Wnt signalling maintains self-renewal of human hepatoblasts without blocking their differentiation

Abstract

Hepatoblasts play a key role in liver organogenesis by differentiating into hepatocytes and cholangiocytes, the main functional cell types of the liver. Mouse studies have demonstrated an association of Wnt signalling with proliferation and differentiation of hepatoblasts. However, the exact function of this pathway in hepatic development has not been fully uncovered, especially in human. Here, we use hepatoblast organoids derived from human foetal livers to investigate the importance of Wnt signalling in self-renewal and cell fate decisions during liver development. We first showed that Wnt plays a key role in hepatoblast self-renewal capacity in vitro by maintaining their proliferative state. However, Wnt was not sufficient to block differentiation of hepatoblast organoids into hepatocytes or cholangiocytes, suggesting that other factors are necessary to maintain hepatoblast bipotency. Finally, single-cell transcriptomic analyses revealed that Wnt signalling activity correlates with hepatoblast proliferation in the human foetal liver, suggesting that the role for Wnt could be conserved in vivo. Taken together, our results support a model in which Wnt signalling acts to preserve the proliferative capacity of hepatoblasts without being sufficient to maintain their bipotent state.

Keywords: Cholangiocyte; Hepatoblast; Hepatocyte; Liver; Organoid.

Fig. 1.

Absence of Wnt signalling does not induce differentiation of HBOs. (A,B) qPCR analyses showing the expression of the denoted genes, including hepatoblast markers (A) and mature hepatic and biliary genes (B) in HBOs grown in culture conditions with enhanced or reduced Wnt signalling. HBOs grown in maintenance conditions were used as a control. (C) Immunofluorescence staining showing the expression of the denoted proteins in HBOs grown in maintenance conditions (HBO Control), in the presence of CHIR and in the absence of WNT3A, RSPO or both combined. (D) Immunofluorescence staining showing the expression of the foetal hepatocyte markers (HPX, MRP2) and biliary markers (SOX9, CK19) in HBOs grown in the different culture conditions. qPCR data are shown as mean±s.d. in three to five independent biological replicates (n=3-5), where *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA). ns, not significant. Images are representative of 3 samples.

Fig. 2.

Wnt signalling is necessary for HBO self-renewal. (A) Brightfield images showing HBOs grown for 7 days, 14 days (passage 1) and 21 days (passage 2) in the presence or absence of WNT3A and/or RSPO. Insets show a zoomed in picture of a representative organoid. Scale bars: 1000 µm. (B) Assay for live (green) and dead (red) cells in HBOs grown in the absence of Wnt signalling for 14 days. Scale bars: 1000 µm. (C) qPCR analyses for the expression of denoted genes in HBOs grown for 14 or 21 days (passage 1 and 2, respectively) in the presence or in the absence of Wnt signalling molecules. (D,E) Immunofluorescence staining for KI67, ALB, AFP, A1AT, MRP2 and CK19 on HBOs grown for 14 days (D) or 21 days (E) in the presence or absence of WNT3A and/or RSPO. Scale bars: 100 µm. qPCR data are shown as mean±s.d. in three independent biological replicates (n=3), where *P<0.05, **P<0.01, ***P<0.001 (two-way ANOVA). ns, not significant. Images are representative of 3 samples.

Fig. 3.

Addition of WNT3A and RSPO does not block HBO differentiation into foetal hepatocytes and biliary cells. (A) Schematic showing the method for differentiating HBOs into foetal hepatocytes in the presence or absence of Wnt signalling. (B) Brightfield images of HBOs grown in culture conditions supporting their self-renewal (HBO control), inducing their differentiation into foetal hepatocytes (HPZ+OSM) and in the same conditions with the presence of WNT3A and/or RSPO (HPZ+WNT+OSM; HPZ+RSPO+OSM; HPZ+WNT+RSPO+OSM). Scale bars: 400 μm. (C) qPCR analyses showing the expression of markers for HBOs (ALB, AFP), the Wnt target gene LGR5 and foetal hepatocytes (G6PC, HPX, and C3). HBOs differentiated into foetal hepatocytes were used as control. (D) Immunofluorescence staining showing the expression of the denoted proteins in HBOs grown in the different conditions described above. Scale bars: 100 μm. (E) Schematic showing the method for differentiating HBOs into biliary cells in the presence or absence of WNT3A. (F) Brightfield images of HBOs grown in culture conditions supporting their self-renewal (control) and in culture conditions inducing their differentiation into biliary cells (−A83+WNT+TGFb) and in the same conditions without WNT3A (−A83−WNT+TGFb). Scale bars: 400 µm. (G) qPCR analyses showing the expression of the denoted genes in HBO differentiated into biliary cells in the presence or absence of WNT3A. Relative expression of hepatoblast markers ALB, AFP and mature biliary markers CK19, CK18, SOX9 and HNF6 on HBOs and biliary differentiation controls were compared to WNT3A removal from the latter. (H) Immunofluorescence staining showing the expression of the denoted proteins in undifferentiated HBOs and in HBOs grown in culture conditions inducing biliary differentiation with and without WNT3A. Scale bars: 100 µm. qPCR data are shown as mean±s.d. in three to four independent biological replicates (n=3-4), where *P<0.05, **P<0.01, ***P<0.001 (two-way ANOVA). ns, not significant. Images are representative of 3 samples.

Fig. 4.

Wnt signalling limits the commitment of HBOs toward the hepatocyte pathway. (A) Schematic of the experimental design. Hepatic organoids were generated ±WNT3A and RSPO and then split in culture conditions driving differentiation or supporting HBO self-renewal. (B) Brightfield images of HBOs grown in the denoted culture conditions; organoids in hepatic differentiation media with WNT3A and RSPO appear healthier (black arrows) than those grown without (red arrows). R, RSPO; W, WBT3A. Insets show a zoomed in picture of a representative organoid. Scale bars: 1000 µm. (C) Immunofluorescence staining for the expression of ALB, AFP, FOXA3, MRP2 and the proliferation marker KI67 on HBOs and hepatic organoids in respective conditions. Scale bars: 100 µm. (D,E) qPCR analyses for the expression of hepatoblast, Wnt target and proliferation markers (D), and hepatic and biliary lineage markers (E) on HBOs treated in hepatic differentiation medium only (HPZ+OSM) or with WNT3A and RSPO added (HPZ+WNT+RSPO) split into their respective media or HBO maintenance medium. qPCR data are shown as mean±s.d. in three independent biological replicates (n=3), where *P<0.05, **P<0.01, ***P<0.001 (two-way ANOVA). ns, not significant. Images are representative of 3 samples.

Fig. 5.

Expression profile of HBOs grown in the presence of the absence of Wnt signalling. (A) PCA showing the impact of WNT3A and/or RSPO on the gene expression profile of HBOs. (B) Venn diagram indicating uniquely and shared DEGs amongst the different comparisons (log2 fold change≥1). (C) Volcano plots showing the top 20-25 genes differentially expressed in HBOs grown in the reduced WNT3A/RSPO culture conditions against the control. Red boxes indicate genes of biological interest and those that are common among comparisons. Each dot represents a separate gene; plotted is Log2FC (fold change) against the −Log10 of the adjusted P-value for each comparison and gene investigated. (D) Gene expression levels, determined by RNA-seq analysis for ATF5 and MYC. Individual data points from n=2-3 biologically independent samples are shown. The bar indicates the mean expression value. (E) Left: Schematic showing ATF5 OE in HBOs using LNPs. Right: Brightfield and GFP images of HBOs treated with GFP-mRNA LNPs (control) and ATF5-GFP-mRNA LNPs (ATF5 OE) 7 days after transfection. Scale bars: 250 µm. d, day. Arrowhead in schematic indicates the day of collection. (F) qPCR analyses for the expression of the denoted genes in HBOs transfected with HBO control and ATF5 OE after 7 days. qPCR data are shown as mean±s.e.m. in three independent technical replicates (n=3); *P<0.05, **P<0.01 (two-tailed t-test). (G) Immunofluorescence staining for ALB, CK19 and AFP on HBO control and ATF5 OE after 7 days. Scale bars: 100 µm. ns, not significant. Images are representative of 3 samples.

Fig. 6.

Active Wnt signalling matches proliferation changes through liver development. (A) UMAP plot of single-cell RNA-seq data (Wesley et al., 2022), clustering cells by gene signature through liver development. Each dot represents one cell. AH, adult hepatocyte cluster (one adult liver sample); FH1/2, foetal hepatocyte clusters 1 and 2 (FH1 from 7, 8, 9 and 11 pcw samples; FH2 from 12, 14, two 16, and 17 pcw samples); HB1/2, hepatoblast clusters 1 and 2 (HB1 derived from a 5 pcw sample; HB2 from a 6 pcw sample). (B,C) UMAP plots showing distribution of gene expression for hepatoblast markers (ALB, AFP, FOXA3), a biliary marker (CK19) and mature hepatic markers (G6PC, C3) (B); and the Wnt target gene LGR5, the proliferation marker MKI67 and the cell cycle gene CDK1 (C). (D) UMAP plots showing co-expression of LGR5, denoting active Wnt signalling, versus the cell cycle/proliferation genes MKI67 and CDK1. (E) Percentage of cells positive for the denoted genes in each cluster. (F) Percentage of cells co-expressing Wnt-targeted genes and MKI67 (left) or CDK1 (right) in each cluster.