Supplementary Figure 8: Signalling in endoderm differentiation and expansion.

(A) To test the role of LIF signalling in nEnd expansion, LIF (1:1,000) or JAKi (either 1 μM or 10 μM) was added to passage 5 nEnd cultures, and to XEN cells. After 6 days, cells were counted and then stained for PDGFRA. Cells were analysed by flow cytometry, and the different numbers of live (DAPI-ve) nEnd cells (PDGFRA + ve) are shown, relative to the DMSO control in each replicate (white bars). N = 3 experimental replicates plotted seperately. (B) nEnd cells were cultured in the presence of LY (10 μM or PD03 (1 μM) for 6 days, then counted. Mean counts (n = 3 biological replicates) of independent E14TG2A-derived cell lines (NanogGFP, SOX2-GFP, and HVMC) are shown, error bars show ± s.d., P values: for LY to DMSO, P = 0.008, for PD03 to DMSO, P = 0.004. (C) The impact of insulin concentration on naïve ESC expansion. SOX2-GFP ESCs were differentiated for 5 days to PrE, then analysed by flow cytometry. On day 1, the concentrations of insulin shown were added to the media. Left graph shows the final percentage of PrE (PDGFRA + ve, SOX2-GFP-ve cells) at the end of differentiation. Three replicates are plotted (n = 3), with the mean shown in red. Both graphs show the same experiment, left graph with scale on the X-axis, right graph without to show effect at very low concentrations. (D) Flow cytometry dot plots of rtO EpiSCs differentiated to ADE for 4 days in Act and Wnt3a, and analysed for induction of definitive endoderm using the combination of CXCR4-PE and c-Kit-APC antibodies. Percentages of live cells shown for each quadrant. Dot plots are representative of 4 differentiations. (E) Insulin inhibits EpiSC to ADE differentiation. rtO EpiSCs were differentiated to ADE for 4 days in standard ADE medium, or medium supplemented with 12.5 ng μl⁻¹ insulin. Flow cytometry dot plots show staining for CXCR4 and c-Kit. Quadrant percentages shown are double negative undifferentiated cells (red) and double positive ADE (black). Dot plots are representative of 7 differentiations (F) HRHnG EpiSCs were differentiated for 4 days to ADE in the presence of LIF (1:1,000), insulin (12.5 μg ml⁻¹), insulin receptor inhibitor GSK 1838705, and analysed by flow cytometry. Populations were gated on HexRS and CXCR4 and categorized as follows: Epi (HexRS⁻/CXCR4⁻), Mesendoderm (HexRS⁻/CXCR4⁺), and ADE (HexRS⁺/CXCR4⁺). Data represented as the mean (n = 4 independent experiments) +/−sd. P values by t test (left to right) = Epi 0.001617, 0.000098, 0.005683, Mes 0.003460, 0.010756, ADE 0.000833, 0.000249. (G) FACS gating strategy example, showing steps taken to set gates for live cell analysis or sorting based on DAPI and an example reporter (GFP) and conjugated antibody (CD31-APC).