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STEM_621_sm_suppinfofig1.eps3689KSupplementary Figure 1: Endogenous WNT activity facilitates baseline activation of GSC in early EBs. a. Effect of Activin A and DKK1 on the percentage of GFP+ cells obtained from dissociated 2-3 day-old GSC-GFP EBs. Bars show fold induction compared to the control, and error bars represent standard errors. n=3. b-d. FACS analysis of dissociated 3 day old EBs. b. Control EBs. c. EBs treated with 67ng/ml ActivinA. d. EBs treated with 67ng/ml ActivinA and 200ng/ml DKK1.
STEM_621_sm_suppinfofig2.eps697KSupplementary Figure 2: Major organizer related genes upregulated in both GSC-GFP+ cells and dorsal pieces obtained from st.10.5 Xenopus laevis embryos. the graph presents the log values of the change in expression of the genes. For each gene, its enrichment is presented in GSC-GFP+ vs. GSC-GFP- hESC derived cells; and in dorsal vs. ventral pieces obtained from st. 10.5 frog embryos.
STEM_621_sm_suppinfofig3.eps6887KSupplementary Figure 3: Genetic labeling and molecular analysis of CER1+ cells. a. A scheme of a BAC containing the CER-GFP reporter construct. The two exons of CER1 ORF were replaced with the sequence coding for eGFP adjacent to the neomycin resistance gene under constitutive SV40 regulation. b. Early EBs made of CER-GFP cells show clusters of GFP expressing cells. Left - bright field; Center - dark field; Right - overlay of bright and dark fields. c. FACS analysis of dissociated 2 days old EBs. Left- CER-GFP cells; Right - CER-GFP cells treated with 67ng/ml Activin A. d. Effect of Activin A concentration on the percentage of GFP+ cells obtained from dissociated 2 days old CER-GFP EBs. Error bars represent standard errors. Asterisk indicates p-value≤0.05 under 2 sided paired t-test (n=2). e-g. CER-GFP cells were aggregated into EBs in the presence of 67ng/μl Activin A and sorted after 2 days. e. Relative CER1 expression in the sorted populations, as deduced from real time PCR. f. Heat map analysis of gene expression in GFP+ and GFP- populations obtained from Activin A treated EBs of GSC-GFP and CER-GFP cells, 2 or 3 days after aggregation. The main organizer related genes upregulated in the GSC+ cell population show a similar expression pattern in the CER1+ cells when mRNA extracted from them is hybridized to Affymetrix Gene ST1.0 microarray. g. Gene expression pattern of CER1+ cells is highly similar to that of GSC+ cells. A dendogram analysis by Pearson correlation was performed on the genes which showed highest substantial differences (>1 log difference, either upregulated or downregulated) between GSC+ and GSC- cells obtained from 2 day old EBs treated with Activin A. CER1+ and GSC+ cells from 2 day old EBs showed the highest similarity. Adult tissue average expression levels are based on data published by Affymetrix.
STEM_621_sm_suppinfotable1.tif88KSupplementary Table 1: a. Analysis of the number of secondary axes obtained from transplantations of EBs into frog embryos. b. Analysis of the number of secondary axes obtained from injection of GSC-GFP cells into frog embryos

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