Hepatocytes, Rather than Cholangiocytes, Can Be the Major Source of Primitive Ductules in the Chronically Injured Mouse Liver
2014; Elsevier BV; Volume: 184; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2014.01.005
ISSN1525-2191
AutoresSayaka Sekiya, Atsushi Suzuki,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoThe proliferation of biliary lineage cells in chronic liver diseases, which leads to formation of primitive ductules in portal areas of the hepatic lobule, may be important not only for liver regeneration, but also for initiation of liver cancer. Thus, understanding how these primitive ductular cells emerge and proliferate in chronically injured liver holds promise for development of therapeutic strategies for liver diseases. However, the origin of these primitive ductular cells remains controversial. Here, we use a method for genetic lineage tracing to determine the origin of cells that form primitive ductules in a mouse model of chronic liver injury. Our results show that hepatocytes, rather than cholangiocytes, are the major source of cells for the primitive ductules formed in response to chronic liver damage. Moreover, activation of the Notch–Hes1 signaling axis is important for conversion of hepatocytes into primitive ductular cells in chronically injured liver. These findings should be valuable in elucidating the mechanism of liver regeneration associated with the fate-conversion of hepatocytes and in developing therapeutic strategies for liver diseases. The proliferation of biliary lineage cells in chronic liver diseases, which leads to formation of primitive ductules in portal areas of the hepatic lobule, may be important not only for liver regeneration, but also for initiation of liver cancer. Thus, understanding how these primitive ductular cells emerge and proliferate in chronically injured liver holds promise for development of therapeutic strategies for liver diseases. However, the origin of these primitive ductular cells remains controversial. Here, we use a method for genetic lineage tracing to determine the origin of cells that form primitive ductules in a mouse model of chronic liver injury. Our results show that hepatocytes, rather than cholangiocytes, are the major source of cells for the primitive ductules formed in response to chronic liver damage. Moreover, activation of the Notch–Hes1 signaling axis is important for conversion of hepatocytes into primitive ductular cells in chronically injured liver. These findings should be valuable in elucidating the mechanism of liver regeneration associated with the fate-conversion of hepatocytes and in developing therapeutic strategies for liver diseases. The small epithelial cells (often called oval cells) that are histologically identified as cells expressing biliary lineage markers and forming primitive ductules in portal areas of the hepatic lobule appear in response to several types of chronic injury in the human liver, including hepatitis C virus infection, hemochromatosis, and alcoholic liver disease.1Lowes K.N. Brennan B.A. Yeoh G.C. Olynyk J.K. Oval cell numbers in human chronic liver diseases are directly related to disease severity.Am J Pathol. 1999; 154: 537-541Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar These liver pathologies are associated with an increased risk of primary liver cancers.2Prior P. Long-term cancer risk in alcoholism.Alcohol Alcohol. 1988; 23: 163-171PubMed Google Scholar, 3Tsukuma H. Hiyama T. Tanaka S. Nakao M. Yabuuchi T. Kitamura T. Nakanishi K. Fujimoto I. Inoue A. Yamazaki H. Kawashima T. Risk factors for hepatocellular carcinoma among patients with chronic liver disease.N Engl J Med. 1993; 328: 1797-1801Crossref PubMed Scopus (1059) Google Scholar, 4Deugnier Y.M. Guyader D. Crantock L. Lopez J.M. Turlin B. Yaouanq J. Jouanolle H. Campion J.P. Launois B. Halliday J.W. Powell L.W. Brissot P. Primary liver cancer in genetic hemochromatosis: a clinical, pathological, and pathogenetic study of 54 cases.Gastroenterology. 1993; 104: 228-234Abstract PubMed Scopus (0) Google Scholar To study such human diseases, experimental rodent models have been developed with chronic liver injury induced by potential carcinogens, including azo dyes, choline-deficient/ethionine-containing (CDE) diet, d-galactosamine, 2-acetylaminofluorene, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), and 1,4-bis[N,N′-di(ethylene)-phosphamide]-piperazine (dipin).5Farber E. Similarities in the sequence of early histological changes induced in the liver of the rat by ethionine, 2-acetylamino-fluorene, and 3'-methyl-4-dimethylaminoazobenzene.Cancer Res. 1956; 16: 142-148PubMed Google Scholar, 6Shinozuka H. Lombardi B. Sell S. Iammarino R.M. Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline-deficient diet.Cancer Res. 1978; 38: 1092-1098PubMed Google Scholar, 7Lemire J.M. Shiojiri N. Fausto N. Oval cell proliferation and the origin of small hepatocytes in liver injury induced by d-galactosamine.Am J Pathol. 1991; 139: 535-552PubMed Google Scholar, 8Tatematsu M. Ho R.H. Kaku T. Ekem J.K. Farber E. Studies on the proliferation and fate of oval cells in the liver of rats treated with 2-acetylaminofluorene and partial hepatectomy.Am J Pathol. 1984; 114: 418-430PubMed Google Scholar, 9Preisegger K.H. Factor V.M. Fuchsbichler A. Stumptner C. Denk H. Thorgeirsson S.S. Atypical ductular proliferation and its inhibition by transforming growth factor beta1 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model for chronic alcoholic liver disease.Lab Invest. 1999; 79: 103-109PubMed Google Scholar, 10Factor V.M. Radaeva S.A. Oval cells–hepatocytes relationships in Dipin-induced hepatocarcinogenesis in mice.Exp Toxicol Pathol. 1993; 45: 239-244Crossref PubMed Scopus (29) Google Scholar In these models, biliary lineage cells that form primitive ductules appear and proliferate around the portal veins to regenerate damaged liver tissues. Moreover, deletion of Tp53 (alias p53) in these cells resulted in formation of malignant tumors on injection into immunodeficient mutant mice, suggesting that these rodent models of chronic liver injury mirror the progression of human liver pathologies.11Dumble M.L. Croager E.J. Yeoh G.C. Quail E.A. Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma.Carcinogenesis. 2002; 23: 435-445Crossref PubMed Scopus (196) Google Scholar, 12Suzuki A. Sekiya S. Onishi M. Oshima N. Kiyonari H. Nakauchi H. Taniguchi H. Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver.Hepatology. 2008; 48: 1964-1978Crossref PubMed Scopus (137) Google Scholar Thus, to develop therapeutic strategies for liver diseases, it is important to understand how biliary lineage cells emerge and form primitive ductules in models of chronic liver injury. Previous studies involving histological observations of liver injury and carcinogenesis have suggested that the portal biliary ductules and the canals of Hering, through which the terminal segments of the biliary system connect with hepatocytes in liver parenchyma, constitute a niche for cells capable of generating biliary lineage cells that form primitive ductules.13Fausto N. Campbell J.S. The role of hepatocytes and oval cells in liver regeneration and repopulation.Mech Dev. 2003; 120: 117-130Crossref PubMed Scopus (560) Google Scholar, 14Michalopoulos G.K. Liver regeneration: alternative epithelial pathways.Int J Biochem Cell Biol. 2011; 43: 173-179Crossref PubMed Scopus (99) Google Scholar Recent evidence from Cre-based genetic lineage-tracing experiments also showed that primitive ductular cells found in the mouse DDC and CDE models of chronic liver injury are, at least in part, derived from cholangiocytes lining the intrahepatic bile ducts.15Furuyama K. Kawaguchi Y. Akiyama H. Horiguchi M. Kodama S. Kuhara T. Hosokawa S. Elbahrawy A. Soeda T. Koizumi M. Masui T. Kawaguchi M. Takaori K. Doi R. Nishi E. Kakinoki R. Deng J.M. Behringer R.R. Nakamura T. Uemoto S. Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine.Nat Genet. 2011; 43: 34-41Crossref PubMed Scopus (651) Google Scholar, 16Dorrell C. Erker L. Schug J. Kopp J.L. Canaday P.S. Fox A.J. Smirnova O. Duncan A.W. Finegold M.J. Sander M. Kaestner K.H. Grompe M. Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice.Genes Dev. 2011; 25: 1193-1203Crossref PubMed Scopus (191) Google Scholar, 17Español-Suñer R. Carpentier R. Van Hul N. Legry V. Achouri Y. Cordi S. Jacquemin P. Lemaigre F. Leclercq I.A. Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice.Gastroenterology. 2012; 143: 1564-1575Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar In addition to this biliary origin scenario, other histological and cell culture-based studies have suggested that mature hepatocytes can transdifferentiate into biliary lineage cells that form ductular structures, in which case hepatocytes could be a potential source of cells that also express biliary lineage markers and form primitive ductular structures in chronically injured liver.18Michalopoulos G.K. Bowen W.C. Mulè K. Lopez-Talavera J.C. Mars W. Hepatocytes undergo phenotypic transformation to biliary epithelium in organoid cultures.Hepatology. 2002; 36: 278-283Crossref PubMed Scopus (72) Google Scholar, 19Michalopoulos G.K. Barua L. Bowen W.C. Transdifferentiation of rat hepatocytes into biliary cells after bile duct ligation and toxic biliary injury.Hepatology. 2005; 41: 535-544Crossref PubMed Scopus (245) Google Scholar, 20Nishikawa Y. Doi Y. Watanabe H. Tokairin T. Omori Y. Su M. Yoshioka T. Enomoto K. Transdifferentiation of mature rat hepatocytes into bile duct-like cells in vitro.Am J Pathol. 2005; 166: 1077-1088Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Moreover, a recent study using adeno-associated virus (AAV) with a thyroid binding globulin (TBG) promoter-driven Cre recombinase showed that hepatocytes can be converted into biliary lineage cells in DDC-induced chronic liver injury.21Yanger K. Zong Y. Maggs L.R. Shapira S.N. Maddipati R. Aiello N.M. Thung S.N. Wells R.G. Greenbaum L.E. Stanger B.Z. Robust cellular reprogramming occurs spontaneously during liver regeneration.Genes Dev. 2013; 27 ([Erratum appeared in Genes Dev 2013, 27:1537]): 719-724Crossref PubMed Scopus (335) Google Scholar In that study, however, the proportion of hepatocyte-derived cytokeratin 19 (CK19)–positive primitive ductular cells (indicative of terminal conversion of hepatocytes to biliary lineage cells) was only approximately 14%; however, several biliary lineage markers were expressed in 30% to 50% of hepatocytes. Thus, it is difficult to conclude whether hepatocytes actively contribute to primitive ductule formation in DDC-treated chronically injured liver. Indeed, other hepatocyte fate-tracing studies using mice with chimeric livers or AAV with a transthyretin (Ttr) promoter-driven Cre recombinase have shown that the primitive ductular cells induced in the mouse DDC model were not derived from hepatocytes.22Wang X. Foster M. Al-Dhalimy M. Lagasse E. Finegold M. Grompe M. The origin and liver repopulating capacity of murine oval cells.Proc Natl Acad Sci USA. 2003; 100: 11881-11888Crossref PubMed Scopus (373) Google Scholar, 23Malato Y. Naqvi S. Schürmann N. Ng R. Wang B. Zape J. Kay M.A. Grimm D. Willenbring H. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration.J Clin Invest. 2011; 121: 4850-4860Crossref PubMed Scopus (330) Google Scholar Furthermore, although it has also been suggested that the primitive ductular cells found in chronic liver diseases originate from bone marrow–derived cells,24Petersen B.E. Bowen W.C. Patrene K.D. Mars W.M. Sullivan A.K. Murase N. Boggs S.S. Greenberger J.S. Goff J.P. Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Crossref PubMed Scopus (2196) Google Scholar evidence from a study combining bone marrow chimeras with the mouse DDC model indicated no contribution of bone marrow–derived cells to primitive ductule formation in liver.22Wang X. Foster M. Al-Dhalimy M. Lagasse E. Finegold M. Grompe M. The origin and liver repopulating capacity of murine oval cells.Proc Natl Acad Sci USA. 2003; 100: 11881-11888Crossref PubMed Scopus (373) Google Scholar Overall, the cellular origin of the biliary lineage cells that appear and form primitive ductules in chronically injured liver remains controversial. In the present study, we performed Cre-based genetic lineage-tracing analyses to determine the fates of hepatocytes and cholangiocytes using more definitive markers for these cells and to identify which cell type is the source of the primitive ductular cells that appear in chronically injured liver. Specifically, hepatocytes expressing albumin or cholangiocytes expressing CK19 in liver were heritably labeled in mutant mice with a tamoxifen (TMX) inducible Cre/loxP system. The mutant mice were then fed a DDC-containing diet to induce chronic liver injury and primitive ductule formation. Our present findings show that hepatocytes, rather than cholangiocytes, give rise to the majority of the biliary lineage cells that form primitive ductules in chronically injured liver, through Notch-mediated cell lineage conversion. Thus, at least in the mouse DDC model, albumin-positive hepatocytes can be the major source of the biliary lineage cells that form primitive ductules around the portal veins with a small number of CK19+ cholangiocyte-derived cells in chronically injured liver. The mice used in this study were C57BL/6 mice (CLEA Japan, Fuji, Japan), Alb-CreERT2 mice [a gift from Drs. Pierre Chambon and Daniel Metzger (Institute of Genetics and Molecular and Cellular Biology, Strasbourg, France)], CK19-CreERT2 mice,25Sekiya S. Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.J Clin Invest. 2012; 122: 3914-3918Crossref PubMed Scopus (246) Google Scholar R26RlacZ/lacZ mice (Jackson Laboratory, Bar Harbor, ME), R26RYFP/YFP mice [a gift from Dr. Frank Costantini (Columbia University, New York, NY)], R26RNotch/+ mice [a gift from Dr. Douglas A. Melton (Harvard University, Cambridge, MA)], and Hes1fl/fl mice [a gift from Dr. Ryoichiro Kageyama (Kyoto University, Kyoto, Japan)]. For induction of Cre activity, 8- to 10-week–old mice were given a single intraperitoneal injection of 6.5 to 8.0 mg TMX per mouse (Sigma-Aldrich, St. Louis, MO) dissolved in olive oil (Nacalai Tesque, Kyoto, Japan) at a concentration of 50 mg/mL.25Sekiya S. Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.J Clin Invest. 2012; 122: 3914-3918Crossref PubMed Scopus (246) Google Scholar To induce chronic liver injury, mice were fed a diet containing 0.1% DDC (Sigma-Aldrich). Liver tissues with yellow fluorescent protein (YFP) expression were fixed with 2% paraformaldehyde at 4°C overnight.25Sekiya S. Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.J Clin Invest. 2012; 122: 3914-3918Crossref PubMed Scopus (246) Google Scholar After treatment with 30% sucrose at 4°C overnight, the tissues were embedded in Tissue-Tek OCT optimal cutting temperature compound (Sakura Finetek, Torrance, CA; Tokyo, Japan), and frozen sections were prepared.25Sekiya S. Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.J Clin Invest. 2012; 122: 3914-3918Crossref PubMed Scopus (246) Google Scholar Liver tissues without YFP expression were directly embedded in the OCT compound, and frozen sections of these tissues were initially fixed with 4% paraformaldehyde for 5 minutes and then fixed with methanol for 5 minutes at room temperature.25Sekiya S. Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.J Clin Invest. 2012; 122: 3914-3918Crossref PubMed Scopus (246) Google Scholar For albumin staining, antigen retrieval was performed by microwave heating in 0.01 mol/L citrate buffer (pH 6.0). After a washing in PBS containing 0.1% Tween 20, followed by blocking, the sections were incubated with the following primary antibodies: rat anti–Ep-CAM (1:200; eBioscience, San Diego, CA), rabbit anti–β-gal (1:2000; Abcam, Cambridge, MA), goat anti-albumin (1:200; Bethyl Laboratories, Montgomery, TX), rabbit anti-keratin (1:2000; Dako, Carpinteria, CA), mouse anti-Hnf4α (1:200; Perseus Proteomics, Tokyo, Japan), rabbit anti-CK19 (1:2000; a rabbit anti–mouse CK19 antibody generated against the peptide HYNNLPTPKAI25Sekiya S. Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.J Clin Invest. 2012; 122: 3914-3918Crossref PubMed Scopus (246) Google Scholar; GeneNet, Fukuoka, Japan), goat anti-Trop2 (1:50; R&D Systems), rabbit anti-Sox9 (1:1000; EMD Millipore, Billerica, MA), rabbit anti-Cre (1:2000; Covance, Princeton, NJ), and rat anti-A6 [1:50; a gift from Dr. Valentina M. Factor (National Cancer Institute, Bethesda, MD)]. After a washing, the sections were incubated with Alexa Fluor 488–conjugated and/or Alexa Fluor 555–conjugated secondary antibodies (1:200; Life Technologies, Carlsbad, CA) and DAPI. Quantitative analyses were performed using the data obtained by counting approximately 1000 cells per field of view in three discontinuous liver tissue slides for three individual mice. Single-cell suspensions of nonparenchymal cells were prepared from liver of TMX-administered CK19-CreERT2;R26RYFP/+ mice after 2 weeks of DDC feeding and were incubated with phycoerythrin-conjugated anti–Ep-CAM monoclonal antibody (eBioscience) as described previously.12Suzuki A. Sekiya S. Onishi M. Oshima N. Kiyonari H. Nakauchi H. Taniguchi H. Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver.Hepatology. 2008; 48: 1964-1978Crossref PubMed Scopus (137) Google Scholar The fluorescence-labeled cells were analyzed and separated with a FACSJazz cell sorter (BD Biosciences, San Jose, CA). PCR analyses were performed to determine whether Cre-mediated recombination occurred in the isolated cells, using their genomic DNA and the primers 5′-AAAGTCGCTCTGAGTTGTTAT-3′ and 5′-TGGTAGCTCAGGTAGTGGTTGT-3′ (forward and reverse, respectively). We crossed mouse lines expressing an inducible form of Cre recombinase (CreERT2) from the albumin genomic locus (Alb-CreERT2 mice)26Schuler M. Dierich A. Chambon P. Metzger D. Efficient temporally controlled targeted somatic mutagenesis in hepatocytes of the mouse.Genesis. 2004; 39: 167-172Crossref PubMed Scopus (114) Google Scholar or the CK19 genomic locus (CK19-CreERT2 mice),25Sekiya S. Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.J Clin Invest. 2012; 122: 3914-3918Crossref PubMed Scopus (246) Google Scholar with R26RlacZ/lacZ or R26RYFP/YFP reporter mouse lines.27Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat Genet. 1999; 21: 70-71Crossref PubMed Scopus (4157) Google Scholar, 28Srinivas S. Watanabe T. Lin C.S. William C.M. Tanabe Y. Jessell T.M. Costantini F. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.BMC Dev Biol. 2001; 1: 4Crossref PubMed Scopus (2313) Google Scholar In the double-mutant mice, administration of TMX allowed permanent marking of albumin-expressing or CK19-expressing cells and enabled us to follow the fate of the progeny of hepatocytes or cholangiocytes, respectively.25Sekiya S. Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.J Clin Invest. 2012; 122: 3914-3918Crossref PubMed Scopus (246) Google Scholar After TMX injection, hepatocytes in liver of Alb-CreERT2;R26RlacZ/+ mice were labeled by lacZ, as assessed by X-gal staining to detect β-galactosidase (β-gal) enzyme activity (Figure 1A). By contrast, no lacZ+ cells were found within intrahepatic bile ducts or blood vessels, even at 4 weeks after TMX injection (Figure 1A). Moreover, in liver of Alb-CreERT2;R26RYFP/+ mice, YFP was expressed in almost all hepatocytes that were marked with the expression of hepatocyte nuclear factor 4α (Hnf4α), but not in any CK19+ cholangiocytes lining the intrahepatic bile ducts (Figure 1B). Thus, TMX injection led to specific labeling of hepatocytes and allowed us to trace the fate of lacZ+ or YFP+ hepatocyte progeny. To induce chronic liver injury and primitive ductule formation in liver, we started feeding a 0.1% DDC-containing diet to Alb-CreERT2;R26RlacZ/+ mice at 1 week after TMX injection. After 3 weeks of DDC feeding, many ductular structures appeared around the portal veins in liver of Alb-CreERT2;R26RlacZ/+ mice, and these primitive ductules consisted mainly of lacZ+ cells (Figure 1C). Moreover, the majority of DDC-induced primitive ductular cells observed in liver of Alb-CreERT2;R26RYFP/+ mice or Alb-CreERT2;R26RlacZ/+ mice expressed YFP or β-gal with CK19, keratin, epithelial cell adhesion molecule (Ep-CAM), Trop2, and A6, which are antigens marking biliary lineage cells in normal and chronically injured livers29de Boer C.J. van Krieken J.H. Janssen-van Rhijn C.M. Litvinov S.V. Expression of Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver.J Pathol. 1999; 188: 201-206Crossref PubMed Scopus (191) Google Scholar, 30Okabe M. Tsukahara Y. Tanaka M. Suzuki K. Saito S. Kamiya Y. Tsujimura T. Nakamura K. Miyajima A. Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver.Development. 2009; 136: 1951-1960Crossref PubMed Scopus (228) Google Scholar, 31Engelhardt N.V. Factor V.M. Yasova A.K. Poltoranina V.S. Baranov V.N. Lasareva M.N. Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes.Differentiation. 1990; 45: 29-37Crossref PubMed Scopus (88) Google Scholar (Figure 1D). On the other hand, in liver of CK19-CreERT2;R26RYFP/+ mice, in which approximately 60% of cholangiocytes were specifically marked by YFP expression after TMX injection (Figure 2A), only a small number of cells within DDC-induced primitive ductules expressed YFP with CK19 and keratin (Figure 2B). To validate these findings, YFP+ and YFP− cells among the Ep-CAM+ biliary lineage cells were isolated from the nonparenchymal cell fraction in liver of TMX-administered CK19-CreERT2;R26RYFP/+ mice after 2 weeks of DDC feeding. PCR analyses using genomic DNA from these cells revealed that Cre-mediated excision of the floxed termination sequence occurred in YFP+ cells, but not in YFP− cells (Supplemental Figure S1). Taken together, these findings demonstrate that both hepatocytes and cholangiocytes can be sources of the biliary lineage cells that form primitive ductules in a mouse model of chronic liver injury, although the majority of these cells arise from hepatocytes, rather than cholangiocytes. We next investigated what occurs in liver tissues after cessation of DDC treatment. In recovery from chronic injury, DDC-induced primitive ductules disappeared from the liver during subsequent feeding of a normal diet for 15 weeks, and only intrahepatic bile ducts remained around the portal veins (Figure 3, A–C). Lineage-tracing analyses using Alb-CreERT2;R26RYFP/+ mice revealed that YFP was still expressed in almost all Hnf4α+ hepatocytes, and that YFP+ hepatocyte-derived cells could also contribute (but rarely) to the formation of bile ducts as CK19+ cholangiocytes, in liver tissues after recovery from DDC-induced chronic injury (Figure 3D). On the other hand, in liver tissues of CK19-CreERT2;R26RYFP/+ mice after recovery from chronic injury, approximately 60% of cholangiocytes still expressed YFP, but any Hnf4α+ hepatocytes did not express YFP (Figure 3E). These findings demonstrate that, in recovery from DDC-induced chronic liver injury, hepatocytes arise from hepatocyte-derived cells, but not from cholangiocyte-derived cells, and bile duct regeneration occurs by self-duplication of cholangiocytes, with only a slight contribution from hepatocyte-derived cells. In the rat 2-acetylaminofluorene liver injury model, biliary lineage cells within the primitive ductules are, at least in part, positive for albumin expression.32Evarts R.P. Nagy P. Nakatsukasa H. Marsden E. Thorgeirsson S.S. In vivo differentiation of rat liver oval cells into hepatocytes.Cancer Res. 1989; 49: 1541-1547PubMed Google Scholar Thus, in the mouse DDC model, newly generated ductular cells might also express albumin in an early phase of ductular structure formation and be labeled by the remnant TMX. However, this possibility can be excluded, for several reasons. First, the efficacy of TMX is rapid and short lived, disappearing within 2 days,33Ahn S. Joyner A.L. Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning.Cell. 2004; 118: 505-516Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 34Danielian P.S. Muccino D. Rowitch D.H. Michael S.K. McMahon A.P. Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.Curr Biol. 1998; 8: 1323-1326Abstract Full Text Full Text PDF PubMed Google Scholar and therefore TMX is no longer effective at the beginning of DDC feeding in this model and just induces labeling of hepatocytes in the normal liver. Indeed, accumulation of Cre recombinase in the nuclei of hepatocytes was not observed in liver of Alb-CreERT2 mice at 1 week after TMX injection, when we normally started feeding the DDC-containing diet to mice (Supplemental Figure S2). Moreover, even though we started DDC feeding at 2 weeks after TMX injection, primitive ductular cells in liver of Alb-CreERT2;R26RYFP/+ mice were YFP+ (Supplemental Figure S3). Second, the A6+ ductular cells that appeared after 1 week of DDC feeding did not express either albumin or Hnf4α, although most of these ductular cells were derived from hepatocytes (Figure 4). Third, all of the primitive ductular cells in DDC-treated mouse liver expressed CD133 (prominin-1) (a five-transmembrane cell surface glycoprotein12Suzuki A. Sekiya S. Onishi M. Oshima N. Kiyonari H. Nakauchi H. Taniguchi H. Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver.Hepatology. 2008; 48: 1964-1978Crossref PubMed Scopus (137) Google Scholar). Our research group has previously isolated these CD133+ ductular cells by flow cytometry and examined the expression of markers for hepatocytes and biliary lineage cells; the results showed that freshly isolated CD133+ cells expressed CK19 and CK7, but did not express any hepatocyte markers, including albumin.12Suzuki A. Sekiya S. Onishi M. Oshima N. Kiyonari H. Nakauchi H. Taniguchi H. Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver.Hepatology. 2008; 48: 1964-1978Crossref PubMed Scopus (137) Google Scholar There is also a possibility that TMX-independent Cre activation can occur in DDC-treated mouse liver, in which case the primitive ductules formed in chronically injured liver would arise from other than hepatocytes. To address this possibility, we fed the DDC-containing diet to Alb-CreERT2;R26RYFP/+ mice without TMX injection. After 3 weeks of DDC feeding, we did not find any YFP+ cells in liver of these mice, which suggests that DDC did not induce TMX-independent Cre activity in the liver (Supplemental Figure S4). Taken together, these findings demonstrate that hepatocytes contribute to primitive ductule formation in DDC-treated chronically injured mouse liver. Given these findings, we further investigated whether hepatocytes contribute to biliary ductule reconstitution during normal liver regeneration after partial hepatectomy (PH) or fulminant hepatic failure caused by administration of hepatotoxic chemicals. At 1 week after TMX injection into Alb-CreERT2;R26RlacZ/+ mice and Alb-CreERT2;R26RYFP/+ mice, we performed PH (removal of 70% of the liver lobe) or subcutaneously injected carbon tetrachloride (CCl4) dissolved in olive oil (2 mL/kg mouse weight). As expected, no ductular structures other than normal bile ducts were observed in regenerated liver tissues analyzed at 2 weeks after PH or chemical injury. After PH treatment, lacZ and YFP were detected only in hepatocytes, similar to the normal liver (Figure 5, A and B). However, in liver tissues regenerated after fulminant hepatic failure, lacZ+ or YFP+ cells resided within bile ducts, although the frequencies of these lacZ+ or YFP+ cholangiocytes were extremely low (Figure 5, C–F). These findings demonstrate that, in addition to DDC-induced chronic injury, CCl4–induced fulminant hepatic failure can be a trigger for the generation of hepatocyte-derived biliary lineage cells (although this effect may be too weak to induce primitive ductule formation from hepatocytes). Next, we focused on the mechanism controlling the conversion of hepatocytes into primitive ductular cells in DDC-induced chronic liver injury. Activation of Notch signaling is important for induction of the biliary program, not only in hepatic progenitor cells during development, but also in postnatal hepatocytes.21Yanger K. Zong Y. Maggs L.R. Shapira S.N. Maddipati R. Aiello N.M. Thung S.N. Wells R.G. Greenbaum L.E. Stanger B.Z. Robust cellular reprogramming occurs spontaneously during liver regeneration.Genes Dev. 2013; 27 ([Erratum appeared in Genes Dev 2013, 27:1537]): 719-724Crossref PubMed Scopus (335) Google Scholar, 25Sekiya S. Suzuki A. Intrahepatic cholangiocarcinoma can arise from Notch-mediated conversion of hepatocytes.J Clin Invest. 2012; 122: 3914-3918Crossref PubMed Scopus (246) Google Scholar, 35McCright B. Lozier J. Gridley T. A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency.Development. 2002; 129: 1075-1082Crossref PubMed Google Scholar, 36Zong Y. Panikkar A. Xu J. Anto
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