Supplementary Methods.

Teratoma formation and histological analysis. hESCs and D3-Oct4 mESCs were cultured at 1 x 105 and 1 x 107 cells/ml, respectively, in their culture media in the absence of LIf for mESCs or bFGF for hESCsplus 2 μM DETA-NO. Male SCID-CB17 mice aged six weeks (Jackson Laboratories, Bar Harbor, ME, USA) were subcutaneously injected in the dorsal flank with 1 x 106 mESCs or 1 x 105 hESCs in 100 µl of cell suspension. Six weeks after the injection of mESCs and eight weeks after that of hESCs, tumours (20 mm diameter) were surgically dissected from the mice. Samples were weighed, fixed in PBS containing 4% formaldehyde and embedded in paraffin. Sections were stained with haematoxylin and eosin and subjected to histological examination.

Supplementary Table 1: List of primers sets used in the study

Mouse genes
Gene / Sequence
β-Actin Forward / TTCCTTCTTGGGTATGGAAT
β-Actin Reverse / GAAGATCAAGATCATTGCTC
Gata 6 Forward / TCATTACCTGTGCAATGCATGCGG
Gata 6 Reverse / ACCACAACCACTACCTTATGGCG
Gata 4 Forward / AGGGTGAGCCTGTATGTAATGCCT
Gata 4 Reverse / AGGACCTGCTGGCGTCTTAGATTT
Oct4 Forward / AGCTGCTGAAGCAGAAGAGGATCA
Oct4 Reverse / AACACCTTTCCAAAGAGAACGCCC
Nanog Forward / AGCAGATGCAAGAACTCTCCTCCA
Nanog Reverse / CCGCTTGCACTTCATCCTTTGGTT
Sox2 Forward / CACATGAAGGAGCACCCGGATTAT
Sox2 Reverse / TCCGGGAAGCGTGTACTTATCCTT
Fgf5 Forward / ATAGCAGTTTCCAGTGGAGCCCTT
Fgf5 Reverse / ACTTAACACACTGGCTTCGTGGGA
Fgf8 Forward / CGAAGCTCATTGTGGAGACCGATA
Fgf8 Reverse / TCTGTGAATACGCAGTCCTTGCCT
eNOS Forward / TACCAGCTGGCCAAAGTGACCATA
eNOS Reverse / CAGAATGGTTGCCTTCACACGCTT
iNOS Forward / TCTTTGACGCTCGGAACTGTAGCA
iNOS Reverse / TAGGTCGATGCACAACTGGGTGAA
Human genes
Nanog Forward / CCCAAAGGCAAACAACCCACTTCT
Nanog Reverse / AGCTGGGTGGAAGAGAACACAGTT
Sox2 Forward / CACATGAAGGAGCACCCGGATTAT
Sox2 Reverse / GTTCATGTGCGCGTAACTGTCCAT
Brachyury Forward / AAAGAGATGATGGAGGAACCCGGA
Brachyury Reverse / AGGATGAGGATTTGCAGGTGGACA
Oct4 Forward / TATGCAAAGCAGAAACCCTCGTGC
Oct4 Reverse / TTCGGGCACTGCAGGAACAAATTC
Gata 4 Forward / AGACGTTCTCAGTCAGTGCGATGT
Gata 4 Reverse / AAGACCAGGCTGTTCCAAGAGT
β-Actin Forward / TCAGAAGGATTCCTATGTGGGCGA
β-Actin Reverse / TTTCTCCATGTCGTCCCAGTTGGT

Supplementary Figure 1. Transfected mES cell lines express Green Fluorescence Protein under control of pOct4 promoter. (A) Fluorescence microscopy of Oct4 expression in mESCs lines D3-pOct4-eGFP and D3-pOct4-eGFP-eNOSafter selection by cell sorting (representative image of five colonies). Scale bars are 20 µm. (B) Comparison of GFP-positive cells by flow cytometry between D3 mESCs, D3-pOct4-eGFP mESCs and D3-pOct4-eGFP-eNOS mESCs. Representative graph of five experiments

Supplementary Figure 2. (A) Confocal images of cells positive for SSEA-1 (red) and Oct4 (green) in non-transfected D3 mESCs cultured for seven days in the presence (left panel) or absence (middle panel) of LIF and in the absence of LIF plus 2 μM DETA-NO (right panel). (B) Immunofluorescence images of cells co-expressing SSEA-1 (red) and Oct4 (green) in D3-eNOS mESCs cultured for seven days in the presence (left panel) or absence (middle panel) of LIF and in the absence of LIF plus 400 μM L-NMMA (right panel). Images are representative of three experiments. Scale bars are 20 µm. C) Effect of staurosporine on NO action. D3 mESCs cultured for seven days in the presence or absence of LIF plus 2 M DETA-NO and 2 nM staurosporine under the indicated conditions. Immunodetection of Nanog, Brachyury and cleaved caspase 3 and -actin was described in Materials and Methods. D). Effect of L-NMMA on Oct4 expression in the presence of LIF. Wild-type D3 mESCs and D3 mESCs that overexpress eNOS were cultured for seven days in the presence of LIF plus 400 M L-NMMA. Immunodetection of Oct4 and -actin was described in Materials and Methods. In C and D, the figures are representative of three independent experiments.

Supplementary Figure 3. mESCs treated with DETA-NO retain their stem morphology R1-E mESCs maintain stem morphology after five days of culture in the absence of LIF plus 2 μM DETA-NO. Optical microscopy. Scale Bar 40µm.

Supplementary Figure 4. Teratoma formation by ESCs cultured in medium supplemented with NO. mESCs from passage 10 grown in the absence of LIF and supplemented with 2 M DETA/NO were injected into immunosuppressed mice. Six weeks after the injection of mESCs, the mice presented multiple teratomas formed from tissues derived from all three germ layers: i) endoderm (glandular tissue); ii) mesoderm (cartilage); and iii) ectoderm (hair); panels A, B and C, respectively. Teratoma sections stained with haematoxylin and eosin. Magnification A 400X, B and C 200X. hESCs from passage 10 cultured in the absence of bFGF but supplemented with 2 M DETA/NO were injected into immunosuppressed mice. Eight weeks after the injection, the mice presented teratomas containing tissues derived from all three germ layers: i) endoderm (glandular tissues); ii) mesoderm (muscle); and iii) ectoderm (keratinocytes); panels D, E and F, respectively. Teratoma sections stained with haematoxylin and eosin. Magnification: D, E and F 100X. Pictures are representative of teratomas formed in five mice from each type of ESC injected.

Supplementary Figure 5. Activation of self-renewal signalling pathways by NO in ESCs. (A) D3-pOct4-eGFP mESCs and (B) D3-pOct4-eGFP-eNOS mESCs. Cells were cultured in the presence of LIF for five days. For transitory phosphorylation detection, cells were maintained for four hours in medium without LIF (A, B and C) or without LIF or serum (D), after which the cells were incubated under the indicated conditions for 15 minutes. Immunodetection of phosphorylated proteins was described in Materials and Methods. (C) hESC. Cells were cultured in the presence of bFGF for ten days. For the detection of transitory phosphorylation, cells were maintained for four hours in medium without bFGF, after which cells were incubated under the indicated conditions for 15 minutes. Immunodetection of phosphorylated proteins is described in Materials and Methods. (E) D3-pOct4-eGFP mESCs were cultured for seven days under the indicated conditions, Oct-4 and -actin were immunodetected by western blotting according to the protocols described in Materials and Methods. The figures are representative of three independent experiments.

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