Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities

The utilization of human hepatocytes for biomedical research, drug discovery, and treatment of liver diseases is hindered by the limited availability of donated livers and the variability of their derived hepatocytes. Human embryonic stem cells (hESCs) are pluripotent and provide a unique, unlimited resource for human hepatocytes. However, differentiation of hESCs to hepatocytes remains a challenge. We have developed a multistage procedure by which hESCs can be directly differentiated to hepatocyte-like cells without embryoid body formation and the requirement of sodium butyrate. The hESC-derived hepatocyte-like cells (HLCs) exhibited characteristic hepatocyte morphology, expressed hepatocyte markers, including alpha-fetoprotein, albumin, and hepatocyte nuclear factor 4alpha, and possessed hepatocyte-specific activities, such as p450 metabolism, albumin production, glycogen storage, and uptake and excretion of indocyanine green. Hepatocyte growth factor was found to play a positive role in promoting hepatocyte differentiation. Our differentiation system has shown that hESCs can be differentiated to hepatocyte-like cells capable of executing a range of hepatocyte functions. Therefore, it presents a proof-of-principle of potential applications of using the hESC-derived hepatocytes. Additionally, the hESC-derived HLCs provide a unique model to study the mechanisms involved in human hepatocyte differentiation and liver function.     

Maintenance of rat hepatocytes under inflammation by coculture with human orbital fat-derived stem cells

Preservation of hepatocyte functions in vitro will undoubtedly help the management of acute liver failure. The coculture system may be able to prevent functional decline of hepatocytes. It has already been shown that hepatocytes, when cocultured with bone marrow mesenchymal stem cells, could undergo long-term culture in vitro without loss of functions. In this study, human orbital fat-derived stem cells were isolated and cocultured with rat hepatocytes. When treated with serum from an acute liver failure patient, rat hepatocyte monoculture showed reduction of cell viability and loss of liverspecific functions. However, rat hepatocytes in the coculture system were still able to secret albumin and synthesize urea. IL-6 was significantly elevated in the coculture of rat hepatocyte with orbital fat-derived stem cells, and it might be the key immunoregulator which protects rat hepatocytes against inflammation. Our data confirmed that orbital fat-derived stem cells, or other adipose tissue-derived stem cells, are an ideal candidate to support rat hepatocyte functions in vitro.     

Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months. Albumin- and fibrinogen-secreting cells were selected and cloned by limiting dilution to obtain homologous cell populations. The established IHH (immortalized human hepatocyte) cell lines were evaluated for their usefulness in studying the regulation of cell growth and of certain differentiated hepatocyte functions.IHH cells retain several differentiated features of normal hepatocytes. They display albumin secretion at a level comparable to cultured primary human hepatocytes (30 µg albumin/ml per day). A portion of the IHH cells are polarized, forming bile canaliculi-like vacuoles where exogeneous organic anions accumulate. The multidrug resistance (MDR) P-glycoprotein, known to be localized at the canalicular membrane, is also present in these vacuoles. The polarized features allowed the use of IHH cells for the study of localization of the newly characterized multidrug resistance protein MRP1. The homologues of MRP were found in hepatocytes, MRP1 and MRP2 (cMOAT), both functioning in ATP-dependent excretion of anionic conjugates. In differentiated hepatocytes, MRP1 expression is extremely low. In contrast, MRP1 is highly expressed in proliferating IHH cells, where it is localized in lateral membranes. A highly differentiated feature of short-term cultured primary hepatocytes which is not detectable in IHH cells is active uptake of the bile salt taurocholate. Furthermore, IHH cells secrete triglyceride (TG)-rich lipoproteins, apolipoprotein B (0.6 µg/ml per day), and apolipoprotein A-I (1 µg/ml per day). However, they secrete apoB-containing TG-rich lipoproteins mainly in the LDL density range, while short-term cultured primary hepatocytes mainly secrete TG-rich lipoproteins in the VLDL density range.In conclusion, functions that are rapidly lost in short-term hepatocyte cultures are, in general, not displayed by IHH cells. Immortalized human hepatocytes provide a valuable tool for studying the regulation of hepatocyte proliferation-related phenomena.     

Stem cells and liver engineering

Human hepatocytes, suitable for treatment of patients with liver failure, for the creation of bioartificial (BAL) devices, or for studies for toxicity and metabolization studies in the pharmaceutical industry, are in short supply due to the lack of donor organs. Therefore, methods that allow ex vivo expansion of hepatocytes with mature function are being pursued. One cell source, believed to be a possible inexhaustible source of hepatocytes, is pluripotent stem cells (PSCs). However, directed differentiation of PSCs to cells with features of adult hepatocytes is not yet possible. Differentiated progeny remains mixed and PSC progeny does not have a number of the functional features of mature hepatocytes. In this review article, we will address tools being developed that allow for the identification of mature hepatocytes, in a non-invasive manner; to perform lineage tracing of PSC progeny; and novel culture systems being created for the in vitro differentiation of PSCs to hepatocyte like cells, and for the maintenance of primary liver derived hepatocytes or PSC-derived hepatic progeny in culture. As conventional two-dimensional (2D) static culture conditions poorly recapitulate the in vivo cellular environment, we will discuss bioreactor systems for liver tissue engineering, both macro-scale and micro-scale culture systems.     

Spheroid formation and expression of liver specific functions of primary rat hepatocytes co-cultured with bone marrow cells

We have developed a new co-culture system consisting of adhesive bone marrow cells (A-BMCs), non-adhesive bone marrow cells (NA-BMCs) and hepatocytes with improved hepatocyte immobilization efficiency and better maintenance of liver specific functions. The composition of the inoculated cells affected the morphology of hepatocytes. Spheroids formed spontaneously when rat hepatocytes were co-cultured with bone marrow cells (BMCs). However, the addition of NA-BMCs to existing hepatocyte monolayers did not change their morphologies to spheroids. On the other hand, NA-BMCs dramatically increased hepatocyte immobilization efficiency. Hepatocytes co-cultured with either NA-BMCs or A-BMCs maintained their albumin production activities significantly better than hepatocyte culture alone. Interestingly, the two fractions of BMCs appear to have combination effects on hepatocytes to maintain albumin production activity. We conclude that co-culture of hepatocytes and BMCs is an effective strategy to enhance hepatocyte immobilization efficiency and functions in vitro.     

Hepatic progenitor cells

Liver diseases are associated with a marked reduction in the viable mass of hepatocytes. The most severe cases of liver disease (liver failure) are treated by orthotopic liver transplantation. One alternative to whole organ transplantation for patients with hepatic failure (and hereditary liver disease) is hepatocyte transplantation. However, there is a serious limitation to the treatment of liver diseases either by whole organ or hepatocyte transplantation, and that is the shortage of organ donors. Therefore, to overcome the problem of organ shortage, additional sources of hepatocytes must be found. Alternative sources of cells for transplantation have been proposed including embryonic stem cells, immortalised liver cells and differentiated cells. One other source of cells for transplantation found in the adult liver is the progeny of stem cells. These cells are termed hepatic progenitor cells (HPCs). The therapeutic potential of HPCs lies in their ability to proliferate and differentiate into hepatocytes and cholangiocytes. However, using HPCs as a cell therapy cannot be exploited fully until the mechanisms governing hepatocyte differentiation are elucidated. Here, we discuss the fundamental cellular and molecular elements required for HPC differentiation to hepatocytes.     

Heterotypic interactions in the preservation of morphology and functionality of porcine hepatocytes by bone marrow mesenchymal stem cells in vitro

Temporary replacement of specific liver functions with extracorporeal bioartificial liver has been hampered by rapid de‐differentiation of porcine hepatocytes in vitro. Co‐cultivation of hepatocytes with non‐parenchymal cells may be beneficial for optimizing cell functions via mimicry of physiological microenvironment consisting of endogenous matrix proteins. However, the underlying mechanisms remain to be elucidated. A randomly distributed co‐culture system composed of porcine hepatocytes and bone marrow mesenchymal stem cells was generated, and the morphological and functional changes of varying degrees of heterotypic interactions were characterized. Furthermore, contributions of extracellular matrix within this co‐culture were evaluated. A rapid attachment and self‐organization of three‐dimensional hepatocyte spheroids were encouraged. Studies on hepatocyte viability showed a metabolically active, viable cell population in all co‐culture configurations with occurrence of few dead cells. The maximal induction of albumin production, urea synthesis, and cytochrome P4503A1 activities was achieved at seeding ratio of 2:1. Immunocytochemical detection of various extracellular matrix confirmed that a high level of matrix proteins synthesis within distinct cells was involved in hepatocyte homeostasis. These results demonstrate for the first time that cell–matrix has synergic effects on the preservation of hepatic morphology and functionality in the co‐culture of porcine hepatocytes with mesenchymal stem cells in vitro, which could represent a promising tool for tissue engineering, cell biology, and bioartificial liver devices. J. Cell. Physiol. 219: 100–108, 2009. © 2008 Wiley‐Liss, Inc.     

IGF-1 induces growth, survival and morphological change of primary hepatocytes on a galactose-bared polymer through both MAPK and beta-catenin pathways

PVLA poly-(N-p-vinylbenzyl-O-beta-D-galactopyranosyl-D-gluconamide) is a glycopolymer composed of hydrophilic carbohydrate side chain and hydrophobic styrene polymer. The hydrophilic carbohydrate residue of PVLA can be recognized as a ligand for hepatocytes asialoglycoprotein receptor (ASGP-R), which is abundant on the hepatocyte cell surface. Adhering to the PVLA coated dishes, hepatocytes try to form aggregates that have a long time survival and also cells in these aggregates exhibit better maintenance of specific hepatocyte functions. Stimulation of the cells with IGF-1 in this culture condition results in the formation of lower aggregates. In addition to the morphological influences of IGF-1 to these cells, we have also found that IGF-1 transmits growth stimulatory responses to hepatocytes on PVLA through both mitogen activated protein kinase (MAPK) pathway and beta-catenin pathways. The phosphorylation of MAPK can take place within 5 min of stimulation with IGF-1 and within at least 10 ng/ml of IGF-1 concentration. Inhibition of MAPK activation by MEK-1 inhibitor PD98059 reduces IGF-1 induced MAPK phosphorylation, and also IGF-1 stimulated DNA synthesis. Similarly, the use of PI3-K inhibitor LY294002 also inhibits IGF-1 stimulated DNA synthesis. IGF-1 stimulation enhances the migration of beta-catenin from the cytoskeleton and cell membrane to the cytoplasm which also is the reason behind formation of spheroids and lower aggregates. IGF-1 stimulation also shows increased translocalization of beta-catenin to the nucleus that is essentially important to produce beta-catenin responsive effects to the cells. These studies thus suggest that IGF-1 can stimulate the growth and survival of hepatocytes on PVLA through both MAPK and beta-catenin signaling pathways, and that the activation of beta-catenin signaling pathway produces the morphological changes of IGF-1 induced cells.     

Hepatic differentiation of mouse iPS cells and analysis of liver engraftment potential of multistage iPS progeny

Hepatocyte transplantation is considered a promising therapy for patients with liver diseases. Induced pluripotent stem cells (iPSCs) are an unlimited source for the generation of functional hepatocytes. While several protocols that direct the differentiation of iPSCs into hepatocyte-like cells have already been reported, the liver engraftment potential of iPSC progeny obtained at each step of hepatic differentiation has not yet been thoroughly investigated. In this study, we present an efficient strategy to differentiate mouse iPSCs into hepatocyte-like cells and evaluate their liver engraftment potential at different time points of the protocol (5, 10, 15, and 20 days of differentiation). iPSCs were differentiated in the presence of cytokines, growth factors, and small molecules to finally generate hepatocyte-like cells. These iPSC-derived hepatocyte-like cells exhibited hepatocyte-associated functions, such as albumin secretion and urea synthesis. When we transplanted iPSC progeny into the spleen, we found that 15- and 20-day iPSC progeny engrafted into the livers and further acquired hepatocyte morphology. In contrast, 5- and 10-day iPSC progeny were also able to engraft but did not generate hepatocyte-like cells in vivo. Our data may aid in improving current protocols geared towards the use of iPSCs as a new source of liver-targeted cell therapies.     

Histochemical and immunohistochemical similarities between hepatic tumors in two chimpanzees and man

A well-differentiated trabecular hepatocellular carcinoma (HCC) and a well-differentiated tumor resembling HCC from each of two chimpanzees were found to have histochemical and immunohistochemical staining characteristics similar to those in human HCCs. Transforming growth factor α was overexpressed in both tumors. Oval cells, thought to be liver stem cell progeny with a possible role in hepatocarcinogenesis, were observed among nontumorous hepatocytes, particularly near the tumors. Hepatic tumors are rare in chimpanzees but their similarities to human HCC provides a useful research model.     

Pluripotential liver stem cells: facultative stem cells located in the biliary tree

Abstract. The ability of the liver to regenerate after parenchymal damage is usually accomplished by the ephemeral entry of normally proliferatively quiescent (G₀) hepatocytes into the cell cycle. However, when hepatocyte regeneration is defective, arborizing ductules which are continuous with the biliary tree, proliferate and migrate into the surrounding parenchyma. In man these biliary cells have variously been referred to as ductular structures, neoductules and neocholangioles, and have been observed in many forms of chronic liver disease, including cancer. In experimental animals similar ductal cells are usually called oval cells, and their association with defective regeneration has led to the belief that these cells represent a progenitor cell population. Oval cells are thought to take over the burden of regenerative growth after substantial hepatocyte loss, suggesting that they are the progeny of facultative stem cells. The liver is not, however, generally considered as a stem cellfed hierarchy, although this is disputed by others. Despite this, the subject of oval cells has aroused intense interest as these cells may represent a target population for hepatic carcinogens, and they may be useful vehicles for ex vivo gene therapy. This review proposes that the liver does harbour stem cells which are located throughout the biliary epithelium, and that oval cells represent the progeny of these stem cells and function as an amplification compartment for the generation of ‘new’hepatocytes. This is a conditional process which only occurs when the regenerative capacity of hepatocytes is overwhelmed and thus, unlike the intestinal epithelium, the liver is not behaving as a classical continually renewing stem cell-fed lineage. We focus on the biliary network, not merely as a conduit for bile, but also as a cell compartment with the potential to proliferate under appropriate conditions and give rise to fully differentiated hepatocytes and other cell types.     

Functional hepatocellular heterogeneity for the production of plasma proteins.

It is now well established that hepatocytes are the main liver cells responsible for the synthesis of plasma proteins produced by the liver. That these cells are not specialized in the production of the different plasma proteins is also well established. Presently the point still debated is whether a functional hepatocellular heterogeneity exists for plasma protein synthesis as for many other hepatocyte functions. Several physiological and pathological situations suggest that this heterogeneity takes place in the hepatocytes of two opposite hepatic lobular zones, the periportal and centrilobular zones. However, this zonal difference, which supposes different regulatory mechanisms, must be confirmed by techniques other than the now classical immunocytochemistry or the in situ hybridization technique recently proposed for the demonstration of mRNAs in hepatocytes. Another hepatocellular heterogeneity, the intercellular heterogeneity, which can be observed in the same lobular zone, is more difficult to analyze, but shows that from hepatocyte to hepatocyte a variation exists in the synthesis of a given plasma protein.     

Mutants of PC12 cells with altered cyclic AMP responses.

PC12 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 microM 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(beta, gamma-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, Ns. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PC12 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PC12 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.     

The morphological and immunocytochemical evaluation of primary rat hepatocytes undergoing spontaneous cell death: modulation by the nitric oxide donor S-nitroso-N-acetylpenicillamine

Nitric oxide (NO) is one of the smallest molecules synthesised in the human body. It is produced by three distinct nitric oxide synthase isoenzymes (NOS) and plays a number of physiological functions in many organs and tissues. Among its numerous properties is the ability to influence programmed cell death. NO can either inhibit or induce apoptosis depending on the context of its production. In the liver, NO is produced in greater amounts especially during inflammation. The effect of NO in liver physiology and pathophysiology can be both beneficial and detrimental. Therefore, the aim of our study was to examine NO effect on cell viability and cell death in primary rat hepatocyte culture. By using NO donor, S-nitroso-N-acetylpenicillamine (SNAP), the potential of exogenously delivered NO to influence spontaneous cell death in culture was examined. The morphological approach was used in order to discriminate between apoptotic and necrotic cell death. The nitrite level, urea production and alanine aminotransferase leakage were determined in the culture medium. The immunocytochemical detection of three apoptotic markers: cleaved caspase-3, cleaved caspase-9 and lamin A, was performed. Immunocytochemical analysis of hepatocyte apoptosis revealed different labelling pattern for each method, while the detection of cleaved caspase-3 best correlated with defined phenotypical criteria. Our data showed that under present conditions NO improved the viability of primary rat hepatocytes compared to untreated cells. This was manifested by the increase of viable hepatocytes in contrast to the decrease of necrotic and apoptotic hepatocytes as assessed by the morphological examination of cell culture. The NO effect was dose-dependent in the range of SNAP concentration between 200-800 microM.     

The role of actin filaments and microtubules in hepatocyte spheroid self-assembly

Cultured rat hepatocytes self-assemble into three-dimensional structures or spheroids that exhibit ultrastructural characteristics of native hepatic tissue and enhanced liver-specific functions. The spheroid formation process involves cell translocation and changes in cell shape, indicative of the reorganization of the cytoskeletal elements. To elucidate the function of the cytoskeleton, hepatocytes undergoing spheroid formation were treated with drugs that disrupt the different cytoskeletal components. Cytochalasin D, which targets the actin filaments, caused inhibition of spheroid formation. The role of microtubules in this process was assessed by incubating the cells with taxol or nocodazole. Perturbation of microtubules had minimal effects on spheroid assembly. Scanning electron micrographs showed no morphological differences between spheroids formed in control cultures and those formed in the presence of taxol or nocodazole. In addition, the effects of those agents on hepatocyte functions were investigated. Albumin secretion and cytochrome P450 2B1/2 activities of hepatocytes were comparable in spheroids formed in the presence of taxol or nocodazole to those formed in control cultures. The levels of these liver-specific activities were lower in cytochalasin D--treated cultures where only dispersed cells or cell clumps were found but spheroids had not found. Thus, hepatocytes require an intact actin network to self-assemble efficiently into functional tissue-like structures. Perturbation of the microtubule lattice does not impair the formation process. Events that transpire during hepatocyte spheroid self-assembly exhibit striking similarities to processes commonly observed in tissue morphogenesis. The results provide insight into the mechanisms that cells employ to organize into tissues and can contribute to our understanding of how to control the cellular assembly in tissue engineering and clinical applications.     

Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions

Specific differentiated gene expression and the morphology of adult rat hepatocytes can be maintained for as long as 8 weeks in vitro only when they are cultured in the presence of biliary epithelial cells; when primary hepatocytes are cultured alone, they lose these functions within 2 to 3 days. We obtained evidence suggesting that contact between hepatocytes and biliary epithelial cells is necessary for maintaining hepatocyte functions. We examined whether junctional communication between and among hepatocytes and biliary epithelial cells is required for long-term maintenance of hepatocyte functions, using a dye-transfer method, in three co-cultures: (1) hepatocytes and biliary epithelial cells prepared from Sprague-Dawley rats; (2) hepatocytes from Sprague-Dawley rats and epithelial cells of the IAR 20 line, originally established from BDVI rats; and (3) hepatocytes from BDVI rats and IAR 20 epithelial cells. The established epithelial cell line (IAR 20) and early-passage cultures of biliary epithelial cells maintained hepatocyte-specific functions in culture for 40 and 70 days, respectively, but the latter induced more stable maintenance of albumin secretion. Hepatocytes cultured alone lost their characteristic morphology within 5 to 8 days, and almost no dye transfer was observed. In co-cultures, the capacity of biliary epithelial cells to communicate among themselves remained relatively high throughout the culture period, whereas hepatocytes showed almost no junctional communication at an early phase of culture and first began to communicate after 2 weeks, communication capacity increasing for at least the next 10 days of culture. The most notable finding was that there was no dye transfer between hepatocytes and biliary epithelial cells in any co-culture system. These results suggest that the maintenance of hepatocyte-specific functions requires intercellular contact but probably not gap-junctional communication between hepatocytes and biliary epithelial cells. This system is useful for studying heterotypic cell-cell interactions and the control of gene expression.     

Encapsulation of rat hepatocyte spheroids for the development of artificial liver

Hepatocyte spheroids and hepatocyte were immobilized in chitosan/alginate capsules formed by the electrostatic interactions between chitosan and alginate. After encapsulation, there was a 10% decrease in the viability of spheroids due to the exposure of the cells to a pH 6 during the encapsulation process. However, the encapsulated hepatocyte spheroids maintained over 50% viability and liver specific functions for 2 weeks while the encapsulated hepatocytes, free hepatocytes and free hepatocyte spheroids showed low viability and liver specific functions. Therefore, encapsulated hepatocyte spheroid might be applied to the development of a bioartificial liver.     

Enhanced liver-specific functions of endothelial cell-covered hepatocyte hetero-spheroids

The performance of an extracorporeal bioartificial liver (BAL) support system depends on the functional activities of the hepatocytes immobilized in the system. One of the most promising techniques in retaining liver-specific functions is co-culturing hepatocytes with other cell types, such as epithelial cells, endothelial cells and dermal fibroblasts. Primary rat hepatocytes were suspension co-cultured with rat prostate endothelial cell line (RPEn) for 20 h in a spinner vessel to form hetero-spheroids, which contain the two types of the cells, i.e., hepatocytes and endothelial cells in the same spheroid. For the subsequent culture, the hetero-spheroids were entrapped in a Ca-alginate gel bead. From the results of incorporation efficiency test, it was found that RPEn cells have a significantly higher attachment affinity to hepatocytes than human dermal fibroblast and rat liver epithelial cells. We clearly found out that RPEn cells located on the surface of the hepatocyte spheroids from immunostained paraffin sections of the hetero-spheroids. Identical with in vivo liver tissue, laminin was stained at the surface of the hetero-spheroids. Ultrastructures of liver tissue, such as bile canaliculus-like and Disse’s space-like structures, were also found at the surface of the hetero-spheroids. In vivo liver tissue, in which hepatocytes were covered with sinusoidal endothelial cells, was partly mimicked by the endothelial cell-covered hepatocyte spheroids. And the hetero-spheroids showed significantly higher and stable albumin secretion and ammonia removal activities than pure spheroids for 12 days of observations.

Therefore, the endothelial cell-covered hepatocyte hetero-spheroids may offer a useful study model of epithelial–mesenchymal interactions and information about liver tissue engineering research as well as a substitute of a cell source of a BAL system.     

Wound healing in the liver with particular reference to stem cells.

The efficiency of liver regeneration in response to the loss of hepatocytes is widely acknowledged, and this is usually accomplished by the triggering of normally proliferatively quiescent hepatocytes into the cell cycle. However, when regeneration is defective, tortuous ductular structures, initially continuous with the biliary tree, proliferate and migrate into the surrounding hepatocyte parenchyma. In humans, these biliary cells have variously been referred to as ductular structures, neoductules and neocholangioles, and have been observed in many forms of chronic liver disease, including cancer. In experimental animals, similar ductal cells are usually called oval cells, and their association with impaired regeneration has led to the conclusion that they are the progeny of facultative stem cells. Oval cells are of considerable biological interest as they may represent a target population for hepatic carcinogens, and they may also be useful vehicles for ex vivo gene therapy for the correction of inborn errors of metabolism. This review proposes that the liver harbours stem cells that are located in the biliary epithelium, that oval cells are the progeny of these stem cells, and that these cells can undergo massive expansion in their numbers before differentiating into hepatocytes. This is a conditional process that only occurs when the regenerative capacity of hepatocytes is overwhelmed, and thus, unlike the intestinal epithelium, the liver is not behaving as a classical, continually renewing, stem cell-fed lineage. We focus on the biliary network, not merely as a conduit for bile, but also as a cell compartment with the ability to proliferate under appropriate conditions and give rise to fully differentiated hepatocytes and other cell types.