Supplementary Figure 3: Additional characterisation of nEnd cultures.

(A) nEnd lines can be frozen and thawed. HRHnG nEnd showing the recovery after thawing, and expansion until confluency at 9 days, scale bars 200 μm. On day 9, the cells were analysed by flow cytometry, and expressed both Hnf4αGFP and PDGFRA (dot plot). Images are representative of 6 differentiations and more than 10 different photographed fields for each. (B) nEnd lines have a normal karyotype. Images show p13 HRHnG-BFP nEnd (left, n = 23 cells counted) and p6 HV5.1 (right, n = 9 cells counted) nEnd. Typical are shown. Both nEnd lines have an average chromosome counts of 40, scale bars 25 μm. The figure shows two lines, but the source data contains a third (p4 HRHnG, n = 10 cells counted). (C) Progression of PDFGRA and Hnf4αGFP expression during PrE differentiation. HRHnG ESCs differentiated from 2i/LIF to PrE over 8 days. PDGFRA expression emerges first on day 3, and this population begins to express both Hn4aGFP and PDGFRA on days 4–5. The proportion of PrE increases between days 5 and 8, and becomes more homogeneous. This increased homogeneity is also apparent by microscopy (lower panel, scale bar 200 μm). Dot plots are representative of 4 differentiations. (D) Expression panels for pluripotency, endoderm and immunity markers based on the set of significantly changing genes defined based on ANOVA. Values indicate intensity values from the microarray (from 0.2 to 5). (E) Pairwise comparisons of microarray expression data, comparing: Undifferentiated ESCs to differentiated cultures (Diff), Late passaged nEnd (Late) to XEN cells, and differentiated cultures (Diff) to Late passaged nEnd (Late). Comparisons were used to carried out using ExAtlas (http://lgsun.grc.nia.nih.gov/exatlas) to generate lists of underexpressed and overexpressed genes (fold change cut off = 2, FDR threshold ≤ 0.05). The position of key markers is indicated in each comparison. Genes lists were uploaded to DAVID for GO analysis, and are shown in Supplementary Table 4. (F) Normalised data was used to generate the heatmap shown in Fig. 3i. This data was grouped into 20 significant expression clusters. Expression values of each cluster, and each triplicate, were averaged, to give the simplified heatmap overview shown here. Data has been sorted according to the expression in ESCs. (G) Key clusters from F and Fig. 3i were analysed further. Clusters were chosen based on high expression. GO analysis of all clusters is shown in Supplementary Table 4. A summary of the top 5 GO terms is shown for each cluster, sorted based on P value as calculated by DAVID (https://david.ncifcrf.gov).