Fig 2: HSEs display altered expression patterns and lineage paths(A–D) Immunostaining of (A) the terminal differentiation markers FLG and LOR, (B) the structural proteins DSG1 and COL17A1, (C) the BAS stem cell markers KRT15 and KRT19, and (D) the HSE-unique markers PSCA and VIM. Shown are human neonatal skin (top), day 28 GelHSEs (center), and day 28 FibHSEs (bottom). Feature plots (right) show RNA expression of the indicated markers for each sample type. Scale bars, 100 μm. Dashed lines denote the epidermal-dermal junction.(E) Pseudotime inference of epidermal keratinocytes from the integrated datasets.(F) Cell lineage diagram of keratinocytes from the integrated datasets. Edge weights denote the probability of transition to each cluster. Dot size denotes number of cells.(G) Splicing kinetics depicted as RNA velocity streams calculated using the Python package scVelo.(H) Quantification of Cellular Entropy (ξ) using the R package SoptSC.

Fig 3: Double BAP1- and PRKAR2A-mutant hepatocyte organoids exclusively adapt and thrive in a ductal cell environment.a, Experimental set-up to test the capacity of the different organoid lines to grow in an environment stimulating ductal cell growth. b, Schematic representing the culture time under which the different organoid lines could be maintained in ductal medium. Dashed line indicates a splitting (expansion) event. Results are derived from observations in n = 3 independent lines per genotype. See Supplementary Fig. 7b. c, Representative brightfield images of (arrested) wild type organoids versus (growing) BAP1KO;PRKAR2AKO organoids in ductal medium at 30 days post medium switching. d, Line plots showing the acute growth following single cell dissociation in hep or ductal medium of the different lines over 15 days as determined by ATP assay. Data represent growth percentage as compared to day 1. Mean ± SD is plotted. n = 2 independent replicates per genotype. **P < 0.01, ***P < 0.001 versus hep medium; two-tailed Student’s t-test. e, Schematic of forskolin-mediated PKA stimulation. f, Experimental set-up to evaluate the effect of forskolin on the growth of the different lines. g, Representative brightfield images of wild type organoids cultured from single cells in hep medium with or without forskolin supplementation at day 50. h, Representative brightfield images of BAP1KO;PRKAR2AKO organoids cultured from single cells in hep medium with or without forskolin supplementation over 50 days. i, Quantification of the area of BAP1KO;PRKAR2AKO organoids when cultured in hep medium with or without forskolin supplementation over 50 days. n = 65, 49 and 72 organoids for hep medium and n = 52, 46 and 49 organoids for hep medium + forskolin at day 4, 15, and 45, respectively. **P < 0.01, ***P < 0.001 versus hep medium; two-tailed Student’s t-test. j, qPCR analysis of expression of selected hepatocyte (AFP, ALB, HNF4A), ductal/progenitor (KRT7, KRT19), and proliferation (MKI67) markers in wild type and BAP1KO;PRKAR2AKO organoids cultured in the indicated medium. n = 3 independent measurements per gene and condition. k, Representative image of KRT staining of a BAP1KO;PRKAR2AKO organoid cultured in ductal medium. c, g, h, k Representative of n = 3, 3, 3 and 2 independent experiments, respectively. Scale bars, 200 μm (low mag) and 50 μm (high mag) (c), 200 μm (g, h) and 50 μm (k). Exact sample size and exact P values for d and i are provided in the source data. Source data are provided as a Source Data file.

Fig 4: Temporal transcriptomic characterization of human fetal hepatocyte organoid growth from tissue.a Experimental strategy to temporally address the transcriptomic changes associated with FH organoid growth from tissue. b Representative brightfield images of FH organoids outgrowing from tissue at day 7 post seeding (left) and an established organoid line. Scale bar = 300 μm. c Representative images of immunofluorescence staining for AFP, ALB, CK7 and CK19 in FH organoids. Scale bar = 150 μm. d Representative image of immunofluorescence staining for Ki-67 and F-actin (left) and MRP2 (right) in FH organoids. Asterisks indicate binucleated hepatocytes. Scale bar = 50 μm. e PCA plot visualizing the temporal transcriptomic changes underlying FH organoid growth from tissue across n = 2 donors. f Heatmap displaying the temporal expression patterns of genes significantly differently expressed at least at one time point versus 0 h (|log2FC| > 0.5, p-adj < 0.05) based on responses of n = 2 donors. The expression patterns are visualized as row Z-scores. The n indicates the number of genes belonging to each cluster. g Temporal Z-score expression of the genes identified in the different fetal tissue gene clusters across n = 2 donors. Mean ± SD is plotted, clusters 1–7: n = 3086, 2509, 3083, 4260, 2872, 2111, and 2330 genes, respectively. h Biological processes/cell types associated with the different temporal gene clusters, based on GO-term enrichment analysis and manual inspection. b–d Representative of characterization of n = 4 expanding FH organoid cultures. Source data are provided as a Source data file.

Fig 5: Characterization of primary human hepatocyte organoid cultures.a Representative low- and high-magnification brightfield images of expanding PHH organoids (passage 4) in the IL6+FXRa condition. Scale bar = 400 μm (low mag) and 100 μm (high mag). b Representative image of H&E staining of PHH organoids. Scale bar = 75 μm. c Representative immunofluorescence staining for Ki-67 of PHH organoids. Scale bar = 100 μm. d, e Representative immunofluorescence staining of PHH organoids for (d) ALB, AFP, CK19, and CK7 and (e) A1AT and HNF4A, CYP3A4, and ZO1. Scale bar = 100 μm (d, e). f Single-cell profiling of PHH organoids. g UMAP plots of the indicated markers. h Single-cell profiles of the FH and PHH organoids (originating from a single library, with the two cultures distinguished based on genotype demultiplexing). i UMAP plots of the indicated fetal and adult hepatocyte markers in the combined single cell datasets. j Schematic illustrating the dynamics underlying human hepatocyte growth and differences between fetal and adult. a–e Representative of characterization of n = 2 expanding PHH organoid cultures.