Spatial transcriptomics of healthy and fibrotic human liver at single-cell resolution

Abstract

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cell types and their heterogeneity within the human liver, but the spatial organization at single-cell resolution has not yet been described. Here we apply multiplexed error robust fluorescent in situ hybridization (MERFISH) to map the zonal distribution of hepatocytes, spatially resolve subsets of macrophage and mesenchymal populations, and investigate the relationship between hepatocyte ploidy and gene expression within the healthy human liver. Integrating spatial information from MERFISH with the more complete transcriptome produced by single-nucleus RNA sequencing (snRNA-seq), also reveals zonally enriched receptor-ligand interactions. Finally, MERFISH and snRNA-seq analysis of fibrotic liver samples identify two hepatocyte populations that expand with injury and do not have clear zonal distributions. Together these spatial maps of the healthy and fibrotic liver provide a deeper understanding of the cellular and spatial remodeling that drives disease which, in turn, could provide new avenues for intervention and further study.

Fig. 1. Mapping hepatocytes within the architecture of the healthy human liver with MERFISH.

a Uniform manifold approximation and projection (UMAP) of all cells measured with MERFISH in the healthy human liver. b Heatmap displaying differentially expressed genes that identify each cluster. Normalized gene expression is indicated by color. Each column represents a single cell, and cells are grouped by cluster as indicated by color at the left of the heatmap. Genes are indicated on the top of the heatmap, and groups of genes characteristic of individual clusters are indicated on the bottom. Hierarchical clustering is shown at the right. Genes enriched in each cluster are shown. Genes may appear more than once if enriched in more than one cluster. c–e Distribution of cells expressing SDS and CYP2A6 (enriched in zone 1, c), ALDOB and ADH1B (most enriched in zone 2, d), and CYP2E1 and CYP1A2 (enriched in zone 3, e) in a section of liver tissue. Scale bars: 1000 μm. Expression level is measured in normalized gene expression. f Dot plot quantifying expression of genes mapped in c–e where the size of the dot represents the percentage of cells expressing a specific gene, and the color intensity indicates mean expression. g mRNA distributions across a single lobule. Each dot represents an individual mRNA transcript for the indicated gene oriented from a portal region at the top to a central region at the bottom. The relative abundance of each transcript across the lobule was then plotted (lower panel) for the same field of view. Scale bars: 50 μm. h Spatial distribution of cells assigned to each cluster of hepatocytes (Hep 1, Hep 2, or Hep 3). Hep 1 cells (purple) map to periportal areas (zone 1), Hep 3 cells (pink) map to pericentral areas (zone 3), and Hep 2 cells (brown) map between Hep 1 and Hep 3 cells (zone 2). Scale bar: 1000 μm.

Fig. 2. MERFISH maps non-parenchymal cells within the healthy liver.

a, b Spatial distribution of all cells defined by MERFISH within the architecture of the healthy liver for tissue from two different donors, donor 1 (a) and donor 2 (b). Scale bars: 1000 μm. c, e, g Spatial distribution of macrophage (c), HSC (e), and cholangiocyte and EC (g) populations mapped in the same tissue section as a. Scale bars: 1000 μm. d, f, h Spatial distribution of cells in the same tissue section as (a) colored by the normalized expression of the indicated marker genes. CD74 and CD5L identify macrophages (d), PDGFRB and LAMB1 identify HSCs (f), KRT7 identifies cholangiocytes, and DNAS1L3 identifies ECs (h). Scale bars: 1000 μm. i Dot plot quantifying expression of genes that identify non-parenchymal cells in the liver. The size of the dot represents the percentage of cells expressing a specific gene, and the color intensity indicates mean expression.

Fig. 3. Combining MERFISH and snRNA-seq to define spatial interactions.

a UMAP of snRNA-seq data for healthy human liver. Annotations for non-parenchymal cells were determined from the snRNA-seq alone while annotations for the hepatocytes were determined by merging data from both MERFISH and snRNA-seq and leveraging both marker gene expression and label-transfer of MERFISH labels. b Heatmap displaying differentially expressed genes that identify each cluster of snRNA-seq nuclei. Normalized expression level is indicated by the color bar. Each column represents a single cell, and cells are grouped by cluster as indicated at the top of the heatmap. Genes are indicated to the left of the heatmap, and groups of genes characteristic of individual cell clusters are indicated on the right. Hierarchical clustering is shown at the bottom. c Putative receptor-ligand interactions identified between zonal hepatocytes and notable non-parenchymal cells. Receptor-ligand interactions were mapped between each zone of hepatocytes (zone (z) 1, z2, z3). Only interactions that are uniquely enriched by hepatocyte zonation are shown. Gray circles indicate no interaction, and colored circles (zone 1 hepatocytes - purple; zone 2 hepatocytes - brown; zone 3 hepatocytes - pink) indicate receptor ligand co-expression.

Fig. 4. Multinucleated hepatocytes do not have unique spatial or gene expression distributions.

a Example MERFISH DAPI images showing hepatocytes containing single and multiple nuclei. Examples of multinucleated cells are indicated by white arrowheads. DAPI staining marks nuclei (white), and cell boundaries (light blue) were determined by Baysor with input from Cellpose. Scale bars: 20 μm. b Distribution of number of nuclei per hepatocyte. c Hepatocyte UMAP, as in Fig. 1, colored by the number of nuclei found within each hepatocyte. Source data are provided as a Source Data file. d Spatial distribution of hepatocytes with 1 or 2 detected nuclei in healthy human liver (donor 1, left and donor 2, right). Scale bar: 1000 μm. e Dot plots of hepatocyte genes for hepatocytes with 1 or 2 nuclei. The size of circles represents the percentage of cells expressing a gene in each group and the color intensity indicates mean expression. f Distribution of the transcripts per cell (left), area per cell, (middle) and RNA density per cell (right) for hepatocytes with the indicated nuclear content. Source data are provided as a Source Data file.

Fig. 5. MERFISH reveals conserved zonation and expansion of hepatocyte populations in fibrotic livers that do not follow zonal distribution.

a UMAP of fibrotic tissue measured with MERFISH. Clusters are indicated by color. Portal Hep and Central Hep show similarities to hepatocytes identified as zone 1, 2, and 3 cells from healthy liver, and Fibrotic Hep 1 and Fibrotic Hep 2 contain hepatocyte clusters that emerge with fibrotic injury. The HSC cluster shows similarities to both HSC 1 and HSC 2 cells from healthy liver, and the Mac cluster has similarities to both Mac 1 and Mac 2 clusters from healthy liver. b Relative abundance of each cell type identified in each sample (left to right: healthy donors 1–3 followed by fibrotic donors 4–6). Cell types are colored as in a. Source data are provided as a Source Data file. c–e Spatial distribution of cell populations in the fibrotic liver (donor 4), highlight hepatocytes that are similar to those seen in healthy samples (c), hepatocyte populations that are identified only when including fibrotic liver samples (d), and key non-parenchymal cells (e). Scale bar: 1000 μm. f–h Dot plot quantifying expression of genes differentially expressed in clusters seen in healthy measurements (Healthy zone 1, 2, or 3) or in fibrotic samples (Portal Hep, Central Hep, Fibrotic Hep 1, or Fibrotic Hep 2) for hepatocytes (f), macrophages (g), and HSCs (h). Macs Healthy 1 and Healthy 2 refer to Mac 1 and Mac 2 cells in healthy liver, and HSCs Healthy 1 and Healthy 2 refer to HSC 1 and HSC 2 cells in healthy liver.

Fig. 6. Combining MERFISH and snRNA-seq to analyze fibrotic livers.

a UMAP of snRNA-seq data for injured human liver. Annotations for the hepatocytes were determined by merging data from both MERFISH and snRNA-seq and leveraging both marker gene expression and label-transfer of MERFISH labels. b Heatmap displaying differentially expressed genes that identify each snRNA-seq cluster. Normalized expression level is indicated by the color bar on the right. Each column represents a single nucleus, and nuclei are grouped by cluster as indicated at the top of the heatmap. Genes are indicated at the bottom of the heatmap, groups of genes characteristics of individual cell clusters are indicated on the top, and cells are grouped by cluster as indicated by color at the left. c Dot plot quantifying expression of genes differentially expressed in clusters seen in healthy measurements (Healthy zone 1, 2, or 3) or in fibrotic samples (Portal Hep, Central Hep, Fibrotic Hep 1, or Fibrotic Hep 2) for hepatocytes. Dot plots quantifying expression of genes differentially expressed in macrophages (d) and HSCs (e) are shown. Macs Healthy 1 and Healthy 2 refer to Mac 1 and Mac 2 cells in healthy liver, and HSCs Healthy 1 and Healthy 2 refer to HSC 1 and HSC 2 cells in healthy liver.