Small cells in hepatoblastoma lack "oval" cell phenotype.

Hepatoblastoma, a childhood tumor of the liver, is composed of epithelial and mesenchymal elements in varying proportions and at various stages of differentiation. The epithelial element recapitulates the stages of hepatocyte development from the primitive blastema through embryonal hepatocytes to fetal hepatocytes. The blastemal or undifferentiated cells have been postulated to represent neoplastic hepatocyte progenitor cells. In this study, we examine the immunophenotype of the various epithelial cells of hepatoblastoma with special emphasis on the small undifferentiated cell component and compare it with that of adult hepatocytes and hepatic stem (oval) cells. Putative stem cells in the liver can express all of the following markers: alpha-feto protein, CK19 (OV-6), chromogranin A, Bcl-2, HepPar-1, and alpha1 microglobulin. The latter, like alpha-feto protein, is a plasma protein synthesized by hepatocytes. Both alpha1 microglobulin and HepPar-1 are expressed in fetal liver cells as early as 7 weeks of intrauterine life. They are also expressed in hepatocellular carcinoma and in hepatocytic cell lines derived from normal fetal or adult liver. Formalin-fixed, paraffin-embedded archival tissues from 10 predominantly epithelial hepatoblastomas were immunostained with antibodies directed against CD 34, alpha1 microglobulin, Bcl-2, HepPar 1, and CK19 using the avidin-biotin-peroxidase method. The undifferentiated small cell component did not express any of the markers studied, namely, Bcl-2, HepPar-1, alpha(1) microglobulin, CD34, or CK19. Hepatocyte-like cells were alpha1 microglobulin- and HepPar-1-positive, with the intensity of staining correlating with the degree of hepatocytic differentiation. Bcl-2 expression was restricted to areas of ductular differentiation. CK19 was detected in foci that showed duct formation. The small cells of hepatoblastoma did not express HepPar-1, Bcl-2, CK19, alpha1 microglobulin, or CD34, markers that characterize the immunophenotype of hepatic stem cells ("oval" cells). Thus, this observation raises the following questions: (1) is "hepatoblastoma" a misnomer? (2) is the expression of tumor antigens dysregulated in hepatoblastoma? (3) does the liver have two different types of progenitor cells, oval cells and blastemal cells, with differing immunophenotypes? and (4) do the blastemal cells, rather than oval cells, represent the more primitive progenitor cells of the liver?     

Analysis of hemopoietic cells in rat bone marrow and fetal liver with monoclonal antibody

The expression of cell surface antigens recognized by UB-12 monoclonal antibody against rat hemopoietic cells was examined in fetal liver, and adult bone marrow using immunohistochemistry, radioimmunoassay, and flow cytofluorometry. Comparisons were made with the expression of the antigens recognized by OX-7 (anti-Thy-1) or W3/13 (anti-leukocyte sialoglycoprotein) antibodies. UB-12 positive cells appeared at earlier stages of gestation and in higher cell frequency than OX-7 or W3/13 positive cells in fetal liver. The antigen detected with UB-12 antibody was positive in fetal liver at day 12 and increased at day 15, reaching about 90% of the positive cells. In bone marrows, UB-12 positive cells started to appear around birth and were about 30-40% of total nucleated cells during adult periods. Thus, the antigen recognized by UB-12 was found only during early stages of blood cells. UB-12 monoclonal antibody recognized a B-cell lymphoma, Y-3, but did not react with a rat T lymphoma cell line. This monoclonal antibody may be important for distinguishing normal and neoplastic B cells and their precursors.     

The GCTM-5 Epitope Associated with the Mucin-Like Glycoprotein FCGBP Marks Progenitor Cells in Tissues of Endodermal Origin

Monoclonal antibodies against cell surface markers are powerful tools in the study of tissue regeneration, repair, and neoplasia, but there is a paucity of specific reagents to identify stem and progenitor cells in tissues of endodermal origin. The epitope defined by the GCTM-5 monoclonal antibody is a putative marker of hepatic progenitors. We sought to analyze further the distribution of the GCTM-5 antigen in normal tissues and disease states and to characterize the antigen biochemically. The GCTM-5 epitope was specifically expressed on tissues derived from the definitive endoderm, in particular the fetal gut, liver, and pancreas. Antibody reactivity was detected in subpopulations of normal adult biliary and pancreatic duct cells, and GCTM-5-positive cells isolated from the nonparenchymal fraction of adult liver expressed markers of progenitor cells. The GCTM-5-positive cell populations in liver and pancreas expanded greatly in numbers in disease states such as biliary atresia, cirrhosis, and pancreatitis. Neoplasms arising in these tissues also expressed the GCTM-5 antigen, with pancreatic adenocarcinoma in particular showing strong and consistent reactivity. The GCTM-5 epitope was also strongly displayed on cells undergoing intestinal metaplasia in Barrett's esophagus, a precursor to esophageal carcinoma. Biochemical, mass spectrometry, and immunochemical studies revealed that the GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP. The GCTM-5 epitope on the mucin-like glycoprotein FCGBP is a cell surface marker for the study of normal differentiation lineages, regeneration, and disease progression in tissues of endodermal origin. Stem Cells2012;30:1999-2009.     

Characterization of a receptor on activated chicken T cells

Chicken lymphoblasts were generated from spleen cells which had been incubated with concanavalin A (Con A) for 48 h. Monoclonal antibodies were produced by immunizing mice with 48-h Con A lymphoblasts. The cellular ELISA, with lymphoblasts and spleen cells as the target cells, was used to select for specific monoclonal antibodies. A monoclonal antibody was found to have strong response against the lymphoblasts but not resting spleen cells. By immunofluorescence staining, this monoclonal antibody reacted strongly with lymphoblasts and did not react with resting spleen cells. This monoclonal antibody was also shown to inhibit the lymphokine activity present in conditioned medium (CM) which was collected from 24-h cultures of Con-A-activated spleen cells. In immunoprecipitation, this monoclonal antibody precipitated a lymphoblast surface antigen with a molecular weight of about 45 kDa.     

Characterization of Tritrichomonas foetus antigens by use of monoclonal antibodies.

The specificity for and function of monoclonal antibodies against Tritrichomonas foetus were characterized. Four monoclonal antibodies generated by immunization of mice with live T. foetus were selected on the basis of enzyme-linked immunosorbent assay reactions. The approximate molecular masses of the predominant proteins were determined by Western blotting (immunoblotting). Monoclonal antibody TF3.8 recognized a predominant band at approximately 155 kilodaltons, whereas TF3.2 reacted with several bands. Monoclonal antibodies TF1.17 and TF1.15 recognized broad bands between 45 and 75 kilodaltons. The first two antibodies (TF3.8 and TF3.2) did not react with the surface of T. foetus, as determined by live-cell immunofluorescence, agglutination, and immobilization, whereas two other monoclonal antibodies (TF1.17 and TF1.15) did react with surface epitopes, as determined by these criteria. The latter two monoclonal antibodies also mediated complement-dependent killing of T. foetus and prevented of adherence of organisms to bovine vaginal epithelial cells. One antibody, TF1.15, also killed in the absence of complement. Since these functions are in vitro correlates of protection, the antigens recognized by these monoclonal antibodies may induce protective immunity.     

Monoclonal antibodies against a preadipose cell line (MC3T3-G2/PA6) which can support hemopoiesis

Monoclonal antibodies against a mouse preadipose cell line (MC3T3-G2/PA6:PA6), which can support hemopoiesis by direct cell-to-cell interaction, were produced and characterized. The antibodies react with PA6 but not PA6-M (a mutant cell line) which has the capacity neither to contact with hemopoietic stem cells (HSCs) nor to support hemopoiesis. Endosteal cells in the bone marrow show positive staining to these antibodies. They inhibit pseudoemperipolesis of PA6 to HSCs, resulting in a significant decrease in hemopoietic cell number. These findings suggest that the monoclonal antibodies bind to the stromal cell receptors for HSCs and block the binding of HSCs to stromal cells leading to suppression of hemopoiesis.     

Differentiation status of rat ductal cells and ethionine-induced hepatic carcinomas defined with surface-reactive monoclonal antibodies

To gain further insight into the differentiation of oval cells and their role in carcinogenesis, we have generated cell surface reactive monoclonal antibodies (MAbs) by a Balb/c nude mouse (nu/nu) immunization protocol. Three MAbs designated OC.4, OC.5, and OC.10 were generated from a mouse immunized with CDE6, an oval cell line established from oval cells induced by feeding a choline-deficient diet containing 0.1% ethionine (CDE). These MAbs demonstrated stage-specific expression in fetal liver and displayed strong reactivity with oval and bile duct epithelial cells. In general, oval cells displayed a more mature phenotype than fetal ductal cells, suggesting the existence in adult liver of more primitive ductal progenitors. A fourth MAb recognized a cytoplasmic antigen (OC.6) expressed by mucus-secreting hepatic ducts induced by CDE diet. Immunocytochemical analysis indicated that OC.4, OC.5, and OC.10 were also expressed on CDE-induced, OV6+ hepatocellular carcinomas (HCC) but not on OV6+ HCC induced by the Solt/Farber protocol. In most cases, CDE-induced, OV6+ HCC expressed early ductal developmental markers such as OC.10 but lacked those expressed at later stages (OC.5, OC.4). These new MAb will be useful for characterizing HCC subpopulations with oval cell characteristics and for isolating biliary cells at antigenically defined stages during differentiation.     

Immunohistochemical study of human pulmonary surfactant apoproteins with monoclonal antibodies. Pathologic application for hyaline membrane disease.

Three monoclonal antibodies, PC6, PE10, and PE12, were used for immunohistochemical studies of human lungs by immunoperoxidase staining. Monoclonal antibodies PC6 and PE10 against pulmonary surfactant apoproteins stained faint granules in the cytoplasm of some alveolar wall cells in adult lung. These stained cells appeared to be alveolar Type II cells. A fetal lung of 20 weeks' gestation had no any positive staining. However, a few scattered positive cells were observed in a newborn lung of 31 weeks' gestation, and the stained cells increased progressively with increasing gestational age. The positively stained cells were very few in the lungs of newborns who died of respiratory distress syndrome (RDS), but the lungs of newborns who died of other causes after recovery from RDS showed many positively stained cells. These results suggest that the immunohistochemical demonstration of the monoclonal antibodies PC6 and PE10 could be a good pathodiagnostic indicator reflecting the localization and development of pulmonary surfactant by alveolar Type II cells. On the other hand, monoclonal antibody PE12 was found to recognize the antigen that occurs on the surfaces of the alveoli of fetal, newborn, and adult lungs as one component of the alveolar lining layer, different from pulmonary surfactant.     

Oncodevelopmental expression of rat placental alkaline phosphatase. Detection in oval cells during liver carcinogenesis.

Oval cells isolated from livers of rats fed a choline-deficient diet containing 0.1% DL-ethionine (CDE) have an alkaline phosphatase (ALKP) isozyme which can be distinguished by its electrophoretic mobility from the enzyme present in parenchymal cells isolated from normal liver or livers of rats fed the CDE diet for 4 weeks. The oval cell ALKP has the same electrophoretic mobility as the enzyme from fetal rat liver and placenta. ALKPs from oval cells, parenchymal cells, and placenta all differ from the intestinal enzyme by their electrophoretic mobility, isoelectric focusing, and the patterns of amino acid inhibition of enzyme activity. Oval cells in preneoplastic livers, fetal hepatocytes, and tumor cells of a primary hepatocellular carcinoma induced by CDE feeding stained with a monoclonal antibody directed against rat placental ALKP. Hepatocytes (in normal or preneoplastic livers) and bile duct cells in normal liver did not stain with the same antibody. Placental ALKP may thus be a useful marker in tracing the origin and fate of oval cells during hepatocarcinogenesis.     

Cell behavior in the acetylaminofluorene-treated regenerating rat liver. Light and electron microscopic observations.

When hepatocyte regeneration is impaired, facultative stem cells and their descendants, also called oval cells, become activated and produce cell progeny that eventually differentiate. We have observed these cells in the rat liver after partial hepatectomy when the animals have been fed 2-acetylaminofluorene. Oval cells emerge from the portal areas and stain strongly with monoclonal antibodies raised against cytokeratins 8 and 19 and vimentin, the intermediate filament traditionally associated with mesenchymal cells. The majority of oval cells appeared to be part of a bile ductular reaction, manifest by their cytokeratin expression, and the bile duct injection of pigmented gelatin confirmed that these oval cells were essentially tortuous, arborizing duct-like structures (cholangioles) branched from and continuous with preexisting bile ducts. In situ hybridization studies showed that hepatocyte growth factor mRNA-expressing sinusoid lining cells were most numerous in the periportal areas during the period of ductular proliferation. At 1 week after partial hepatectomy, we observed morphological evidence of areas of in situ focal differentiation in the ductular structures, either to a columnar intestinal-type epithelia or to a hepatocyte phenotype, with abundant large mitochondria and membranous cytokeratin 8 immunoreactivity contrasting with the diffuse staining of the ductular cells. By following the fate of oval cells the authors conclude that in this model proliferated bile ductules represent the oval cell compartment capable of producing pluripotential progenitor cells.     

肝卵圆细胞的研究进展

肝卵圆细胞（OCs）是肝脏的干细胞,能够分化为肝细胞、胆管上皮细胞及其他细胞,亦与肝再生和肝脏疾病密切相关。本文简要地总结了近几年有关肝卵圆细胞的来源、定位、增殖、分化、凋亡、移植及在肝再生和肝脏疾病中作用等研究进展。     

合成多肽为半抗原制备抗SCF受体单克隆抗体

本文成功地建立了以合成的小分子肽与蛋白质交联的偶联物为免疫原，制备单克隆抗体（ｍＡｂ）的技术路线。选定ＳＣＦ受体特异性肽段进行人工合成，将其偶联于ＢＳＡ免疫动物，应用细胞融合技术制备了抗两肽段的杂交瘤细胞７株。ＥＬＩＳＡ法和免疫印迹结果显示，其分泌的ｍＡｂ特异性强，与偶联载体及多种多肽生长因子均无交叉反应。经ＡＰＡＡＰ法鉴定，抗胞内羧基端肽的ｍＡｂＳＢ３０４与ＳＣＦ受体表达细胞系ＨＥＬ有弱的阳性反应；免疫组化测定与胎儿皮肤基底层色素细胞及胃粘膜上皮细胞呈阳性反应，提示ｍＡｂＳＢ３０４可识别天然状态的ＳＣＦ受体。     

A new monoclonal antibody to human subcapsular thymic epithelial cells

Summary A monoclonal antibody, termed K-20, was generated against an anaplastic thymic carcinoma cell line, Ty-82. Subcapsular thymic epithelial cells of the thymus and blood vessels in various organs were shown to react with the K-20 monoclonal antibody by immunohistochemical staining. Immunofluorescent study revealed that various haematopoietic fresh cells and cell lines did not show any significant reactivity with K-20, except for one Epstein-Barr-virus-carrying lymphoma cell line (SP-50B). Western immunoblotting and affinity purification procedure revealed that K-20 was directed to a protein with a molecular weight of 28 kDa. K-20 is unique in its restrictive reactivity with human subcapsular thymic epithelial cells.     

Expression of Fc epsilon receptors and surface and cytoplasmic IgE on human fetal and adult lymphopoietic tissue

The appearance during ontogeny of IgE-positive and Fc epsilon receptor (FcER)-bearing cells was studied. Monoclonal antibody to the constant region (Fc) of IgE (CIA-E-7.12) was used to detect cytoplasmic and surface IgE. A monoclonal antibody to the low-affinity Fc epsilon receptor (FcER-II = CD23) and immune complexes composed of human IgE and mouse monoclonal anti-human IgE Fc were used to detect FcER. Cryostat sections of human fetal tissues (liver, lung, spleen, and thymus) from 11 to 22 weeks gestation as well as adult tonsil tissues were examined for IgE, FcER, and other lymphoid markers by immunoperoxidase staining. Although both IgE- and FcER-positive cells were present in adult tissues, we found that, in contrast to an earlier report, such cells were not present in the fetal tissues examined. The in situ location of FcER on cells in human lymphoid tissues revealed that the FcER-bearing cells were localized predominantly in the germinal centers (mature B cell and macrophage areas) of the tonsil follicles with some staining in the mantle (resting and less mature B cell areas).     

Common neuroectodermal antigens on human melanoma,neuroblastoma, retinoblastoma,glioblastoma and fetal brain revealed by hybridoma antibodies raised against melanoma cells

The hybridoma system has been utilized to produce antibodies to characterize the cell surface antigens on human melanoma cells. On initial screening, two antibodies derived by the fusion of mouse myeloma cells (SP2/0-Ag14) and splenocytes from a mouse immunized with a melanoma cell line (CaCL 78-1) showed cross-reactivity with 10 melanoma cell lines and did not react with any of 4 epithelial cancer lines, or 4 normal adult fibroblast lines. However, because of reactivity with 2 neuroblastoma cell lines, additional testing with other neuroectodermal derivatives was carried out and revealed a broad cross-reactivity among melanomas, neuroblastomas, retinoblastomas and glioblastomas and against antigens shared by fetal but not adult brain. Thus, these results indicate the existence of common neuroectodermal antigens on melanoma cells. Before melanoma specificity can be claimed for, an antibody reactivity with nonmelanoma cells bearing these neuroectodermal antigen should be excluded.     

Isolation and characterization of a stem cell population from adult human liver

Several studies suggested the presence of stem cells in the adult normal human liver; however, a population with stem cell properties has not yet been isolated. The purpose of the present study was to identify and characterize progenitor cells in normal adult human liver. By stringent conditions of liver cell cultures, we isolated and characterized a population of human liver stem cells (HLSCs). HLSCs expressed the mesenchymal stem cell markers CD29, CD73, CD44, and CD90 but not the hematopoietic stem cell markers CD34, CD45, CD117, and CD133. HLSCs were also positive for vimentin and nestin, a stem cell marker. The absence of staining for cytokeratin-19, CD117, and CD34 indicated that HLSCs were not oval stem cells. In addition, HLSCs expressed albumin, alpha-fetoprotein, and in a small percentage of cells, cytokeratin-8 and cytokeratin-18, indicating a partial commitment to hepatic cells. HLSCs differentiated in mature hepatocytes when cultured in the presence of hepatocyte growth factor and fibroblast growth factor 4, as indicated by the expression of functional cytochrome P450, albumin, and urea production. Under this condition, HLSCs downregulated alpha-fetoprotein and expressed cytokeratin-8 and cytokeratin-18. HLSCs were also able to undergo osteogenic and endothelial differentiation when cultured in the appropriated differentiation media, but they did not undergo lipogenic differentiation. Moreover, HLSCs differentiated in insulin-producing islet-like structures. In vivo, HLSCs contributed to regeneration of the liver parenchyma in severe-combined immunodeficient mice. In conclusion, we here identified a pluripotent progenitor population in adult human liver that could provide a basis for cell therapy strategies.     

Hepatic stem cells: a review

The existence of a liver stem cell population has only gained credence recently, following the results of animal experiments. These cells are thought to reside in the terminal bile ductules (canals of Hering). Hepatocyte division is responsible for liver regeneration after most causes of injury. However, stem cells may contribute to hepatocyte regeneration, or even take over this role if the liver injury is severe and associated with an impairment of hepatocyte proliferation as in cirrhosis or submassive/massive necrosis, due to drugs, toxins or viruses. "Oval" cells are the descendants of the stem cells and are found in the portal and periportal regions in experimental animals within days of the liver injury. These cells proliferate to form narrow ductules, which may stain positively for biliary cytokeratins CK 19, and radiate out into the damaged parenchyma. Both in vitro and in vivo animal studies now suggest that oval cells can differentiate into bile ductular cells or hepatocytes to allow repopulation of the injured liver. As the oval cells differentiate into hepatocytes they may show positive staining for pyruvate kinase isoenzyme L-PK, albumin and alpha-fetoprotein. There is also growing evidence that bone marrow stem cells may contribute to liver regeneration. The possible involvement of hepatic stem cells in the development of dysplastic nodules, hepatocellular carcinoma and cholangiocarcinoma has been suggested but remains highly controversial. Oval cell isolation and culture techniques, together with stem cell transplantation strategies, may in the future provide novel treatments for individuals with inherited and acquired hepatic disorders.     

Hepatic progenitor cell represents a transitioning cell population between liver epithelium and stroma

Following an acute injury, the liver may maintain its structure and function through mitotic division of mature hepatocytes (i.e. hepatic regeneration). However, the regeneration ability of hepatocytes can be impaired in chronic liver diseases including chronic viral infection and alcohol abuse. Hepatic progenitor cells/oval cells (HPCs/OCs), capable of differentiation into both hepatocytes and cholangiocytes, occur and proliferate during chronic injury. Unfortunately, a use of HPCs for clinical therapy is blocked by the difficulty of exact identity of HPCs in liver. Focusing on the links between phenotype of HPCs and real stem cells originating from fetal liver or bone marrow (BM), the recent studies of HPCs neglect functional analysis and the close relationship between activation of HPCs and extracellular matrix (ECM) remodeling. It is currently widely accepted that mesenchymal-epithelial transition (EMT) and epithelial-mesenchymal transition (MET) play important roles not only in liver development but also in healing of chronic injured adult liver. Co-expression of epithelial/mesenchymal and HPCs markers has been demonstrated in cells undergoing EMT/MET. These cells led to hepatic regeneration after transplanted into rats with chronic liver injury. Notably, there is an increased expression of mesenchymal markers in HPCs after exposure to transforming growth factor-beta1 (TGF-β1). Based on these evidences, we hypothesize that HPCs represent a transitioning cell population undergoing EMT/MET, both parenchymal and mesenchymal cells of liver may be the direct sources of HPCs.     

Dexamethasone inhibits the development of mast cells from dispersed human fetal liver cells cultured in the presence of recombinant human stem cell factor.

Human fetal liver cells cultured in the presence of recombinant human stem cell factor (rhuSCF) give rise to highly purified mast cell populations. This study examined the effect of steroid hormones on mast cell differentiation. Dispersed fetal liver cells cultured in the presence of rhuSCF at 50 ng/ml and in the presence or absence of various steroid hormones for 4 weeks, were analysed for the presence of mast cells by metachromatic staining with toluidine blue, by immunohistochemistry with a monoclonal antibody against tryptase, and by immunofluorescent flow cytometry with a monoclonal antibody against Kit. Dexamethasone added to the cultures at day 0 resulted in a dose-dependent inhibition of rhuSCF-induced mast cell differentiation with > 85% inhibition seen at a dose of 10(-6) M. A similar effect was seen with hydrocortisone, but not with oestradiol or progesterone. The addition of dexamethasone resulted in decreased DNA synthesis in 14-day-old cultured cells, as assessed by incorporation of bromodeoxyuridine. Addition of dexamethasone to 3-week-old SCF-dependent fetal liver mast cells had no significant effect on mast cell survival. Removal of dexamethasone after 3 weeks of culture with SCF did not result in mast cell development. Thus, dexamethasone inhibits SCF-induced development of mast cells from fetal liver cells, but shows no appreciable effect on developed mast cells.