Supplementary Figure 1: Characterisation of the HRHnG reporter cell line and PrE induction in vitro.

(A) Hnf4α and Hhex targeting constructs showing genomic locus, linearised and digested constructs with targeting arms, and incorporation into the locus by homologous recombination, both before and after treatment with Cre recombinase. Southern blot probes are shown in blue, and the respective size of the digested DNA is shown with two headed arrows. (B) Southern blot of HRHnG cell line showing the wild type gene size (22–23 kb) and the labelled targeted fragment (6 kb) before selection cassette removal. Clones HRHnG 44 and 45 are correctly targeted. HRHnG_44 ESCs were treated with Cre recombinase to give rise to the sub-clones 44_6, 44_7, 44_18, 44_19 and 44_22. The labelled band changes to 23 kb after successful cassette excision. 44_18 still contains the selection cassette and was not used. Targeted sub-clones were karyotyped: the chromosome count for each is 40 (average result of 10 cell counts, from 6 independent clones), scale bars 25 μm. (C) HRHnG ESCs were differentiated to ADE using our previously published protocol, shown in the cartoon. ADE (left) was differentiated for 5 further days to hepatic. Cells were not sorted before plating into hepatic differentiation, and hepatic colonies grew in small clusters surrounded by non-hepatic cells (right). Cells express Hnf4αGFP (green) or HexRS (red), scale bars 200 μm. Images are representative of 3 differentiations and more than 10 different photographed fields. (D) Dot plot illustrating qRT–PCR of HRHnG ESCs differentiated for 4 days in Activin and Wnt3a. Differentiated cells were sorted for RNA based on GFP expression and analysed for key markers: Oct4 (ESC), Pdgfra, Hnf4α Sox7, Gata6 and Hhex (PrE), each normalised to unsorted expression and log2 transformed. N = 3 biological replicates and the mean value is shown by the horizontal line and standard error shown. Source data provided in Supplementary Table 10. (E) Western blot showing Stat3^−/− HexVenus ESCs generated using a high-fidelity CRISPR nuclease. Clones Nu1c1, Nu1c15, and Nu2c6 were used for the experiments in Fig. 1. Cells were cultured in 2i/LIF. (F) Dot plot showing qRT–PCR time course of HRHnG ESCs cultured in 2i/LIF and then differentiated to PrE over 7 days. XEN and definitive endoderm (ADE) were included for comparison. Key lineage markers are shown for pluripotency (Oct4, Nanog, Klf4), primitive streak/mesendoderm (Foxa2, Gsc, T), and endoderm (Sox17, Gata6, Gata4, Hnf4α, PDGFRA, Sox7). All expression data is normalised to ESC controls, with the mean and standard error shown for n = 3. The definitive endoderm samples are those shown in Supplementary Fig. 2C, but renormalized to ESCs for comparison. Source data provided in Supplementary Table 10.