The biliary epithelium gives rise to liver progenitor cells

HepatologyVolume 60, Issue 4 p. 1367-1377 Liver Injury/RegenerationFree Access The biliary epithelium gives rise to liver progenitor cells Daniel Rodrigo-Torres, Daniel Rodrigo-Torres Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorSilvia Affò, Silvia Affò Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorMar Coll, Mar Coll Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorOriol Morales-Ibanez, Oriol Morales-Ibanez Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorCristina Millán, Cristina Millán Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorDelia Blaya, Delia Blaya Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorAnna Alvarez-Guaita, Anna Alvarez-Guaita Departament de Biologia Cel·lular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, Barcelona, SpainSearch for more papers by this authorCarles Rentero, Carles Rentero Departament de Biologia Cel·lular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, Barcelona, SpainSearch for more papers by this authorJuan José Lozano, Juan José Lozano Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorMiguel Angel Maestro, Miguel Angel Maestro Genomic Programming of Beta Cells Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, SpainSearch for more papers by this authorMyriam Solar, Myriam Solar Genomic Programming of Beta Cells Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, SpainSearch for more papers by this authorVicente Arroyo, Vicente Arroyo Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorJoan Caballería, Joan Caballería Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorLeo A. van Grunsven, Leo A. van Grunsven Liver Cell Biology Lab, Department of Cell Biology, Vrije Universiteit Brussel, Brussels, BelgiumSearch for more papers by this authorCarlos Enrich, Carlos Enrich Departament de Biologia Cel·lular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, Barcelona, SpainSearch for more papers by this authorPere Ginès, Pere Ginès Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain Instituto Reina Sofía por la Investigación Nefrología (IRSIN, FRIAT)Search for more papers by this authorRamon Bataller, Ramon Bataller Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain Division of Gastroenterology and Hepatology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC, USASearch for more papers by this authorPau Sancho-Bru, Corresponding Author Pau Sancho-Bru Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainAddress reprint requests to: Pau Sancho-Bru, Ph.D., Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) C/ Roselló, 149-153, Planta 3, 08036 Barcelona, Spain. E-mail: [email protected].Search for more papers by this author Daniel Rodrigo-Torres, Daniel Rodrigo-Torres Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorSilvia Affò, Silvia Affò Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorMar Coll, Mar Coll Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorOriol Morales-Ibanez, Oriol Morales-Ibanez Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorCristina Millán, Cristina Millán Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorDelia Blaya, Delia Blaya Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorAnna Alvarez-Guaita, Anna Alvarez-Guaita Departament de Biologia Cel·lular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, Barcelona, SpainSearch for more papers by this authorCarles Rentero, Carles Rentero Departament de Biologia Cel·lular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, Barcelona, SpainSearch for more papers by this authorJuan José Lozano, Juan José Lozano Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorMiguel Angel Maestro, Miguel Angel Maestro Genomic Programming of Beta Cells Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, SpainSearch for more papers by this authorMyriam Solar, Myriam Solar Genomic Programming of Beta Cells Laboratory, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, SpainSearch for more papers by this authorVicente Arroyo, Vicente Arroyo Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorJoan Caballería, Joan Caballería Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainSearch for more papers by this authorLeo A. van Grunsven, Leo A. van Grunsven Liver Cell Biology Lab, Department of Cell Biology, Vrije Universiteit Brussel, Brussels, BelgiumSearch for more papers by this authorCarlos Enrich, Carlos Enrich Departament de Biologia Cel·lular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, Barcelona, SpainSearch for more papers by this authorPere Ginès, Pere Ginès Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain Instituto Reina Sofía por la Investigación Nefrología (IRSIN, FRIAT)Search for more papers by this authorRamon Bataller, Ramon Bataller Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, Spain Division of Gastroenterology and Hepatology, Departments of Medicine and Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC, USASearch for more papers by this authorPau Sancho-Bru, Corresponding Author Pau Sancho-Bru Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), University of Barcelona, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Barcelona, SpainAddress reprint requests to: Pau Sancho-Bru, Ph.D., Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS) C/ Roselló, 149-153, Planta 3, 08036 Barcelona, Spain. E-mail: [email protected].Search for more papers by this author First published: 20 February 2014 https://doi.org/10.1002/hep.27078Citations: 137 Potential conflict of interest: Nothing to report. Supported by grants from the Instituto de Salud Carlos III (FIS PI041538, FIS PI12/01274, FIS PS09/01164 and FIS PI080126 to Dr. Bataller, Dr. Arroyo, Dr. Caballería and Dr. Ginès, respectively) and from the National Institute on Alcohol Abuse and Alcoholism (NIAA)(1U01AA021908-01 [33490]) to Pau Sancho-Bru. Dr. Sancho-Bru is funded by Instituto de Salud Carlos III, Miguel Servet (CP11/00071) and co-financed by Fondo Europeo de Desarrollo Europeo (FEDER), Unión Europea, “Una manera de hacer Europa”. D. Rodrigo-Torres received a grant from the Ministerio de Educación, Cultura y Deporte, FPU program. S. Affò received a grant from IDIBAPS. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Severe liver diseases are characterized by expansion of liver progenitor cells (LPC), which correlates with disease severity. However, the origin and role of LPC in liver physiology and in hepatic injury remains a contentious topic. We found that ductular reaction cells in human cirrhotic livers express hepatocyte nuclear factor 1 homeobox B (HNF1β). However, HNF1β expression was not present in newly generated epithelial cell adhesion molecule (EpCAM)-positive hepatocytes. In order to investigate the role of HNF1β-expressing cells we used a tamoxifen-inducible Hnf1βCreER/R26RYfp/LacZ mouse to lineage-trace Hnf1β+ biliary duct cells and to assess their contribution to LPC expansion and hepatocyte generation. Lineage tracing demonstrated no contribution of HNF1β+ cells to hepatocytes during liver homeostasis in healthy mice or after loss of liver mass. After acute acetaminophen or carbon tetrachloride injury no contribution of HNF1β+ cells to hepatocyte was detected. We next assessed the contribution of Hnf1β+-derived cells following two liver injury models with LPC expansion, a diethoxycarbonyl-1,4-dihydro-collidin (DDC)-diet and a choline-deficient ethionine-supplemented (CDE)-diet. The contribution of Hnf1β+ cells to liver regeneration was dependent on the liver injury model. While no contribution was observed after DDC-diet treatment, mice fed with a CDE-diet showed a small population of hepatocytes derived from Hnf1β+ cells that were expanded to 1.86% of total hepatocytes after injury recovery. Genome-wide expression profile of Hnf1β+-derived cells from the DDC and CDE models indicated that no contribution of LPC to hepatocytes was associated with LPC expression of genes related to telomere maintenance, inflammation, and chemokine signaling pathways. Conclusion: HNF1β+ biliary duct cells are the origin of LPC. HNF1β+ cells do not contribute to hepatocyte turnover in the healthy liver, but after certain liver injury, they can differentiate to hepatocytes contributing to liver regeneration. (Hepatology 2014;60:1367–1377) Abbreviations APAP acetaminophen β-GAL beta galactosidase CCl4 carbon tetrachloride CD133 (PROM1) prominin-1 CDE choline-deficient, ethionine-supplemented DAPI 4',6-diamidino-2-phenylindole DDC 3,5-diethoxycarbonyl-1,4-dihydro-collidin EpCAM epithelial cell adhesion molecule HEP PAR-1 hepatocyte paraffin-1 HNF1β hepatocyte nuclear factor 1 homeobox B HNF4α hepatocyte nuclear factor 4 alpha Krt keratin LPC liver progenitor cells PH partial hepatectomy SOX9 sex determining region Y-box 9 TROP2 tumor-associated calcium signal transducer 2 YFP yellow fluorescent protein Liver injury from any etiology induces mature liver cells to proliferate in order to replace the damaged tissue, allowing the recovery of the parenchymal function. In most situations, this process takes place without a clear involvement of liver progenitor cells (LPCs).1, 2 LPC expansion has been described in several liver diseases, and correlates with the degree of liver injury.3, 4 We have recently shown that in alcoholic hepatitis LPC markers correlate with liver injury and predict short-term mortality.3 This observation raises the question whether LPC expansion is a marker of liver injury or an incomplete attempt to regenerate the damaged liver. Moreover, it highlights the need for identifying the pattern of liver injury that favors LPC contribution to liver regeneration. Ductular reaction constitutes a heterogeneous population of proliferating cells ranging from cells expressing stem cell markers with an immature phenotype, to more committed cells with an intermediate hepatobiliary phenotype.5-8 One of the most widely investigated markers is epithelial cell adhesion molecule (EpCAM), which is expressed in ductular reaction cells but also in newly generated hepatocytes, suggesting that EpCAM-positive hepatocytes may derive from progenitor cells.2, 9, 10 Several studies have shown the capacity of LPC to differentiate in vitro to hepatocyte-like and cholangiocyte-like cells.10-13 However, the role of LPC in liver diseases is not well understood and whether LPCs derive from the biliary compartment and how they contribute to liver homeostasis and repair is still controversial. Moreover, it is largely unknown how the environment within the injured liver influences LPC differentiation.3, 14, 15 Genetic lineage-tracing has become a gold standard to evaluate the contribution of any given cell type to cells that arise during organ development, tissue homeostasis or disease. Recent studies aimed at evaluating the contribution of LPC to liver regeneration using this strategy have yielded disparate results. Using a sex-determining region Y-box 9 (SOX9) lineage-tracing model, Furuyama et al.16 showed an important contribution of SOX9 progeny to hepatocyte regeneration, supporting a model of liver homeostasis and regeneration based on a permanent supply of liver cells from LPC. By contrast, other recent studies showed that SOX9-positive embryonic ductal epithelium cells and osteopontin-labeled adult liver cells have the potential to give rise to transit-amplifying progenitor cells and mature hepatocytes, although to a much lesser extent.17, 18 Moreover, lineage-tracing studies of markers not expressed in intact liver but in ductular reaction cells have shown the potential of LPC to differentiate to hepatocytes and cholangiocytes.13, 19, 20 In summary, there are conflicting evidences concerning the possible contribution of biliary duct cells and LPC to hepatocyte regeneration in response to liver injury. Hepatocyte nuclear factor (HNF)1β is a homeobox transcription factor that plays a pivotal role during organogenesis and regulates gene expression in the adult liver and other epithelial organs.21-23 In liver development, HNF1β is involved in the hepatobiliary specification of hepatoblasts to cholangiocytes, and it is strongly expressed throughout the embryonic and adult biliary epithelium.21-24 However, little is known about the expression of HNF1β during liver injury and regeneration, and particularly its expression in LPC. In this study we traced HNF1β+ cells to assess the contribution of the biliary epithelium to LPC and hepatocytes during healthy liver homeostasis, liver regeneration, and in animal models of acute and repeated liver injury. We show that under physiological conditions, hepatocytes do not derive from HNF1β+ cells. Only after liver injury do adult HNF1β+ cells give rise to the expansion of cells with an LPC phenotype and to periportal hepatocytes. However, the population of HNF1β+ cell-derived hepatocytes is small under these experimental conditions, suggesting that LPC do not substantially contribute to liver parenchymal regeneration under most liver injury insults. Materials and Methods Human Biopsies and Samples Liver tissue samples were obtained from fragments of normal tissue surrounding colon metastasis collected at the moment of liver resection or from explants from liver transplantation due to alcohol-induced liver cirrhosis. The study was approved by the Ethics Committee of the Hospital Clinic of Barcelona and all patients included in this study gave written informed consent. Animal Protocols Hnf1bCreER transgenic mice were generated and genotyped as previously described.25 Mice were crossed with mice bearing Cre-inducible Rosa26R reporters LacZ (β-galactosidase [β-GAL] or yellow fluorescent protein [YFP]). To induce Cre-recombination, 12 to 24-week-old mice were treated with three tamoxifen (Sigma-Aldrich, St. Louis, MO) doses (20 mg, 20 mg, and 10 mg) by gavage over 1 week. Tamoxifen was dissolved at 100 mg/mL in 0.9% NaCl and 10% ethanol absolute in order to facilitate sonication. All animal models of liver injury were started 1 week after the last tamoxifen treatment. Two control groups were used for each experimental setting: mice treated with tamoxifen without liver injury, and mice with liver injury but without tamoxifen treatment. The animal models of liver injury used in this study were: acute acetaminophen (APAP) (Sigma-Aldrich); acute carbon tetrachloride injection (CCl4, Sigma-Aldrich); two-thirds partial hepatectomy (PH); 0.1% 3,5-diethoxycarbonyl-1,4-dihydro-collidin (DDC) diet26 (Sigma-Aldrich); choline-deficient (MP Biomedicals, Santa Ana, CA) ethionine-supplemented (0.15% in water, Sigma-Aldrich) (CDE) diet,27 and chronic CCl4 administration. All experimental models are described in the Supporting Material. All animal experiments were approved by the Ethics Committee of Animal Experimentation of the University of Barcelona and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Histochemical Procedures and Immunostaining The staining procedure is described in the Supporting Material. Isolation of YFP+ Cells YFP+ cells were obtained by liver perfusion method followed by flow cytometry cell sorting from uninjured mice (n = 3), and from mice treated with a DDC (n = 3) or CDE diet (n = 3) for 3 weeks. A detailed procedure of isolation and following RNA extraction and gene expression analysis are described in the Supporting Material. Statistical Analysis Continuous variables were described as means (± standard error) and were compared using the Student t test. All statistical analyses were performed using SPSS v. 14.0 for Windows (Chicago, IL). Results Expression of HNF1β in Normal and Cirrhotic Human Livers We first investigated the expression pattern of HNF1β in human liver tissue. HNF1β expression was restricted to biliary duct cells in healthy livers (Fig. 1A). Immunostaining of liver samples from patients with advanced alcoholic liver disease showed that ductular reaction cells were positive for HNF1β (Fig. 1B), whereas HNF1β was not expressed in mature hepatocytes as assessed by double staining of HEP PAR-1 and HNF1β (Fig. 1C,D). To further determine HNF1β expression in immature hepatocytes, we performed a double staining of HNF1β and EpCAM in healthy and alcohol-induced cirrhotic livers. Double-positive HNF1β/EpCAM cells were detected in the biliary epithelia from healthy tissue and in ductular reaction cells from cirrhotic tissue (Fig. 1E,F). Importantly, EpCAM-positive hepatocytes did not show HNF1β expression (Fig. 1F) suggesting that immature hepatocytes lost the expression of biliary markers such as HNF1β, while still retaining the LPC marker EpCAM. Figure 1Open in figure viewerPowerPoint HNF1β is expressed in biliary cells and in ductular reaction cells in healthy and diseased human liver. Representative images in healthy liver of (A) HNF1β immunostaining; (C) double immunostaining for HNF1β and hepatocyte marker HEP PAR-1; and (E) double immunostaining for HNF1β and EpCAM. Representative pictures in cirrhotic liver of (B) HNF1β immunostaining; (D) double immunostaining for HNF1β and HEP PAR-1; and (F) double immunostaining for HNF1β and EpCAM. Nuclei counterstaining was performed with DAPI (blue) (magnification ×200 and ×400). Tamoxifen Induction of Cre-Recombinase in Hnf1βCreER/R26R Mice In order to assess the specificity of Cre expression in HNF1β+ biliary cells, Hnf1βCreER/R26RYfp/LacZ mice were treated with tamoxifen and analyzed for Cre expression. The expression of Cre was assessed 16 hours posttamoxifen administration since Cre is transiently located in the nucleus for only 6-36 hours (Supporting Fig. 1A). Immunostaining analysis showed Cre expression restricted to the bile duct cells and colocalized with YFP expression (Supporting Fig. 1B). Importantly, Cre recombinase expression was restricted to HNF1β+ cells, with an 88.47 ± 4.5% of HNF1β+ cells also positive for Cre (Supporting Fig. 1C). Moreover, a 98.35 ± 0.4% of HNF1β+ cells also showed expression of SOX9 (Supporting Fig. 1D). Cre expression colocalized with KRT19 and A626 (Supporting Fig. 1E,F). An overdose of tamoxifen is known to generate liver damage that could induce ectopic expression of HNF1β and the reporter. Tamoxifen treatment induced a mild increase in aspartate transaminase levels (AST) 42 ± 5.2 U/L versus 95.4 ± 30.2 U/L and alanine transaminase levels (ALT) 36 ± 16.9 U/L versus 76.4 ± 28.8 U/L. However, immunostaining results demonstrate that tamoxifen did not induce the expression of HNF1β or CRE in HNF4α+ hepatocytes (Supporting Fig. 1G,H). Induction of reporter gene YFP or β-GAL showed a labeling efficiency of 28.7 ± 10% as assessed by KRT19/YFP or KRT19/β-GAL. These results indicate that the Hnf1βCreER/R26RYfp/LacZ model can be used to trace the fate of HNF1β+ biliary duct cells. Contribution of HNF1β+ Cells to Hepatocyte Turnover During Normal Liver Homeostasis To evaluate the contribution of HNF1β+ cells in a situation of normal liver homeostasis, mice were treated with tamoxifen and sacrificed 2 months later. We assessed the phenotype of the cellular progeny of HNF1β+ cells by performing double staining with the reporter gene β-GAL and cell lineage markers HNF4α and KRT19. As shown in Supporting Fig. 2, we failed to find any hepatocytes that stained with the reporter marker, indicating that HNF1β+ cells give rise to biliary cells but do not contribute to physiological hepatocyte turnover. Figure 2Open in figure viewerPowerPoint HNF1β+ cells do not contribute to liver regeneration after partial hepatectomy. (A) Scheme of experimental design. Double immunostaining with YFP and KRT19 (magnification ×200) or HNF4α (magnification ×400) were performed at different timepoints: (B) Liver excised during PH (0d); (C) 7 days after PH (7d); and (D) 28 days after PH (28d). Few hepatocytes were double stained with HNF4α/YFP (white arrow). (E) Krt7, EpCAM, Trop2, CD133 and Krt19 gene expression in mice with partial hepatectomy (PH) at 7 days (PH 7d) (n = 4) or 28 days after surgery (PH 28d) (n = 4) compared to control liver (PH 0d) (n = 4) (*P < 0.05; **P < 0.005). Nuclei counterstaining was performed with DAPI (blue) (magnification ×200 and ×400). d; days; w, weeks before or after tamoxifen administration; PH, partial hepatectomy. Contribution of HNF1β+ Cells to Liver Regeneration To trace the fate of HNF1β+ cells in a model of liver regeneration, we performed a two-thirds PH in Hnf1βCreERYfp mice (Fig. 2A). Before PH, expression of YFP was restricted to the biliary epithelia as shown by KRT19/YFP and HNF4α/YFP staining (Fig. 2B). At 7 days after surgery mice showed a ductular reaction with HNF1β+ cells restricted to bile ducts (Fig. 2C). Sporadic single hepatocytes located in periportal areas were positive for HNF4α and the reporter gene YFP (Fig. 2C). We also evaluated the contribution of HNF1β+ cells in mice at 28 days after surgery to allow the complete regeneration of the liver (Fig. 2D). No contribution of HNF1β+ cells to hepatocytes was detected at 28 days after surgery, demonstrating that the biliary epithelium does not make a significant contribution to hepatocyte regeneration after PH. Gene expression of Krt7, EpCAM, CD133, Trop2, and Krt199, 28 was also evaluated in mice with PH at time 0 (excised liver), 7 days, and 28 days after surgery. As shown in Fig. 2E, gene expression of LPC markers was increased at 7 days compared to time 0 and returned to basal levels at 28 days. These results suggest that resolution of the liver regenerative process is accompanied with a reduction of cells with an LPC phenotype. Fate of HNF1β+ Cells in Acute Liver Injury In order to evaluate the proliferation and contribution of HNF1β+ cells after acute liver injury, we used two well-known models of acute damage, acute APAP and acute CCl4 administration (Fig. 3A). As shown in Fig. 3B-D,F-H, dual staining of KRT19/YFP, SOX9/YFP, and A6/YFP showed a mild induction of LPC expansion. Moreover, no duct-derived hepatocyte generation was observed based on dual staining of HNF4α/YFP (Fig. 3E,I). Figure 3Open in figure viewerPowerPoint HNF1β+ cells do not contribute to hepatocyte regeneration in acute injury models. (A) Scheme of APAP and CCl4 single injection experimental design. Representative pictures of double staining with YFP and (B,F) KRT19, (C,G) SOX9, (D) HNF1β, (E,I) HNF4α, or (H) A6. HNF1β+ cells did not contribute to hepatocyte population after injury damage induced by (B-E) APAP administration or (F-I) CCl4 single injection. Nuclei counterstaining was performed with DAPI (blue) (magnification × 400). w, weeks before or after tamoxifen administration. Contribution of Hnf1β+ Cells to Chronically Damaged Livers LPC expansion is associated with liver damage compromising hepatocyte proliferative capacity. To determine the contribution of HNF1β+ cells to LPC expansion and hepatocyte regeneration, we performed three different types of chronic liver injury: a DDC diet, which induces cholangiocytic injury, as well as chronic CCl4 treatment and a CDE diet, both known for their hepatocyte toxic effect. We first investigated the progeny of HNF1β+ cells in a DDC diet model, which typically stimulates progenitor cell proliferation (Fig. 4A). As expected, after 4 weeks of DDC diet an important ductular reaction was observed accompanied by a significant increase in Krt7, CD133, and Trop2 messenger RNA (mRNA) expression, but not in EpCAM and Krt19 (Fig. 4B). Double immunostaining with LPC marker A6 or biliary markers SOX9 or KRT19 together with the