Ductular Reaction in the Livers

Path. Res. Pract. 191,513-524 (1995)

Ductular Reaction in the Liver1 V. Desmet, T. Roskams and P. Van Eyken Laboratory of Histo-and Cytochemistry, University Hospital St. Rafael, University of Leuven, Leuven, Belgium

Introduction Many liver diseases in man and diverse experimental conditions in rodents are associated with an increase in the amount of bile ductular profiles forming a labyrinth at the periphery of portal tracts. The increase in ductular structures is accompanied by a predominantly neutrophil inflammatory cell infiltrate and periportal fibrosis. This sequence of changes has been referred to as 'ductular reaction'35, 36, 51, 77, 133. Since the first description of this histological feature by Waldeyer in 1868 148 , a number of studies have attempted to unveil its etiopathogenesis and physiopathological significance. Some progress has been recently made in understanding both the histogenesis and development capacity of the reactive bile ductular epithelial cells. Depending on the nature and duration of human liver disease and on the conditions used in the animal models, 3 types of ductular reactions have been described, referred to as "typical", "atypical" 85, 105, 127, 151 and "oval cell proliferation"42, 44, 90. Typical bile ductular reaction is seen in acute biliary obstruction in man and after bile duct ligation or alpha-naphtilisothiocyanate (ANIT) intoxication in rat49 , 55,69, 85, 105, 117, 119, 123, 127, 135, 151. Atypical bile ductular reaction is seen in human chronic cholestatic diseases, in regenerating human liver after submassive necrosis, in alcoholic liver disease and in focal nodular hyperplasia 31 - 33 ,51, 133. Atypical bile ductular reaction associated with Oval cell proliferation is seen in rodents after parenchymal damage induced by chemicals like D-galactosamine and carbon tetrachloride 75 , 121, 129. Considerable controversy exists considering the origin of the proliferating bile duct( -like) cells, especially in atypical ductular reaction. One of the problems with reviewing earlier literature is that often no distinction was made between different animal models in inter-

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Dedicated to the memory of Professor Pierre Dustin

(1914-1993).

© 1995 by Gustav Fischer Verlag, Stuttgart

preting the experimental results (e.g. species differences, regeneration after partial hepatectomy versus regeneration induced by widespread hepatocyte necrosis). It is imperative to take into consideration the context of each specific clinical or experimental situation. Three different mechanisms may be responsible for ductular reaction: 1) proliferation of preexisting bile ductular cells49 , 55, 123; 2) ductular metaplasia of hepatocytes 69, 136, 139, 140; and 3) activation, proliferation and differentiation of hepatic "stem cells" or progenitor cells 7. Probably, different mechanisms may operate together synchronously of asynchronously and at different rates in different conditions 32 , depending, among other factors, on the presence or absence of injury, the type and extent of injury and the capacity of hepatocytes to proliferate rapidly in response to growth stimuli. The question whether a stem cell compartment is present in a quiescent organ such as the adult liver is of great importance for fundamental biological processes such as embryogenesis, carcinogenesis and regeneration. Multiple animal studies have provided experimental evidence for the existence of such stem cell compartment. Moreover, since several recent immunophenotypic and ultrastructural studies support the existence of equivalents of these stem cells in man and suggest that these are present in the finest ramifications of the biliary tree 27, 51, 65, 102, the process of ductular reaction gained renewed interest. For this reason, this review on the different possible mechanisms of ductular reaction, will focus especially on data in support for the stem cell hypothesis and discuss its physiopathological significance. Ductular Reaction in Cholestasis

Proliferation of Preexisting Bile Ducts: Typical Ductular Proliferation In "typical" ductular proliferation, the new ductules are well formed, have well defined lumina and are confined to the portal tracts. The reactive ductules create 0344-0338/95/0191-0513 $3.50/0

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further extensions of the finest ramifications of the intrahepatic biliary tree. This type of ductular reaction is seen in acute severe cholestasis (acute extrahepatic biliary obstruction in man and after bile duct ligation or alpha-naphtilisothiocyanate (ANIT) intoxication in the rat)69, 85, 105, 117, 119, 123, 127, 135, 151. It seems quite well established that typical ductular reaction is the result of multiplication of preexisting bile ducts. This conclusion is based on the observation that the proliferating ducts in the rat models referred to above, retain the (immuno )histochemicaI4, 21, 50, 55, 119, 123, ultrastructural 4, 110, 120 and functional 3, 4 characteristics of their normal counterparts. Also, the results of thymidine labeling studies were consistent with proliferation of preexisting ductules 3, 4, 50, 59, 79, 123. The ductular epithelial cell proliferation is associated with an increased expression of cytosolic (H-ras) and nuclear (c-myc) proto oncogene mRNA's130. It has been further shown that after biliary obstruction in the rat, the hyperplasia involves elongation of preexisting bile ductules and ducts and that this is mainly induced by increased pressure 113, 123. James et al. 69 showed that in human extrahepatic biliary obstruction of recent onset, typical ductular reaction is present and both parenchymal and bile ductular cells retain their respective normal cytokeratin profiles. However, in bile duct obstruction of longer duration, hepatocytes in acinar zone 1 gradually become positive for cytokeratin 7, a type of cytokeratin that is normally confined to bile duct cells 141, suggesting incipient ductular metaplasia. This will be discussed in the following section. The functional meaning of ductular proliferation is not clear. Alpini et al. 3,4 have shown that bile ducts proliferating in response to ANIT or bile duct ligation still secrete a bicarbonate rich fluid and are responsive to secretin. The ductular cells reabsorb bile acids15 and may thus provide the hepatocyte with a means of getting rid of accumulating bile acids. This process could lead to increased cholehepatic cycling36 . Upon removal of the proliferative stimulus in the bile duct ligation model, the proliferating ductules disappear by apoptosis 6, 10,50. Atypical Ductular Reaction

Atypical bile ductular reaction consists of bile ductules located adjacent to the parenchyma and arranged in anastomosing cords with poorly defined lumina and small cytoplasmic volume of the lining cells. The ductules correspond to flattened cylinders, and their threedimensional anatomy is quite similar to hepatic muralia 71. Atypical bile ductular reaction is seen in human chronic cholestatic diseases like primary biliary cirrhosis, primary sclerosing cholangitis or longstanding extrahepatic biliary obstruction, in regenerating human liver after submassive necrosis, in alcoholic liver disease and in focal nodular hyperplasia31 - 33 , 51, 133. In rodents, atypical duct-like structures, associated with oval cell proliferation, are seen after administration

of chemicals like D-galactosamine and carbon tetrachloride 75 , 121, 129. This section will mainly focus on atypical bile ductular reaction in human liver. In contrast to typical bile ductular reaction, where the proliferating cells retain the characteristics of their normal counterparts, in atypical bile ductular reaction, many studies have documented the presence of transitional cells or intermediate cells - cells that exhibit phenotypic characteristic of both hepatocytes and bile duct cells2, 14,49,51,61,85,93,119,128,133,136,139,140,144,145. Wegmann et al. studied atypical ductular reaction in human cirrhotic livers of various etiologies using enzyme histochemistry149. They found cells with the morphological appearance of hepatocytes, displaying histochemical staining characteristics of bile duct cells and bile duct cells displaying histochemical staining characteristics of hepatocytes (like glucose-6-phosphatase activity). Glycogen and glucose-6-phosphatase were also demonstrated in proliferating ductules in alcoholic human liver disease by Uchida and Peters 133 • In human liver diseases associated with atypical ductular reaction, several studies showed that acinar zone 1 hepatocytes can express several antigens that are normally confined to bile duct cells, such as tissue polypeptide antigen 14,37, S100 protein144,145, Le and Lb blood group antigens 86 , VLA 2,3,6 integrins 146, 147, a 41 kD antigen termed 17-1A Ag that is also expressed in human colon carcinoma cells 107, an undefined biliary antigen identified by its reactivity with a monoclonal antibody designated clone 58 and the gamma subunit of the glycolytic enzyme enolase 48 . In longstanding cholestasis, acinar zone 1 hepatocytes, seemingly in continuity with bile duct cells, gradually become immunoreactive for cytokeratin 7 and 1969,136,140 which normally are restricted to bile duct cells141. Also, at the ultrastructural level, a range of cells intermediate between hepatocytes and bile duct cells were recognized in chronic cholestatic human liver disease 84,104. The presence of transitional cells is thought to support the concept that hepatocytes can transform or "transdifferentiate"109 into bile duct type cells, a hypothesis also referred to as "ductular metaplasia of hepatocytes 35 ." Three-dimensional reconstruction studies also provide evidence in favour of this concept 16, 84, 151. Moreover, Hillan et al. 61 demonstrated that isolated rat hepatocytes can give rise to bile ductular structures when transplanted into the spleen of bile duct ligated rats and not in control rats, suggesting a hormonal interaction leading to metaplasia. However, the presence of intermediate or transitional cells could also be explained by a mechanism other than "ductular metaplasia of hepatocytes." With the growing evidence for the existence of a "stem cell" compartment in the human liver, a pathway involving these stem cells, transforming into intermediate cells and further differentiating into hepatocytes, has to be considered13, 109, 122. Whether atypical ductules exhibit functional characteristics of normal biliary epithelium is not known. Electron-microscopic observations suggest that they

Ductular Reaction in the Liver

Fig. 1. Focal nodular hyperplasia stained for chromogranin A. x 250. Atypical reactive bile ductules forming anastomosing cords are immunoreactive for chromogranin A. Smaller hepatocyte-like cells (intermediate cells), adjacent to fibrous septa (arrow) sometimes forming liver cell rosettes (arrowhead) and often in continuity with bile ductules show immunoreactivity of varying intensity for chromogranin A. The presence of these transitional cells is thought to support the concept of "ductular metaplasia of hepatocytes." However, the presence of these cells can also be explained by a pathway involving "stem-cells," transforming into intermediate cells and further differentiating into hepatocytes. Reproduced with permission from "Reactive human bile ductules express Parathyroid Hormone-related Peptide." Histopathology, 1993, 23: 11-19.

participate in reabsorption 108 • Also, the presence of bilirubin and lipofuscin in ductular cells indicates that they can reabsorb biliary constituents 3o • As in typical ductular reaction, this capacity would provide the parenchymal cells with an efficient mechanism of getting rid of extensive bile acid overload36 • The precise mechanisms that lead to induction of atypical ductular reaction are unknown. Humoral factors may playa role as suggested by the transplantation experiments of Hillan et ai., referred to above 61 • The

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Fig. 2. Partial extrahepatic obstruction in cirrhotic stage, stained for Parathyroid Hormone-related peptide (PTHrP). x 100. PTHrP-positive reactive bile ductules, surrounded by an increase in extracellular matrix, resulting in progressive periportal fibrogenesis leading to cirrhosis. Reproduced with permission from "Reactive human bile ductules express Parathyroid Hormone-related Peptide." Histopathology, 1993, 23: 11-19.

changes in acinar zone 1 hepatocytes in chronic cholestasis have led to hypothesize that retention of bile constituents may also be involved35 • A recent study demonstrated that reactive human bile ductules in chronic cholestatic liver diseases express the neuroendocrine markers chromogranin A (Fig. 1) and Neural Cell Adhesion Molecule (NCAM) and contain neuroendocrine granules. These features are not found in interlobular bile ducts 104 • It was speculated that the reactive bile ductular cells might contain peptide growth factors playing an autocrine or paracrine role in the control of liver cell proliferation and differentiation 104 . Moreover, further studies reported that reactive human bile ductules, but not normal bile ducts, express Parathyroid Hormone-related Peptide (PTHrP) (Fig. 2), a peptide that is encoded by a growth factor regulated "early response" gene and that plays an impor-

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tant role in growth and differentiation of neoplastic as well as non-neoplastic cells. As such, PTHrP probably acts as a growth factor in the process of atypical bile ductular reaction 99 . The presence of endothelin in reactive bile ductules also lends further support for their neuroendocrine nature 64 . The role of growth factors will be further discussed in the following section on liver regeneration after (sub)massive hepatic necrosis. Ductular reaction also comprises an increase in extracellular matrix, resulting in progressive periportal fibrosis (Fig. 2). Increased matrix production is due to the secretion of basement membrane components (collagen type IV, laminin) by ductular cells, and the stimulation of mesenchymal cells1. In acinar zone 1, Kupffer cells proliferate and monocytes are recruited 62 . In addition, near the extending front of reactive bile ductules, also in acinar zone 1, transformation of perisinusoidal Ito cells into myofibroblasts occurs 17, 18. Immunoelectronmicroscopy studies and, more recently, in situ hybridization, have shown that these transformed Ito cells are probably the main producers of matrix proteins and a major source of fibrosis22, 82, 87, 125. The proliferation, transition into myofibroblasts and matrix production of Ito cells is modulated by cytokines, e.g. transforming growth factor 13. Bile ductular cells have recently been shown to express transforming growth factor ~2 83. As such, bile duct cells are thought to be the "pacemaker" in the development of the progressive fibrosis leading to cirrhosis in diseases such as primary biliary cirrhosis and primary sclerosing cholangitis 34 . The complex interactions between bile ductular cells, mesenchymal cells, the surrounding matrix and growth factors are not yet fully elucidated. To address this problem Roskams et al. recently studied the expression of integral membrane (syndecans) and matrix (perlecan) heparan sulfate proteoglycans (HSPGs) in normal human liver lOO and cholestatic human liver disease101. In comparison to what is seen in normal liver, chronic cholestasis is marked by an increase in synd1 and synd4 expression in reactive bile ductules, accompanied by an increase in perlecan expression around bile ductules. In acinar zone 1, large stimulated macrophages and an increased number of Ito cells show strong positivity for synd3. Syndecans are known to be essential co-factors for the binding of Fibroblast Growth Factors (FGF) to their high affinity receptors 1S2 and for binding extracellular matrix molecules9. They are also important in cell-cell interactions and playa role in epithelial organization. The distribution pattern of the different syndecans in chronic cholestasis is very well compatible with these functions. The strong expression of perlecan around reactive bile ductules probably also has an important function in binding growth factors. It is known that perlecan functions as a slow release reservoir for basic FGF47. Also Platelet Derived Growth Factor (PDGF) has been shown to be associated with matrix HSPGs73,94. Moreover, a recent in vitro study suggested that Hepatocyte Growth Factor (HGF) binds to perlecan in Disse's space and that this

may playa role in stabilizing and maintaining the pool of HGF80. As such, perlecan can retain these growth factors in the vicinity of the cell, modify their diffusion properties, increase their stability and protect them from proteolytic degradation 46, 47,94, hence playing an important role in the cell-growth factor interactions. Ductular Reaction in (Sub)massive Hepatic Necrosis Activation of Stem Cells? One of the most commonly used rat models to study liver regeneration is a two-third hepatectomy. The nonresected part of the liver is intact and restoration of the liver parenchyma is due to rapid compensatory hyperplasia of both liver parenchymal cells and bile duct cells until the original liver mass is restored 78 • In this model of regeneration, no ductular reaction is seen. This animal model correctly reflects what happens in the human liver after partial resection 72 or after liver transplantation when the recipient is larger than the donor142. However, hepatocyte replication in the human liver occurs much more frequently as a response to hepatic injury caused by chemicals, viruses or anoxia. Human liver damage with panlobular or multilobular necrosis, is accompanied by an (atypical) bile ductular reaction at the periphery of necrotic ghost lobules. Such reactive bile ductules have been termed pseudo ducts, neocholangioles, and ductular or biliary hepatocytes 92 • As in atypical bile ductular reaction seen in chronic cholestatic disease, the ductular structures seen after (sub)massive necrosis also contain cells intermediate between hepatocytes and bile duct cells. They are immunoreactive for secretory component28 , carcinoembryonic antigenS1 and bile duct type cytokeratins 138, but they also stain positively for alpha-1-antitrypsins1, a hepatocyte marker. Electron microscopically, the reactive ductules also contain cells that are intermediate between hepatocytes and bile duct cellss1 , 102. In a case of end-stage cirrhosis of presumably viral origin, cells expressing both Hepatitis B surface antigen (hepatocyte marker) and bile duct-type cytokeratin were observed143. The presence of these intermediate cells was interpreted as indicative of a gradual transformation of the ductular sprouts into true functioning hepatocytes. As already mentioned, intermediate cells could derive from (bile ductule-related) facultative stem cells. At the earliest stage of regeneration after (sub)massive hepatic necrosis in human liver, there is not yet a clearly discernable ductular reaction (Fig. 3). This earliest stage is characterized by the presence of small single cells with an oval nucleus and scanty cytoplasm at the periphery of portal tracts and in the vicinity of necrotic areas (Fig. 3)102. These cells are immunoreactive for bile duct-type cytokeratin 19 and chromogranin-A (Fig. 3) and contain neuroendocrine granules, which suggests that these cells can produce substances that might playa role in the growth and/or differentiation

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Fig. 3. Liver tissue in an early stage of regeneration after submassive necrosis, stained for chromogranin A. Original magnification x 80 (3a) and x 625 (3b). 3a: Immunoreactive bile ductules as well as small, singular cells with oval nuclei and scanty cytoplasm are seen. N: are of necrosis; P: portal tract, 3b: High magnification, illustrating the small, singular cells. Reproduced with permission from "Cells with neuroendocrine features in regenerating human liver," APMIS, 1991, supp!. 23: 32-39.

of liver cells lo2 • In later stages of regeneration, a florid bile ductular reaction is recognizable and both bile ductular epithelial cells and clusters of periportal hepatocytes are positive for cytokeratin 19 and chromogranin A. This chronological sequence suggests a differentiation of the 'small cells' into both bile duct cells and hepatocytes lo2 • Similar to what is seen in chronic cholestatic liver disease, the reactive ductules in human regenerating liver contain PTHrP, while normal bile ducts do not 99 • PTHrP has been shown to stimulate DNA synthesis in different cell types in the presence of Epidermal Growth Factor or Transforming Growth Factor and has been suggested to participate in multifactorial growth factor 100ps l9, 68. Several growth factors have been described as important in liver regeneration (reviewed in ref8l ). Various serum factors (e.g. Epidermal Growth Factor, Hepatocyte Growth Factor) induce DNA synthesis in hepatocytes as well as in bile duct epithelial cells 70 • A sharp increase in Hepatocyte Growth Factor in the plasma is thought to be the earliest event leading to liver regeneration after partial hepatectomy in the rat. This event

triggers a cascade of phenomena, leading to early changes in gene expression, including expression of several growth factors by hepatocytes (Transforming Growth Factor-alpha and acidic Fibroblast Growth Factor) and Ito cells (Transforming Growth Factorbeta and Hepatocyte Growth Factor). Moreover, both mature bile duct cells and also ductular cells in rat and human liver express Transforming Growth Factor-alpha 12,24,66, suggesting that they might function as a pool of Transforming Growth Factor-alpha to be released into periportal zones of the parenchyma 12. Similarly, PTHrP produced by ductular cells may be involved in complex growth factor loops which may have an important role in liver regeneration following inj ury 99. None of the above described conditions in human liver associated with hepatocellular necrosis can be exactly reproduced in experimental models, but a large number of both necrogenic and non-necrogenic chemicals cause hepatic injury and induce liver growth and a ductular reaction in laboratory animals. Most commonly used are necrogenic agents like carbon-

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tetrachloride and D-galactosamine43 , 75, 121, 129. In contrast to the partial hepatectomy model, liver regeneration after chemical injury takes place in damaged tissue and differentiated hepatocytes are unable to assure regeneration, either because too many hepatocytes are lost or because they are unable to respond because of toxic injury of because of an unknown mechanism that inhibits their replication. Under these conditions, 'reserve' cells may take over. This hypothesis was first postulated by Wilson and Leduc in 1958, based on experiments involving liver regeneration after severe nutritionat injury150. They concluded that bile ductules (cholangioles) gave rise to hepatocytes in the recovery phase of severe nutritional injury in mice. They proposed that cells of bile ductules formed a reserve compartment that could expand and generate hepatocytes after severe hepatic injury but would not do so after partial hepatectomy. The portal population of nonparenchymal epithelial cells that proliferates after chemical injury43, 75, 121, 129, often referred to as oval cells, has been reported to display traits of both hepatocytes and biliary epithelium 67, 75, 76, 88, 89, 91,111 and to differentiate into normal and neoplastic liver cells29, 41. It has thus been proposed that these cells are the progeny of 'facultative stem cells,29, 41, 43, 56, 78, 112.

Further Considerations on Liver Stem Cells Stem cells can be defined as pluripotent cells that after division give rise to two daughter cells. One daughter cell remains a stem cell, while the other cell expresses a differentiated phenotype 112 . In continually renewing systems such as the skin, intestinal epithelia and the hematopoietic bone marrow, progenitor or stem cells continually divide to generate transit cells, which in turn mature into terminally differentiated cells that lack proliferative capacity60. The identification of this stem cell population is extremely difficult. Under normal circumstances, stem cells replace senescent cells and restore destroyed tissues in a variety of disease states 112 . In static cell populations such as the brain, no cell division occurs. The liver has traditionally been considered as an example of a conditional renewal system. Under normal circumstances, there is little cell turnover, but in response to injury, the principally stable cells, the hepatocytes, proliferate rapidly60. It is usually claimed that conditional renewal systems do not contain stem cells60 . There is however growing evidence pointing towards the existence of stem or progenitor cells in the liver. Support for the stem cell hypothesis has come mainly from cell culture data (for review see 44 ) and from the finding that during chemical hepatocarcinogenesis42, 43, 58, 90,112 or liver cell regeneration after chemical injury43, 75,150, a periportal population of small 'primitive' epithelial cells proliferates in association with or before hepatocyte multiplication. These cells are also called oval cells because of their shape42,90.

An extensive review of the currently available data on oval cells is beyond the scope of this review. It is generally believed that oval cells are related to terminal biliary ductules, the so-called canals of Hering 35 , 52, 54, 56, 75, 76. However, they constitute a heterogenous population of non-parenchymal epithelial cells and a proportion of these cells expresses phenotypic markers of both (immature) hepatocytes (like alpha-fetoprotein) and bile duct cells25, 26, 39, 40, 53, 63. At least a subset of oval cells is pluripotent and has the capacity to differentiate towards hepatocytes, bile ductular cells, pancreas and intestinal epithelium and can give rise to hepatocellular carcinoma and cholangiocellular carcinoma29, 41, 54, 56, 74, 112, 126, 131. Support for the existence of stem cells and their location in the canal of Hering has also come from a better understanding of normal embryonic development of the liver. The early embryonic liver is composed of progenitor cells (hepatoblasts) that ~enerate both mature hepatocytes and bile duct cells 3 ,124,135. The hepatoblasts near vascular spaces (equivalents of portal tracts in mature liver) form primitive ductular structures, so called "ductal plates." The "ductal plates" are phenotypically equivalent to oval cells and express markers of both hepatocytes (alpha-fetoprotein and albumin) and bile duct cells (cytokeratin 7 and 19)45,114, 137. It is likely that a small number of progenitor cells persist in the adult liver and that they are located in the smallest units of the biliary tree, at the transition between portal tracts and the parenchyma. Grisham proved that normal hepatocytes in culture are non-clonogenic and suggested that the liver epithelial cell lines that display some hepatocyte-like functions in vitro originate from stem cells 56,132. Moreover, he introduced the concept that liver stem cells are "facultative". This means that they are only activated and only proliferate when severe and prolonged damage to hepatocytes is coupled with inhibition of hepatocyte replication56 • As mentioned in the previous section, the early experiments of Wilson and Leduc on regeneration after severe nutritional damage 150 perfectly fit with this "facultative stem cell" theory and with the concept that these "stem cells" are related to biliary ductules. Subsequent work by other groups using experimental protocols that lead simultaneously to hepatocyte damage and impaired regeneration confirmed the hypothesis. For example treatment of rats with D-galactosamine induces proliferation of oval cells, small hepatocytes and atypical duct-like structures25 , 26, 75, 129. Following the fate of ductular cells by labelling them with 3H -thymidine, Lemire et al. could demonstrate a precursor product relationship between these bile ductular cells and both oval cells and small hepatocytes 75 . After bile duct ligation followed by carbon tetrachloride administration 121 or after severely hepatotoxic treatment with furan 118, hepatic cell cholangioles appear, consisting of both bile ductular epithelial cells and ductular hepatocytes. They are thought to indicate generation of hepatocytes by bile ductular cells 121 •

Ductular Reaction in the Liver . 519

The most convincing example of the generation of hepatocytes from ductular cells after toxic injury is found in the pancreas rather than the liver. After feeding rats a copper deficient diet for 8 - 9 weeks, a widespread injury of the exocrine pancreas is elicited. When the animals are refed a normal diet, the ductular structures that remain in the tissue give rise to cells with all the phenotypic traits and biological properties of hepatocytes instead of pancreatic acinar cells 95 - 98 . If acinar destruction is less extensive, acinar structures regenerate and repopulate the organ, rather than hepatocytes 95 . Thus both the pancreatic and hepatic ductal system which share similar embryological origins, retain the capacity to generate hepatocytes. Based on thymidine labelling experiments and autoradiography, Zajicek et al. proposed the "streaming liver" conceptS, 153. They suggest that even under normal circumstances there is a continuous, slow proliferation of uncommitted stem cells, located in the canals of Hering, that differentiate in two committed stem cells: one a hepatocyte and the other one a biliary epithelial celIS. The periportal hepatocytes then progressively move towards the perivenous end of the lobule, bringing along associated extracellular matrix and non-parenchymal cells. In the perivenous region, they undergo apoptosiS 153 . This implies that liver stem cells are not just "facultative" stem cells, but that the liver is a slow continually renewing system. Sigal et al. suggested that the liver is a "stem cell and lineage system" with three compartments: 1) a stem cell compartment in which cells have a fetal phenotype, have a slow rate of cell cycling and respond slowly to injury; 2) an amplification compartment in which cells have an intermediate phenotype and respond more rapidly to injury and 3) a terminal differentiation compartment with increasingly differentiated cells which that largely lost their capacity for division 115. The phenotype of cells within the different compartments is mainly determined by their age. The composition of the surrounding matrix is not uniform, however, and through cell-matrix interactions, the extracellular matrix might also contribute to alterations in phenotype as cells progress through the lineage115. The "streaming liver" and the liver as a "stem cell and lineage system" are provocative concepts and are not universally accepted (for discussion see reP, 11,44, 57). The precise mechanisms that trigger stem cell activation are not known. That cell-matrix interactions are involved is nicely demonstrated in a recent study of Coleman et al. 23. They used a cell line with oval cell characteristics (designated WB-F344), previously isolated by Tsao et al.132 which can be chemically transformed into tumorigenic cell lines l3l. Coleman et al. introduced genetic markers into WB-F344 cells that were either untransformed or obtained from a tumorigenic clone. After injection into the livers of syngeneic rats, both types of cells became integrated in lobules as normal hepatocytes. These results show that the microenvironment of the liver parenchyma exerts a regulating

influence that suppresses or reduces the tumorigenicity of some highly tumorigenic variants of WB-F344 cells, perha ps by inducing terminal differentiation23 . Several recent studies on human liver support the existence of progenitor cells in human liver as well, and suggest that they are present in the periportal region. As already discussed, immunohistochemical studies on the normal embryonic development of the human liver indicate that the cell layers surrounding the portal vein branches, the ductal plates, consitute a population of cells with phenotypic characteristics of both hepatocytes and bile duct cells137. Ductal plates and incorporated bile ducts in fetal and neonatal human liver express PTHrPl03. This expression disappears at the age of 4 to 5 years and is absent from adult liver. The reexpression of PTHrP in reactive ductules also fits with the concept of stem cells and their location in the biliary ductules (canals of Heringj99. The demonstration of bcl-2-proto-oncogene protein in human normal bile ductules and small ducts and in proliferating ductules in a variety of liver injuries also lends support for the localization of a stem cell compartment in the biliary ductules 20 . By preventing apoptosis, the bcl-2 protein prolongs cell survival, an essential property for a stem cell population. Older studies on experimental liver carcinogenesis proposed already in 1963 that hepatocellular carcinomas could from either hepatocytes or oval cells29 . Subsequent immunohistochemical work using anti-cytokeratin antibodies showed that up to 50 percent of human primary hepatocellular carcinomas express cytokeratins that are normally seen only in bile duct cells, suggesting that these tumours may originate from cells with a biliary epithelial phenotype, possibly oval cell equivalents 135 • The above mentioned study on regenerating human liver that demonstrated the presence of small single cells with scant cytoplasm, expressing chromogranin-A (Fig. 3) and cytokeratin 19, in the earliest stages of regeneration highly suggests the activation of a progenitor compartment. In later stages of regeneration, reactive bile ductular cells and groups of periportal hepatocytes expressed chromogranin-A and cytokeratin 19, suggesting subsequent differentiation of these small cells into hepatocytes and bile duct cells 102 . In a recent ultrastructural study of ductular reaction in human chronic cholestatic liver disease, a novel small epithelial cell was identified, which might be a candidate progenitor cell capable of differentiating into either parenchymal or bile duct cells27. Hsia et al. observed oval-type cells in non-neoplastic human liver tissue from patients with hepatitis B virus-associated hepatocellular carcinoma 65 ,6 . These cells were immunoreactive for cytokeratin 19 (ductular phenotype) and displayed heterogeneity in alpha-fetoprotein and albumin expression (hepatocellular phenotype)65. In cases with hepatitis and liver cirrhosis, alpha-fetoprotein producing cells were already described as having the appearance of "oval cells" and transitional cells by Sakamoto et al. in 1975 106 .

520 . V. Desmet et al.

Conclusions and Perspectives for the Future The growing body of evidence that there exists a progenitor cell in the liver and that it is related to the finest ramifications of the biliary tree, has shed new light on the process of ductular reaction. The different cell types in the liver, including (pluripotent) progenitor cells appear to exhibit significant differentiation plasticity. Hepatocytes appear to be capable to "transdifferentiate" to bile duct epithelial cells. Vice versa, some biliary epithelial cells appear capable of altering their differentiation commitment so as to give rise to hepatocytes and even small intestinal mucosa cell types. At present, we do not understand how phenotypic differentiation "switches" are regulated. Neither do we understand which factors regulate the differentiation of progenitor cells along hepatocyte and bile duct (or even intestinal) cell lineages and at which stage they become commited to a particular pathway. Furthermore, we do not fully understand what triggers progenitor cells to proliferate after hepatic injury. Hormones and serum growth factors certainly playa role in these processes. However, one would expect these to have a generalized effect throughout the liver, but ductular reaction does not generally occur at the same extent in all lobules. Hence, it is unlikely that circulating serum factors have a major triggering effect. Instead, localized changes taking place in the areas of injury ought to be considered. These changes may include alterations in cell-cell and cell-matrix contact as complex changes in the interactions between the different cell compartments, the surrounding matrix and growth factors. Although some progress has been made in this area of growing interest, the story of ductular reaction is far from being unraveled. Observations indicating the existence of a human counterpart of rat oval cells are at present still limited. Moreover, these observations do not prove that these phenotypically oval-like cells also function as true progenitor cells. Hence, data obtained from in vitro work, with cell culture conditions which mimic the in vivo situation as closely as possible, will be important, but they need to be confirmed as much as possible with data obtained in vivo, both in animal models as in man134. To date, no pure progenitor cell populations have been isolated from adult livers. However, new and better techniques for enriching cell cultures are being developed using immunoabsorption ("panning") and fluorescence-activated cell sorting 116 • Further identification of stem cells in the liver is a top priority. If their presence is confirmed and their nature better understood, these stem cells might eventually be used to introduce new genes into the liver and to repopulate the organ in some cases of fulminant hepatic failure.

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Key words: Ductular reaction - Oval cells - Stem cells - Biliary fibrosis Professor Dr. Valeer Desmet, Laboratory of Histo- and Cytochemistry, University Hospital St. Rafael, Minderbroederstraat 12, B-3000 Leuven, Belgium, Tel: 3216336550, Fax: 3216336548