Biliary Obstruction Promotes Multilineage Differentiation of Hepatic Stem Cells.

Because of their high regenerative potential, stem cells are an ideal resource for development of therapies that replace injured tissue mass and restore function in patients with end-stage liver diseases. Using a rat model of bile duct ligation (BDL) and biliary fibrosis, we investigated cell engraftment, liver repopulation, and ectopic tissue formation after intrasplenic transplantation of epithelial stem/progenitor cells. Fetal liver cells were infused into the spleens of Fisher 344 rats with progressing biliary fibrosis induced by common BDL or rats without BDL. Cell delivery was well tolerated. After migration to the liver, donor-derived stem/progenitor cells engrafted, differentiated into hepatocytes and cholangiocytes, and formed large cell clusters at 2 months in BDL rats but not controls. Substantial numbers of donor cells were also detected at the splenic injection site where they generated hepatic and nonhepatic tissue. Transplanted cells differentiated into phenotypes other than hepato/cholangiocytic cells only in rats that underwent BDL. Quantitative reverse-transcription polymerase chain reaction analyses demonstrated marked up-regulation of tissue-specific genes of nonhepatic endodermal lineages (e.g., caudal type homeobox 2 [Cdx2], pancreatic and duodenal homeobox 1 [Pdx1], keratin 13 [CK-13]), confirmed by immunohistochemistry. Conclusion: BDL and its induced fibrosis promote liver repopulation by ectopically transplanted fetal liver-derived cells. These cell fractions contain multipotent stem cells that colonize the spleen of BDL rats and differentiate into multiple gastrointestinal tissues, including liver, pancreas, intestine, and esophagus. The splenic microenvironment, therefore, represents an ideal niche to assess the differentiation of these stem cells, while BDL provides a stimulus that induces their differentiation.

Activin A is a prominent autocrine regulator of hepatocyte growth arrest.

Activin A, a multifunctional cytokine, plays an important role in hepatocyte growth suppression and is involved in liver size control. The present study was aimed to determine the cell location of activin A in the normal rat liver microenvironment and the contribution of activin A signaling to the hepatocyte phenotype to obtain insight into molecular mechanisms. Immunohistochemical and in situ hybridization analyses identified hepatocytes as the major activin A-positive cell population in normal liver and identified mast cells as an additional activin A source. To investigate paracrine and autocrine activin A-stimulated effects, hepatocytes were cocultured with engineered activin A-secreting cell lines (RF1, TL8) or transduced with an adeno-associated virus vector encoding activin ßA, which led to strikingly altered expression of cell cycle-related genes (Ki-67, E2F transcription factor 1 [E2F1], minichromosome maintenance complex component 2 [Mcm2], forkhead box M1 [FoxM1]) and senescence-related genes (cyclin-dependent kinase inhibitor 2B [p15INK4b/CDKN2B], differentiated embryo-chondrocyte expressed gene 1 [DEC1]) and reduced proliferation and induction of senescence. Microarray analyses identified 453 differentially expressed genes, many of which were not yet recognized as activin A downstream targets (e.g., ADAM metallopeptidase domain 12 [Adam12], semaphorin 7A [Sema7a], LIM and cysteine-rich domains-1 [Lmcd1], DAB2, clathrin adaptor protein [Dab2]). Among the main activin A-mediated molecular/cellular functions are cellular growth/proliferation and movement, molecular transport, and metabolic processes containing highly down-regulated genes, such as cytochrome P450, subfamily 2, polypeptide 11 (Cyp2C11), sulfotransferase family 1A, member 1 (Sult1a1), glycine-N-acyltransferase (Glyat), and bile acid-CoA:amino acid N-acyltransferase (Baat). Moreover, Ingenuity Pathway Analyses identified particular gene networks regulated by hepatocyte nuclear factor (HNF)-4α and peroxisome proliferator-activated receptor gamma (PPARγ) as key targets of activin A signaling. Conclusion: Our in vitro models demonstrated that activin A-stimulated growth inhibition and cellular senescence is mediated through p15INK4b/CDKN2B and is associated with up- and down-regulation of numerous target genes involved in multiple biological processes performed by hepatocytes, suggesting that activin A fulfills a critical role in normal liver function. (Hepatology Communications 2017;1:852-870).

Regeneration and Cell Recruitment in an Improved Heterotopic Auxiliary Partial Liver Transplantation Model in the Rat.

BACKGROUND: Auxiliary partial liver transplantation (APLT) in humans is a therapeutic modality used especially to treat liver failure in children or congenital metabolic disease. Animal models of APLT have helped to explore therapeutic options. Though many groups have suggested improvements, standardizing the surgical procedure has been challenging. Additionally, the question of whether graft livers are reconstituted by recipient-derived cells after transplantation has been controversial. The aim of this study was to improve experimental APLT in rats and to assess cell recruitment in the liver grafts. METHODS: To inhibit recipient liver regeneration and to promote graft regeneration, we treated recipients with retrorsine and added arterial anastomosis. Using green fluorescence protein transgenic rats as recipients, we examined liver resident cell recruitment within graft livers by immunofluorescence costaining. RESULTS: In the improved APLT model, we achieved well-regenerated grafts that could maintain regeneration for at least 4 weeks. Regarding the cell recruitment, there was no evidence of recipient-derived hepatocyte, cholangiocyte, or hepatic stellate cell recruitment into the graft. Macrophages/monocytes, however, were consistently recruited into the graft and increased over time, which might be related to inflammatory responses. Very few endothelial cells showed colocalization of markers. CONCLUSIONS: We have successfully established an improved rat APLT model with arterial anastomosis as a standard technique. Using this model, we have characterized cell recruitment into the regenerating grafts.

Fetal Liver Stem/Progenitor Cell Transplantation: A Model to Study Tissue Mass Replacement and Cell-Based Therapies.

Liver transplantation is the only therapeutic treatment for patients with end-stage liver diseases. However, donor organ scarcity is the major limitation, and therefore, alternative strategies are urgently needed. The ultimate goal for successful cell-based therapies is the ability of transplanted cells to efficiently engraft and reconstitute injured liver mass. To evaluate the repopulation capacity of transplanted cells, it is essential to identify their specific characteristics, as well as to study the mechanism(s) Through which transplanted donor cells replace tissue mass in hepatic microenvironments, using well-established cell transplantation models. To date, rat fetal liver stem/progenitor cells represent the most efficient cell population to reconstitute the near-normal liver and the liver microenvironment with advanced fibrosis/cirrhosis, and therefore, can be used for developing strategies in engineering potential donor cells in the future that will be useful for clinical application in hepatic cell therapy.The present protocol describes the isolation of epithelial stem/progenitor cells derived from ED14/15 fetal livers of DPPIV+ F344 or F344-Tg(EGFP) F455/Rrrc rats, the immunohistochemical staining method to detect E-cadherin-positive epithelial cells within unfractionated cell isolates, their transplantation into different DPPIV- liver microenvironments (near-normal, retrorsine-treated, and TAA-induced fibrotic/cirrhotic liver), as well as detection methods to follow the fate of transplanted cells in the recipient liver (see Fig. 1).

Wnt signaling regulates hepatobiliary repair following cholestatic liver injury in mice.

Hepatic repair is directed chiefly by the proliferation of resident mature epithelial cells. Furthermore, if predominant injury is to cholangiocytes, the hepatocytes can transdifferentiate to cholangiocytes to assist in the repair and vice versa, as shown by various fate-tracing studies. However, the molecular bases of reprogramming remain elusive. Using two models of biliary injury where repair occurs through cholangiocyte proliferation and hepatocyte transdifferentiation to cholangiocytes, we identify an important role of Wnt signaling. First we identify up-regulation of specific Wnt proteins in the cholangiocytes. Next, using conditional knockouts of Wntless and Wnt coreceptors low-density lipoprotein-related protein 5/6, transgenic mice expressing stable ß-catenin, and in vitro studies, we show a role of Wnt signaling through ß-catenin in hepatocyte to biliary transdifferentiation. Last, we show that specific Wnts regulate cholangiocyte proliferation, but in a ß-catenin-independent manner. CONCLUSION: Wnt signaling regulates hepatobiliary repair after cholestatic injury in both ß-catenin-dependent and -independent manners. (Hepatology 2016;64:1652-1666).

Biliary fibrosis drives liver repopulation and phenotype transition of transplanted hepatocytes.

BACKGROUND & AIMS: Current research focuses on developing alternative strategies to restore decreased liver mass prior to the onset of end-stage liver disease. Cell engraftment/repopulation requires regeneration in normal liver, but we have shown that severe liver injury stimulates repopulation without partial hepatectomy (PH). We have now investigated whether a less severe injury, secondary biliary fibrosis, would drive engraftment/repopulation of ectopically transplanted mature hepatocytes. METHODS: Ductular proliferation and progressive fibrosis in dipeptidyl-peptidase IV (DPPIV)(-) F344 rats was induced by common bile duct ligation (BDL). Purified DPPIV(+)/green fluorescent protein (GFP)(+) hepatocytes were infused without PH into the spleen of BDL rats and compared to rats without BDL. RESULTS: Within one week, transplanted hepatocytes were detected in hepatic portal areas and at the periphery of expanding portal regions. DPPIV(+)/GFP(+) repopulating cell clusters of different sizes were observed in BDL rats but not untreated normal recipients. Surprisingly, some engrafted hepatocytes formed CK-19/claudin-7 expressing epithelial cells resembling cholangiocytes within repopulating clusters. In addition, substantial numbers of hepatocytes engrafted at the intrasplenic injection site assembled into multicellular groups. These also showed biliary "transdifferentiation" in the majority of intrasplenic injection sites of rats that received BDL but not in untreated recipients. PCR array analysis showed upregulation of osteopontin (SPP1). Cell culture studies demonstrated increased Itgß4, HNF1ß, HNF6, Sox-9, and CK-19 mRNA expression in hepatocytes incubated with osteopontin, suggesting that this secreted protein promotes dedifferentiation of hepatocytes. CONCLUSIONS: Our studies show that biliary fibrosis stimulates liver repopulation by ectopically transplanted hepatocytes and also stimulates hepatocyte transition towards a biliary epithelial phenotype.

Experimental Model for Successful Liver Cell Therapy by Lenti TTR-YapERT2 Transduced Hepatocytes with Tamoxifen Control of Yap Subcellular Location.

Liver repopulation by transplanted hepatocytes has not been achieved previously in a normal liver microenvironment. Here we report that adult rat hepatocytes transduced ex vivo with a lentivirus expressing a human YapERT2 fusion protein (hYapERT2) under control of the hepatocyte-specific transthyretin (TTR) promoter repopulate normal rat liver in a tamoxifen-dependent manner. Transplanted hepatocytes expand very slowly but progressively to produce 10% repopulation at 6 months, showing clusters of mature hepatocytes that are fully integrated into hepatic parenchyma, with no evidence for dedifferentiation, dysplasia or malignant transformation. Thus, we have developed the first vector designed to regulate the growth control properties of Yap that renders it capable of producing effective cell therapy. The level of liver repopulation achieved has significant translational implications, as it is 2-3x the level required to cure many monogenic disorders of liver function that have no underlying hepatic pathology and is potentially applicable to diseases of other tissues and organs.

Identification and characterization of mesenchymal-epithelial progenitor-like cells in normal and injured rat liver.

In normal rat liver, thymocyte antigen 1 (Thy1) is expressed in fibroblasts/myofibroblasts and in some blood progenitor cells. Thy1-expressing cells also accumulate in the liver during impaired liver regeneration. The origin and nature of these cells are not well understood. By using RT-PCR analysis and immunofluorescence microscopy, we describe the presence of rare Thy1(+) cells in the liver lobule of normal animals, occasionally forming small collections of up to 20 cells. These cells constitute a small portion (1.7% to 1.8%) of nonparenchymal cells and reveal a mixed mesenchymal-epithelial phenotype, expressing E-cadherin, cytokeratin 18, and desmin. The most potent mitogens for mesenchymal-epithelial Thy1(+) cells in vitro are the inflammatory cytokines interferon γ, IL-1, and platelet-derived growth factor-BB, which are not produced by Thy1(+) cells. Thy1(+) cells express all typical mesenchymal stem cell and hepatic progenitor cell markers and produce growth factor and cytokine mRNA (Hgf, Il6, Tgfa, and Tweak) for proteins that maintain oval cell growth and differentiation. Under appropriate conditions, mesenchymal-epithelial cells differentiate in vitro into hepatocyte-like cells. In this study, we show that the adult rat liver harbors a small pool of endogenous mesenchymal-epithelial cells not recognized previously. In the quiescent state, these cells express both mesenchymal and epithelial cell markers. They behave like hepatic stem cells/progenitors with dual phenotype, exhibiting high plasticity and long-lasting proliferative activity.

A switch in the source of ATP production and a loss in capacity to perform glycolysis are hallmarks of hepatocyte failure in advance liver disease.

BACKGROUND & AIMS: The cause of hepatic failure in the terminal stages of chronic injury is unknown. Cellular metabolic adaptations in response to the microenvironment have been implicated in cellular breakdown. METHODS: To address the role of energy metabolism in this process we studied mitochondrial number, respiration, and functional reserve, as well as cellular adenosine-5'-triphosphate (ATP) production, glycolytic flux, and expression of glycolysis related genes in isolated hepatocytes from early and terminal stages of cirrhosis using a model that produces hepatic failure from irreversible cirrhosis in rats. To study the clinical relevance of energy metabolism in terminal stages of chronic liver failure, we analyzed glycolysis and energy metabolism related gene expression in liver tissue from patients at different stages of chronic liver failure according to Child-Pugh classification. Additionally, to determine whether the expression of these genes in early-stage cirrhosis (Child-Pugh Class A) is related to patient outcome, we performed network analysis of publicly available microarray data obtained from biopsies of 216 patients with hepatitis C-related Child-Pugh A cirrhosis who were prospectively followed up for a median of 10years. RESULTS: In the early phase of cirrhosis, mitochondrial function and ATP generation are maintained by increasing energy production from glycolytic flux as production from oxidative phosphorylation falls. At the terminal stage of hepatic injury, mitochondria respiration and ATP production are significantly compromised, as the hepatocytes are unable to sustain the increased demand for high levels of ATP generation from glycolysis. This impairment corresponds to a decrease in glucose-6-phosphatase catalytic subunit and phosphoglucomutase 1. Similar decreased gene expression was observed in liver tissue from patients at different stages of chronic liver injury. Further, unbiased network analysis of microarray data revealed that expression of these genes was down regulated in the group of patients with poor outcome. CONCLUSIONS: An adaptive metabolic shift, from generating energy predominantly from oxidative phosphorylation to glycolysis, allows maintenance of energy homeostasis during early stages of liver injury, but leads to hepatocyte dysfunction during terminal stages of chronic liver disease because hepatocytes are unable to sustain high levels of energy production from glycolysis.

Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes.

UNLABELLED: Considerable progress has been made in developing antifibrotic agents and other strategies to treat liver fibrosis; however, significant long-term restoration of functional liver mass has not yet been achieved. Therefore, we investigated whether transplanted hepatic stem/progenitor cells can effectively repopulate the liver with advanced fibrosis/cirrhosis. Stem/progenitor cells derived from fetal livers or mature hepatocytes from DPPIV(+) F344 rats were transplanted into DPPIV(-) rats with thioacetamide (TAA)-induced fibrosis/cirrhosis; rats were sacrificed 1, 2, or 4 months later. Liver tissues were analyzed by histochemistry, hydroxyproline determination, reverse-transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. After chronic TAA administration, DPPIV(-) F344 rats exhibited progressive fibrosis, cirrhosis, and severe hepatocyte damage. Besides stellate cell activation, increased numbers of stem/progenitor cells (Dlk-1(+), AFP(+), CD133(+), Sox-9(+), FoxJ1(+)) were observed. In conjunction with partial hepatectomy (PH), transplanted stem/progenitor cells engrafted, proliferated competitively compared to host hepatocytes, differentiated into hepatocytic and biliary epithelial cells, and generated new liver mass with extensive long-term liver repopulation (40.8 ± 10.3%). Remarkably, more than 20% liver repopulation was achieved in the absence of PH, associated with reduced fibrogenic activity (e.g., expression of alpha smooth muscle actin, platelet-derived growth factor receptor ß, desmin, vimentin, tissue inhibitor of metalloproteinase-1) and fibrosis (reduced collagen). Furthermore, hepatocytes can also replace liver mass with advanced fibrosis/cirrhosis, but to a lesser extent than fetal liver stem/progenitor cells. CONCLUSION: This study is a proof of principle demonstration that transplanted epithelial stem/progenitor cells can restore injured parenchyma in a liver environment with advanced fibrosis/cirrhosis and exhibit antifibrotic effects.

Isolation, characterization, and transplantation of adult liver progenitor cells.

Many chronic liver diseases are life-threatening. When the liver loses the ability to repair itself the only treatment currently available is liver transplant. However, there are not enough donors to treat all the patients. This requires the search of alternative therapies utilizing stem and progenitor cells for treatment of these patients and restoration of their normal liver function.Hepatic progenitor cells can be isolated from livers at different developmental stages including adult liver. In the adult rat liver, there is clear evidence that progenitor cells (also called "oval cells") derive from precursors in the canals of Herring that are capable to differentiate into hepatocytes and bile duct cells. In experimental models, hepatic progenitor cells can be isolated and propagated in vitro and used for restoration of the diseased liver. The first step in utilization of progenitor cells is their identification in the liver, isolation of purified progenitor cell fractions, which are subsequently transplanted in the diseased liver for evaluation of liver repopulation by transplanted cells, and evaluation their potentials for clinical application.The present protocol describes the isolation of non-parenchymal cells (NPCs) from wt DPPIV(+) F344 rats, followed by purification of "oval cells", immunohistochemical staining techniques to characterize these cells, their transplantation into retrorsine-treated mutant DPPIV(-) rats, as well as the enzyme histochemical staining for DPPIV to detect transplanted cells in the host liver.

Activin A, p15INK4b signaling, and cell competition promote stem/progenitor cell repopulation of livers in aging rats.

BACKGROUND & AIMS: Highly proliferative fetal liver stem/progenitor cells (FLSPCs) repopulate livers of normal recipients by cell competition. We investigated the mechanisms by which FLSPCs repopulate livers of older compared with younger rats. METHODS: Fetal liver cells were transplanted from DPPIV(+) F344 rats into DPPIV(-) rats of different ages (2, 6, 14, or 18 months); liver tissues were analyzed 6 months later. Cultured cells and liver tissues were analyzed by reverse transcription polymerase chain reaction, immunoblot, histochemistry, laser-capture microscopy, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling analyses. RESULTS: We observed 4- to 5-fold increases in liver repopulation when FLSPCs were transplanted into older compared with younger rats. Messenger RNA levels of cyclin-dependent kinase inhibitors increased progressively in livers of older rats; hepatocytes from 20-month-old rats had 6.1-fold higher expression of p15INK4b and were less proliferative in vitro than hepatocytes from 2-month-old rats. Expression of p15INK4b in cultured hepatocytes was up-regulated by activin A, which increased in liver during aging. Activin A inhibited proliferation of adult hepatocytes, whereas FLSPCs were unresponsive because they had reduced expression of activin receptors (eg, ALK-4). In vivo, expanding cell clusters derived from transplanted FLSPCs had lower levels of ALK-4 and p15INK4b and increased levels of Ki-67 compared with the host parenchyma. Liver tissue of older rats had 3-fold more apoptotic cells than that of younger rats. CONCLUSIONS: FLSPCs, resistant to activin A signaling, repopulate livers of older rats; hepatocytes in older rats have less proliferation because of increased activin A and p15INK4b levels and increased apoptosis than younger rats. These factors and cell types might be manipulated to improve liver cell transplantation strategies in patients with liver diseases in which activin A levels are increased.

Thymus cell antigen-1-expressing cells in the oval cell compartment.

UNLABELLED: Thymus cell antigen-1 (Thy-1)-expressing cells proliferate in the liver during oval cell (OC)-mediated liver regeneration. We characterized these cells in normal liver, in carbon tetrachloride-injured liver, and in several models of OC activation. The gene expression analyses were performed using reverse-transcriptase polymerase chain reaction (RT-PCR), quantitative RT-PCR (Q-RT-PCR) of cells isolated by fluorescence-activated cell sorting (FACS), and by immunofluorescent microscopy of tissue sections and isolated cells. In normal liver, Thy-1(+) cells are a heterogeneous population: those located in the periportal region do not coexpress desmin or alpha smooth muscle actin (alpha-SMA). The majority of Thy-1(+) cells located at the lobular interface and in the parenchyma coexpress desmin but not alpha-SMA, i.e., they are not resident myofibroblasts. Although Thy-1(+) cells proliferate moderately after carbon tetrachloride injury, in all models of OC-mediated liver regeneration they proliferate quickly and expand significantly and disappear from the liver when the OC response subsides. Activated Thy-1(+) cells do not express OC genes but they express genes known to be expressed in mesenchymal stem cells (CD105, CD73, CD29), genes considered specific for activated stellate cells (desmin, collagen I-a2, Mmp2, Mmp14) and myofibroblasts (alpha-SMA, fibulin-2), as well as growth factors and cytokines (Hgf, Tweak, IL-1b, IL-6, IL-15) that can affect OC growth. Activated in vitro stellate cells do not express Thy-1. Subcloning of Thy-1(+) cells from OC-activated livers yield Thy-1(+) fibroblastic cells and a population of E-cadherin(+) mesenchymal cells that gradually discontinue expression of Thy-1 and begin to express cytokeratins. However, upon transplantation these cells do not differentiate into hepatocytes or cholangiocytes. Activated Thy-1(+) cells produce predominantly latent transforming growth factor beta. CONCLUSION: Thy-1(+) cells in the OC niche are activated mesenchymal-epithelial cells that are distinct from resident stellate cells, myofibroblasts, and oval cells.

Identification of adult hepatic progenitor cells capable of repopulating injured rat liver.

UNLABELLED: Oval cells appear and expand in the liver when hepatocyte proliferation is compromised. Many different markers have been attributed to these cells, but their nature still remains obscure. This study is a detailed gene expression analysis aimed at revealing their identity and repopulating in vivo capacity. Oval cells were activated in 2-acetylaminofluorene-treated rats subjected to partial hepatectomy or in D-galactosamine-treated rats. Two surface markers [epithelial cell adhesion molecule (EpCAM) and thymus cell antigen 1 (Thy-1)] were used for purification of freshly isolated cells. Their gene expression analysis was studied with Affymetrix Rat Expression Array 230 2.0, reverse-transcriptase polymerase chain reaction, and immunofluorescent microscopy. We found that EpCAM(+) and Thy-1(+) cells represent two different populations of cells in the oval cell niche. EpCAM(+) cells express the classical oval cell markers (alpha-fetoprotein, cytokeratin-19, OV-1 antigen, a6 integrin, and connexin 43), cell surface markers recently identified by us (CD44, CD24, EpCAM, aquaporin 5, claudin-4, secretin receptor, claudin-7, V-ros sarcoma virus oncogene homolog 1, cadherin 22, mucin-1, and CD133), and liver-enriched transcription factors (forkhead box q, forkhead box a2, onecut 1, and transcription factor 2). Oval cells do not express previously reported hematopoietic stem cell markers Thy-1, c-kit, and CD34 or the neuroepithelial marker neural cell adhesion molecule 1. However, oval cells express a number of mesenchymal markers including vimentin, mesothelin, bone morphogenetic protein 7, and Tweak receptor (tumor necrosis factor receptor superfamily, member 12A). A group of novel differentially expressed oval cell genes is also presented. It is shown that Thy-1(+) cells are mesenchymal cells with characteristics of myofibroblasts/activated stellate cells. Transplantation experiments reveal that EpCAM(+) cells are true progenitors capable of repopulating injured rat liver. CONCLUSION: We have shown that EpCAM(+) oval cells are bipotential adult hepatic epithelial progenitors. These cells display a mixed epithelial/mesenchymal phenotype that has not been recognized previously. They are valuable candidates for liver cell therapy.

Novel hepatic progenitor cell surface markers in the adult rat liver.

UNLABELLED: Hepatic progenitor/oval cells appear in injured livers when hepatocyte proliferation is impaired. These cells can differentiate into hepatocytes and cholangiocytes and could be useful for cell and gene therapy applications. In this work, we studied progenitor/oval cell surface markers in the liver of rats subjected to 2-acetylaminofluorene treatment followed by partial hepatectomy (2-AAF/PH) by using rat genome 230 2.0 Array chips and subsequent RT-PCR, immunofluorescent (IF), immunohistochemical (IHC) and in situ hybridization (ISH) analyses. We also studied expression of the identified novel cell surface markers in fetal rat liver progenitor cells and FAO-1 hepatoma cells. Novel cell surface markers in adult progenitor cells included tight junction proteins, integrins, cadherins, cell adhesion molecules, receptors, membrane channels and other transmembrane proteins. From the panel of 21 cell surface markers, 9 were overexpressed in fetal progenitor cells, 6 in FAO-1 cells and 6 are unique for the adult progenitors (CD133, claudin-7, cadherin 22, mucin-1, ros-1, Gabrp). The specificity of progenitor/oval cell surface markers was confirmed by ISH and double IF analyses. Moreover, study of progenitor cells purified with Ep-CAM antibodies from D-galactosamine injured rat liver, a noncarcinogenic model of progenitor cell activation, verified that progenitor cells expressed these markers. CONCLUSION: We identified novel cell surface markers specific for hepatic progenitor/oval cells, which offers powerful tool for their identification, isolation and studies of their physiology and pathophysiology. Our studies also reveal the mesenchymal/epithelial phenotype of these cells and the existence of species diversity in the hepatic progenitor cell identity.

The oncofetal protein glypican-3 is a novel marker of hepatic progenitor/oval cells.

Glypican-3 (Gpc3), a cell surface-linked heparan sulfate proteoglycan is highly expressed during embryogenesis and is involved in organogenesis. Its exact biological function remains unknown. We have studied the expression of Gpc3 in fetal and adult liver, in liver injury models of activation of liver progenitor cells: D-galactosamine and 2-acetylaminofluorene (2-AAF) administration followed by partial hepatectomy (PH) (2-AAF/PH); and in the Solt-Farber carcinogenic model: by initiation with a single dose of diethylnitrosamine and promotion with 2-AAF followed by PH treatment. Gpc3 expression was studied using complementary DNA microarrays, reverse transcriptase-polymerase chain reaction, in situ hybridization (ISH); ISH combined with immunohistochemistry (IHC) and immunofluorescent microscopy. We found that Gpc3 is highly expressed in fetal hepatoblasts from embryonic days 13 through 16 and its expression gradually decreases towards birth. Dual ISH with Gpc3 and alpha-fetoprotein (AFP) probes confirmed that only hepatoblasts and no other fetal liver cells express Gpc3. At 3 weeks after birth the expression of Gpc3 mRNA and protein was hardly detected in the liver. Gpc3 expression was highly induced in oval cell of D-gal and 2-AAF/PH treated animals. Dual ISH/IHC with Gpc3 riboprobe and cytokeratin-19 (CK-19) antibody revealed that Gpc3 is expressed in activated liver progenitor cells. ISH for Gpc3 and AFP performed on serial liver sections also showed coexpression of the two-oncofetal proteins. FACS isolated oval cells with anti-rat Thy1 revealed expression of Gpc3. Gpc3 expression persists in atypical duct-like structures and liver lesions of animals subjected to the Solt-Farber model of initiation and promotion of liver cancer expressing CK-19. In this work we report for the first time that the oncofetal protein Gpc3 is a marker of hepatic progenitor cells and of early liver lesions. Our findings show further that hepatic progenitor/oval cells are the target for malignant transformation in the Solt-Farber model of hepatic carcinogenesis.