Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration

Abstract

After liver injury, regeneration occurs through self-replication of hepatocytes. In severe liver injury, hepatocyte proliferation is impaired-a feature of human chronic liver disease. It is unclear whether other liver cell types can regenerate hepatocytes. Here we use two independent systems to impair hepatocyte proliferation during liver injury to evaluate the contribution of non-hepatocytes to parenchymal regeneration. First, loss of β1-integrin in hepatocytes with liver injury triggered a ductular reaction of cholangiocyte origin, with approximately 25% of hepatocytes being derived from a non-hepatocyte origin. Second, cholangiocytes were lineage traced with concurrent inhibition of hepatocyte proliferation by β1-integrin knockdown or p21 overexpression, resulting in the significant emergence of cholangiocyte-derived hepatocytes. We describe a model of combined liver injury and inhibition of hepatocyte proliferation that causes physiologically significant levels of regeneration of functional hepatocytes from biliary cells.

Extended Data Figure 1. AAV8.TBG.Cre labelled and deleted β1-Integrin in 99.5% of hepatocytes.

a, tdTom, biliary specific cytokeratin 19 (CK19) and hepatocyte specific HNF4α immunofluorescence in AAV8.TBG.Cre/null treated livers 2 weeks post AAV administration. Cre treated HNF4α^pos hepatocytes are tdTom^pos in contrast to the null treated; CK19^pos ductal cells are located at the portal tract (PT), confocal images. b, Liver tissue analysis quantifying tdTom^pos, HNF4α^pos hepatocytes 2 weeks post AAV8.TBG.Cre/null administration, AAV8.TBG.Cre n=7, AAV8.TBG.null n=3. c, Hepatocellular β1-Integrin immunofluorescence grayscale image and dual image with membranous β-catenin, 2 weeks post AAV8.TBG.Cre administration, arrows identify β1-Integrin in the hepatocyte cell membrane, arrowheads identify non-parenchymal β1-Integrin staining; confocal images. d, 2 weeks post AAV8.TBG.Cre administration; whole liver β1-Integrin Western Blot. e, 2 weeks post AAV8.TBG.Cre administration; whole liver QPCR for β1-Integrin, n=3. n=number of mice per group, experiment was repeated twice. Scale bars: 100µM. Data are mean ± s.e.m.; two-tailed unpaired t-test; * P=0.05.

Extended Data Figure 2. Ablation of β1-Integrin caused liver damage, ductular reaction and small patches of un-labelled periportal hepatocytes.

a, Serum ELISA for markers associated with liver function 9 weeks post AAV8.TBG.Cre administration; n=3. b, 9 weeks post AAV8.TBG.Cre administration; anti-HMGB1 DAB immunohistochemistry. Arrowheads identify nuclear HMGB1, arrows highlight cytoplasmic HMGB1. c, 9 week post AAV8.TBG.Cre; α smooth muscle actin (αSMA)/CK19, tdTom/HNF4α and glutamine synthetase (GS)/tdTom immunofluorescence, arrows point to areas with DR. d, Liver tissue analysis quantifying CK19^pos cells 9 weeks post AAV8.TBG.Cre administration; n=3. e, Liver tissue analysis quantifying αSMA^pos cells 9 weeks post AAV8.TBG.Cre administration; n=3. f, Liver tissue analysis quantifying tdTom^pos, HNF4α^pos hepatocytes in livers 9 weeks post AAV8.TBG.Cre administration; n=3. g, Liver tissue analysis quantifying Ki67^pos, HNF4α^pos hepatocytes 9 weeks post AAV8.TBG.Cre administration; n=3. h, Liver tissue analysis quantifying p21^pos, HNF4α^pos hepatocytes 9 weeks post AAV8.TBG.Cre administration; n=3. n=number of mice per condition, the experiment was performed once. Scale bars: 100µM. Data are mean ± s.e.m.; two-tailed unpaired t-test; * P=0.05, ** P=0.01.

Extended Data Figure 3. Hepatocyte β1-Integrin deletion delayed and altered the regenerative response after treatment with the liver injury diet DDC.

a, DDC diet and recovery; serum ELISA for markers associated with liver function, n=5. b Body weight measurements during DDC diet and the subsequent recovery; n=5. c, Liver tissue analysis quantifying prolonged Ki67^pos/HNF4α^pos hepatocytes after DDC injury; n=5. d, Liver tissue analysis quantifying p21^pos/HNF4α^pos hepatocytes after DDC injury; n=5, and representative p21/HNF4α immunofluorescence in samples 7 days after DDC injury. e, p21/Ki67 immunofluorescence 7 days after DDC injury. f, Representative Haematoxylin & Eosin (H&E) histochemical stain of sections after DDC injury; asterisks highlight necrotic areas; arrows highlight small cellular infiltrate. g, Morphometric quantification of necrotic area from H&E sections in DDC treated samples at peak injury and recovery. h, fibrosis analysis, percentage of pixels PicroSirius Red (PSR) positive, before, during and after DDC injury; n=5, except pre-injury PSR which is n=3. i, Representative images of PSR histochemistry on samples from day 42 of recovery after DDC diet. n=number of mice per condition, the experiment was performed 1-2 times. Scale bars: 100µM. Data are mean ± s.e.m.; two-tailed unpaired t-test; 2way ANOVA, Bonferroni post-tests. * P=<0.05, ** P=<0.01, *** P=<0.001.

Extended Data Figure 4. β1-Integrin ablation and DDC induced injury resulted in large areas of tdTom^neg hepatocytes.

a, tdTom/CK19 immunofluorescence, tiled images of β1-Integrin^fl/fl and β1-Integrin WT livers after DDC diet and 14 days of recovery. (1-4) Enlarged regions of interest; (1) invasive CK19 positive cells (arrows), (2) tdTom negative regenerative nodule, (3, 4) fully recovered β1-Integrin WT liver with typical biliary ducts (arrowheads). b, Separate channels from a merged CYP2D and tdTom immunofluorescent confocal image in Fig 1g, showing CYP2D expression between tdTom^pos and tdTom^neg hepatocytes. c, d and e, Liver tissue analysis and a representative PCNA/HNF4α/tdTom immunofluorescent image in β1-Integrin^fl/fl mice at day 14 recovery post DDC injury; quantifying PCNA expression and nucleus size in both tdTom^pos and tdTom^neg hepatocytes (HNF4α^pos); n=8 mice analysed, the experiment was performed once. f, Liver tissue analysis in β1-Integrin^fl/fl mice at day 14 recovery post DDC injury; quantifying tdTom^pos HNF4α^pos hepatocytes adjacent to the GS^pos CV. n=7 mice analysed, the experiment was performed once. g, tdTom/GS/CK19 immunofluorescence in mice at day 14 recovery post DDC diet, β1-Integrin^fl/fl liver has patches of tdTom^neg hepatocytes connected to CK19^pos biliary epithelium, tdTom^pos hepatocytes remain arranged around the GS^pos central veins (CV). Data are mean ± s.e.m.; ** P=<0.01, paired t-test.

Extended Data Figure 5. K19^CreERT tdTomato^LSL strictly labels 40% of the biliary epithelium.

Three X 4mg doses of tamoxifen followed by a 14day wash out period. a. Representative images of CK19/tdTom dual immunofluorescence. b. Quantification of CK19pos biliary epithelial cells which are tdTomato positive n=8 mice analysed, the experiment was performed once.Scale Bars: 100µM. Data are shown as mean ± s.e.m.

Extended Data Figure 6. Analysis of AAV8-p21 injected livers.

a. Schematic showing the experimental design of the AAV8-p21 injection. b. Haematoxylin and Eosin staining on the livers of AAV8 injected mice. c. Quantification and representative images of immunostaining for PanCK^pos cells of the AAV8 vectors injected liver. d. Picro Sirius Red analysis of the AAV8 vectors injected livers. Scale Bars: 100µM. n=5 mice. Experiments were performed twice. Data are shown as mean ± s.e.m. Mann-Whitney U-test, *P < 0.05

Extended Data Figure 7. Analysis of AAV8-p21 injected livers following CDE injury regime.

a. Schematic representation of the CDE recovery regime. b. Immunostaining of Ki67 (red), HNF4α (green) on AAV8 injected mice during CDE injury. White arrows denote Ki67 and HNF4α double positive hepatocytes. c. Split channel images of DAPI, Ki67, and HNF4α on liver sections of AAV8-p21 injected mice following CDE injury. d. Ki67 (red) and HNF4α (green) immunohistochemistry of the liver of mice injected with AAV8-p21 and control 2 weeks after CDE injury e. PanCK immunohistochemistry and quantification of PanCK^pos cells of the liver of AAV8 injected mice during and 2 weeks after CDE diet induced injury. f. p21 immunohistochemistry on AAV8 injected mice during and after CDE injury. Insets showing high magnification images. g. Whole liver Cdkn1a, Igfbp1, Tgfb1, Krt19 mRNA expression of AAV8 vectors injected mice 2 weeks following CDE injury. Scale Bars: 100µM. n=4 mice. Experiments were performed once. Data are shown as mean ± s.e.m. Mann-Whitney U-test, *P < 0.05

Extended Data Figure 8. Analysis of the Krt19^CretdTomato^LSL mice which received AAV8-p21 injection followed by DDC or CDE recovery regime.

a. Schematic showing the experimental design of the AAV8-p21 injection followed by DDC recovery regime. b,c. tdTom staining of Krt19^Cre tdTomato^LSL mice that received AAV8-p21 injection followed by DDC recovery regime. d. HNF4α (Green) and tdTom (red) staining on the Krt19^CretdTomato^LSL mice that received AAV8 injections and treated with the DDC recovery regime. e. SOX9 (Green) and tdTom (red) staining on the Krt19^Cre tdTomato^LSL mice that received AAV8 injections and treated with the DDC recovery regime. f. Schematic representation of the experimental design of the AAV8-p21 injection followed by CDE recovery regime. g. Epifluorescence images of the liver of the mice received AAV8-ctrl or AAV8-p21 injections followed by the CDE recovery regime. h. Immunostaining for tdTomato of mice received AAV8-ctrl or AAV8-p21 injections followed by the CDE recovery regime. i. tdTomato (red) and HNF4α (green) immunofluorescent double staining of mice received AAV8-ctrl or AAV8-p21 injections followed by the CDE recovery regime. n=4 mice, experiments were performed once. Scale Bars: 100µM.

Extended Data Figure 9. Analysis of the Krt19^Cre tdTomato^LSL mice which received AAV8-p21 injection followed by MCD recovery regime.

a. Representative immunofluorescent images showing split channels of tdTomato, HNF4α, and DAPI of mice received AAV8-ctrl or AAV8-p21 injections followed by the MCD recovery regime. b. Schematic representation showing the experimental design of the AAV8-p21 injection followed by a MCD long recovery regime. c,d. tdTom staining of Krt19^Cre tdTomato^LSL mice that received AAV8-p21 injection followed by MCD long recovery regime. e. tdTomato (red) and HNF4α (green) immunofluorescent double staining and quantification of tdTom^pos HNF4α^pos hepatocytes in that mice received AAV8-ctrl or AAV8-p21 injections followed by the MCD long recovery regime. f. Schematic representation of partial hepatectomy performed after the MCD recovery regime. g. Quantification and Immunofluorescent double staining of HNF4α (red), and Ki67(green) of mice received 50% partial hepatectomy. h. Confocal microscopy images of mice received 50% partial hepatectomy. tdTomato (red), Ki67 (green), E-Cadherin (grey). n=4 mice. Experiments were performed twice. Scale Bars: 100µM. Data are shown as mean ± s.e.m.

Extended Data Figure 10. Comparison between ductular derived hepatocytes and hepatocytes regenerated through self-duplication.

a. Experimental design to isolate wildtype hepatocytes/ducts and ductular derived hepatocytes from a regenerated liver. b. FACS gating strategy to isolate tdTom^pos and tdTom^neg hepatocytes. c. Cytospins and tdTom/HNF4α immunofluorescence of FACS sorted cells. d. Visual examples of average RNA-seq transcriptional reads in sample groups across select loci. Scales referring to normalised read counts are displayed on the right of each plot. RNAseq annotated genes are plotted n black below. e. RNAseq generated Z-score heatmaps with hierarchical clustering across specific gene sets. Heatmaps display gene expression levels normalised to each gene, yellow = higher expression, blue = lower expression, N=3. f. qPCR array for phase 1 drug metabolism enzymes n=3 mice. Experiments were performed once. Scale Bars: 100µM.

Figure 1. Hepatocyte β1-Integrin ablation combined with liver injury amplifies ductular reaction and hepatocytes regenerate from a non-hepatocyte source.

a, Experimental strategy to stimulate liver regeneration with hepatotoxic agents. b-d, Quantification of CK19^pos cells or αSMA^pos cells; before, during and after injury (n=5 DDC, n=3 MCD and n=2 TAA; n=3 for preinjury αSMA analysis. e, Confocal CK19/αSMA immunofluorescence; invasive biliary cells(arrowheads), ducts with lumens(arrows). f,h, Quantification of tdTom^pos/HNF4α^pos hepatocytes before, during and after injury (n=5 DDC, n=3 MCD and n=2 TAA). g Confocal tdTom immunofluorescence of CYP2D and HNF4α hepatocytes. PFV-per field of view. n=number of mice per condition; experiments were performed 1-2 times. Scale bars:100µM. Data are mean ± s.e.m.; * P=<0.05; ** P=<0.01; *** P=<0.001; 2way ANOVA, Bonferroni post-tests.

Figure 2. Non-hepatocyte derived hepatocytes originated from the portal tract, Keratin19 biliary lineage tracing confirmed their ductal origin.

a, tdTom /HNF4α/CK19 and tdTom/HNF4α/Sox9 immunofluorescent confocal images at day 7 recovery post DDC diet. tdTom^neg hepatocytes(HNF4α^pos) adjacent to CK19^pos and Sox9^pos ductular reactions (white arrows) and rare tdTom^neg hepatocytes Sox9^pos/HNF4α^pos(yellow arrows). b, Quantification of tdTom^neg/HNF4α^pos hepatocytes adjacent to the CK19^pos DRs post DDC injury; n=6 mice analysed, the experiment was performed twice. c, Experimental strategy to lineage trace biliary epithelial cells on a background of liver injury and hepatocyte Itgβ1 silencing. d, Immunofluorescent confocal images and immunohistochemistry 14 days after DDC and MCD diet combined with RNAi mediated β1 Integrin or luciferase mRNA suppression, bile ducts(arrowheads). Images are representative of n = 4 mice per condition, experiments were performed once. Scale bars:100µM Data are mean ± s.e.m.

Figure 3. Inhibition of hepatocyte proliferation following AAV8-p21 injection.

a, Schematic representing the AAV8-p21 construct. b, Cdkn1a whole liver RNA expression after AAV8 injection. n=3 mice c, p21 immunohistochemistry of AAV8 injected mice. d, Ki67 immunostaining of AAV8 injected livers. e, Experimental design of the AAV8-p21 with CCl₄ regime. f, Ki67/HNF4α immunofluorescence following AAV8 and CCl₄ injection, upper panel. p21 immunohistochemistry following CCl₄ injection. g, Quantification of Ki67^pos hepatocytes in mice injected with CCl₄ after AAV8 injection. n=3 mice h, Necrotic area 48-hours post CCl₄ injection n=7 mice I, Mouse serum ALT and AST after CCl₄. Scale bars:100µM. n=3 mice. Data are representative of 1-2 independent experiments. Experiments were performed twice. Data are shown as mean ± s.e.m. Mann-Whitney U-test *P < 0.05.

Figure 4. Emergence of ductular derived hepatocytes following chronic liver injury when hepatocyte proliferation is impaired.

a, Schematic of the MCD/recovery injury diet regime. b, tdTom immunohistochemistry on Krt19^CretdTomato^LSL livers following MCD recovery regime. High magnification images and quantification of tdTom positive cells (insets) n=4 mice. c, Immunostaining of tdTom and Periodic acid-Schiff staining on serial liver sections of AAV8 injected mice. d, Immunofluorescent double staining of HNF4α (green, upper panel), Glutamine Synthetase (green, middle panel), CYP2D (bottom, lower panel) and tdTom (red). e, DoubleImmunohistochemistry of tdTom (red), and SOX9 (green, upper panel), PanCK (green, lower panel). f, Confocal images of serial liver sections of AAV8 injected mice. tdTomato (red), HNF4α (green, left panel), PanCK (green, right panel), E-cadherin (grey). n=4 mice g,h, Pearson’s correlation plot with hierarchical clustering and 3D Principal Component Analysis (PCA) for total transcriptional landscapes n=3 mice. Scale Bars:100µM. Data are representative of 1–2 independent experiments, experiments were repeated at least twice except g,h were only performed once. Data are shown as mean ± s.e.m. Mann-Whitney U-test, *P < 0.05.