Gene name forward primer 5->3 reverse primer 5->3 product GCCCATA. Nfatc1 AGGTGCAGCCCAAGTCTCAC GTGGCCATCTGGAGCCTTC CTGT GATGC GCT ACCAA

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Morgan Abigail Manning
5 years ago
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1 Supplementary able 1 is presenting the PCR primer list used. ene name forward primer 5->3 reverse primer 5->3 product size cdna VE-Cadherin AACCCAAAAA CACCCCAAAAC 260bp CCA CCCAA PECAM-1 CACCACAA CCCCCACC 260bp AA Flk1 CAAAACAACCCCA CAAAAACCACCA 133bp A CC Nfatc1 ACACCCAACCAC CCACACCC 620bp Nrg1 CCAACCACACCAAA ACCCCAACC 229bp C Isl-1 CACCCACACACAAC ACCAACAC 253bp CCA AC Nkx2.5 ACCCCCCCACAACC CAAAACACC 178bp C C ata4 CAAAACAAA AACACAA 230bp C cn ACCCACACCCAAA CCCCCCCCAA 115bp C MLC2v ACCACCCCCACA CCAAA 254bp ACCAA 1

2 MLC2a AAAACCCACAAC AACACCACCCA 202bp CA CAA AFP CCCCCACCCCCAC ACAAACAAAA 234bp FOXA2 CCAAAAACAC CACCACCCCACAA 210bp Pdx1 CCCCCAAAACCA CAACAACACAC 264bp CC Nestin CAACAACCCA ACCCCA 130bp ubb3 CCCCACCC CCCAAACCCCCA 94bp Pax6 AAAAACCAC CACCCCACC 214bp NANO CCAAACCCACA CCAC 159bp SOX2 AAACC AAACAAACCACAAAAC 156bp b-actin ACACACCAAA CCACCACACC 244bp gapdh ACAACA CACAACA 94bp 2

3 Supplementary Figure S1. Supplementary Figure S1: Characterization of Pvec- and Pvec-EFP- genetically modified ESCs. (A-E) Undifferentiated cells from clone A11 and 11 express pluripotency markers Oct3/4, Sox2, PECAM-1, SSEA-1 and E-cadherin but not EFP, as shown by immunostaining. 3

4 DNA was stained with OPRO-1. (F) Mesodermal Bry + cells and Isl1 + cardiovascular progenitor cells in A11 derived-ebs at d4. () Endothelial VE-cadherin + cells were typically located adjacent to Isl1+ progenitor cells in A11 derived-ebs at d7. (H) Statistical analysis of beating activity in clones A11, A12, 11, 12 and control (E14 ESCs) during differentiation days 7-15 (an EB was considered as beating if contained one or more beating areas). *** P <0,001 vs control. Data were evaluated by Repeated-measures two-way ANOVA, followed by Bonferroni post-hoc analysis for multiple comparisons. Probability values P <0.05 were considered significant. Scale bars: 20 μm Supplementary Figure S2. 4

Supplementary Figure S2: Pvec+ cells express VE-cadherin but not vwf or CD39 after double immunostaining with anti-ve-cadherin and anti- vwf (A) or anti- CD39 (B).

5 Supplementary Figure S2: Pvec+ cells express VE-cadherin but not vwf or CD39 after double immunostaining with anti-ve-cadherin and anti- vwf (A) or anti- CD39 (B). Right panels are the immunofluorescence images superimposed on the background of phase-contrast optics. Scale bars: (A-B) 20 μm Supplementary Figure S3. Supplementary Figure S3: (A) ypical image of spheres formed in the presence of SB after 5 days of CEDPs differentiation. (B) cn + cells did not form under these conditions after 10 days of CEDPs differentiation, after immunostaining with anti-cn and anti-ve-cadherin. Scale bar: (B) 20 μm Supplemental video of a beating area after 15 days of CEDPs differentiation. 5

6 Supplementary Figure S4. Supplementary Figure S4: R-PCR analysis of CEDPs differentiation products at d10 showed absence of neuroectodermal and endodermal markers. Supplementary Figure S5. 6

Supplementary Figure S5: Optical microscope image of spheres formed in gelatinised alginate hydrogel at day 5 of CEDPs differentiation (magnification

7 Supplementary Figure S5: Optical microscope image of spheres formed in gelatinised alginate hydrogel at day 5 of CEDPs differentiation (magnification corresponds to marked area). Supplementary Figure S6. Supplementary Figure S6: Absence of pluripotency markers Oct3/4 and Nanog in transplanted animals by R-PCR analysis. 7

8 Supplementary Figure S7. Supplementary Figure S7: Sequence alignment data supporting species-selectivity of the MLC2v primer set used to identify putative mouse-derived ventricular myocytes in the rat heart. 8

9 Supplementary able S2 is presenting the mean of Isl1 + cells counted in two non-sequential sections. sampled sections # Isl1 + number of cells counted # mean 110 (108 and 112) (138 and 142) (188 and 192) (238 and 242) (278 and 282) (328 and 332) (368 and 372) 71 9

Supplementary Data. Supplementary Table S1.

Supplementary Data. Supplementary Table S1. Supplementary Data Supplementary Table S1. Primer Sequences for Real-Time Reverse Transcription-Polymerase Chain Reaction Gene Forward 5?3 Reverse 3?5 Housekeeping gene 36B4 TCCAGGCTTTGGGCATCA CTTTATCAGCTGCACATCACTCAGA