were determined by normalization against expression of GAPDH using the ΔΔCT method. Fold difference in gene expression was calculated as 2-(ΔΔCT), where ΔΔCT represents the cycle difference between the gene of interest and GAPDH.
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Table S1. Antibodies
P R I M A R Y
Antibody Origin Supplier Catalog No. Dilution
(Application)
α-actinin Mouse IgG1 Sigma A7811 4 µg/ml (FC, IC)
Active β-catenin Mouse IgG1k EMD Millipore 05-665 1:500 (FC, IC)
CD31 Mouse IgG1 R&D systems BBA7 5 µg/ml (IC)
Ki-67 Mouse IgG1 BD Biosciences 550609 5 µg/ml (FC)
LGR5 Rat IgG2bκ BD Biosciences 562731 1:50 (IC)
NANOG Rabbit IgG EMD Millipore AB9220 1:200 (IC)
NKX2-5 Rabbit IgG Santa Cruz Biotech
sc-14033 1:1000 (IC)
OCT4 Mouse IgG1 EMD Millipore MAB4401 1:1000 (IC)
SOX2 Rabbit IgG Biolegend 630801 1:200 (IC)
SSEA4 Mouse IgG3 DSHB MC-813-70 1:200 (IC)
TRA1-60 Mouse IgM EMD Millipore MAB4360 1:200 (IC)
TRA1-81 Mouse IgM EMD Millipore MAB4381 1:200 (IC)
VE-cadherin Mouse IgG2b R&D systems MAB9381 5 µg/ml (IC)
S E C O N D A R Y
Alexa 488, Goat anti-mouse IgG1 Life Technologies A-21121 1:1000 (FC, IC)
Alexa 488, Goat anti-mouse IgG3 Life Technologies A-21151 1:1000 (FC, IC)
Alexa 488, Goat anti-mouse IgM Life Technologies A-21042 1:1000 (FC, IC)
Alexa 594, Goat anti-rabbit IgG Life Technologies A-11012 1:1000 (FC, IC)
Alexa 594, Goat anti-mouse IgM Life Technologies A-21044 1:1000 (FC, IC)
Alexa 488, Goat anti-rabbit IgG Life Technologies A-11034 1:1000 (FC, IC)
C O N J
APC-CD31 (Mouse-IgG1) Miltenyi Biotech 130-092-652 1:11 (FC)
PE-LGR5 (Rat-IgG2b) Miltenyi Biotech 130-100-849 1:11 (FC)
Alexa 488-VE-cadherin eBioscience 53-1449-42 1:20 (FC)
I S O
APC-Mouse-IgG1 Miltenyi Biotech 130-098-846 1:11 (FC)
FITC-Mouse-IgG2a Miltenyi Biotech 130-098-877 1:11 (FC)
Mouse-IgG1k BD Biosciences 554121 1:200 (FC)
PE-Rat-IgG2b Miltenyi Biotech 130-102-663 1:11 (FC)
Note: FC, flow cytometry; IC, immunocytochemistry; CONJ, conjugated; ISO; isotype.
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Table S2. Primers for qRT-PCR
Gene Accession no. Forward primer (5’-3’) Reverse primer (5’-3’)
AXIN2 NM_004655 CTGGTGCAAAGACATAGCCA AGTGTGAGGTCCACGGAAAC
CCND1 NM_053056.2 GGCGGATTGGAAATGAACTT TCCTCTCCAAAATGCCAGAG
CD31 NM_000442 GGGTCAGGTTCTTCCCATTT CCTTCTGCTCTGTTCAAGCC
CD34 NM_001773 ATTTGAAAATGTTCCCTGGGT TTTGCTTGCTGAGTTTGCTG
CDH5 NM_001795 CATCTTCCCAGGAGGAACAG AGAGCTCCACTCACGCTCAG
CDX1 NM_001804 TGTAGACCACGCGGTACTTG GACGCCCTACGAGTGGATG
CDX4 NM_005193 GGAATTCCTTTTCCAGCTCC CAGTTCCCCCAGCAGGA
EOMES NM_005442 CACATTGTAGTGGGCAGTGG CGCCACCAAACTGAGATGAT
GAPDH NM_001256799 CTGGGCTACACTGAGCACC AAGTGGTCGTTGAGGGCAATG
GSC NM_173849 CCACGTTCATGTAGGGCAG CAGCAGTGCTCCTGCGT
HAND1 NM_004821 AATCCTCTTCTCGACTGGGC CCTTCAAGGCTGAACTCAAGA
HHEX NM_002729 GGGGCTCCAGAGTAGAGGTT CCTCTGTACCCCTTCCCG
HNF3B NM_021784 TACGTGTTCATGCCGTTCAT CGACTGGAGCAGCTACTATGC
ISL1 NM_002202 TCACGAAGTCGTTCTTGCTG CATGCTTTGTTAGGGATGGG
LEF1 NM_016269 TCACTGTAAGTGATGAGGGGG CGGGTACATAATGATGCCAA
LGR5 NM_003667 GTTTCCCGCAAGACGTAACT CAGCGTCTTCACCTCCTACC
LMO2 NM_005574 CTTCCTCCTCTCTCGGGAA GCAGTAGCTAGTGGAGAGGCA
MEF2C NM_002397 TTTCCTGTTTCCTCCAAACAA CCAAGGACTAATCTGATCGGG
MESP1 NM_018670 CTCAGGCAGCCACTCCAG GATGGAGCCAAGCCCAC
MESP2 NM_001039958 CACGACCACTGGATCTTCGC AACCCGACGAATCGGAGGA
MIXL1 NM_031944 GAGACTTGGCACGCCTGT GGTACCCCGACATCCACTT
MKI67 NM_002417 TGACTTCCTTCCATTCTGAAGAC TGGGTCTGTTATTGATGAGCC
MYH6 NM_002471 CTTCTCCACCTTAGCCCTGG GCTGGCCCTTCAACTACAGA
MYH7 NM_000257 CGCACCTTCTTCTCTTGCTC GAGGACAAGGTCAACACCCT
MYL2 NM_000432 CGTTCTTGTCAATGAAGCCA CAACGTGTTCTCCATGTTCG
MYL7 NM_021223 CTTGTAGTCGATGTTCCCCG TCAAGCAGCTTCTCCTGACC
NANOG NM_024865 TTTGTGGGCCTGAAGAAAACT AGGGCTGTCCTGAATAAGCAG
NKX2-5 NM_004387 CTGTCTTCTCCAGCTCCACC TTCTATCCACGTGCCTACAGC
OCT4 NM_002701 CTTCTGCTTCAGGAGCTTGG GAAGGAGAAGCTGGAGCAAA
PCNA NM_182649 TCAGGTACCTCAGTGCAAAAG TGCAAGTGGAGAACTTGGAA
SOX2 NM_003106 GCTTAGCCTCGTCGATGAAC AACCCCAAGATGCACAACTC
SOX7 NM_031439 CTCTTCTGGGACAGCGTCA GCTTAGCCTCGTCGATGAAC
GCCAAGGACGAGAGGAAAC
SOX17 NM_022454 TCTGCCTCCTCCACGAAG CAGAATCCAGACCTGCACAA
T NM_003181 CGTTGCTCACAGACCACAG ATGACAATTGGTCCAGCCTT
TAL1 NM_003189 NM_003181
AAGATACGCCGCACAACTTT CGTTGCTCACAGACCACAG
GCCTTCCCTATGTTCACCAC ATGACAATTGGTCCAGCCTT TBX5 NM_181486 AGACTCGCTGCTGAAAGGAC GGAGCTGCACAGAATGTCAA
TBX6 NM_004608 CCGCAGTTTCCTCTTCACAC AGACTCGCTGCTGAAAGGAC
CAACTGAAGATTGCAGCCAA
TNNT2 NM_001001431 GCGGGTCTTGGAGACTTTCT TTCGACCTGCAGGAGAAGTT
WNT3A NM_033131 NM_001001431
ATGAGCGTGTCACTGCAAAG GCGGGTCTTGGAGACTTTCT
ACAGCCTGGCCATCTTCG TTCGACCTGCAGGAGAAGTT WNT8A
WNT3A NM_003392 NM_033131
CAAAGCAACTCCTGGGCTTA ATGAGCGTGTCACTGCAAAG
GCTCGGATTCCTCGGCT ACAGCCTGGCCATCTTCG WNT5A
WNT8A NM_031933 NM_003392
TCGAAGTCACCCATGCTACA CAAAGCAACTCCTGGGCTTA
ACCCACAACAGGCTGAGAAG GCTCGGATTCCTCGGCT WNT11 NM_004626 ACCTGTGCAGACACCAGACC CGTGTGCTATGGCATCAAGT
Note: primers were retrieved from open access websites (http://primerdepot.nci.nih.gov/ or http://pga.mgh.harvard.edu/primerbank/)
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Figure S1. Knockdown of LGR5 Inhibits Differentiation of Cardiomyocytes from hPSCs. Related to Figures 1 and 3. (A-E) Relative RNA and protein expression of LGR5 and cardiomyocyte differentiation in IMR90 iPSCs. (F-J) Relative RNA and protein expression of LGR5 and cardiomyocyte differentiation in H9 hESCs. (A & F) Relative expression levels of LGR5 analyzed using qRT-PCR. (B & G) Flow cytometry of LGR5 protein at day 2. (C & H) qRT-PCR analysis of LGR5 at day 5. (D & I) Flow cytometry of LGR5 protein at day 5. (E & J) Flow cytometry of α-actinin at day 14. n = 3 independent experiments. Data are presented as mean ± SEM. ***, p<0.001, ****, p<0.0001. Please note: At differentiation day 2, the expression of LGR5 increased ~50-fold in H9 hESCs and ~250-450-fold in IMR90 iPSCs compared with those at day 0 (Figures S1A, S1G and 2C). The temporal expression of LGR5 also showed the highest expression in day 4 or 5 differentiated cells (>500-fold higher compared with cells at day 0) for both cell
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lines (Figures S1A, S1G and 2C). At the protein level, ~53% and 49% of the day-5 cells derived from IMR90 iPSCs and H9 hESCs, respectively, were positive for LGR5 protein as detected by flow cytometry (Figures S1D and S1I). From parallel cultures, IMR90
iPSCs and H9 hESCs generated >55% -actinin+ cells at day 14 (Figures S1E and S1J).
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Figure S2. Knockdown of LGR5 Does Not Affect the Growth and Maintenance of Undifferentiated hPSCs. Related to Figure 2. (A) LGR5 shRNA led to the significant reduction in relative LGR5 mRNA levels but did not affect the expression of pluripotent genes NANOG, POU5F1 and SOX2 as measured by qRT-PCR in IMR90 iPSCs. (B) Knockdown of LGR5 did not affect undifferentiated cell morphology maintained in stem cell medium at low density (3 days before cardiomyocyte induction/day -3) and high
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density (at the day of induction/day 0) in IMR90 iPSCs. Scale bar = 200 μm. (C) Cell growth curve of IMR90 iPSCs showing similar growth in control shRNA cells and LGR5 shRNA cells at indicated days post plating in the culture condition for undifferentiated hPSCs. (D) Knockdown of LGR5 did not affect the expression of pluripotent stem cell markers detected by immunocytochemical analysis IMR90 iPSCs. Scale bar = 50 μm. n = 3 independent experiments. Data are presented as mean ± SEM. ****, p<0.0001.
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Figure S3. Knockdown of LGR5 Promotes Differentiation of Endothelial Cells from hPSCs. Related to Figure 4. (A) Detection of endothelial cell markers CD31 and VE-cadherin in H9 hESC cultures at day 14 by immunocytochemetry. (B) Tube-forming ability upon VEGF treatment in LGR5 shRNA IMR90 iPSC cultures but not in control shRNA cultures. Scale bar = 200 μm. (C) Ac-LDL uptake in LGR5 shRNA IMR90 iPSC cultures. Scale bar = 100 μm.
Tube formation assay Ac-LDL uptake assay
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Figure S4. Effect of LGR5 Knockdown on Cell Proliferation during Cardiomyocyte Differentiation. Related to Figure 5. (A) Representative flow cytometry analysis of cell proliferation marker Ki-67 in IMR90 iPSCs at differentiation day 8. (B) Summary of percentage of Ki-67+ cells in control shRNA and LGR5 shRNA IMR90 iPSC cultures at differentiation day 8. (C) Representative flow cytometry of cell proliferation marker Ki-67 in IMR90 iPSCs at differentiation day 14. (D) Summary of percentage of Ki-67+ cells in control shRNA and LGR5 shRNA IMR90 iPSC cultures at differentiation day 14. n = 3 independent experiments. Data are presented as mean ± SEM.
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Figure S5. Upregulation of the Expression of Wnt Signaling-related Genes during Cardiomyocyte Differentiation. Related to Figure 6. IMR90 iPSCs were induced for cardiomyocyte differentiation with activin A (100 ng/ml) at day 0 and BMP4 (10 ng/ml) at day 1 in RPMI/B27 without insulin medium. At day 5, medium was replaced with RPMI/B27 medium and cells were harvested for qRT-PCR analysis at various time points. (A) Relative expression levels of Wnt signaling target genes. (B) Relative expression levels of canonical Wnt signaling-related genes. (C) Relative expression levels of non-canonical Wnt signaling-related genes. n = 3 independent experiments. Data are presented as mean ± SEM.
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