Hepatic progenitor cells of biliary origin with liver repopulation capacity

Abstract

Hepatocytes and cholangiocytes self-renew following liver injury. Following severe injury hepatocytes are increasingly senescent, but whether hepatic progenitor cells (HPCs) then contribute to liver regeneration is unclear. Here, we describe a mouse model where the E3 ubiquitin ligase Mdm2 is inducibly deleted in more than 98% of hepatocytes, causing apoptosis, necrosis and senescence with nearly all hepatocytes expressing p21. This results in florid HPC activation, which is necessary for survival, followed by complete, functional liver reconstitution. HPCs isolated from genetically normal mice, using cell surface markers, were highly expandable and phenotypically stable in vitro. These HPCs were transplanted into adult mouse livers where hepatocyte Mdm2 was repeatedly deleted, creating a non-competitive repopulation assay. Transplanted HPCs contributed significantly to restoration of liver parenchyma, regenerating hepatocytes and biliary epithelia, highlighting their in vivo lineage potency. HPCs are therefore a potential future alternative to hepatocyte or liver transplantation for liver disease.

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Figure 1: Induction of hepatocyte damage following AhCre-mediated loss of Mdm2.
Figure 2: Hepatocyte Mdm2 loss results in rapid activation of HPCs.
Figure 3: Mdm2 deletion in the Mdm2flox/− model leads to HPC expansion and subsequent recovery.
Figure 4: Loss of recombined ΔMdm2 hepatocytes over time during recovery.
Figure 5: Fn14/TWEAK-regulated HPCs are necessary for liver regeneration following hepatocyte Mdm2 deletion.
Figure 6: TWEAK enhances the ductular reaction through activation of HPCs.
Figure 7: In vitro expanded HPCs are genetically and phenotypically stable.
Figure 8: Relationship between activated HPCs and hepatocytes.

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Acknowledgements

The authors would like to thank Biogen Idec for supplying the Fn14KO mouse line. We would also like to thank V. Factor for kindly supplying the A6 antibody, and M. Alison for proofreading the manuscript. We would like to thank F. Rossi, C. Cryer, O. Rodrigues and S. Monard for assistance with flow cytometry. T.G.B. is financially supported by the Wellcome Trust and the Academy of Medical Sciences. W.Y.L. was supported by the University of Edinburgh, Charles Darwin Scholarship, Edinburgh Overseas Research Scholarship, and the UK Regenerative Medicine Platform. J.P.I is supported by the MRC. O.J.S. is financially supported by Cancer Research UK and the European Research Council. S.J.F. is supported by the Sir Jules Thorn Charitable Trust, the Medical Research Council, and the UK Regenerative Medicine Platform.

Author information

W.Y.L.: experimental design, data generation, data analysis, manuscript preparation, critiqued manuscript. T.G.B.: experimental design, data generation, data analysis, manuscript preparation, critiqued manuscript. L.B.: experimental design, data generation, data analysis, critiqued manuscript. A.T.: experimental design, data generation, data analysis. A.M.C.: experimental design, data generation. T.H.: experimental design, data generation, data analysis. R.V.G.: experimental design, data generation, critiqued manuscript. D.W.: data generation. T.Y.M.: data generation. A.M.: data generation, critiqued manuscript. R.A.R.: data generation. T.K.: data analysis, critiqued manuscript. M.J.W.: data generation. T.J.: data generation. A.R.: data generation, critiqued manuscript. D.C.H.: critiqued manuscript. J.P.I.: experimental design, critiqued manuscript. A.R.C.: experimental design. O.J.S.: experimental design, critiqued manuscript. S.J.F.: experimental design, manuscript preparation, critiqued manuscript.

Correspondence to Stuart J. Forbes.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 3 Lineage tracing experiments to investigate the differentiation ability of HPCs.

Schematic representation showing experimental design of lineage tracing experiments using the Krt19CreERTLSLTdTomato mice. (b) Immunohistochemistry analysis for tdTomato and p21 on injured and uninjured tamoxifen induced Krt19CreERTLSLTdTomato mice. (c) Immunohistochemistry for CYP2D (green), TdTomato (red), and DAPI (blue) on liver of tamoxifen induced Krt19CreERTLSLTdTomato mice after CDE–recovery. The results shown are representative of 2 experiments with 5-8 mice each group. Scale bars = 50 μm.

Supplementary Figure 4 Administration of BNF induce hepatocyte damage.

Morphology by H&E and (b) expression of CYP2D6 by isolated purified hepatocytes following liver perfusion and digestion. Arrows denote examples of multinucleated hepatocytes. (c) Expression of nuclear p53 following extraction and purification of hepatocytes from AhCre+ Mdm2flox/flox mice (n = 3) 2 days following induction with 80 mg kg−1βNF compared to AhCreMdm2flox/flox controls; arrow highlights low nuclear p53 expression. (d) Modified representation of Mdm2flox construct outlining primer targets for qPCR assessment of recombination efficiency. (e) Serum alkaline phosphatase (f) and ALT following induction in with 80 mg kg−1 in AhCre+ Mdm2flox/flox animals (mean ± s.e.m., Kruskal Wallis Test, n = 3 mice each group, except day 8 where n = 1 mouse due to cohort morbidity). (g) Expression of apoptosis associated p53-dependent gene Bax in whole liver over time following induction with 80 mg kg−1βNF in AhCre+ Mdm2flox/flox mice (mean ± s.e.m., One-way ANOVA.; n = 3 mice each control time point and n = 3,3,6,5 for experimental time points). (h) Immunohistochemistry for lactate dehydrogenase of healthy mice, βNF induced AhCreMdm2flox/flox controls and βNF induced AhCre+ Mdm2flox/flox mice. Representative images shown are representative of 2 experiments with 12 mice in total. Scale bars = 50 μm.

Supplementary Figure 5 Activation of ductular reaction following hepatocyte damage.

Detection of (a) EpCAM (b) DLK1 (c) A6 (inset, AhCre control) expressing cells following ΔMdm2 in hepatocytes. (d) Immunohistochemistry for CD24 (red), EpCAM (green), DAPI (blue) on CDE treated and βNF induced AhCre+Mdm2flox/flox mice. Representative images are shown are representative of 3 experiments with 5-8 mice each group (e) Ck19 expression in the whole liver of the induced Mdm2flox/flox mice versus uninduced control over time (mean ± s.e.m., One-way ANOVA with Bonferroni correction. P = 0.0058 day 8; n = 3 mice each control time point and n = 3,3,6,5 for experimental time points, repeated twice). (f) Ascl2 expression of Mdm2flox/flox mice over time following induction (mean ± s.e.m., One-way ANOVA with Bonferroni correction. P = 0.05; n = 3 mice, repeated twice). (g) Quantitative comparison of the panCK positive cells between the uninduced control, Mdm2flox/−Mdm2flox/flox, and the choline deficient ethionine supplemented diet (CDE) model (mean ± s.e.m., One-way ANOVA with Bonferroni correction. n = 4,6,5,5 mice each group respectively). (h) BrdU and panCK co-expressing cells can be observed 2 days after ΔMdm2. Representative images are shown are representative of 3 mice each group. Scale bars = 50 μm.

Supplementary Figure 6 Expandability of EpCAM+CD24+CD133+population in vitro.

(a) Morphology of cdHPCs after passaging with trypsin (left) or diluted trypsin (right). Insets show high magnification pictures. (b) Percentage of total EpCAM+CD24+CD133+ cells after in vitro expansion. (mean ± s.e.m., n = 6 biological replicates, Mann–Whitney test). (c) mRNA expression in relative to housekeeping gene (Ppia) of expanded cdHPCs (mean ± s.e.m., n = 3 biological replicates). (d) Heat map representation comparing mRNA expression in relative to housekeeping gene Ppia of cdHPC clones and primary hepatocytes. (e) Relative mRNA expression for HPC related genes Lgr5, EpCAM, Albumin, Ck19, Spp1, Sox9 on early and late passages cdHPCs (mean ± s.e.m., Kruskal Wallis test. P > 0.05, except Lgr5 P = 0.0286; n = 4 biological replicates). (f) Immunocytochemistry for HPC markers Sox9, CK19, OPN, and HNF1β on early and late passages cdHPC. (g) Cell area, roundness, and width to length ratio of early and late passages cdHPCs (mean ± s.e.m., Kruskal Wallis test P = 0.0107; n = 4 biological replicates). (h) Calculation for the average number of cell division after 10 passages; n = 3 biological replicates. Representative images represent data obtained form 3 individual experiments. Scale Bar = 100 μm.

Supplementary Figure 7 Ability to differentiate towards both hepatic and biliary lineage in vitro.

(a) FACS analysis of LGR5, CD31 and CD45 expression on in vitro expanded cdHPCs. Isotype control (blue line). (b) Immunocytochemistry for desmin and GFAP in in vitro expanded HPCs. (isolated stellate cells as positive control). (c) In vitro differentiation of expanded HPCs into cholangiocytes stained with activated bile duct marker MIC1C3 (green) and Hnf1β (red), DAPI (blue). (d) In vitro differentiation of expanded HPCs into hepatocytes dotted line demarcates a hepatocyte like colony, upper panel. Increase Glycogen storage detected by Periodic acid-Schiff staining on differentiated cdHPCs. (e) Alb mRNA expression and secreted protein following hepatocyte differentiation (mean ± s.e.m., P = 0.007 Mann–Whitney test; n = 5 biological replicates). Lower histograms demonstrate expression of cholangiocyte related genes, and hepatocyte transcription factor Hnf1α. Representative images are shown as representative of 3 individual experiments. Scale Bars = 50 μm.

Supplementary Figure 8 Secondary clone sorting assay for the in vitro expanded HPCs.

(a) EpCAM and CD24 expression of in vitro expanded HPCs. (b) EpCAM and CD24 expression analysis of secondary clones 7 days after replating. (c) Percentage of EpCAM + CD24hi population in secondary clone cultures (mean ± s.e.m., Kruskal Wallis test; P = 0.0076). Data are represented as mean ± s.e.m., n = 5 biological replicates. Representative images represents 3 individual experiments.

Supplementary Figure 9 Liver repopulating capacity of the in vitro expanded HPCs.

(a) Stitched image of GFP expressing cells in CAG-GFP HPCs transplanted animals. (b) Detection of GFP, ductular marker (panCK) and hepatocyte marker (HNF4α) in transplanted animals and non-transplanted controls (white arrow, GFP panCK+; red arrow, GFP+ panCK+; green arrow, GFP+ HNF4α− ; yellow arrow, GFP+ HNF4α+). (c) Detection of GFP and proliferation marker (Ki67) or senescence marker (p21) in transplanted animals and non-transplanted controls (insets, higher magnification) three months after HPC transplantation (upper panel). (White arrows show GFP+ Ki67 + hepatocytes; Yellow arrow shows p21- GFP+ hepatocytes). Data shown here are representative of 3 experiments with 8–10 mice each group. Scale Bars = 50 μm, except stitched image (a) where scale bar = 200 μm.

Supplementary Figure 10 Schematic representation of experimental design for the AhCreΔMdm2 mice.

Supplementary Table 1 Recombination efficiency of the ΔMdm system and summary of experimental design.
Supplementary Table 2 List of antibodies used.

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Lu, W., Bird, T., Boulter, L. et al. Hepatic progenitor cells of biliary origin with liver repopulation capacity. Nat Cell Biol 17, 971–983 (2015). https://doi.org/10.1038/ncb3203

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