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Fig. S1. Characterisation of hESCs derived HLCs. A. Semi quantitative PCR was used to determine the emergence of hepatic endoderm during the human embryonic stem cell differentiation protocol. Cells were collected daily during the differentiation and RNA was extracted, the resulting cDNAs were analysed for hepatic gene expression. α fetoprotein (AFP), albumin (ALB), tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) were first detected by day 6, whilst transthyretin (TTR) was first detected by day 5. B. Flow cytometry plots are provided for days 6, 12 and 18 in the differentiation procedure. The secondary antibody controls are shown in black and the Ki67 labelled cells are shown in grey C. hESC-derived HLCs were stained a 3 time points following differentiation. At day 7, 94% (±2.5) HLCs stained positive for AFP; at day 9, 94% (±2) HLCs stained positive for AFP; and at day 12, 95% (±3) HLCs stained positive for AFP.

Fig. S2. QPCR was carried out to verify the increasing presence of human mesenchymal cells in the spleen at 7 days and 31 days post-transplant. Human TAQMAN primers were used to detect human alpha smooth muscle actin (α SMA) and collagen-1. Human αSMA and collagen-1 were detected in mice transplanted with differentiated HLCs. Results are displayed as fold expression over mouse control RNA.

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