Fig. 5: ARPKD HOs have an expanded population of myofibroblasts that resemble those in fibrotic human liver tissue.

a KEGG pathway enrichment analysis identifies 3 gene networks whose expression is most increased in cluster 0 cells: protein digestion and adsorption, JAK-STAT signaling, and extracellular matrix (ECM)-receptor interactions. b A volcano plot showing the differentially expressed genes (fold change > 1.5, adjust p-value < 0.05) when the transcriptomes of cluster 0 and cluster 3 are compared. The 20 genes whose mRNAs exhibit the highest level of differential expression are highlighted, which include STAT3, PDGFRB, and SOCS3. The gray dashed line indicates the 1.5-fold cutoff for a differentially expressed gene. A red (or blue) color indicates that a gene is up (or down) regulated in cluster 0 (vs. cluster 3); a black color indicates that the expression level was not significantly different. The differentially expressed genes among the groups were determined using the Wald test in DESeq2. c Violin plots showing three genes (PDGFRB, STAT3, SOCS3) whose mRNAs were markedly increased in cluster 0 cells relative to cluster 3. d Day 21 control and ARPKD organoids were immunostained with an anti-PDGFRB antibody. PDGFRB is highly expressed in ARPKD, but not in control, HOs. Scale bar, 50 μm. e A scatter plot of CyToF data performed with anti-PDGRFΒ and anti-CD56 antibodies shows that ARPKD organoids have a markedly increased amount of PDGFRB⁺ cells (9.0%) relative to control organoids (2.1%). f bhSNE maps (upper) generated from CyToF data indicate that ARPKD HOs have an increased amount of SMA⁺ cells. (Lower) Representative images of HOs immunostained with anti-smooth muscle actin (SMA) and anti-CK8 antibodies. The yellow dotted lines indicate ductal structures. While only a few SMA⁺ cells were present at a limited number of sites within control organoids (indicated by an arrow); large clusters of SMA⁺ cells were present within ARPKD organoids (indicated by arrowhead) and were located near ductal structures. Scale bar, 10 μm. g Normal and ARPKD liver tissue was immunostained with antibodies to PDGFRB, CK8, and SMA. PDGFRB expression was much higher in ARPKD than in control liver tissue. Also, SMA⁺ cells were only located within perivascular regions of normal liver tissue; but they infiltrate the parenchyma of ARPKD liver tissue (as shown in the inset). The white dashed line separates epithelial and mesenchymal areas. Scale bar, 50 μm. h ARPKD and Control HOs were immunostained with anti-CK8 and anti-phospo-STAT3 (Tyr705) antibodies. Phospho-STAT3 was extensively expressed in bile duct and mesenchymal cells in ARPKD HOs. The right panels are enlargements of the boxed yellow area in the ARPKD organoid; the arrows indicate mesenchymal cells with Phospho-STAT3 within the nucleus. Scale bar, 5 μm. i, j ARPKD and normal liver tissues were immunostained with antibodies to CK8, Phospho-Stat3, and CK8, and were counterstained with Hoechst. i Low (top) and high (bottom) power views of ARPKD liver tissue immunostained with antibodies to CK8, PDGFRB, and Phospho-STAT3. A myofibroblast area with is within the dotted line. A high-power view of the yellow boxed area shows phospho-STAT3 is co-localized within PDGFRB⁺ myofibroblasts. Scale bar, 5 μm. j Phospho-STAT3 is extensively expressed in an enlarged bile duct within ARPKD liver tissue, but its level of expression is much weaker in normal bile ducts. k The bar graph shows the GSEA results (normalized enrichment score (NES)) obtained from each analysis; and the false discovery rate (FDR) for each comparison is shown at the top of each bar. The myofibroblast gene expression signature was very strongly associated with cirrhotic and NASH liver tissue, but this signature was not induced by obesity alone or by the presence of a HCC. GSEA was also performed by comparing the cluster 0 cell transcriptome with overlapping genes present in the expression signatures defined from the scRNA-Seq analysis of myofibroblasts and B cells present in cirrhotic human livers³⁹. Bottom: GSEA results for the correlation of the ARPKD myofibroblast gene expression signature with that in liver tissues obtained from: 10 normal and 10 cirrhotic subjects (Cirrhosis); 98 normal and 126 NASH subjects (NASH).