Extended Data Figure 7: Cell composition in human longer-term LBs, adult and fetal human liver, and mouse embryonic liver.

a, scRNA-seq was performed on LBs grown for an extended period in culture (LB1 = 11 days in 3D, LB2 = 12 days, LB3 = 7 days, LB4 = 13 days, LB5 = 10 days; 173 cells total). PCA was used to identify genes describing cell populations, and tSNE was used to cluster cells. We identified hepatic, endothelial, and MC clusters. b, Expression of genes marking each cluster is coloured (log₂(FPKM scale)). c, Scaled heatmap (yellow, high; purple, low) of genes specifically expressed (average difference >2, power to discriminate >0.5) in each cluster (labelled at the bottom), with exemplary genes from each cluster labelled on the right. Cells are in columns, genes in rows. d, scRNA-seq was performed on human adult (5 donors, age 21 – 65, 256 cells) and fetal (gestation weeks 10.5 and 17.5, 238 cells) after enrichment for hepatic lineage cells. PCA was used to identify genes describing cell populations, and tSNE was used to cluster cells. We identified hepatic, endothelial, mesenchymal, and immune lineage cells both in fetal and in adult tissues. e, Cluster assignments are highlighted on the tSNE plot and expression of genes marking each cluster is coloured (log₂(FPKM scale)). f, Scaled heatmap (yellow, high; purple, low) of genes marking each cluster (numbered at the bottom), with exemplary genes from each cluster labelled on the right. Cells are in columns, genes in rows. g, Marker genes (average difference >2, power to discriminate cluster >0.5) were analysed for GO enrichments, and representative top enrichments are shown with the P value (−log₁₀). h, scRNA-seq was performed on mouse hepatoblasts isolated from mouse liver at E14.5, E15.5, and E16.5 (92 cells total). PCA was used to identify genes describing cell populations, and tSNE was used to cluster cells. We identified hepatic, mesenchymal, and immune cell clusters. i, Expression of genes marking each cluster is coloured (log₂(FPKM scale)). j, Scaled heatmap (yellow, high; purple, low) of genes specifically expressed (average difference >2, power to discriminate >0.5) in each cluster (labelled at the bottom), with exemplary genes from each cluster labelled on the right. Cells are in columns, genes in rows. k, Heatmap showing normalized expression (z-score) of genes differentially expressed between fetal and adult human hepatocytes. Column, gene; row, cell. Heatmap shows that fetal hepatocyte marker genes are highly expressed in mouse AFP⁺, ALB⁺, DLK⁺ early hepatocytes/hepatoblasts, corroborating the human fetal data.