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Additional Supporting Information may be found in the online version of this article.

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sc-12-0422_sm_SupplFigure1.tif1356KFigure S1. Extended analysis of EC differentiation. (A): Flow cytometric analysis of the effects of various BMP4 concentrations on KDR expression on day 6 of differentiation. (B): Flow cytometric analysis of the mature EC differentiation state on day 6 before KDR+ cell sorting using PECAM and VE-Cadherin. The control population consisted of cells labeled with isotype control antibodies. (C): Flow cytometric analysis for EPHB2 staining on PECAM / VE-Cadherin double positive cells on day 14. HAEC, HSVEC, and HLEC were used as positive and negative controls and H9 ECs were differentiated from ES cells.
sc-12-0422_sm_SupplFigure2.pdf673KFigure S2. Control immunofluorescence staining. (A) Staining of positive control (HMVECs) and negative control (H1 ES) with PECAM1 and eNOS primary antibodies (green). The same alexa-488 secondary was used to visualize both primary antibodies. Negative control no primary wells were stained with secondary antibody only. (B) Staining for VE-Cadherin or vWF (red). The same alexa-594 secondary was used to visualize both primary antibodies. (C) Dil-Ac- LDL alexa 594 uptake (red) in positive control (HMVEC) or negative control (H1 ES) cells. Negative control cells received no Alexa 594 conjugated LDL. Nuclei in all panels visualized with DAPI (blue).
sc-12-0422_sm_SupplFigure3.pdf192KFigure S3. Extended microarray analysis related to Fig. 4. (A): Log2 intensity plots of all samples from four groups of cells; ESC ECs (H1, H7, H9, H9 cont.), iPSC ECs (iPS1, iPS2A, iPS2B, iPS3, iPS3 Cont.), primary ECs (HAEC, HSVEC, HLEC )and ESCs (H9 ES). Red points are between group comparisons. Values in lower left are Pearson's R values of log transformed values. (B): Heat map and clustering analysis of genes specifically expressed in endothelial cells. Scale extends from 2.5 to 13. Red indicates log2 intensity higher than 7.5, and green lower than 7.5. (C): Heat map and clustering analysis of genes culled from the literature that are specifically expressed in different endothelial cell subtypes. Overlapping bars indicate genes expressed in two of the three primary cell lines. Scale extends from 2.5 to 13. Red indicates log2 intensity higher than 7.5, and green lower than 7.5.
sc-12-0422_sm_SupplFigure4.tif112KFigure S4. Microarray data validation by qRT-PCR of genes differentially expressed between ES-ECs and iPS-ECs by at least 4 fold. Average gene expression from three biological replicates is plotted and error bars show standard deviation. Primary EC bar is average of HAECs, HSVECs and HLECs. RNA samples from H9 hES cells (ES), day 6 H9 differentiated cells (KDR–), or endothelial precursors (KDR+), are included for reference. Statistical significance was determined using one-way ANOVA with Bonferroni's multiple comparison post hoc test(* P< 0.05, ** P< 0.01, *** P< 0.001, **** P< 0.0001).
sc-12-0422_sm_SupplFigure5.tif239KFigure S5. Microarray data validation by qRT-PCR of genes differentially expressed between primary-ECs and pluripotent derived-ECs (ES-ECs and iPSECs). Average gene expression from three biological replicates is plotted and error bars show standard deviation. RNA samples from H9 hES cells (ES), day 6 H9 differentiated cells (KDR–), or endothelial precursors (KDR+), are included for reference but were not included in the statistical analysis because of the null hypothesis being tested, i.e., that there is no difference in expression between primary ECs and pluripotent derived ECs. Statistical significance was determined using one-way ANOVA with Bonferroni's multiple comparison post hoc test(* P< 0.05, ** P< 0.01, **** P< 0.0001).
sc-12-0422_sm_SupplFigure6.tif242KFigure S6. EC specific genes expressed in indicated cell types. Average gene expression from three biological replicates is plotted and error bars show standard deviation. Statistical significance was determined using one-way ANOVA with Bonferroni's multiple comparison post hoc test(* P< 0.05, ** P< 0.01, *** P< 0.001, **** P< 0.0001).
sc-12-0422_sm_SupplFigure7.tif241KFigure S7. Microarray data validation by qRT-PCR of genes differentially expressed between (A) Arterial (B) Venous and (C) Lymphatic EC subtypes . Grey bars show the mean expression level from each of the primary EC types and error bars show technical standard deviation as a reference to compare the pluripotent derived ECs. Black bars show the average of three biological replicates of each EC type (ES or iPS derived) and error bars show standard deviation. RNA samples from H9 hES cells (ES), day 6 H9 differentiated cells (KDR-), or endothelial precursors (KDR+), are included for reference but were not included in the statistical analysis. ES-ECs and iPS ECs were compared with students t test and no genes showed statistical differences in expression between these two pairs.
sc-12-0422_sm_SupplInfor.pdf28KSupplementary Data
sc-12-0422_sm_SupplTable1.pdf7KTable S1. Differential Gene Expression Method A vs Method B. Twofold Cutoff (P<0.05)- 7 Protein Coding Genes.
sc-12-0422_sm_SupplTable2.pdf10KTable S2. Differential Gene Expression iPS-ECs vs. ES-ECs. Threefold Cutoff (P<0.05) - 18 Protein Coding Genes.
sc-12-0422_sm_SupplTable3.pdf27KTable S3. Differential Gene Expression iPS-ECs vs. ES-ECs. Twofold Cutoff (P<0.05) - 147 Protein Coding Genes.
sc-12-0422_sm_SupplTable4.pdf38KTable S4. iPS-EC Specific Gene Signatures Compared to Three Other Reports of iPS Specific Gene Signatures (Gupta et al., Chin et al., Marchetto et al.). Comparison of Chin et al. done with late iPS vs ES cell data set. Green= iPS-EC Gene Expression Fold Change Matches Direction of Fold Change in Report Cited, Red cells= iPS-EC Gene Expression Fold Change is Opposite to Direction of Fold Change in Report Cited.
sc-12-0422_sm_SupplTable5.pdf40KTable S5. Summary of Top 25 GO Categories for Genes Differentially Regulated by Twofold or Greater Between iPS-ECs and ES-ECs Including Gene Lists for Each GO Category.
sc-12-0422_sm_SupplTable6.pdf138KTable S6. Differential Gene Expression pcdECs vs. Primary ECs. Threefold Cutoff (P<0.05) – 839 Protein Coding Genes

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