Figure 1: Endogenous Wnt signalling reveals distinct subpopulations of hESCs.

Analysis of transgenic 7xTCF-GFP hESCs in standard self-renewing conditions. (a) TCF-GFP expression pattern in live hESC colonies. Scale bar, 200 μm. (b) FACS analysis of TCF-GFP expression in live cells after 4 days of IWP2, Wnt3a or CHIR99021 treatment. Pre-gated for live, non-mouse cells. (c) Quantitative Clonogenic Potential Assay. Double FACS-sorted populations of TCF-GFP^high and TCF-GFP^low cells were plated as single cells in standard hESC. Clonogenic potential was determined by counting the number of alkaline phosphatase (AP)-positive (left) and OCT4-positive (right) colonies 7 days after plating. Error bars show the range of two biological replicates and are representative of three independent experiments. (d) Gene expression analysis in double FACS-sorted populations of >99.5% purity. The ratio of glyceraldehyde 3-phosphate dehydrogenase-normalized gene expression level in TCF-GFP^high hESCs divided by TCF-GFP^low hESCs is shown. ND, not detectable. ±s.d of three biological replicates and are representative of two independent experiments. (e) GFP expression pattern of live colonies derived from plating single cells of >99.5% pure populations at clonal density (500 cells per cm²) in standard self-renewing conditions. The starting populations are indicated on the x axis, examples of the three GFP expression patterns counted are shown in a. (f) FACS analysis of GFP expression in hESC cultures 9 days after plating single cells from the indicated population of sorted cells. %GFP^pos is based on live, non-mouse cells. (g) FACS analysis of GFP expression in hESC passage 2 cultures initiated from single colonies that were entirely GFP^low or GFP^high. %GFP^pos is based on live, non-mouse cells.