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• W2005876661 abstract "Background & Aims: Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are 2 distinct mesenchymal cells in adult liver. HSCs in sinusoids accumulate lipids and express p75 neurotrophin receptor (p75NTR). HSCs and PFs play pivotal roles in liver regeneration and fibrosis. However, the roles of mesenchymal cells in fetal liver remain poorly understood. In this study, we aimed to characterize mesenchymal cells in mouse fetal liver. Methods: We prepared an anti-p75NTR monoclonal antibody applicable for flow cytometry and immunohistochemistry. p75NTR+ cells isolated from fetal liver by flow cytometry were characterized by reverse-transcription polymerase chain reaction, immunohistochemistry, and cell cultivation. Lipid-containing cells were visualized by Oil-red O staining. Results: p75NTR+ cells in fetal liver were clearly distinct from endothelial cells and showed characteristics of mesenchymal cells. At embryonic day (E) 10.5, p75NTR+ cells were present at the periphery of the liver bud in close contact with endothelial cells, and spread over the liver at E11.5. With the formation of the liver architecture, they began to localize to 2 distinct areas, parenchymal and portal areas, and lipid-containing p75NTR+ cells increased accordingly. p75NTR+ cells around portal veins were adjacent to cholangiocytes and expressed Jagged1, a crucial factor for the commitment of hepatoblasts to cholangiocytes. By cultivation, p75NTR+ cells showed features of adult HSCs with markedly increased expression of glial fibrillary acidic protein and α-smooth muscle actin. Conclusions: p75NTR+ mesenchymal cells in fetal liver include progenitors for HSCs and PFs, and the anti-p75NTR monoclonal antibody is useful for their isolation. Background & Aims: Hepatic stellate cells (HSCs) and portal fibroblasts (PFs) are 2 distinct mesenchymal cells in adult liver. HSCs in sinusoids accumulate lipids and express p75 neurotrophin receptor (p75NTR). HSCs and PFs play pivotal roles in liver regeneration and fibrosis. However, the roles of mesenchymal cells in fetal liver remain poorly understood. In this study, we aimed to characterize mesenchymal cells in mouse fetal liver. Methods: We prepared an anti-p75NTR monoclonal antibody applicable for flow cytometry and immunohistochemistry. p75NTR+ cells isolated from fetal liver by flow cytometry were characterized by reverse-transcription polymerase chain reaction, immunohistochemistry, and cell cultivation. Lipid-containing cells were visualized by Oil-red O staining. Results: p75NTR+ cells in fetal liver were clearly distinct from endothelial cells and showed characteristics of mesenchymal cells. At embryonic day (E) 10.5, p75NTR+ cells were present at the periphery of the liver bud in close contact with endothelial cells, and spread over the liver at E11.5. With the formation of the liver architecture, they began to localize to 2 distinct areas, parenchymal and portal areas, and lipid-containing p75NTR+ cells increased accordingly. p75NTR+ cells around portal veins were adjacent to cholangiocytes and expressed Jagged1, a crucial factor for the commitment of hepatoblasts to cholangiocytes. By cultivation, p75NTR+ cells showed features of adult HSCs with markedly increased expression of glial fibrillary acidic protein and α-smooth muscle actin. Conclusions: p75NTR+ mesenchymal cells in fetal liver include progenitors for HSCs and PFs, and the anti-p75NTR monoclonal antibody is useful for their isolation. The liver consists of 2 types of epithelial cells, hepatocytes and cholangiocytes (biliary epithelial cells), and various nonparenchymal cells that include hematopoietic, endothelial, and mesenchymal cells. There are 3 major mesenchymal cells in the liver (ie, hepatic stellate cells [HSCs], portal fibroblasts [PFs], and vascular smooth muscle cells residing in the walls of portal veins, arteries, and central veins). HSCs, also known as Ito cells, fat-storing cells, or lipocytes, are pericytes found in the perisinusoidal space between sinusoids and hepatocytes in adult liver. Protrusions extending from HSCs wrap sinusoidal endothelial cells that constitute the sinusoidal capillary network in the liver. By contrast, PFs are present in the connective tissue around portal vessels and bile ducts. In normal liver, HSCs are in a quiescent state and contain vitamin A–rich lipid droplets, constituting the largest reservoir of vitamin A in the body,1Hautekeete M.L. Geerts A. The hepatic stellate (Ito) cell: its role in human liver disease.Virchows Arch. 1997; 430: 195-207Crossref PubMed Scopus (263) Google Scholar whereas PFs do not store lipid droplets. Although HSCs express desmin but not elastin, PFs show an inverse expression pattern.2Li Z. Dranoff J.A. Chan E.P. et al.Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture.Hepatology. 2007; 46: 1246-1256Crossref PubMed Scopus (266) Google Scholar In injured liver, HSCs become activated and transform into myofibroblast-like cells that secrete various cytokines to contribute to liver regeneration.3Knittel T. Kobold D. Piscaglia F. et al.Localization of liver myofibroblasts and hepatic stellate cells in normal and diseased rat livers: distinct roles of (myo-)fibroblast subpopulations in hepatic tissue repair.Histochem Cell Biol. 1999; 112: 387-401Crossref PubMed Scopus (162) Google Scholar, 4Taub R. Liver regeneration: from myth to mechanism.Nat Rev Mol Cell Biol. 2004; 5: 836-847Crossref PubMed Scopus (1243) Google Scholar Those activated HSCs are enlarged, lack lipid droplets,5Friedman S.L. Wei S. Blaner W.S. Retinol release by activated rat hepatic lipocytes: regulation by Kupffer cell-conditioned medium and PDGF.Am J Physiol. 1993; 264: G947-G952PubMed Google Scholar, 6Tsukamoto H. Cheng S. Blaner W.S. Effects of dietary polyunsaturated fat on ethanol-induced Ito cell activation.Am J Physiol. 1996; 270: G581-G586PubMed Google Scholar and express α-smooth muscle actin (α-SMA).7Schmitt-Graff A. Kruger S. Bochard F. et al.Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers.Am J Pathol. 1991; 138: 1233-1242PubMed Google Scholar, 8Yamaoka K. Nouchi T. Marumo F. et al.Alpha-smooth-muscle actin expression in normal and fibrotic human livers.Dig Dis Sci. 1993; 38: 1473-1479Crossref PubMed Scopus (74) Google Scholar Upon prolonged liver injury, the transformed myofibroblast-like cells produce a large amount of extracellular matrix, leading to fibrosis.1Hautekeete M.L. Geerts A. The hepatic stellate (Ito) cell: its role in human liver disease.Virchows Arch. 1997; 430: 195-207Crossref PubMed Scopus (263) Google Scholar, 9Friedman S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury.J Biol Chem. 2000; 275: 2247-2250Crossref PubMed Scopus (1877) Google Scholar Similarly, the activated PFs express α-SMA and differentiate into myofibroblasts, which are involved in the early stages of biliary fibrosis.10Kinnman N. Francoz C. Barbu V. et al.The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis.Lab Invest. 2003; 83: 163-173Crossref PubMed Scopus (198) Google Scholar, 11Guyot C. Lepreux S. Combe C. et al.Hepatic fibrosis and cirrhosis: the (myo)fibroblastic cell subpopulations involved.Int J Biochem Cell Biol. 2006; 38: 135-151PubMed Google Scholar Because HSCs and PFs play pivotal roles in liver repair and diseases, the nature of these intriguing cell types has been a subject of intense research. HSCs show features of both mesenchymal cells and neural/neuroendocrine cells by expressing vimentin, desmin,12Burt A.D. Robertson J.L. Heir J. et al.Desmin-containing stellate cells in rat liver; distribution in normal animals and response to experimental acute liver injury.J Pathol. 1986; 150: 29-35Crossref PubMed Scopus (103) Google Scholar and α-SMA, as well as glial fibrillary acidic protein (GFAP),13Neubauer K. Knittel T. Aurisch S. et al.Glial fibrillary acidic protein–a cell type specific marker for Ito cells in vivo and in vitro.J Hepatol. 1996; 24: 719-730Abstract Full Text PDF PubMed Scopus (160) Google Scholar nestin,14Niki T. Pekny M. Hellemans K. et al.Class VI intermediate filament protein nestin is induced during activation of rat hepatic stellate cells.Hepatology. 1999; 29: 520-527Crossref PubMed Scopus (239) Google Scholar neural cell adhesion molecule,15Knittel T. Aurisch S. Neubauer K. et al.Cell-type-specific expression of neural cell adhesion molecule (N-CAM) in Ito cells of rat liver Up-regulation during in vitro activation and in hepatic tissue repair.Am J Pathol. 1996; 149: 449-462PubMed Google Scholar and synaptophysin,16Cassiman D. van Pelt J. de Vos R. et al.Synaptophysin: a novel marker for human and rat hepatic stellate cells.Am J Pathol. 1999; 155: 1831-1839Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar respectively. Because HSCs show mesenchymal and neuronal features, it has been postulated that their origin is related to the neuronal lineage (ie, neural crest cells). However, Cassiman et al17Cassiman D. Barlow A. Vander Borght S. et al.Hepatic stellate cells do not derive from the neural crest.J Hepatol. 2006; 44: 1098-1104Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar reported that HSCs are not derived from the neural crest. Moreover, it was recently reported that bone marrow cells become HSCs in adult liver.18Baba S. Fujii H. Hirose T. et al.Commitment of bone marrow cells to hepatic stellate cells in mouse.J Hepatol. 2004; 40: 255-260Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar Although the distinction between adult HSCs and PFs has now been shown,2Li Z. Dranoff J.A. Chan E.P. et al.Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture.Hepatology. 2007; 46: 1246-1256Crossref PubMed Scopus (266) Google Scholar the nature and origin of these mesenchymal precursors remain unclear.19Geerts A. On the origin of stellate cells: mesodermal, endodermal or neuro-ectodermal?.J Hepatol. 2004; 40: 331-334Abstract Full Text Full Text PDF PubMed Scopus (71) Google ScholarHepatocytes and cholangiocytes derive from hepatoblasts, which emerge from the foregut endoderm and invade into the septum transversum mesenchyme to form the liver bud.20Duncan S.A. Mechanisms controlling early development of the liver.Mech Dev. 2003; 120: 19-33Crossref PubMed Scopus (145) Google Scholar It was shown that the proliferation of hepatoblasts requires endothelial cells using explant cultures from mutant mouse embryos defective in the development of endothelial cells.21Matsumoto K. Yoshitomi H. Rossant J. et al.Liver organogenesis promoted by endothelial cells prior to vascular function.Science. 2001; 294: 559-563Crossref PubMed Scopus (686) Google Scholar Because hepatocytes and HSCs interact intimately in adult liver and bile ducts are formed along with the portal veins consisting of endothelial cells and mesenchymal cells, they are speculated to interact at a certain stage of development. However, it still remains unknown when mesenchymal cells develop and whether they affect the proliferation and/or differentiation of hepatoblasts. Because adult HSCs contain numerous vitamin A–rich lipid droplets, low-density cells separated from the nonparenchymal cells in normal liver by centrifugation through various density media have been used to study HSCs.22Knook D.L. Seffelaar A.M. de Leeuw A.M. Fat-storing cells of the rat liver Their isolation and purification.Exp Cell Res. 1982; 139: 468-471Crossref PubMed Scopus (221) Google Scholar, 23Geerts A. Vrijsen R. Rauterberg J. et al.In vitro differentiation of fat-storing cells parallels marked increase of collagen synthesis and secretion.J Hepatol. 1989; 9: 59-68Abstract Full Text PDF PubMed Scopus (167) Google Scholar, 24Blomhoff R. Berg T. Isolation and cultivation of rat liver stellate cells.Methods Enzymol. 1990; 190: 58-71Crossref PubMed Scopus (60) Google Scholar, 25Greenwel P. Schwartz M. Rosas M. et al.Characterization of fat-storing cell lines derived from normal and CCl4-cirrhotic livers Differences in the production of interleukin-6.Lab Invest. 1991; 65: 644-653PubMed Google Scholar, 26Friedman S.L. Rockey D.C. McGuire R.F. et al.Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture.Hepatology. 1992; 15: 234-243Crossref PubMed Scopus (240) Google Scholar However, this method is not applicable to the preparation of fetal HSC precursors because they do not store enough lipid droplets for separation. In addition, a lack of HSC markers for cell sorting has prevented the investigation of HSCs in liver development. Therefore, little information is available on fetal HSCs and/or their precursors and their characterization requires the prospective isolation of the fetal liver cell populations. To this end, we generated many monoclonal antibodies (mAbs) against fetal liver cells and selected those that recognize cell surface proteins. One of those mAbs recognized p75 neurotrophin receptor (p75NTR), a receptor for neurotrophic factors such as nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5. Although p75NTR is expressed mainly in the nervous system,27Bibel M. Barde Y.A. Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system.Genes Dev. 2000; 14: 2919-2937Crossref PubMed Scopus (873) Google Scholar it is also expressed in nonneural tissues, some tumor cells, and normal mesenchymal precursor cells of myoid cells in testis.28Campagnolo L. Russo M.A. Puglianiello A. et al.Mesenchymal cell precursors of peritubular smooth muscle cells of the mouse testis can be identified by the presence of the p75 neurotrophin receptor.Biol Reprod. 2001; 64: 464-472Crossref PubMed Scopus (43) Google Scholar In addition, it was shown previously that human and rat HSCs expressed p75NTR29Cassiman D. Denef C. Desmet V.J. et al.Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors.Hepatology. 2001; 33: 148-158Crossref PubMed Scopus (192) Google Scholar and that activated HSCs expressing p75NTR undergo apoptosis in response to nerve growth factor in vitro.30Trim N. Morgan S. Evans M. et al.Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation.Am J Pathol. 2000; 156: 1235-1243Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 31Oakley F. Trim N. Constandinou C.M. et al.Hepatocytes express nerve growth factor during liver injury: evidence for paracrine regulation of hepatic stellate cell apoptosis.Am J Pathol. 2003; 163: 1849-1858Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 32Asai K. Tamakawa S. Yamamoto M. et al.Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation.Liver Int. 2006; 26: 595-603Crossref PubMed Scopus (57) Google Scholar Recently, Passino et al33Passino M.A. Adams R.A. Sikorski S.L. et al.Regulation of hepatic stellate cell differentiation by the neurotrophin receptor p75NTR.Science. 2007; 315: 1853-1856Crossref PubMed Scopus (151) Google Scholar, 34Geerts A. The simple truth is seldom true and never simple: dual role for p75(NTR) in transdifferentiation and cell death of hepatic stellate cells.Hepatology. 2007; 46: 600-601Crossref PubMed Scopus (7) Google Scholar reported that depletion of p75NTR in mice exacerbated liver pathology and inhibited hepatocyte proliferation in vivo. Furthermore, they showed that ligand-independent p75NTR signaling to Rho enhanced the differentiation of HSCs into regeneration-promoting cells that support hepatocyte proliferation in diseased liver. However, the expression of p75NTR in fetal liver and the nature of p75NTR+ cells remained unknown. Here, we show p75NTR expression in fetal liver at an early stage of development, and characterize the p75NTR+ cells isolated from mouse fetal liver. Our data suggest that the p75NTR+ cell population contains mesenchymal cell precursors for HSCs and PFs. Furthermore, we show that the p75NTR+ cells in the periportal area could contribute to the formation of intrahepatic bile ducts in the late gestational stage.Materials and MethodsMiceC57BL/6 mice (Nihon SLC, Hamamatsu, Japan) were used for all the experiments. All experiments with animals were approved by the Institutional Animal Care and Use Committee of the University of Tokyo.Generation of mAbs and Identification of Their AntigensAnti-p75NTR (clone 25-8), anti–Delta-like protein (Dlk) and antilymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) mAbs were generated by immunization of a rat with mouse fetal hepatic cells. Hepatic cells were dissociated by collagenase from embryonic day (E) 14.5 mouse liver,35Kamiya A. Kinoshita T. Ito Y. et al.Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer.EMBO J. 1999; 18: 2127-2136Crossref PubMed Scopus (357) Google Scholar and used for the immunization after the depletion of CD45+/TER119+ hematopoietic cells by magnetic beads (Dynabeads; Invitrogen Corp, Carlsbad, CA). Hybridomas were established by the fusion of lymphocytes and P3X myeloma cells with polyethylene glycol as described previously.36Hara T. Nakano Y. Tanaka M. et al.Identification of podocalyxin-like protein 1 as a novel cell surface marker for hemangioblasts in the murine aorta-gonad-mesonephros region.Immunity. 1999; 11: 567-578Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar To identify antigens, retrovirus-mediated expression cloning was performed as described previously.37Kitamura T. Onishi M. Kinoshita S. et al.Efficient screening of retroviral cDNA expression libraries.Proc Natl Acad Sci U S A. 1995; 92: 9146-9150Crossref PubMed Scopus (223) Google Scholar To confirm the specific reactivity, mouse p75NTR, Dlk, or Lyve1 complementary DNA (cDNA) were expressed in Ba/F3 cells by a retroviral vector, pMX/IRES-GFP,38Chida D. Miura O. Yoshimura A. et al.Role of cytokine signaling molecules in erythroid differentiation of mouse fetal liver hematopoietic cells: functional analysis of signaling molecules by retrovirus-mediated expression.Blood. 1999; 93: 1567-1578Crossref PubMed Google Scholar and the transfectants were stained with each purified antibody, and analyzed with flow cytometry (FCM).AntibodiesIn addition to the mAbs we prepared, we used 2 kinds of polyclonal antibodies against p75NTR; rabbit anti-mouse p75NTR polyclonal antibody (AB1554) (CHEMICON International Inc, Temecula, CA) and goat anti-mouse nerve growth factor receptor/tumor necrosis factor receptor superfamily, member 16 polyclonal antibody (AF1557) (R&D Systems, Minneapolis, MN) for immunocytochemistry and immunohistochemistry (IHC). The characteristics of each anti-p75NTR antibody and information of the other antibodies used are described in supplementary Figure 1 and the supplementary Materials and Methods section (see supplementary material online at www.gastrojournal.org).Preparation of Liver Cells and Flow CytometrySingle-cell suspensions were prepared from mouse E11.5 and E14.5 liver according to the method of Kamiya et al.35Kamiya A. Kinoshita T. Ito Y. et al.Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer.EMBO J. 1999; 18: 2127-2136Crossref PubMed Scopus (357) Google Scholar Aliquots of cells were blocked with anti-Fc gamma receptor III antibody, co-stained with fluorescein- and biotin-conjugated antibodies, washed, incubated with allophycocyamin-conjugated streptavidin, and analyzed by FACSCalibur (Beckon Dickinson, San Jose, CA). Dead cells were excluded by propidium iodide staining. Cell sorting was performed by FACSVantage (Beckon Dickinson), or by autoMACS (Miltenyi Biotec GmbH, Bergisch, Gladbach, Germany) after incubation with biotinylated anti-p75NTR antibody (25-8) and streptavidin-microbeads. A single-cell suspension and HSCs from adult liver were prepared as described previously.39Nakamura K. Nonaka H. Saito H. et al.Hepatocyte proliferation and tissue remodeling is impaired after liver injury in oncostatin M receptor knockout mice.Hepatology. 2004; 39: 635-644Crossref PubMed Scopus (124) Google Scholar In short, liver cells were isolated by the 2-step collagenase perfusion method. The cell suspension was centrifuged at 500 rpm for 1 minute twice. Nonparenchymal cells were prepared from the supernatant and subjected to 11% Nycodenz (Axis-Shield PoC, Rodelokka, Oslo, Norway) gradient centrifugation to collect HSCs.18Baba S. Fujii H. Hirose T. et al.Commitment of bone marrow cells to hepatic stellate cells in mouse.J Hepatol. 2004; 40: 255-260Abstract Full Text Full Text PDF PubMed Scopus (164) Google ScholarImmunohistochemistry and ImmunocytochemistryFrozen sections (5 μm) were prepared with a Microm HM505E cryostat (MICROM International GmbH, Randburg, Germany) and mounted on glass slides coated with aminopropylsilane (Matsunami Glass Ind, Ltd, Osaka, Japan). They were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) or in cold acetone. Cells were permeabilized with a 0.2% Triton-X solution if required. After a blocking procedure with 5% skim milk in PBS, sections were incubated with primary antibodies, followed by biotin- or fluorescent-conjugated secondary antibody. To visualize signals, sections were treated with 0.3% H2O2 in PBS after fixation, and a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and 3,3-diaminobenzidine tetrahydrochloride were used. Counterstaining was performed with hematoxylin or Hoechst. For immunocytochemistry, cells were mounted on glass slides with Shandon Cytospin (Thermo Fisher Scientific Inc, Waltham, MA).RNA Extraction and Reverse-Transcription Polymerase Chain ReactionTotal RNA was extracted with TRIZOL (Invitrogen). Total RNA and random hexamer primers were used to synthesize cDNA using SuperScript III reverse transcriptase (Invitrogen). The PCR primers used are described in the supplementary Materials and Methods.Cell CultureCells were cultured at 37°C in a humidified atmosphere containing 5% CO2. p75NTR+ cells isolated from E11.5 liver by autoMACS were suspended in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics. For the induction of GFAP expression, cells were cultured on gelatin-coated plastic dishes in the presence of oncostatin M (10 ng/mL). For activation, cells were cultured on noncoated plastic dishes.Oil-Red O Stainingp75NTR+ cells were collected from E11.5, E14.5, E18.5, and neonatal liver with autoMACS. The cells were mounted on glass slides and stained with Oil-red O staining solution.40Miyaoka Y. Tanaka M. Naiki T. et al.Oncostatin M inhibits adipogenesis through the RAS/ERK and STAT5 signaling pathways.J Biol Chem. 2006; 281: 37913-37920Crossref PubMed Scopus (64) Google ScholarResultsMonoclonal Antibodies Against Mouse Fetal Liver CellsTo characterize various cell compartments in mouse fetal liver, we immunized a rat with E14.5 liver cells and established a total of 627 hybridomas (supplementary Figure 2; see supplementary material online at www.gastrojournal.org). By FCM and IHC of fetal liver cells using each antibody, we found that the 25-8 antibody showed a staining pattern apparently distinct from that of hepatoblasts, which were stained with anti-Dlk antibody41Tanimizu N. Nishikawa M. Saito H. et al.Isolation of hepatoblasts based on the expression of Dlk/Pref-1.J Cell Sci. 2003; 116: 1775-1786Crossref PubMed Scopus (290) Google Scholar (Figure 1A, C, and D). The pattern of staining by the 25-8 antibody was similar to that of sinusoidal endothelial cells, which were stained with the Lyve142Nonaka H. Tanaka M. Suzuki K. et al.Development of murine hepatic sinusoidal endothelial cells characterized by the expression of hyaluronan receptors.Dev Dyn. 2007; 236: 2258-2267Crossref PubMed Scopus (67) Google Scholar antibody (Figure 1B). However, the 25-8 antibody also stained several cells around large blood vessels (Figure 1D), suggesting that it recognized a cell type distinct from hepatoblasts and sinusoidal endothelial cells. By expression cloning of cDNA, we successfully identified p75NTR as the 25-8 antigen. To confirm the specificity of the 25-8 antibody, we expressed p75NTR cDNA or Lyve1 cDNA in Ba/F3 cells and performed FCM using the 25-8 antibody (Figure 1E). The 25-8 antibody bound Ba/F3 cells expressing p75NTR but not parental Ba/F3 cells and Ba/F3 cells expressing Lyve1, indicating that it recognized p75NTR specifically. To confirm the expression of p75NTR in adult mouse HSCs, we isolated HSCs by centrifugation through 11% Nycodenz and examined the expression of p75NTR by FCM using the 25-8 antibody (Figure 1F). Almost all the lipid-storing cells in mouse liver expressed p75NTR. Reverse-transcription polymerase chain reaction (RT-PCR) analysis also showed that HSCs isolated by density centrifugation expressed p75NTR as well as GFAP, a well-known marker for HSCs (Figure 1G).Localization of p75NTR+ Cells in the LiverWe studied the expression of p75NTR in fetal liver cells. In the early stage around E9, the prehepatic cells delaminate from the foregut and migrate as cords into the surrounding septum transversum mesenchyme. However, it remained unknown whether p75NTR+ cells are present at the early stage of mouse liver development. To address this issue, we performed IHC of mouse liver at E10.5, E11.5, and E12.5. Hepatoblasts were clearly detected by anti-Dlk antibody at these stages (Figure 2A–F). Consistent with a previous study,21Matsumoto K. Yoshitomi H. Rossant J. et al.Liver organogenesis promoted by endothelial cells prior to vascular function.Science. 2001; 294: 559-563Crossref PubMed Scopus (686) Google Scholar fetal liver kinase-1+ (Flk-1+) endothelial cells were present in E10.5 liver and formed primitive sinusoids (Figure 2A and J). Interestingly, p75NTR+ cells were found at the periphery of liver, the primordium adjacent to the septum transversum mesenchyme and the part of the primitive sinusoids (Figure 2J). Then, p75NTR+ cells were observed uniformly throughout the liver at E11.5 and E12.5 (Figure 2E and F). In addition, IHC showed that a majority of the p75NTR+ cells were in contact with Flk-1+ endothelial cells as well as Dlk+ hepatoblasts (Figure 2D–K). Although IHC with 25-8 antibody showed a pattern similar to that of sinusoidal endothelial cells (Figure 1), these results strongly suggest that p75NTR+ cells and endothelial cells are distinct populations and interact with each other at an early stage in the development of liver before vascular function.Figure 2IHC of liver sections at the early stage of liver development. Liver sections were prepared from (A, D, G, and J) E10.5, (B, E, H, and K) E11.5, and (C, F, and I) E12.5 embryos. (A–F) Hepatoblasts were detected as a green color with anti-Dlk antibody. Endothelial cells were stained red with (A–C, G–K) anti–fetal liver kinase-1 (flk-1) antibody. p75NTR+ cells were stained (D–F) red or (G–K) green with anti-p75NTR antibody. (K) Image with confocal microscopy. Original magnification, A–I, 200×; J, 100×; K, 400×.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of p75NTR+ Cells in Fetal LiverTo characterize the p75NTR+ cells in E11.5 mouse liver, we performed FCM using the 25-8 antibody and revealed that approximately 1.5% of all fetal liver cells expressed p75NTR (Figure 3B). Neither CD45 nor TER119 was expressed in p75NTR+ cells, indicating that they were a nonhematopoietic lineage (Figure 3C and D). Consistent with the result of IHC in Figure 2, p75NTR+ cells were negative for Lyve1, platelet/endothelial cell adhesion molecule 1, and Flk-1 by FCM, confirming that p75NTR+ cells were not endothelial cells (Figure 3E and supplementary Figure 3; see supplementary material online at www.gastrojournal.org). This result is in accordance with numerous studies that have characterized the expression of p75NTR in the mouse, rat, and human liver and have shown that HSCs are a major source of p75NTR in the adult liver.29Cassiman D. Denef C. Desmet V.J. et al.Human and rat hepatic stellate cells express neurotrophins and neurotrophin receptors.Hepatology. 2001; 33: 148-158Crossref PubMed Scopus (192) Google Scholar, 30Trim N. Morgan S. Evans M. et al.Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation.Am J Pathol. 2000; 156: 1235-1243Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 33Passino M.A. Adams R.A. Sikorski S.L. et al.Regulation of hepatic stellate cell differentiation by the neurotrophin receptor p75NTR.Science. 2007; 315: 1853-1856Crossref PubMed Scopus (151) Google Scholar Considering the localization of mesenchymal cells in adult liver, 2 distinct populations of p75NTR+ cells in E14.5 liver might correspond to HSCs and PFs in developing liver. To address this possibility, we sorted p75NTR+ cells from fetal livers at E11.5 and E14.5, and analyzed the expression of known mesenchymal markers by RT-PCR (Figure 3F). Vimentin, desmin, and α-SMA were expressed in p75NTR+ cells at E11.5. These markers were expressed abundantly in p75NTR+ cells and poorly in p75NTR− cells at E14.5. These results clearly indicated that p75NTR+ cells in fetal liver were mesenchymal cells. Although GFAP was" @default.
• W2005876661 created "2016-06-24" @default.
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• W2005876661 date "2008-07-01" @default.
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• W2005876661 title "p75 Neurotrophin Receptor Is a Marker for Precursors of Stellate Cells and Portal Fibroblasts in Mouse Fetal Liver" @default.
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