Bile Duct-like Structures Research Articles

Time course of primary liver cell reorganization in three-dimensional high-density bioreactors for extracorporeal liver support: an immunohistochemical and ultrastructural study.

To enable extracorporeal liver support based on the use of primary liver cells, culture models supporting the maintenance of cell integrity and function in vitro are required. In this study the cell organization and ultrastructure of primary porcine hepatocytes cocultured with nonparenchymal cells in three-dimensional high-density bioreactors were analyzed after 10, 20, and 30 days of culture by immunohistochemistry and transmission electron microscopy. Biochemical data showed that metabolic activity of the cells in the system was relatively stable over at least 20 days. Immunohistochemical studies were performed in comparison with donor organ biopsies. They showed that hepatocytes and nonparenchymal cells reaggregated in bioreactors, forming structures partly resembling natural liver parenchyma. Bile duct-like structures characterized by cytokeratin 7 (CK-7) immunoreactivity (IR) were regularly detected. Nonparenchymal cells (vimentin IR) formed sinusoidal-like structures within parenchymal cell aggregates. Proliferative activity (Ki-67 IR) increased over time. The detection of collagen I and laminin indicated the production of extracellular matrix components within bioreactors. The results showed that primary liver cell reorganization and long-term maintenance of their differentiated state were achieved within the bioreactors The findings on cell proliferation indicated that the culture model is also of interest for further in vitro studies on cell regeneration and tissue formation.

The generation of functionally differentiated, three-dimensional hepatic tissue from two-dimensional sheets of progenitor small hepatocytes and nonparenchymal cells.

The authors' laboratory has investigated tissue engineering of the liver as a novel approach for treating end-stage liver disease. Fabrication of thick, viable, three-dimensional liver tissue is limited by the lack of vascularity in the tissue-engineered constructs. To overcome this limitation, the authors fabricated three-dimensional, vascularized liver tissue in vivo from two-dimensional cell sheets created from small hepatocytes (SHC) and nonparenchymal cells (NPC) implanted into rat omentum. SHC and NPC were cultured on a silicon wafer and lifted as monolayer cell sheets. After maximal hepatotrophic stimulation was induced in the host by injecting retrorsine, creating a portacaval shunt, and performing a partial hepatectomy, these sheets were placed onto the omentum and then rolled into a three-dimensional cylinder. New tissue consisting of both hepatocytes and bile duct-like structures formed by 2 weeks, and the mass of hepatocytes increased in size up to 2 months. The hepatocytes in these constructs were immunohistochemically positive for albumin and transferrin, and bile duct-like structures were positive for gamma-glutamyl transpeptidase, which suggests that they possess liver-specific function. Electron microscopy also revealed structures resembling bile canaliculi. Functional, morphologically complex new tissue was generated from morphologically simple monolayer cell sheets of SHC and NPC. These results represent an essential step toward the design of tissue-engineered complex vascularized thick tissue.

Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT.

Cholangiocytes perform an essential role in important pathophysiologic functions in the liver. Establishment of a human cholangiocyte line facilitates advances in cholangiocyte research and clinical applications for cell therapies. Here, we describe the immortalization of human cholangiocytes using serial transfection of simian virus 40 large T (SV40T) followed by human telomerase reverse transcriptase (hTERT). SV40T-transduced human liver OUMS-21 cells were superinfected with a retroviral vector SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs. Resulting cell lines were evaluated for gene expression, functional cholangiogenic characteristics in vitro and in vivo, and response to lipopolysaccharide (LPS). One of the SV40T- and hTERT-immortalized cholangiocyte clones, MMNK-1, was established. MMNK-1 expressed cholangiocyte markers, including cytokeratin (CK)-7 and -19 and exhibited cholangiogenic tubule formation in a Matrigel assay. When transplanted into the immunodeficient mice, MMNK-1 cells developed bile duct-like structures in the spleen. After LPS treatment, MMNK-1 cells produced interleukin-6 and failed to form well-developed tubular structures in Matrigel. We have established an immortalized cholangiocyte cell line, MMNK-1, using SV40T and hTERT transduction.

Up-regulated expression of fractalkine and its receptor CX3CR1 during liver injury in humans

Background/Aims : Little is known about the role of fractalkine (CX3CL1) in the liver. The aim of this study was to investigate the expression patterns of fractalkine and its receptor CX3CR1 in normal human liver and in conditions of injury. Methods : Distribution and expression of fractalkine and its receptor were investigated using immunohistochemistry, in situ hybridization, flow cytometry and reverse transcriptase–polymerase chain reaction. In vitro experiments were conducted in HepG2 cells. Results : Both fractalkine and CX3CR1 were up-regulated during chronic injury, in areas of portal and lobular inflammation. In severe acute hepatitis, fractalkine and CX3CR1 were expressed at high levels not only in areas of inflammation but also in regenerating epithelial cells within bile duct-like structures, which showed co-expression of fractalkine and cytokeratin-7 or CX3CR1. The human hepatocarcinoma cell line HepG2 expressed fractalkine at the gene and protein level, and HepG2-conditioned medium was chemotactic for cells overexpressing CX3CR1. Transcripts for CX3CR1 were detected in HepG2, and exposure of these cells to recombinant fractalkine induced cell migration. Conclusions : This study shows that the fractalkine system is up-regulated during liver damage, and suggests that fractalkine may play a role in the recruitment and adhesion of inflammatory cells and in the biology of liver epithelial cells.

Inhibition of MMH (Met murine hepatocyte) cell differentiation by TGF(beta) is abrogated by pre-treatment with the heritable differentiation effector FGF1.

MMH (Met murine hepatocyte) liver cells derived from transgenic mice expressing a truncated constitutively active form of human c-Met are non-transformed immortalized cell lines. We have previously shown that they harbor: (1) epithelial cells that express the liver-enriched transcription factors HNF4 and HNF1(alpha), and that can be stably induced by FGF1 to express liver functions, and (2) fibroblast-like bi-potential palmate cells that can differentiate into bile duct-like structures in Matrigel cultures, or into epithelial cells competent to express hepatic functions. Low concentrations of TGF(beta) have been found to inhibit growth and differentiation of MMH cells. The factor stabilized the palmate cell phenotype, and it provoked epithelial cells to acquire palmate-like morphological characteristics, in parallel with down-regulation of expression of HNF4 and HNF1(alpha) and activation of Snail transcripts. The effects of TGF(beta) were dominant if it was added with FGF1, but the effects on differentiation were abrogated if cells had been pre-treated with FGF1. This work identifies TGF(beta) as a factor that could be implicated in maintaining bi-potential precursor cells in the liver, FGF1 as one that could over-ride the TGF(beta) effects and Snail as a candidate for mediation of the signal.

Induction of three-dimensional assembly of human liver cells by simulated microgravity

The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

Bile duct cells: a novel in vitro model for the study of lipid metabolism and bile acid production

Immortalized bile duct cells (BDC), derived from transgenic mice harboring the SV40 thermosensitive immortalizing mutant gene ts458, were utilized to investigate the role of the biliary epithelium in lipid and sterol metabolism. This cell model closely resembles the in vivo situation because it expresses the specific phenotypic marker cytokeratin 19 (CK-19), exhibits the formation of bile duct-like structures, and displays well-formed microvilli projected from the apical side to central lumen. The BDC were found to incorporate [14C]oleic acid (in nmol/mg protein) into triglycerides (121 +/- 6), phospholipids (PL; 59 +/- 3), and cholesteryl ester (16 +/- 1). The medium lipid content represented 5.90 +/- 0.16% (P < 0. 005) of the total intracellular production, indicating a limited lipid export capacity. Analysis of PL composition demonstrated the synthesis of all classes of polar lipids, with phosphatidylcholine and phosphatidylethanolamine accounting for 60 +/- 1 and 24 +/- 1%, respectively, of the total. Differences in PL distribution were apparent between cells and media. Substantial cholesterol synthesis was observed in BDC, as determined by the incorporation of [14C]acetate suggesting the presence of hydroxymethylglutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. With the use of [14C]acetate and [14C]cholesterol as precursors, both tauro- and glycoconjugates of bile acids were synthesized, indicating the presence of cholesterol 7alpha- and 26R-hydroxylases, the key enzymes involved in bile acid formation. The transport of bile acids was not limited, as shown by their marked accumulation in the medium (>6-fold of cell content). HMG-CoA reductase (53.0 +/- 6.7), cholesterol 7alpha-hydroxylase (15. 5 +/- 0.5), and acyl-CoA:cholesterol acyltransferase (ACAT; 201.7 +/- 10.2) activities (in pmol. min-1. mg protein-1) were present in the microsomal fractions. Our data show that biliary epithelial cells actively synthesize lipids and may directly contribute bile acids to the biliary fluid in vivo. This BDC line thus represents an efficient experimental tool to evaluate biliary epithelium sterol metabolism and to study biliary physiology.

Cultures of human liver cells in simulated microgravity environment

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

Identification of a bipotential precursor cell in hepatic cell lines derived from transgenic mice expressing cyto-Met in the liver.

Met murine hepatocyte (MMH) lines were established from livers of transgenic mice expressing constitutively active human Met. These lines harbor two cell types: epithelial cells resembling the parental populations and flattened cells with multiple projections and a dispersed growth habit that are designated palmate. Epithelial cells express the liver-enriched transcription factors HNF4 and HNF1alpha, and proteins associated with epithelial cell differentiation. Treatments that modulate their differentiation state, including acidic FGF, induce hepatic functions. Palmate cells show none of these properties. However, they can differentiate along the hepatic cell lineage, giving rise to: (a) epithelial cells that express hepatic transcription factors and are competent to express hepatic functions; (b) bile duct-like structures in three-dimensional Matrigel cultures. Derivation of epithelial from palmate cells is confirmed by characterization of the progeny of individually fished cells. Furthermore, karyotype analysis confirms the direction of the phenotypic transition: palmate cells are diploid and the epithelial cells are hypotetraploid. The clonal isolation of the palmate cell, an immortalized nontransformed bipotential cell that does not yet express the liver-enriched transcription factors and is a precursor of the epithelial-hepatocyte in MMH lines, provides a new tool for the study of mechanisms controlling liver development.

Immunohistochemical Study on Hepatic Component Cells and Extracellular Matrix in Wistar Rats with Galactosamine-induced Acute Hepatitis.

After a single administration of D-galactosamine hydrochloride (GalN) in Wistar rats, hepatocyte apoptosis was followed by proliferation of oval cells, resulting in formation of small bile duct-like structures. An activation of Ito cells seems to participate in the formation of small bile duct-like structures through the production of laminin and fibronectin which are primary components of basement membrane.

Hepatocytic Cells Form Bile Duct-like Structures within a Three-Dimensional Collagen Gel Matrix

It has been suggested that hepatocytes have the ability to form bile ductal structures during normal development and in various pathological conditions of the liver. In the present study, we attempted to establish anin vitromodel of ductal morphogenesis of hepatocytic cells by combining an aggregate culture and a type I collagen gel culture. When spheroidal aggregates of rat or mouse primary hepatocytes were embedded within the collagen gel matrix and then cultured with a medium containing a fibroblast-conditioned medium, the aggregates extended many dendritic processes composed of a trabecular arrangement of cells. Dendritic morphogenesis was also seen in embedded aggregates of immortal liver epithelial cell lines, which spontaneously emerged during long-term cultures of mouse primary hepatocytes. A similar morphogenesis was induced by the presence of insulin in the medium. Although epidermal growth factor (EGF) and hepatocyte growth factor (HGF) showed only a small effect on the morphogenesis of most of the hepatocytic cells when used alone, these factors, especially EGF, enhanced the morphogenetic effect of insulin. Electron microscopical observations revealed luminal structures lined by microvilli within these dendritic processes, indicating ductal differentiation. Immunocytochemically, the dendritic processes were positive for cytokeratin 19, a marker for bile duct cells. On the other hand, an H-ras-transformed mouse liver epithelial cell line and rat hepatocellular carcinoma cell lines did not demonstrate the organized morphogenesis. Our results indicate that hepatocytic cells can produce bile duct-like structures in the presence of the type I collagenous matrix and soluble morphogenetic factors.

Biliary proliferation and adaptation in furan-induced rat liver injury and carcinogenesis.

Distinctive intrahepatic biliary adaptation responses occur in the liver of rats subjected to select hepatotoxic and/or carcinogenic treatments with the nongenotoxic cholangiocarcinogenic agent furan. Specifically, metaplastic small intestinal-like glands closely resembling in their cellular composition the crypts of Lieberkühn of normal rat small intestine were selectively derived from putative hyperplastic bile ductule-like progenitor structures in the right and caudate liver lobes of young adult Fischer-344 male rats given furan by gavage at a daily dose of 30-45 mg/kg body weight, 5 times weekly, over a 2-6-wk treatment period. Longer term chronic administration of furan at 30 mg/kg/day for 9-19 wk resulted in the preferential development of primary hepatic adenocarcinomas, which arose at 70-100% incidences from right/caudate liver lobes and which were characterized by small intestine mucosal cell differentiation. Interestingly, the neoplastic glands of these "intestinal-type" hapatic tumors demonstrated strongly positive immunochemical reactions for both hepatocyte growth factor/scatter factor and its c-met encoded receptor and were immunohistochemically positive for transforming growth factor beta 1 (TGF-beta 1) and for mannose-6-phosphate/insulin-like growth factor II receptor, implicated in the activation of latent TGF-beta 1. In contrast, a different pattern of aberrant bile ductular cell differentiation was noted to occur in the atrophied right liver lobe of moribund Fischer-344 male rats that were chronically exposed over a 10-14-day period to a severely hepatotoxic dose of furan (60 mg/kg/day). Under this latter experimental condition, rare yet distinct cholangiolar-like structures composed of biliary epithelial cells and typically a single ductular hepatocytic cell in various stages of maturation specifically formed in association with an extensive hyperplastic bile ductular reaction. Very similar cholangiolar-like structures also appeared in areas of preexisting hyperplastic bile ductule tissue at 3-5 days following the administration of a single hepatonecrogenic dose of CCl4 to rats that 4-6 wk earlier had been subjected to a bile duct ligation. In addition, a novel rat model was developed in which furan combined in a unique synergistic manner with bile duct ligation to induce the replacement of almost all of liver with well-differentiated hyperplastic bile ductules without evidence of differentiation along either metaplastic small intestine mucosal cell or ductular hepatocyte lineages. Bile ductular epithelial cell isolated from bile duct-ligated/furan-treated rats were further observed to be organized in the form of bile duct-like structures in vitro under specific conditions of primary cell culture and in vivo following their cell transplantation into the inguinal fat pads of sygeneic recipient rats. Overall, these findings serve to exemplify the remarkable plasticity that may be exhibited by certain proliferating biliary cell populations in liver in response to specific types of severe hepatic injury and/or during cholangiocarcinogenesis.

Ductular Proliferation and Hepatic Secretory Function in Experimental Fascioliasis

Proliferation of bile duct-like epithelial cells is a common feature of extrahepatic biliary obstruction and it is also present in other forms of liver disease. Experimental infestation with Fasciola hepatica induces hyperplasia of ductular cells together with different functional alterations, such as hypercholeresis, in the rat. We have investigated the contribution of ductular biliary secretion to the hypercholeretic phenomenon in rats with experimental fascioliasis. Infestation was induced by oral administration of 20 metacercariae of F. hepatica. Twelve weeks later, histological study of the liver showed a hyperplastic reaction with proliferation of bile duct-like structures. Basal bile flow in infested animals was significantly higher than that of control animals (+ 17%), while biliary secretion of bile acids did not significantly differ between both groups. Bile flow increase was associated in parasitized animals with a significantly lower [ 14C]mannitol bile:plasma ratio (− 23%). Intravenous secretin administration (0.3 CU/l00 g body wt min) induced a significant increase in the biliary concentration of bicarbonate (+ 21%) and an enhancement of bile flow (+ 21%) only in the animals with ductular proliferation. Taurocholate infusion (0.5 nmol/l00 g body wt min) produced a similar bile flow increase both in control rats and in rats with proliferated biliary ductules. These results indicate that proliferated bile ductules and ducts spontaneously secrete bile in response to infestation by F. hepatica and that secretin-stimulated bile flow originates at the proliferated biliary structures.

Origin, pattern, and mechanism of bile duct proliferation following biliary obstruction in the rat

Proliferation of bile duct-like structures is a hepatic cellular reaction observed in most forms of human liver disease and in a variety of experimental conditions associated with liver injury. Yet the origin, means of initiation, and significance of this hyperplasia are unknown. To clarify these issues we induced bile duct proliferation in rats by ligating the common bile duct and studied (a) hepatic incorporation of [3H]thymidine by histoautoradiography, (b) hepatic morphometry, (c) biliary tree volume using [3H]taurocholate as a marker of biliary transit time, (d) immunohistochemical expression of cytokeratin no. 19, (e) the effect of indomethacin, and (f) the role of increased biliary pressure, in the absence of physiological and biochemical evidence of cholestasis, on [3H]thymidine incorporation by the bile-duct cells. The results have demonstrated that (a) the proliferating bile duct-like cells are products of the extant biliary epithelium and retain its characteristics; (b) bile duct cells divide irrespective of the size of the duct in which they are located and form a system with a lumen continuous with the preexisting one; (c) bile duct proliferation results mainly in elongation, not in circumferential enlargement or sprouting of side branches; (d) portal macrophage infiltration does not play a role in the hyperplastic reaction, and (e) increased biliary pressure is the initiating factor in bile duct cell division. Our results provide evidence that under the present conditions, ductular metaplasia of hepatocytes does not occur and there is no functioning stem cell for biliary epithelial growth segregated in any particular duct size or within the portal connective tissue.

The Ultrastructure and Histology of Cholangiocellular Carcinomas in English Sole (Parophrys vetulus) from Puget Sound, Washington

The ultrastructure and histology of cholangiocellular carcinomas from feral English sole (Parophyrs vetulus) living in polluted waterways of Puget Sound, WA. are described. Electron microscopy confirmed that biliary epithelial cells were the main proliferative cell type composing this variety of neoplasm. The arrangement of these cells varied from well-organized multiple bile duct-like structures to disorganized multilayered sheets of poorly differentiated biliary epithelial cells. A fibrous stroma consisting of multiple layers of collagen fibers and fibroblasts, with macrophages and various blood cell types scattered among these layers occurred between bile duct-like structures or aggregates of biliary epithelial cells. Hepatocytes were not apparent in these neoplasms except within small necrotic regions surrounded by neoplastic biliary epithelial cells. No virus-like particles were observed among the cases examined in this study.

Isolation of a nonparenchymal liver cell fraction enriched in cells with biliary epithelial phenotypes

In the present study we have isolated and purified fractions of nonparenchymal liver cells enriched in biliary epithelial cells. Nonparenchymal liver cells were isolated by collagenase-pronase digestion of the biliary and connective hepatic tissue, which remained undissociated after collagenase perfusion of the liver. Fractionation of the nonparenchymal fractions was then achieved by centrifugal elutriation. Both normal rats and rats with proliferated bile duct-like structures, which were induced either by a 14-day bile duct ligation or by feeding 0.1% α-naphthylisothiocyanate for 28 days, were used in these studies. Using a normal rat liver, the fraction richest in biliary epithelial cells was that obtained at a pump flow rate of 36–40 ml/min. In this fraction 1.8–3.8 × 106 cells per liver were recovered and up to 55% of them were positive for γ-glutamyl transpeptidase and cytokeratins 7 and 19, all of which were histochemically or immunohistochemically detected solely in the biliary structures in the intact rat liver. When the nonparenchymal cells were isolated from hyperplastic livers, the number of cells recovered in such a fraction ranged from 12 to 19 × 106 per liver, and as many as 60%–85% of the cells expressed phenotypes of biliary epithelial cells. These results indicate that (a) by centrifugal elutriation a fraction of nonparenchymal cells enriched in cells with biliary epithelial phenotypes can be obtained from rat liver and (b) the hepatic hyperplasia induced by biliary obstruction or α-naphthylisothiocyanate feeding is a useful and valid strategy for improving both the yield and the purity of the isolated biliary epithelial cells.

Monoclonal antibodies directed against rat liver epithelial cell lines selectively recognize bile duct epithelium in livers of adult rats

Monoclonal antibodies directed against antigens on rat liver epithelial cell lines were prepared. Three antibodies, 4C3, 19C6, and 3C2, recognized surface antigens present (although in different quantities) on eight epithelial cell lines tested, irrespective of whether they were normal or transformed. For MAb 3C2, the primary antigen common to all but one cell line showed a Mr of 135 kD. In paraffin sections of liver tissue, two antibodies, 4C3 and 19C6, reacted exclusively with bile duct epithelium, whereas the MAb 3C2 additionally reacted with sinusoidal endothelium and the endothelium of the portal venules. In sections of livers from rats exposed to diethylnitrosamine, the MAb 19C6 selectively stained bile duct-like structures in cholangiomas, while other preneoplastic and neoplastic lesions were not stained. These results demonstrate that the monoclonal antibodies obtained may prove useful for investigating cell lineages related to propagable liver epithelial cell lines and suggest that these cells may be derived from terminal bile ductular cells.

Liver scarring induced by polychlorinated biphenyl administration to mice previously treated with diethylnitrosamine.

Exposure of mice for 8 weeks to drinking water containing diethylnitrosamine (DEN) was accompanied by alterations in hepatocyte structure and varying degrees of liver nonparenchymal cell (NPC) proliferation. Eighteen and a half weeks after cessation of DEN exposure, there was a 47% incidence of hepatocellular nodules. Centrilobular hepatocyte hypertrophy occurred consistently in mice given intraperitoneal injections of the polychlorinated biphenyl (PCB) mixture Aroclor 1254. PCB administration to mice previously treated with DEN was not accompanied by increases in gross liver nodule incidence above that induced by DEN, but many more developing microscopic nodules within the liver were observed in DEN-treated mice given Aroclor 1254 than in mice treated only with DEN. Aroclor 1254 administration over a 16-week period to mice previously treated with DEN was accompanied by an 83% incidence of severe distortion of liver structure resulting from nodule formation, uneven patterns of hepatocyte growth, and extensive deposition of scar tissue containing proliferating bile ducts. Morphological evidence of intestinal metaplasia was observed in proliferating bile duct-like structures during an early stage of liver adenofibrosis.

Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.

Liver cells isolated from newborn rats and seeded on a non-adherent plastic substratum were found to spontaneously re-aggregate and to form, within a few days, spheroidal aggregates that eventually reached a plateaued diameter of 150-175 micron. Analyses on frozen sections from these spheroids by immunofluorescence microscopy using antibodies to various cytoskeletal elements and extracellular matrix components revealed a sorting out and a histotypic reorganization of three major cell types. A first type consisted of cells that segregated out on the aggregate surface forming a monolayer cell lining; a second type was identified as hepatocytes that regrouped in small islands often defining a central lumen; and a third group of cells reorganized into bile duct-like structures. This intercellular organization in the aggregates was paralleled by the accumulation of extracellular matrix components (laminin, fibronectin, and collagen) and their deposition following a specific pattern around each cell population structure. Determinations of albumin secretion and tyrosine aminotransferase induction by dexamethasone and glucagon at various times after the initiation of the cultures revealed a maintenance of the hepatocyte-differentiated functions for at least up to 2 mo at the levels measured at 3-5 d. It is concluded that cells dispersed as single cells from newborn rat liver conserve in part the necessary information to reconstruct a proper three-dimensional cyto-architecture and that the microenvironment so generated most likely represents a basic requirement for the optimal functioning of these differentiated cells.

Undifferentiated (embryonal) sarcoma of the liver.Report of 31 cases

Thirty-one cases of undifferentiated (embryonal) sarcoma of the liver are presented. The tumor is found predominantly in the pediatric age group, the majority of patients (51.6%) being between 6 and 10 years of age. An abdominal mass and pain are the usual presenting symptoms. Radiographic examination is nonspecific except to demonstrate a space-occupying lesion of the liver. The tumors are large, single, usually globular and well demarcated, and have multiple cystic areas of hemorrhage, necrosis, and gelatinous degeneration. Histologic examination shows a pseudocapsule partially separating the normal liver from undifferentiated sarcomatous cells that, near the periphery of the tumor, surround entrapped hyperplastic or degenerating bile duct-like structures. Eosinophilic globules that are PAS positive are usually found within and adjacent to tumor cells. Areas of necrosis and hemorrhage are prominent. The prognosis is poor, with a median survival of less than 1 year following diagnosis.