Letter

De novo formation of the biliary system by TGFβ-mediated hepatocyte transdifferentiation

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Published:

Abstract

Transdifferentiation is a complete and stable change in cell identity that serves as an alternative to stem-cell-mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to the replenishment of cells lost from preexisting structures, in the presence of a fully developed scaffold and niche1. Here we show that transdifferentiation of hepatocytes in the mouse liver can build a structure that failed to form in development—the biliary system in a mouse model that mimics the hepatic phenotype of human Alagille syndrome (ALGS)2. In these mice, hepatocytes convert into mature cholangiocytes and form bile ducts that are effective in draining bile and persist after the cholestatic liver injury is reversed, consistent with transdifferentiation. These findings redefine hepatocyte plasticity, which appeared to be limited to metaplasia, that is, incomplete and transient biliary differentiation as an adaptation to cell injury, based on previous studies in mice with a fully developed biliary system3,4,5,6. In contrast to bile duct development7,8,9, we show that de novo bile duct formation by hepatocyte transdifferentiation is independent of NOTCH signalling. We identify TGFβ signalling as the driver of this compensatory mechanism and show that it is active in some patients with ALGS. Furthermore, we show that TGFβ signalling can be targeted to enhance the formation of the biliary system from hepatocytes, and that the transdifferentiation-inducing signals and remodelling capacity of the bile-duct-deficient liver can be harnessed with transplanted hepatocytes. Our results define the regenerative potential of mammalian transdifferentiation and reveal opportunities for the treatment of ALGS and other cholestatic liver diseases.

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Acknowledgements

The authors received the following support: H.W.: NIH R01 DK107553, CIRM DISC1-08792, NIH P30 DK026743. S.S.H.: NIH R01 DK078640, NIH R01 DK107553, NIH P30 DK078392. J.R.S.: NIH T32 DK060414, A. P. Giannini Foundation. S.N.T.K. and B.Y.H.: NIH T32 GM008568. M.R.: Deutsche Forschungsgemeinschaft RE 3749/1-1. F.C.: NIH T32 DK060414, Jane Coffin Childs Memorial Fund. H.Y.L.: Eli and Edythe Broad Regeneration Medicine and Stem Cell Fellowship. The authors thank Donghui Wang (UCSF Preclinical Therapeutics Core), Chris Her (UCSF Liver Center Cell Biology Core), Vinh Nguyen (UCSF Flow Cytometry Core) and Matt Kofron and Mike Muntifering (CCHMC Nikon Center of Excellence Confocal Imaging Core) for technical support, Nick Timchenko, Maria Shrower and Abigail Roebker (CCHMC) for reagents and technical assistance, Rik Derynck (UCSF) for advice and Pamela Derish (UCSF) for manuscript editing.

Reviewer information

Nature thanks S. Forbes, W. Goessling and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Johanna R. Schaub, Kari A. Huppert, Simone N. T. Kurial.

  2. These authors jointly supervised this work: Stacey S. Huppert, Holger Willenbring

Affiliations

  1. Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California San Francisco, San Francisco, CA, USA

    • Johanna R. Schaub
    • , Simone N. T. Kurial
    • , Bernadette Y. Hsu
    • , Feng Chen
    • , Milad Rezvani
    •  & Holger Willenbring
  2. Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    • Kari A. Huppert
    • , Ashley E. Cast
    • , Rebekah A. Karns
    •  & Stacey S. Huppert
  3. Biomedical Sciences Graduate Program, University of California San Francisco, San Francisco, CA, USA

    • Simone N. T. Kurial
    •  & Bernadette Y. Hsu
  4. Department of Pediatric General and Thoracic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    • Bryan Donnelly
  5. Department of Surgery, Division of General Surgery, University of California San Francisco, San Francisco, CA, USA

    • Hubert Y. Luu
  6. Department of Pathology, University of California San Francisco, San Francisco, CA, USA

    • Aras N. Mattis
  7. Liver Center, University of California San Francisco, San Francisco, CA, USA

    • Aras N. Mattis
    • , Philip Rosenthal
    •  & Holger Willenbring
  8. Department of Genetic and Laboratory Medicine, Division of Clinical Pathology, Geneva University Hospital, Geneva, Switzerland

    • Anne-Laure Rougemont
  9. Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, University of California San Francisco, San Francisco, CA, USA

    • Philip Rosenthal
  10. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA

    • Stacey S. Huppert
  11. Department of Surgery, Division of Transplant Surgery, University of California San Francisco, San Francisco, CA, USA

    • Holger Willenbring

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Contributions

H.W. and S.S.H. conceived the study. The authors contributed to designing and executing the experiments and data analysis as follows: J.R.S.: Figs. 14, Extended Data Figs. 16. K.A.H.: Figs. 1, 3, 4, Extended Data Figs. 3, 5, 6. S.N.T.K.: Figs. 14, Extended Data Fig. 1, 36, Extended Data Table 1. B.Y.H.: Fig. 4. A.E.C.: Extended Data Fig. 6. B.D.: Extended Data Fig. 6. R.A.K.: Fig. 2, Extended Data Fig. 4. F.C.: Extended Data Fig. 2. M.R.: Fig. 4. H.Y.L.: Fig. 3, Extended Data Fig. 3. A.N.M.: Fig. 4, Extended Data Table 1. A.-L.R.: Fig. 4, Extended Data Table 1. P.R.: Fig. 4, Extended Data Table 1. S.S.H.: Figs. 14, Extended Data Figs. 3, 5. H.W.: Fig. 2, Extended Data Fig. 4, Extended Data Table 1. H.W., S.S.H., J.R.S. and S.N.T.K. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Stacey S. Huppert or Holger Willenbring.

Extended data figures and tables

  1. Extended Data Fig. 1 Flp-based hepatocyte fate tracing.

    a, R26ZG allele. b, Experimental design for establishing efficient, specific and constitutive labelling of hepatocytes in normal adult R26ZG+/+ mice. cf, Immunofluorescence of R26ZG+/+ mouse liver (n = 2) for GFP and the hepatocyte marker major urinary protein (MUP) (c), peripheral and hilar cholangiocyte marker CK19 (d, e), hilar cholangiocyte marker DBA (e), hepatic stellate cell marker desmin (DES), macrophage marker F4/80 and endothelial cell marker LYVE1 (f) 2 weeks after intravenous injection of 1 × 1012 viral genomes (vg) of AAV8-Ttr-Flp. g, Reporter activation in R26ZG+/+ mice 1 and 2 weeks after intravenous injection of the indicated dose of AAV8-Ttr-Flp (n = 1 for each dose and time point). Scale bars, 100 µm.

  2. Extended Data Fig. 2 Efficiency of hepatocyte fate tracing in mice born with or without pBDs.

    a, b, Experimental design for hepatocyte fate tracing at P17 and immunofluorescence at P120 in Rbpjf/fHnf6f/fR26ZG+/+ mice (n = 4). c, Correlation of GFP labelling efficiency between hepatocytes and peripheral cholangiocytes in hepatocyte-fate-traced P120 Alb-cre+/−Rbpjf/fHnf6f/fR26ZG+/+ mice (n = 5; top and bottom). Horizontal line denotes the mean value. d, e, Experimental design for hepatocyte fate tracing at P39 and immunofluorescence at P120 in Alb-cre+/−Rbpjf/fHnf6f/fR26ZG+/+ mice (n = 3). Scale bars, 100 µm. Source Data.

  3. Extended Data Fig. 3 HpBDs relieve cholestasis and liver injury.

    a, Serum total bilirubin levels in P20–P29 (n = 6), P30–P39 (n = 33), P40–P49 (n = 35), P50–P59 (n = 8), P60–P69 (n = 46), P70–P79 (n = 22), P80–P89 (n = 13), P90–P119 (n = 20), P120–P149 (n = 52) and ≥P150 (n = 40) Alb-cre+/−Rbpjf/fHnf6f/f and P20–P29 (n = 5), P30–P39 (n = 24), P40–P49 (n = 19), P50–P59 (n = 7), P60–P69 (n = 27), P70–P79 (n = 10), P80–P89 (n = 11), P90–P119 (n = 10), P120–P149 (n = 41) and ≥P150 (n = 25) Rbpjf/fHnf6f/f mice. **P = 0.0011 at P20–P29, ****P = 1.7 × 10−13 at P30–P39, ****P = 8.2 × 10−12 at P40–P49, ***P = 0.00019 at P50–P59, ****P = 8.4 × 10−10 at P60–P69, ***P = 0.00055 at P70–P79, not significant (ns; P = 0.090) at P80–P89, not significant (P = 0.050) at P90–P119, not significant (P = 0.052) at P120–P149 and not significant (P = 0.064) at ≥P150, two-sided Welch’s t-test. b, Serial measurements of serum total bilirubin levels in Alb-cre+/−Rbpjf/fHnf6f/f (n = 14) and Rbpjf/fHnf6f/f (n = 5) mice. P = 0.11, two-sided Welch’s t-test. ce, Serum alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in P43–P45 (n = 13), P69–P82 (n = 13), P120 (n = 14) and P150 (n = 11) Alb-cre+/−Rbpjf/fHnf6f/f and P43–P45 (n = 5), P69–P82 (n = 4), P120 (n = 9) and P150 (n = 6) Rbpjf/fHnf6f/f mice. In c: ****P = 0.000058 at P43–P45, ****P = 0.000019 at P69–P82, **P = 0.0064 at P120, not significant (P = 0.22) at P150 and ***P = 0.00010 at P120 versus P69–P82, two-way ANOVA followed by Holm–Sidak multiple comparison test. In d: **P = 0.0050 at P43–P45, not significant (P = 0.47) at P69–P82, not significant (P = 0.30) at P120 and not significant (P = 0.47) at P150, two-way ANOVA followed by Holm–Sidak multiple comparison test. In e: **P = 0.0024 at P43–P45, *P = 0.015 at P69–P82, ****P = 0.000073 at P120 and not significant (P = 0.061) at P150, two-way ANOVA followed by Holm–Sidak multiple comparison test. f, Sirius red staining in P15 (n = 9), P70–P90 (n = 7) and P120 (n = 6) Alb-cre+/−Rbpjf/fHnf6f/f and P15 (n = 5), P70–P90 (n = 3) and P120 (n = 3) Rbpjf/fHnf6f/f mice with quantification. Not significant (P = 0.94) at P15, ****P = 0.000095 at P70–P90, **P = 0.0074 at P120 and *P = 0.027 at P120 versus P70–90, two-way ANOVA followed by Holm–Sidak multiple comparison test. g, Immunohistochemistry and Sirius red staining in P313 Alb-cre+/−Rbpjf/fHnf6f/f mice with persistent or resolved cholestasis (n = 1 each). Horizontal lines in af denote mean values. Scale bars, 100 µm. Source Data.

  4. Extended Data Fig. 4 Isolation and gene expression profiling of hepatocyte-derived peripheral cholangiocytes.

    a, FACS gates for peripheral cholangiocyte (pC; EPCAM+DBA) and hilar cholangiocyte (hC; EPCAM+DBA+) isolation from Alb-cre+/−Rbpjf/fHnf6f/f and Rbpjf/fHnf6f/f mice. b, qPCR analysis of floxed Rbpj (Rbpjf/f) genomic DNA in hepatocyte-derived pC (HpC) and hC isolated from Alb-cre+/−Rbpjf/fHnf6f/f mice relative to hepatocytes isolated from Rbpjf/fHnf6f/f mice (dashed line; n = 3 each). Data were normalized to a downstream genomic region of Rbpj to control for gene copy number. Data are mean ± s.e.m. c, d, RNA-seq analysis of normal pC (n = 3 mice), HpC (n = 4 mice) and RBPJ- and HNF6-deficient hepatocytes (H; n = 3 mice). c, Heat map of genes reflecting deletion of Rbpj and Hnf6 (also known as Onecut1). Rbpj mRNA is present in this knockout mouse as a truncated transcript that does not produce a functional protein26. d, Heat map of all differentially expressed CYP genes distinguishing genes associated with mature (M), adolescent (A) and immature (I) hepatocyte differentiation or low expression in the liver (L)29. P < 0.05, one-way ANOVA, FDR-corrected; fold change > 3 (cd, except Rbpj and Notch1Notch4). Bold genes denote P < 0.05 for HpC versus pC, two-sided Student’s t-test (c, d). Source Data.

  5. Extended Data Fig. 5 Proliferation in HpBDs and reactive ductules.

    a, Size distribution of wsCK-positive DBA-positive hilar cholangiocyte clones in P90 Alb-cre+/−Rbpjf/fHnf6f/fR26R-Confetti+/− (n = 2) and Alb-cre+/−R26R-Confetti+/− (n = 2) mouse livers. **P = 0.0079 for three cells and ***P = 0.00092 for seven cells, two-sided Student’s t-test. b, Immunofluorescence of reactive ductules in hepatocyte-fate-traced P32 Alb-cre+/−Rbpjf/fHnf6f/fR26ZG+/+ mouse liver (n = 3). c, Size distribution of wsCK-positive DBA-negative peripheral cholangiocyte clones in P90 Alb-cre+/−Rbpjf/fHnf6f/fR26R-Confetti+/− (n = 2) and Alb-cre+/−R26R-Confetti+/− (n = 2) mouse livers. *P = 0.032 for one cell, *P = 0.024 for two cells, *P = 0.020 for three cells and *P = 0.014 for four cells, two-sided Student’s t-test. Horizontal lines in a and c denote mean values. d, Immunofluorescence and breakdown of OPN-positive KI67-positive cells based on CK19 expression in P54 Alb-cre+/−Rbpjf/fHnf6f/f mouse liver (n = 4). Arrowheads indicate OPN-positive KI67-positive CK19-negative cells. e, Immunofluorescence of liver of >P120 Alb-cre+/−Rbpjf/fHnf6f/f and Rbpjf/fHnf6f/f mice after DDC diet feeding for 2 (n = 1 each), 4 (n = 3 each) and 6 (n = 1 each) weeks. f, Immunofluorescence of liver and breakdown of OPN-positive cells based on hepatocyte fate tracing in >P120 Alb-cre+/−Rbpjf/fHnf6f/fR26ZG+/+ (n = 4) and Rbpjf/fHnf6f/fR26ZG+/+ (n = 3) mice fed DDC diet for 5 weeks starting 1 week after hepatocyte fate tracing was induced. Data in d and f are mean ± s.e.m. Scale bars, 100 µm (b, e, f), 50 µm (d). Source Data.

  6. Extended Data Fig. 6 TGFβ signalling in hepatocyte transdifferentiation.

    a, Ink visualization of biliary tree of P32 Alb-cre+/−Tgfbr2f/f mouse (n = 2). b, Immunofluorescence of P60 Alb-cre+/−Rbpjf/fHnf6f/f and Rbpjf/fHnf6f/f mouse livers (n = 2 each). Arrowheads indicate pSMAD3-positive HNF1-positive nuclei. c, Western blot with quantification of pSMAD3 in nuclear extracts from Alb-cre+/−Rbpjf/fHnf6f/f, Rbpjf/fHnf6f/f and Alb-cre+/−Rbpjf/fHnf6f/fTgfbr2f/f mouse livers (n = 2 each). Source data are shown in Supplementary Fig. 1. df, Experimental design (d) and results of analysis of the effect of TGFβ signalling on biliary differentiation of adult RBPJ- and HNF6-deficient hepatocytes cultured in 3D for the indicated number of days (d). e, Phase-contrast images of RPBJ- and HNF6-deficient hepatocyte spheroids embedded in collagen gels and cultured in the presence or absence of the TGFβ inhibitor SB-431542 (SB) for the indicated number of days. f, Relative expression levels of cholangiocyte and hepatocyte genes in freshly isolated hepatocytes and spheroids before and after embedding in collagen gels. Gene expression in the liver of a mouse fed choline-deficient ethionine-supplemented (CDE) diet was used as a positive control. Data are from three independent cultures per treatment in a representative experiment (n = 2). *P = 0.038 for Sox9 5 days, *P = 0.044 for Sox9 10 days, **P = 0.0034 for Krt19 5 days and **P = 0.0071 for Spp1 5 days, two-sided Welch’s t-test. g, Serum total bilirubin levels in P34–P53 Alb-cre+/−Rbpjf/fHnf6f/fTgfbr2f/f (n = 16) and Rbpjf/fHnf6f/fTgfbr2f/f (n = 7) mice. ****P = 0.000024, two-sided Welch’s t-test. h, Quantification of Sirius red staining in P58–P100 Rbpjf/fHnf6f/f mice after intravenous injection of AAV8-Eef1a1-caTgfbr1 at P20 (n = 4). Grey area represents the range of liver collagen in the indicated Rbpjf/fHnf6f/f mice from Extended Data Fig. 3f. Horizontal lines in c, g and h denote mean values. Data in f are mean ± s.e.m. Scale bars, 2 mm (a), 100 µm (e), 50 µm (b) and 10 µm (b, inset). Source Data.

  7. Extended Data Table 1 Characterization of human subjects and samples

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Tables 2–4 and Supplementary Figures 1–2

  2. Reporting Summary

  3. Supplementary Table 1

    RNA-seq analysis. Excel file showing differentially expressed genes, including analysis of pathway and biological process enrichment, between normal peripheral cholangiocytes (pC; n = 3 mice), hepatocytes (H; n = 3 mice) and hepatocyte-derived peripheral cholangiocytes (HpC; n = 4 mice) as defined by fold-change > 3 for at least 1 of the 3 pairwise comparisons and FDR-corrected P < 0.05 (one-way ANOVA).

  4. Video 1: HpBD connecting to a preexisting hBD

    3D projection and surface rendering of a confocal z-stack from a hepatocyte-fate-traced P120 Alb-cre+/-Rbpjf/fHnf6f/fR26ZG+/+ mouse liver (n = 2) showing hepatocyte fate tracing (GFP, green) and labelling of both peripheral and hilar cholangiocytes (wsCK, red) and specifically hilar cholangiocytes (DBA, blue).

  5. Video 2: Clones of hepatocyte-derived peripheral cholangiocytes

    3D projection of a confocal z-stack from a P150 Alb-cre+/-Rbpjf/fHnf6f/fR26R-Confetti+/- mouse liver (n = 4) showing clonally labeled (yellow, cyan and red) peripheral cholangiocytes (wsCK, purple) and hepatocytes.

Source data

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