Comparative analysis of cell lineage differentiation during hepatogenesis in humans and mice at the single-cell transcriptome level

Abstract

During embryogenesis, the liver is the site of hepatogenesis and hematopoiesis and contains many cell lineages derived from the endoderm and mesoderm. However, the characteristics and developmental programs of many of these cell lineages remain unclear, especially in humans. Here, we performed single-cell RNA sequencing of whole human and mouse fetal livers throughout development. We identified four cell lineage families of endoderm-derived, erythroid, non-erythroid hematopoietic, and mesoderm-derived non-hematopoietic cells, and defined the developmental pathways of the major cell lineage families. In both humans and mice, we identified novel markers of hepatic lineages and an ID3⁺ subpopulation of hepatoblasts as well as verified that hepatoblast differentiation follows the "default-directed" model. Additionally, we found that human but not mouse fetal hepatocytes display heterogeneity associated with expression of metabolism-related genes. We described the developmental process of erythroid progenitor cells during human and mouse hematopoiesis. Moreover, despite the general conservation of cell differentiation programs between species, we observed different cell lineage compositions during hematopoiesis in the human and mouse fetal livers. Taken together, these results reveal the dynamic cell landscape of fetal liver development and illustrate the similarities and differences in liver development between species, providing an extensive resource for inducing various liver cell lineages in vitro.

Fig. 1. scRNA-seq identified major cell types and cell lineage families in the human and mouse fetal livers.

a t-SNE plots showing the developmental stages (left) and cell clusters (right) of human (H) and mouse (M) fetal liver development. b t-SNE plots showing the expression levels of marker genes for each cell population. c Heatmaps showing the Pearson correlations of 13 major cell types between human (H) and mouse (M). d Differentially expressed genes in human (H) and mouse (M) cell populations. Each column represents a cell type and each row represents a gene. The TFs associated with each cell type are listed on the right. The color scheme is the same as a.

Fig. 2. Identification of novel markers of hepatic cells.

a t-SNE plots showing the expression levels of hepatobiliary marker genes. b Immunofluorescence showing the expression and distribution of FXYD1 and HNF4A in the W12 human (H-W12) and E17.5 mouse (M-E17.5) fetal livers. Scale bars, 20 μm. c Immunofluorescence showing the expression and distribution of GJB1 and HNF4A in the W12 human (H-W12) and E17.5 mouse (M-E17.5) fetal livers. Scale bars, 20 μm. d Immunofluorescence showing the expression and distribution of FGB and HNF4A in the W7 human (H-W7) fetal liver. The yellow arrowhead indicates the FGB⁺HNF4A⁺ hepatoblasts. The white arrowhead indicates the FGB^–HNF4A^– cells. Scale bars, 20 μm. e Schematics of strategies for the generation of Fgb-Cre^ERT2 transgenic mice. f Morphologies and tdTomato signals in the livers of E17.5 WT and Fgb-Cre^ERT2;Rosa26-tdTomato mice. Scale bars, 5 mm. g FACS gating and statistical analysis showing the percentage of tdTomato⁺DLK⁺/DLK⁺ (upper) and tdTomato⁺EpCAM⁺/EpCAM⁺ (lower) cells in E17.5 WT and Fgb-Cre^ERT2;Rosa26-tdTomato mice. n, number of embryos.

Fig. 3. Identification of two hepatoblast subpopulations.

a t-SNE plots showing the developmental stages (left) and clusters (right) of human (H) and mouse (M) endoderm-derived cells. b t-SNE plots showing the distinct clusters of ID3⁺ and ID3^– hepatoblasts in the W5 human (H-W5) and E11.5 mouse (M-E11.5) fetal livers. c The proportion of ID3⁺ cells in human (H) and mouse (M) hepatoblasts/hepatocytes at different developmental time points. d Differentially expressed genes in W5 human (H-W5) and E11.5 mouse (M-E11.5) ID3⁺ and ID3^– hepatoblasts. Each column represents a cell type and each row represents a gene. The TFs associated with each cell type are listed on the right. The color scheme is the same as b. e t-SNE plots showing the expression levels of marker genes. f Immunofluorescence showing the expression and distribution of ID3 and HNF4A in the W5 human (H-W5) and E11.5 mouse (M-E11.5) fetal livers. The arrowheads indicate ID3⁺ hepatoblasts. Scale bars, 60 μm. g The morphology of cultured hepatocytes (after 6-day culture from NCAM1⁺DLK⁺ hepatoblasts) and cholangiocyte tissue (after 10-day culture from NCAM1⁺DLK⁺ hepatoblasts). Scale bars, 20 μm. h Immunofluorescence showing the expression and distribution of HNF4A and SOX9 in cultured hepatocytes (upper) and cholangiocytes (lower), respectively. Scale bars, 15 μm.

Fig. 4. Conserved hepatoblast differentiation pathways between species.

a Upper: PCA plots showing the differentiation pathways of human (H) and mouse (M) hepatobiliary cells. 250 and 80 hepatoblasts/hepatocytes were collected for human and mouse samples at each time point, respectively. Lower: PCA plots showing the proliferation of human (H) and mouse (M) hepatobiliary cells. Arrows indicate the directions of differentiation. b Heatmaps showing differentially expressed genes during human (H) and mouse (M) hepatoblast differentiation. Each column represents a cell and each row represents a gene. The TFs of each gene group are listed on the right. The color scheme is the same as a. c Changes in the proportion of proliferative cells (S and G2/M phases) in human (H) and mouse (M) hepatoblasts/hepatocytes. d Heatmaps showing differentially expressed genes and expression switches during human (H) and mouse (M) hepatoblast-to-hepatocyte development. Each column represents a stage and each row represents a gene. The color scheme is the same as a. Dendrograms showing the results of hierarchical clustering of different stages. Black arrows indicate the time point of cell fate transition.

Fig. 5. Heterogeneity of quiescent human hepatocytes.

a Left: PCA plots of human (H) and mouse (M) quiescent hepatoblasts/hepatocytes. Right: Schematic summary of quiescent hepatoblast/hepatocyte development based on the PCA plots. b PCA plots showing the clustering of human VTN^low and VTN^high hepatocytes based on PC2-related genes. c Heatmap showing the expression of PC2-related genes in human hepatoblasts/hepatocytes ordered by PC2 values. Each column represents a cell and each row represents a gene. The color scheme of cell stages is the same as a. d Differentially expressed genes in VTN^low and VTN^high hepatocytes. The TFs of each gene group are listed on the right. e Expression levels of the marker genes VTN and RPL13 in human (H) and mouse (M) hepatoblasts/hepatocytes. f In situ hybridization of sequential sections showing the distribution of VTN and RPL13 in the W19 human (H-W19) and E17.5 mouse (M-E17.5) fetal livers. Scale bars, 50 μm. g GO analysis of genes differentially expressed in W19 human VTN^low and VTN^high hepatocytes.

Fig. 6. Erythropoiesis in the fetal liver.

a The proportion of primitive erythrocytes in human (H) and mouse (M) whole erythrocytes at different developmental time points. b UMAP plots showing developmental stages and cell types during erythropoiesis in the human (H) and mouse (M) fetal livers. c 3D-PCA plots showing the maturation of human (H) and mouse (M) erythroid progenitors. d PCA plots of mSTRT-seq data showing the maturation of E11.5–E17.5 mouse cKit⁺CD71^high cells. e Heatmap of mSTRT-seq data showing differentially expressed genes in E11.5–E17.5 mouse erythroid progenitors. Each column represents a stage and each row represents a gene. The TFs of each stage are listed on the right. f GO analysis of genes differentially expressed in cKit⁺CD71^high E11.5 and E17.5 cells. g Violin plots showing the expression levels of marker genes of E11.5–E17.5 mouse cKit⁺CD71^high cells validated by single-cell RT-qPCR. The y-axis represents the relative expression values normalized to Gapdh expression. Each dot represents a single cell. The black line within each violin plot indicates the median of the expression levels. n, number of single cells. h The morphology of E11.5 and E17.5 mouse cKit⁺CD71^high cells cultured for 3 days. Scale bars, 30 μm. i Statistical analysis showing the number of colonies for cultured E11.5 and E17.5 mouse cKit⁺CD71^high cells. n, number of biological replicates. j Statistical analysis showing the size of the colonies for cultured E11.5 and E17.5 mouse cKit⁺CD71^high cells. The white line indicates the median size of colonies. #, number of randomly selected colonies used to determine the size statistics.

Fig. 7. Hematopoietic pathways in the fetal liver.

a–c FDL (a), RNA velocities (b) and Monocle3 UMAP plots (c) showing the developmental trajectories of hematopoiesis in the human (H) and mouse (M) fetal livers. Lines indicate the directions of differentiation. d FDL plots showing the expression levels of hematopoietic lineage marker genes.

Fig. 8. Developmental pathways of mesoderm-derived cells.

a Diffusion maps showing the developmental pathways of Kupffer cells in the human (H) and mouse (M) fetal livers. b Heatmaps showing the differentially expressed genes of Kupffer cells in the human (H) and mouse (M) fetal livers. Each column represents a cell and each row represents a gene. The color scheme is the same as a. The TFs of each gene group are listed on the right. c Diffusion maps showing the developmental pathways of liver endothelial cells in the human (H) and mouse (M) fetal livers. d Heatmaps showing the differentially expressed genes of liver endothelial cells in the human (H) and mouse (M) fetal livers. The color scheme is the same as c. e Diffusion maps showing the developmental pathways of hepatic stellate cells, septum transversumal cells, and mesothelial cells in the human (H) and mouse (M) fetal livers. f Heatmaps showing the differentially expressed genes during mesenchymal cell differentiation in the human (H) and mouse (M) fetal livers. The color scheme is the same as e.