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Table S1. Genes up- and down-regulated upon SB431542 treatment. Gene expression profiling comparing hESCs grown in CDM supplemented with Activin and FGF with hESCs grown for 48 hours in CDM supplemented with FGF and SB431542.
Fig. S1. The expression of Nanog in hESCs and in mEpiSCs is controlled by Activin/Nodal signalling. (A) Inhibition of Activin/Nodal signalling results in decreased Nanog expression. H9 hESCs were grown for 96 hours in CDM in the presence of SB431542 and FGF2. RNAs were extracted every hour for the first 9 nine hours and then after 24, 36, 48, 72 and 96 hours, then real-time PCR was performed to detect the expression of the genes denoted. Data represent the mean of three independent experiments and error bars indicate standard deviation. (B) Addition of Activin is sufficient to re-activate the expression of NANOG in hESCs grown for 24 hours in the presence of SB431542. H9 cells were grown for 12 hours in the presence of SB431542 (SB 12H) and returned to CDM supplemented with 10 ng/ml Activin ±100 µg/ml cycloheximide, and then RNAs were extracted every 2 hours for 6 hours. H9 cells grown for 24 hours in CDM supplemented with 10 ng/ml Activin without SB431542 treatment were used as a positive control (Activin). Data represents the mean of three independent experiments and error bars indicate standard deviation. (C) Smad3 activity (but not Smad2) controls Nanog expression. H9 cells were transfected with an expression vector for wild-type SMAD2 or SMAD3 or for a dominant-negative mutant form of SMAD2 or SMAD3 in CDM supplemented with Activin. Expression of NANOG and OCT4 was then analysed using real-time PCR. (D) Inhibition of Activin/Nodal signalling results in decreased Nanog expression in mEpiSCs. 129-EpiSCs were grown for 10 days in CDM in the presence of SB431542. RNAs were extracted every day and then real-time PCR was performed to detect the expression of the genes denoted. (E) Addition of Activin is sufficient to induce the expression of Nanog in mEpiSCs grown for 24 hours in the presence of SB431542. 129-EpiSCs cells were grown for 12 hours in the presence of SB431542 (SB 12H) and returned to CDM supplemented with 10 ng/ml Activin ±100 µg/ml cycloheximide, and then RNAs were extracted every 2 hours for 6 hours. mEpiSCs cells grown for 24 hours in CDM supplemented with 10 ng/ml Activin without SB431542 treatment were used as positive controls (Activin). (F) Sequence alignment of the human NANOG promoter with the mouse, dog and cow Nanog promoter. A comparative annotation of the human, dog, mouse and cow orthologs of Nanog was performed using MULTI-LAGAN (http://lagan.stanford.edu/lagan_web/index.shtml) and SynPlot (http://www.sanger.ac.uk/Users/jgrg/SynPlot/) to define evolutionarily conserved regions in the Nanog gene. Pair base marks with an asterisk are conserved in the four species. (G) Smad2/3-binding sites located in NANOG promoter are functional. Luciferase reporter genes containing the promoter of the human NANOG gene (−379 to +18) with or without mutated Smad2/3-binding sites were co-transfected into H9 cells along with the renilla expression vector (control for normalisation) and an expression vector for SMAD3 in the presence of Activin and FGF (A + F), or in the presence of SB431542 (negative control). Firefly luciferase activity (normalised to renilla luciferase activity) is expressed as mean±s.d. from three independent experiments. (H) SMAD2/3 and NANOG bind the same genomic region in Lefty and SMAD7 promoters. ChIP assays were performed using antibodies directed against SMAD2/3 or NANOG. The immunoprecipitated DNA was then amplified by Q-PCR using specific primers to detect enrichment in the denoted genomic regions. Results were normalised against a control region contained in the 3′UTR of the Smad7 (not shown) and are expressed as ±s.d. from three experiments.
Fig. S2. Constitutive expression of Nanog in hESCs. (A) Map of the hrGFPpTP6 and NanogpTP6 vectors. pTP6 vector contains the CAGG promoter followed by the hrGFP or NANOG cDNA and an IRES puromycin resistance gene that allows selection for transgene expression. NANOG-pTP6 or hrGFP-pTP6 were transfected into H9 and hSF6 hESC lines and into mEpiSCs of 129/S2 and NOD genetic background (129-EpiSCs and NOD-EpiSCs). NANOG-overexpressing stable clones were isolated after selection with puromycin and expanded for further analyses. (B) Number of hESC and mEpiSC colonies generated after stable transfection of pTP6 expression vectors for the humanized recombinant Green Fluorescent Protein (hrGFP) and for Nanog. hESCs and mEpiSCs were transfected using Lipofectamine 2000 (Vallier et al., 2004) and the numbers of colonies generated were determined after 10 days of puromycin selection. In the pTP6 vector, expression of the transgene of interest is linked to the expression of the puromycin resistance gene though an IRES (internal ribosome entry site). Therefore, all the hESC colonies that were resistant to puromycin also expressed the relevant transgene. Notably, there was a significantly greater number of NANOG-overexpressing clones than control green fluorescent protein (GFP)-overexpressing clones suggesting that Nanog has a positive effect on hESC and mEpiSCs growth. (C) Screening of NANOG-expressing hESCs. Expression of human NANOG in H9 sub-lines generated by transfection of Nanog-pTP6, as determined by western blot. Protein extracts from non-transfected hESCs were used as negative controls. Actin was used as a loading control. The level of NANOG overexpression in each H9 subclone was found to be 1- to10-fold higher than wild-type hESCs. The experiments were all initially carried out using NANOG-H9 hESCs overexpressing NANOG (sub-lines 1 to 6) with levels of NANOG overexpression 2- to 5-fold higher than controls (GFP expressing cells or/and wild-type cells). A second series of experiments were performed using NANOG-H9 hESCs constitutively expressing NANOG at a physiological level to exclude any artefactual results provoked by higher levels of Nanog expression (sub-line 11 expressing similar NANOG levels to control GFP-expressing cells; Fig. 2A,B,D; Fig. 4B). Similar results were obtained with both overexpressing and constitutively expressing sub-lines. In addition, these results were validated on hSF-6 (data not shown). GFP-expressing hESCs and mEpiSCs and wild-type hESCs were used as controls for all of the experiments. (D) Proliferation curves of hrGFP- and NANOG-expressing hESC lines. H9 cells expressing green fluorescent protein or NANOG (sub-lines 4,5,8) were grown for 6 days and cells were counted every two days. The only notable difference between wild-type hESCs and NANOG-expressing hESCs was the capacity of NANOG-hESCs to reach confluency before their wild-type counterparts. To confirm this observation, we performed cell counting assays over 8 days, showing that the proliferation rate of the NANOG clones was approximately 2- to 5-fold higher than that of GFP clones, suggesting that NANOG could promote self renewal of hESCs. The results were normalised to the number of cells initially plated for each sub-line. Data represent the mean of three independent experiments and error bars indicate s.d. (E) Constitutive expression of NANOG maintains the expression of pluripotency markers and blocks the expression of neuroectoderm markers in hESCs grown in the absence of Activin/Nodal signalling. H9 cells (hESCs) and NANOG-hESC sub-line 11 (Nanog) were grown for 7 days in the presence of SB431542 and FGF2 and then real-time PCR was performed to detect the expression of the genes denoted. H9 cells grown in Activin and FGF2 were used as normalisation controls. Data represents the mean of three independent experiments and error bars indicate s.d.
Fig. S3. Constitutive expression of Nanog blocks neuroectoderm differentiation of mEpiSCs. (A) Immunofluorescence analysis for the expression of the pluripotent markers Oct4 and Nanog in NOD-EpiSCs (top panels) and Nanog-NOD-EpiSCs (Nanog-EpiSCs; bottom panels) grown for 7 days in the presence of SB431542 (10 µM) and FGF2 (12 ng/ml). Scale bar: 50 µm. (B) RT-PCR analysis for the expression of pluripotency and neuroectoderm markers in wild-type and Nanog-NOD-EpiSCs (sub-lines 1, 2, 3) grown for 7 days in standard culture conditions (Activin and FGF2) or in the presence of SB431542 (10 µM) and FGF2 (12 ng/ml).
Fig. S4. Constitutive expression of Nanog is sufficient to prevent neuroectoderm differentiation of embryoid bodies (EBs) grown in CDM. (A) Photomicrographs showing phase-contrast morphology of wild-type (WT; left panels) and NANOG-overexpressing (Nanog, right panels) EBs grown for 15 days in CDM in the absence of any additional growth factors or serum. Scale bar: 50 µm. In these culture conditions, wild-type EBs display a compact morphology (left panels) and mostly differentiate into neuroectoderm cells (expressing SOX1, SOX2 and NEUROD1) and to a lesser extent into extraembryonic endoderm cells (expressing αFP; see B) (Vallier et al., 2004; Smith et al., 2008). NANOG-expressing EBs grown in CDM (right panels) adopted a homogeneous morphology without any sign of differentiated tissues, notably devoid of the neuronal rosettes that were commonly observed in wild-type EBs. (B) RT-PCR analysis for the expression of pluripotency and differentiation markers in wild-type and NANOG-expressing EBs. H9 cells (WT) and NANOG-hESCs (Na4 and Na5) were grown for 15 days in non-adherent condition to form EBs. Then RT-PCR analyses were performed to detect the gene denoted. Contrary their wild-type counterpart, NANOG-expressing EBs grown in CDM maintained the expression of OCT4, SOX2 and SSEA3, while they showed low levels of neuroectoderm differentiation (SOX1, NEUROD1). (C) Immunofluorescence analysis for the expression of the pluripotency markers NANOG, OCT4 and SSEA3 in wild-type EBs (WT EBs) and in NANOG-EBs (Nanog EBs) differentiated for 20 days in CDM. Scale bar: 50 µm. H9 cells and NANOG-hESCs were grown for 15 days in non-adherent conditions and then the resulting EBs were returned to adherent conditions for 5 additional days before performing immunofluorescence analyses. Pluripotency markers (NANOG, OCT4 and SSEA3) were not expressed in wild-type cells, whereas they were maintained in Nanog EBs.
Fig. S5. Nanog does not block differentiation of hESCs and mEpiSCs into extraembryonic tissues. (A) Real-time PCR analysis for the expression of OCT4 and NANOG in cells generated by BMP4 treatment of hESCs and NANOG-hESCs. Wild-type H9 (hESCs) and NANOG-hESCs (sub-lines 1 and 2) were grown for 7 days in CDM supplemented with BMP4 (10 ng/ml), and then OCT4 and NANOG combined endogenous and transgene-derived expression were analysed using real-time PCR. (B) FACS analysis showing the percentage of cells expressing the pluripotency marker TRA-1-60 after differentiation into extra-embryonic tissues. H9 (hESCs) and NANOG-hESCs (sub-lines 1, 2, 3) were grown for 7 days in CDM supplemented with BMP4 (10 ng/ml) and then the fraction of cells expressing TRA-1-60 was determined by FACS. Data represents the mean of three independent experiments and error bars indicate standard deviation. (C) Expression of extraembryonic markers in wild-type and NANOG-hESCs differentiated into mesendoderm-like cells. H9 cells (hESCs) and hESCs constitutively expressing NANOG (sub-line 11) were grown for 4 days in culture conditions maintaining pluripotency, or for 8 days in the presence of BMP4 to induce extraembryonic differentiation. Then, real-time PCR was performed to detect the expression of the genes denoted. Data represents the mean of three independent experiments and error bars indicate standard deviation. (D) Immunofluorescence analysis showing expression of the primitive endoderm marker Sox7 and the trophoblast marker Cdx2 in NOD-mEpiSCs (top panels) and Nanog-NOD-mEpiSCs (bottom panels) grown for 7 days in the presence of 10 ng/ml BMP4 and 10 µM SB431542. Scale bar: 50 µm. (E) RT-PCR analysis for the expression of extraembryonic tissue markers and pluripotency markers in wild-type and Nanog-NOD-mEpiSCs (Nanog sub-lines 1, 2, 3) grown for 7 days in the presence of 10 ng/ml BMP4 and 10 µM SB431542.
Fig. S6. Constitutive expression of Nanog is unable to prevent extraembryonic and mesendodermal differentiation of embryoid bodies grown in medium containing serum. (A) Photomicrographs showing phase-contrast morphology of wild-type (WT, left panel) and NANOG (Nanog, right panel) overexpressing EBs grown for 15 days in FBS-containing medium. Scale bar: 50 µm. NANOG-expressing EBs quickly generated differentiated tissues, suggesting that they were able to differentiate as readily as their wild-type counterparts. Interestingly, NANOG EBs rarely contained compact rosette structures signalling neuroectoderm differentiation (Vallier et al., 2004; Smith et al., 2008). (B) RT-PCR analysis for the expression of pluripotency and differentiation markers in wild-type and Nanog-expressing EBs. H9 cells (WT) and NANOG-hESCs (Na1 and Na7) were grown for 15 days in non-adherent conditions to form EBs in medium containing FBS. RNAs were extracted every 5 days and then RT-PCR analyses were performed to detect the genes denoted. Wild-type EBs grown for 15 days in these culture conditions differentiated (as monitored by the progressive decrease in OCT4 and NANOG expression) into various cell types expressing extraembryonic markers (CDX2, GATA6, AFP), neuronal markers (SOX1, SOX2, NEUROD1), mesendoderm markers (brachyury) and endoderm markers (SOX17), confirming the efficiency of this approach to drive differentiation of hESCs into a wide number of tissues. Constitutive expression of NANOG during EB-induced differentiation was sufficient neither to maintain the expression of the pluripotency markers OCT4 and SOX2 nor to block the expression of markers of extraembryonic (CDX2, GATA6, AFP) and mesendoderm (brachyury) differentiation. However, the expression of neuroectoderm markers was decreased (SOX1) or absent (SOX2 and NEUROD1) in NANOG EBs, confirming that Nanog expression was able to specifically prevent neuroectoderm differentiation of hESCs regardless of the culture conditions used to induce their differentiation.
Fig. S7. Constitutive expression of Nanog blocks further progression of mesendoderm differentiation of mEpiSCs along the endoderm lineage. (A) Immunofluorescence analysis for the expression of the pluripotency marker Oct4 and the mesendoderm markers brachyury and Sox17 in NOD-mEpiSCs (top panels) and Nanog-NOD-mEpiSCs (bottom panels) grown for 7 days in culture conditions inducing mesendoderm differentiation. Scale bar: 50 µm. (B) RT-PCR analysis for the expression of pluripotency and mesendoderm markers in wild-type and Nanog-NOD-mEpiSCs (sublines 1, 2, 3) grown for 7 days in culture conditions inducing mesendoderm differentiation.
Fig. S8. Nanog protein interacts with Smad2/3 in mEpiSCs. Co-IP of endogenous Nanog (Nanog) with Smad2/3. (A) Effect of Nanog on Smad transcriptional activity. A reporter gene for the transcriptional activity of Activin/Nodal signalling (containing four Smad-binding elements, SBE4) was co-transfected into mEpiSCs cells along with a renilla expression vector (control for normalisation) and with the expression vectors listed below the chart in the presence of Activin A or in the presence of SB431542 (negative control). Firefly luciferase activity (normalised to renilla luciferase activity) is expressed as mean ±s.d. from three independent experiments. (B) Immunoprecipitations (IPs) were performed with αSmad2/3 and control rabbit IgG antibodies on mEpiSCs nuclear extracts. Input nuclear extracts (Input NE) and IP lanes were probed with αNanog and αSox2 antibodies. Loading percentage of the total material is indicated.
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