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Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration

A Corrigendum to this article was published on 15 March 2018

This article has been updated

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 disease1,2. It is unclear whether other liver cell types can regenerate hepatocytes3,4,5. 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.

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Figure 1: Hepatocyte β1-integrin ablation combined with liver injury amplifies ductular reaction, and hepatocytes regenerate from a non-hepatocyte source.
Figure 2: Non-hepatocyte-derived hepatocytes originate from the portal tract, and CK19 biliary lineage tracing confirms their ductal origin.
Figure 3: Inhibition of hepatocyte proliferation after AAV8-p21 injection.
Figure 4: Emergence of ductular-derived hepatocytes after chronic liver injury when hepatocyte proliferation is impaired.

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  • 14 March 2018

    Please see accompanying Corrigendum (http://doi.org/10.1038/nature25996). The axes of the FACS plots in Extended Data Fig. 10b have been corrected; see Supplementary Information to the Corrigendum for the original Extended Data Fig. 10b. In addition, a new ref. 25 (Choi, 2014) has been added to the sentence ‘Biliary cells in zebrafish models have been shown to regenerate the liver after massive hepatocytes loss’, and refs 26–28 in the Methods have been renumbered.

References

  1. Richardson, M. M. et al. Progressive fibrosis in nonalcoholic steatohepatitis: association with altered regeneration and a ductular reaction. Gastroenterology 133, 80–90 (2007)

    Article  Google Scholar 

  2. Marshall, A. et al. Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and fibrosis in chronic hepatitis C virus infection. Gastroenterology 128, 33–42 (2005)

    Article  Google Scholar 

  3. Yanger, K. et al. Adult hepatocytes are generated by self-duplication rather than stem cell differentiation. Cell Stem Cell 15, 340–349 (2014)

    Article  CAS  Google Scholar 

  4. Schaub, J. R., Malato, Y., Gormond, C. & Willenbring, H. Evidence against a stem cell origin of new hepatocytes in a common mouse model of chronic liver injury. Cell Reports 8, 933–939 (2014)

    Article  CAS  Google Scholar 

  5. Tarlow, B. D., Finegold, M. J. & Grompe, M. Clonal tracing of Sox9+ liver progenitors in mouse oval cell injury. Hepatology 60, 278–289 (2014)

    Article  CAS  Google Scholar 

  6. Michalopoulos, G. K. & Khan, Z. Liver stem cells: experimental findings and implications for human liver disease. Gastroenterology 149, 876–882 (2015)

    Article  Google Scholar 

  7. Lu, W.-Y. et al. Hepatic progenitor cells of biliary origin with liver repopulation capacity. Nat. Cell Biol. 17, 971–983 (2015)

    Article  CAS  Google Scholar 

  8. Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013)

    Article  ADS  CAS  Google Scholar 

  9. He, J., Lu, H., Zou, Q. & Luo, L. Regeneration of liver after extreme hepatocyte loss occurs mainly via biliary transdifferentiation in zebrafish. Gastroenterology 146, 789–800.e8 (2014)

    Article  CAS  Google Scholar 

  10. Petersen, B. E., Zajac, V. F. & Michalopoulos, G. K. Hepatic oval cell activation in response to injury following chemically induced periportal or pericentral damage in rats. Hepatology 27, 1030–1038 (1998)

    Article  CAS  Google Scholar 

  11. Bird, T. G., Lorenzini, S. & Forbes, S. J. Activation of stem cells in hepatic diseases. Cell Tissue Res. 331, 283–300 (2008)

    Article  CAS  Google Scholar 

  12. Lin, W.-R. et al. The histogenesis of regenerative nodules in human liver cirrhosis. Hepatology 51, 1017–1026 (2010)

    Article  CAS  Google Scholar 

  13. Yanger, K. et al. Robust cellular reprogramming occurs spontaneously during liver regeneration. Genes Dev. 27, 719–724 (2013)

    Article  CAS  Google Scholar 

  14. Font-Burgada, J. et al. Hybrid periportal hepatocytes regenerate the injured liver without giving rise to cancer. Cell 162, 766–779 (2015)

    Article  CAS  Google Scholar 

  15. Shin, S., Upadhyay, N., Greenbaum, L. E. & Kaestner, K. H. Ablation of Foxl1-Cre-labeled hepatic progenitor cells and their descendants impairs recovery of mice from liver injury. Gastroenterology 148, 192–202.e3 (2015)

    Article  CAS  Google Scholar 

  16. Español-Suñer, R. et al. Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice. Gastroenterology 143, 1564–1575.e7 (2012)

    Article  Google Scholar 

  17. Raghavan, S., Bauer, C., Mundschau, G., Li, Q. & Fuchs, E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination. J. Cell Biol. 150, 1149–1160 (2000)

    Article  CAS  Google Scholar 

  18. Speicher, T. et al. Knockdown and knockout of β1-integrin in hepatocytes impairs liver regeneration through inhibition of growth factor signalling. Nat. Commun. 5, 3862 (2014)

    Article  ADS  CAS  Google Scholar 

  19. Huebener, P. et al. The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis. J. Clin. Invest. 125, 539–550 (2015)

    Article  Google Scholar 

  20. Liu, Y. et al. Animal models of chronic liver diseases. Am. J. Physiol. Gastrointest. Liver Physiol. 304, G449–G468 (2013)

    Article  ADS  CAS  Google Scholar 

  21. Bogorad, R. L. et al. Nanoparticle-formulated siRNA targeting integrins inhibits hepatocellular carcinoma progression in mice. Nat. Commun. 5, 3869 (2014)

    Article  ADS  CAS  Google Scholar 

  22. Huppi, K. et al. Molecular cloning, sequencing, chromosomal localization and expression of mouse p21 (Waf1). Oncogene 9, 3017–3020 (1994)

    CAS  PubMed  Google Scholar 

  23. Wang, B., Zhao, L., Fish, M., Logan, C. Y. & Nusse, R. Self-renewing diploid Axin2(+) cells fuel homeostatic renewal of the liver. Nature 524, 180–185 (2015)

    Article  ADS  CAS  Google Scholar 

  24. Planas-Paz, L. et al. The RSPO–LGR4/5–ZNRF3/RNF43 module controls liver zonation and size. Nat. Cell Biol. 18, 467–479 (2016)

    Article  CAS  Google Scholar 

  25. Choi, T. Y., Ninov, N., Stainier, D. Y. & Shin, D. Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish. Gastroenterology 146, 776–788 (2014)

    Article  CAS  Google Scholar 

  26. Mitchell, C. & Willenbring, H. A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nat. Protocols 3, 1167–1170 (2008)

    Article  CAS  Google Scholar 

  27. Preibisch, S., Saalfeld, S. & Tomancak, P. Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25, 1463–1465 (2009)

    Article  CAS  Google Scholar 

  28. Kreamer, B. L. et al. Use of a low-speed, iso-density percoll centrifugation method to increase the viability of isolated rat hepatocyte preparations. In Vitro Cell. Dev. Biol. 22, 201–211 (1986)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank B. Vernay for assistance with microscopy, F. Rossi and C. Cryer with fluorescence-activated cell sorting (FACS), S. Werner for advice, and F. Mushinski for providing the p21 plasmid. Work in the laboratory of R.R.M. was supported by the UK Medical Research Council (MRC) (MC_PC_U127574433). This work was supported by the MRC (MR/L012766/1, MR/K017047/1) and the UK Regenerative Medicine Platform (MR/K026666/1).

Author information

Authors and Affiliations

Authors

Contributions

A.R. performed the β1-integrin experiments, experimental design, data generation, data analysis and interpretation, and manuscript preparation. W.Y.L. performed the AAV8-p21 experiments, experimental design, data generation, data analysis and interpretation, and manuscript preparation. S.G. performed partial hepatectomy, and critiqued the manuscript. T.Y.M. performed immunostaining. E.O.D. analysed data. B.D. critiqued the manuscript and performed western blot. J.P.T. and R.R.M. performed the RNA-seq and analysis. R.B., V.K., and Y.K. provided the nanoparticles. C.ff.-C. critiqued the manuscript. L.B. performed experimental and study design, and critiqued the manuscript. S.J.F. conceived and designed the study, interpreted data, and prepared the manuscript.

Corresponding author

Correspondence to Stuart J. Forbes.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks K. Kaestner and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

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 after AAV administration. Cre-treated HNF4αpos hepatocytes are tdTompos in contrast to the null-treated; CK19pos ductal cells are located at the portal tract (PT). Confocal images. b, Liver tissue analysis quantifying tdTompos, HNF4αpos hepatocytes 2 weeks after AAV8.TBG.Cre/null administration. AAV8.TBG.Cre, n = 7; AAV8.TBG.null, n = 3. c, Hepatocellular β1-integrin immunofluorescence greyscale image and dual image with membranous β-catenin, 2 weeks after AAV8.TBG.Cre administration; arrows identify β1-integrin in the hepatocyte cell membrane, arrowheads identify non-parenchymal β1-integrin staining. Confocal images. d, Two weeks after AAV8.TBG.Cre administration; whole-liver β1-integrin. Western blot. e, Two weeks after AAV8.TBG.Cre administration; whole-liver QPCR for β1-integrin (n = 3; n, number of mice per group, experiment was repeated twice). Scale bars, 100 μm. 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 unlabelled periportal hepatocytes.

a, Serum enzyme-linked immunosorbent assay for markers associated with liver function 9 weeks after AAV8.TBG.Cre administration (n = 3). b, Nine weeks after AAV8.TBG.Cre administration; anti-HMGB1 DAB immunohistochemistry. Arrowheads identify nuclear HMGB1, arrows highlight cytoplasmic HMGB1. c, Nine weeks after AAV8.TBG.Cre; αSMA/CK19, tdTom/HNF4α, and glutamine synthetase/tdTom immunofluorescence; arrows point to areas with ductular reaction. d, Liver tissue analysis quantifying CK19pos cells 9 weeks after AAV8.TBG.Cre administration (n = 3). e, Liver tissue analysis quantifying αSMApos cells 9 weeks after AAV8.TBG.Cre administration (n = 3). f, Liver tissue analysis quantifying tdTompos, HNF4αpos hepatocytes in livers 9 weeks after AAV8.TBG.Cre administration (n = 3). g, Liver tissue analysis quantifying Ki67pos, HNF4αpos hepatocytes 9 weeks after AAV8.TBG.Cre administration (n = 3). h, Liver tissue analysis quantifying p21pos, HNF4αpos hepatocytes 9 weeks after AAV8.TBG.Cre administration (n = 3; n, number of mice per condition, the experiment was performed once). Scale bars, 100 μm. 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 enzyme-linked immunosorbent assay 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 Ki67pos/HNF4αpos hepatocytes after DDC injury (n = 5). d, Liver tissue analysis quantifying p21pos/HNF4αpos 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 and eosin histochemical stain of sections after DDC injury; asterisks highlight necrotic areas, arrows highlight small cellular infiltrate. g, Morphometric quantification of necrotic area from haematoxylin and eosin 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 PicroSirius Red, which is n = 3). i, Representative images of PicroSirius Red histochemistry on samples from day 42 of recovery after DDC diet (n, number of mice per condition, the experiment was performed once or twice). Scale bars, 100 μm. Data are mean ± s.e.m.; two-tailed unpaired t-test; two-way 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 tdTomneg hepatocytes.

a, tdTom/CK19 immunofluorescence; tiled images of β1-integrinfl/fl and β1-integrinWT livers after DDC diet and 14 days of recovery. 1–4, Enlarged regions of interest: 1, invasive CK19-positive cells (arrows); 2, tdTomneg regenerative nodule; 3, 4, fully recovered β1-integrinWT 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 tdTompos and tdTomneg hepatocytes. ce, Liver tissue analysis and a representative PCNA/HNF4α/tdTom immunofluorescent image in β1-integrinfl/fl mice at day 14 recovery after DDC injury; quantifying PCNA expression and nucleus size in both tdTompos and tdTomneg hepatocytes (HNF4αpos) (n = 8 mice analysed, the experiment was performed once). f, Liver tissue analysis in β1-integrinfl/fl mice at day 14 recovery after DDC injury; quantifying tdTompos HNF4αpos hepatocytes adjacent to the glutamine-synthetase-positive central veins (GSpos CV) (n = 7 mice analysed, the experiment was performed once). g, tdTom/glutamine synthetase/CK19 immunofluorescence in mice at day 14 recovery after DDC diet; β1-integrinfl/fl liver has patches of tdTomneg hepatocytes connected to CK19pos biliary epithelium, tdTompos hepatocytes remain arranged around the glutamine-synthetase-positive central veins. Data are mean ± s.e.m.; **P < 0.01, paired t-test.

Extended Data Figure 5 K19CreERTtdTomatoLSL strictly labelled 40% of the biliary epithelium.

Three 4 mg doses of tamoxifen followed by a 14-day wash out period. a, Representative images of CK19/tdTom dual immunofluorescence. b, Quantification of CK19pos biliary epithelial cells that were tdTom-positive (n = 8 mice analysed, the experiment was performed once). Scale bars, 100 μm. Data are mean ± s.e.m.

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

a, 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 PanCKpos cells of the AAV8 vector-injected liver. d, PicroSirius Red analysis of the AAV8 vector-injected livers. Scale bars, 100 μm; n = 5 mice. Experiments were performed twice. Data are mean ± s.e.m. Mann–Whitney U-test, *P < 0.05.

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

a, Schematic representation of the CDE recovery regime. b, Immunostaining of Ki67 (red) and 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 after 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 PanCKpos cells of the liver of AAV8-injected mice during and 2 weeks after CDE diet-induced injury. f, The p21 immunohistochemistry on AAV8-injected mice during and after CDE injury. Insets show high-magnification images. g, Whole-liver Cdkn1a, Igfbp1, Tgfb1, and Krt19 mRNA expression of AAV8 vector-injected mice 2 weeks after CDE injury. Scale bars, 100 μm; n = 4 mice. Experiments were performed once. Data are mean ± s.e.m. Mann–Whitney U-test, *P < 0.05.

Extended Data Figure 8 Analysis of the Krt19CretdTomatoLSL mice which received AAV8-p21 injection followed by DDC or CDE recovery regime.

a, Experimental design of the AAV8-p21 injection followed by DDC recovery regime. b, c, tdTom staining of Krt19Cre tdTomatoLSL mice that received AAV8-p21 injection followed by DDC recovery regime. d, HNF4α (green) and tdTom (red) staining on the Krt19CretdTomatoLSL mice that received AAV8 injections and were treated with the DDC recovery regime. e, SOX9 (green) and tdTom (red) staining on the Krt19Cre tdTomatoLSL mice that received AAV8 injections and were 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 that received AAV8-ctrl or AAV8-p21 injections followed by the CDE recovery regime. h, Immunostaining for tdTom of mice that received AAV8-ctrl or AAV8-p21 injections followed by the CDE recovery regime. i, tdTom (red) and HNF4α (green) immunofluorescent double staining of mice that received AAV8-ctrl or AAV8-p21 injections followed by the CDE recovery regime. N = 4 mice, experiments were performed once. Scale bars, 100 μm.

Extended Data Figure 9 Analysis of the Krt19Cre tdTomatoLSL mice that received AAV8-p21 injection followed by MCD recovery regime.

a, Representative immunofluorescent images showing split channels of tdTom, HNF4α, and DAPI of mice that 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 Krt19Cre tdTomatoLSL mice that received AAV8-p21 injection followed by MCD long recovery regime. e, tdTom (red) and HNF4α (green) immunofluorescent double staining and quantification of tdTompos HNF4αpos hepatocytes in that mice that received AAV8-ctrl or AAV8-p21 injections followed by the MCD long recovery regime. f, Representation of partial hepatectomy performed after the MCD recovery regime. g, Quantification and immunofluorescent double staining of HNF4α (red) and Ki67(green) of mice that received 50% partial hepatectomy. h, Confocal microscopy images of mice that received 50% partial hepatectomy; tdTom (red), Ki67 (green), E-cadherin (grey). N = 4 mice, experiments were performed twice. Scale bars, 100 μm. Data are mean ± s.e.m.

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

a, Experimental design to isolate WT hepatocytes/ducts and ductular-derived hepatocytes from a regenerated liver. b, FACS gating strategy to isolate tdTompos and tdTomneg 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 normalized read counts are displayed on the right of each plot. RNA-seq annotated genes are plotted in black below. e, RNA-seq generated Z-score heatmaps with hierarchical clustering across specific gene sets. Heatmaps display gene expression levels normalized 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, 100 μm.

Supplementary information

Supplementary Table 1

This file contains the primary antibody list. (XLSX 10 kb)

Supplementary Data

This file contains a data sheet split into two tabs. Sheet 1 contains data β1 integrin fl-fl vs Control. Sheet 2 contains data on AAV8-p21 v Control. (XLSX 92 kb)

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Raven, A., Lu, WY., Man, T. et al. Cholangiocytes act as facultative liver stem cells during impaired hepatocyte regeneration. Nature 547, 350–354 (2017). https://doi.org/10.1038/nature23015

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