Differentiation Of Oval Cells Research Articles

C3G down-regulation enhances pro-migratory and stemness properties of oval cells by promoting an epithelial-mesenchymal-like process.

Previous data indicate that C3G (RapGEF1) main isoform is highly expressed in liver progenitor cells (or oval cells) compared to adult mature hepatocytes, suggesting it may play an important role in oval cell biology. Hence, we have explored C3G function in the regulation of oval cell properties by permanent gene silencing using shRNAs. We found that C3G knock-down enhanced migratory and invasive ability of oval cells by promoting a partial epithelial to mesenchymal transition (EMT). This is likely mediated by upregulation of mRNA expression of the EMT-inducing transcription factors, Snail1, Zeb1 and Zeb2, induced in C3G-silenced oval cells. This EMT is associated to a higher expression of the stemness markers, CD133 and CD44. Moreover, C3G down-regulation increased oval cells clonogenic capacity by enhancing cell scattering. However, C3G knock-down did not impair oval cell differentiation into hepatocyte lineage. Mechanistic studies revealed that HGF/MET signaling and its pro-invasive activity was impaired in oval cells with low levels of C3G, while TGF-β signaling was increased. Altogether, these data suggest that C3G might be tightly regulated to ensure liver repair in chronic liver diseases such as non-alcoholic steatohepatitis. Hence, reduced C3G levels could facilitate oval cell expansion, after the proliferation peak, by enhancing migration.

Kangxianruangan granule‑containing serum mediated inhibition of hepatic oval cell differentiation into hepatocellular carcinoma cells via the Wnt‑1/β‑catenin signaling pathway.

Hepatocellular carcinoma is a malignancy with poor clinical prognosis. Hepatic oval cells (HOCs) tend to differentiate into cancerous hepatocellular carcinoma cells (HCCs) in the tumor microenvironment. The purpose of the present study was to explore the role of kangxianruangan granule (KXRG)-containing serum in inhibiting the differentiation of HOCs into HCCs via the Wnt-1/β-catenin signaling pathway. N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) was applied to induce the transformation of the rat HOC cell line WB-F344 into HCCs. The overexpression plasmid, Wnt-1-up, was utilized to increase Wnt-1 expression. Subsequently, high, medium and low concentrations of KXRG were applied to MNNG-treated WB-F344 cells to assess the inhibitory effect of KXRG on cell differentiation. Flow cytometry was conducted to detect the cell cycle distribution, apoptotic rate and expression of cytokeratin-19 (CK-19) protein in cells. An immunofluorescence double staining protocol was used to detect the expression of Wnt-1 and β-catenin. ELISAs were performed to detect α fetoprotein in the cell supernatants. Reverse transcription-quantitative PCR and western blotting were conducted to detect the mRNA and protein expression levels of Wnt-1, β-catenin, Cyclin D1, C-myc, matrix metalloproteinase-7 (MMP-7), Axin2 and epithelial cell adhesion molecule (EpCAM) in cells. Compared with the normal group, the apoptotic rate, proportion of S phase cells, concentration of AFP in the cell supernatant, level of CK-19 protein, and mRNA and protein expression levels of Wnt-1, β-catenin, Cyclin D1, C-myc, MMP-7, Axin2 and EpCAM were all significantly increased in the model group. Addition of KXRG significantly reduced the aforementioned indicators compared with the model group. Moreover, Wnt-1 overexpression further increased the aforementioned indicators compared with the model group, whereas KXRG significantly inhibited these effects. The results indicated that KXRG inhibited the differentiation of HOCs into HCCs via the Wnt-1/β-catenin signaling pathway, which suggested the potential clinical application of KXRG for the prevention of hepatocellular carcinoma.

Analysis of CYP2R1 and CYP26A1 Expression Patterns in Regeneration in Mice with Liver Injury.

Cytochrome P450 enzymes (CYPs) are involved in the metabolism of various substances in the liver and small intestine and show markedly higher expression levels in the liver compared to other organs. The liver exhibits a remarkable capacity to regenerate. After excision of 70% of the liver, the organ can regenerate to its original size in approximately 1 week. Unlike the normal liver, in the injured liver, hepatic stem cells known as oval cells are considered to play an important role in regeneration. However, the role of CYPs in liver regeneration remains unclear. In the present study, we investigated the role of CYPs in the regeneration of injured liver. Liver injury was induced by 4-week repeated doses of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in the diet. Next, both DDC-fed mice and control diet (containing no DDC)-fed mice were subjected to 70% hepatectomy, and the hepatic gene expression patterns measured during regeneration were analyzed. Mice with DDC-induced liver injury expressed the oval cell markers cytokeratin 19 (CK19) and epithelial cell adhesion molecule (EpCAM), and partial hepatectomy increased the expression levels of CYP2R1 and CYP26A1 as well as the hepatoblast marker alpha-fetoprotein (AFP) in these mice. The results of this study suggest that CYP2R1 and CYP26A1 are important in the differentiation of oval cells into hepatoblast-like cells in the injured liver.

Oval Cell Response Is Attenuated by Depletion of Liver Resident Macrophages in the 2-AAF/Partial Hepatectomy Rat

Background/AimsMacrophages are known to play an important role in hepatocyte mediated liver regeneration by secreting inflammatory mediators. However, there is little information available on the role of resident macrophages in oval cell mediated liver regeneration. In the present study we aimed to investigate the role of macrophages in oval cell expansion induced by 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) in rats.Methodology/Principal FindingsWe depleted macrophages in the liver of 2-AAF/PH treated rats by injecting liposome encapsulated clodronate 48 hours before PH. Regeneration of remnant liver mass, as well as proliferation and differentiation of oval cells were measured. We found that macrophage-depleted rats suffered higher mortality and liver transaminase levels. We also showed that depletion of macrophages yielded a significant decrease of EPCAM and PCK positive oval cells in immunohistochemical stained liver sections 9 days after PH. Meanwhile, oval cell differentiation was also attenuated as a result of macrophage depletion, as large foci of small basophilic hepatocytes were observed by day 9 following hepatectomy in control rats whereas they were almost absent in macrophage depleted rats. Accordingly, real-time polymerase chain reaction analysis showed lower expression of albumin mRNA in macrophage depleted livers. Then we assessed whether macrophage depletion may affect hepatic production of stimulating cytokines for liver regeneration. We showed that macrophage-depletion significantly inhibited hepatic expression of tumor necrosis factor-α and interleukin-6, along with a lack of signal transducer and activator of transcription 3 phosphorylation during the early period following hepatectomy.ConclusionsThese data indicate that macrophages play an important role in oval cell mediated liver regeneration in the 2-AAF/PH model.

The role of canonical Wnt signaling pathway in the proliferation and differentiation of rat hepatic oval cell

Objective To investigate the role of canonical Wnt signaling pathway on the proliferation and differentiation of hepatic oval cells in vitro.Methods WB­F344 cells were treated with recombinant Wnt3a(20,40,80,160,200 ng/ml) in the serum­free medium for 24 hours.Cell proliferation was measured by Brdu incorporation analysis，un­treated WB­F344 cells were taken as control.After treated with Wnt3a(160 ng/ml) for 24 hours，the subcellular localization and protein expression of β­catenin in WB­F344 cells were examined by immunofluorescence staining and Western­blot analysis.CyclinD1 mRNA expression was determined by semi­quantitative RT­PCR. The mRNA levels of some phenotypic markers，including AFP，CK­19，ALB and two hepatic nuclear factors(HNF­4α，HNF­6) were determined by RT­PCR.CK­19 and AFP protein expression were examined by Western­blot analysis.Results We found that Wnt3a promoted proliferation of WB­F344 cells.Stimulating of WB­F344 cells with recombinant Wnt3a resulted in the accumulation of the transcriptional activator β­catenin，together with its translocation into the nucleus，and the upregulation of typical Wnt target genes cyclin D1.After 3 days of Wnt3a treatment in the absence of any serum，these cells retained their bipotential to express several specific hepatocyte and cholangiocyte phenotypic markers，such as AFP，CK­19 following activation of canonical Wnt signaling pathway.Conclusions The canonical Wnt signaling pathway promotes proliferation and self­renewal of rat hepatic oval cells.

The Role of the Wnt Family of Secreted Proteins in Rat Oval “Stem” Cell-Based Liver Regeneration: Wnt1 Drives Differentiation

To date the molecular signals regulating activation, proliferation, and differentiation of hepatic oval cells are not fully understood. The Wnt family is essential in hepatic embryogenesis and implicated in hepatic carcinogenesis. This study elucidates novel findings implicating Wnt1 in directing oval cell differentiation during the rat 2-acetylaminofluorene (2AAF) and 2/3 partial hepatectomy (PHx) liver regeneration model. Proteins of Wnt family members were predominantly localized in pericentral hepatocytes during liver injury, oval cell activation, and hepatocyte regeneration. In addition, Wnt message increased coinciding with the rise in oval cell number, whereas protein levels peaked immediately after the height of oval cell proliferation. Immunohistochemical analysis demonstrated nuclear translocation of beta-catenin within oval cells throughout the 2AAF/PHx protocol. Furthermore, RNA interference was used in vivo to confirm the physiological requirement of Wnt1 during the oval cell induction. Ultimately, inhibition of Wnt1 resulted in failure of oval cells to differentiate into hepatocytes and alternatively induced atypical ductular hyperplasia. Taken together, these data indicate that in vivo exposure to Wnt1 shRNA inhibited rat oval cell liver regeneration. In the absence of Wnt1 signaling, oval cells failed to differentiate into hepatocytes and underwent atypical ductular hyperplasia, exhibiting epithelial metaplasia and mucin production. Furthermore, changes in Wnt1 levels are required for the efficient regeneration of the liver by oval cells during massive hepatic injury.

Differentiation of hepatic oval cell into mature hepatocyte induced by hepatic stellate cells

To investigate the role of hepatic stellate cells in the differentiation of hepatic oval cells into adult hepatocyte. The oval cell were cocultured with primary hepatic stellate cells (HSC) in the same well (M-coculture) or separately cultured with HSC by millIcell (S-coculture). Oval cells were cultured alone as control; the expression of adult hepatocyte marker HNF-4alpha, albumin, and oval cell marker AFP, CK-19 in each group were detected by real-time PCR and western-blot. Phenotype changes were observed by transmission electron microscope (TEM); PAS staining was used to detect the quantity of glycogen granule in oval cell. Albumin level in supernatant was detected using ELISA kit. (1) The relative level of HNF-4alpha and albumin mRNA expression compared with pre-coculture: M-coculture: HNF-4a: 1.9+/-0.2, 10.7+/-1.2, 12.0+/-1.3; albumin: 5.7+/-1.6, 110.7+/-13.7, 173.6+/-22.3. S-coculture: 1.4+/-0.1, 3.2+/-0.6, 8.9+/-1.4 times; albumin: 2.9+/-1.4, 22.3+/-8.5, 96.3+/-16.3. The relative level of HNF-4a and albumin mRNA expression in coculture group (M- and S-coculture) were higher than control group (LSD-t: 32.98, 10.08, 13.38, 7.96; P less than 0.01); and a higher level of HNF-4a and albumin was found in M-coculture group compared to S-coculture group (LSD-t: 32.98, 25.65; P less than 0.01). The relative level of AFP and CK-19 mRNA expression compared with pre-coculture: M-coculture: 1.1+/-0.2, 0.2+/-0.0, 0.0+/-0.0; S-coculture group: AFP: 1.0+/-0.2, 0.2+/-0.1, 0.1+/-0.0; CK-19: 0.6+/-0.1, 0.1+/-0.0, 0.0+/-0.0; control group: AFP: 1.0+/-0.1, 1.0+/-0.1, 1.1+/-0.1, CK-19: 1.0+/-0.1, 1.1+/-0.1, 1.0+/-0.1. The relative level of AFP and CK-19 mRNA expression in coculture group (M- and S-coculture) were lower than that in control group (LSD-t: 37.99, 34.50, 13.59, 22.46; P less than 0.01). (2) The albumin secretion was detected in M-coculture: 14 day: (15.30+/-0.09) ng/ml, 21: (20.98+/-0.12) ng/ml; S-coculture: 14 day: (11.41+/-0.13) ng/ml, 21 day:(15.12+/-0.17) ng/ml. (3) It showed more organelles such as endoplasmic reticulum, mitochondrion and Golgi apparatus in oval cells cocultured with HSC. And cholangiole-like structure appeared between oval cells cocultured with HSC. (4) PAS staining showed glycogen granules could be observed in coculture groups. HSC can induce differentiation of oval cell into mature hepatocyte.

Potential role for beta‐catenin in oval cell differentiation during liver injury repair

Atypical ductular proliferation (ADP) occurs in the liver following DDC‐induced liver injury. We previously noted a blunted ADP in mice liver lacking β‐catenin after short‐term DDC exposure. To examine whether the presence of active‐β ‐catenin would provide a growth or differentiation advantage to atypical ductular cells in mice, liver‐specific β‐catenin over expressing mice (TG) with a stabilizing mutation at ser45 were fed the DDC diet. No significant differences in appearance and numbers of A6‐positive cells were observed, but we did observe increased numbers of A6‐positive cells with hepatocyte‐like morphology in TG mice as early as 2 weeks and a dramatic difference at 5 months. These cells may indicate an increased potential for proliferating cells to differentiate in the setting of increased stable β‐catenin. We hypothesized that such a differentiation advantage would allow the liver to recover more quickly following a change from DDC back to a normal diet. Indeed, TG mice exposed to DDC diet for 4 weeks followed by 2 days of normal chow had significantly less A6‐positive cells and had recovered close to normal liver histology compared to what is observed in WT. In sum, these findings indicate diverse roles Wnt/β ‐catenin signaling during oval cell response in mice including a role in the differentiation process during liver repair.

Increased oval cell response and fibrosis in beta‐catenin null livers after long‐term DDC exposure

An oval cell response, or ductular reaction, occurs in the mouse liver following DDC‐induced liver injury. We previously reported a potential role for β‐catenin in oval cell induction and expansion. After short term exposure (2‐4 weeks) to DDC, β‐catenin knockout liver had less A6‐positive oval cells than wild type liver. To examine the role of β‐catenin in the response to long‐term injury, we exposed WT and KO mice to DDC for 5 months. The livers from these animals were examined histologically to examine the extent of biliary injury and oval cell response. At 5 months in both groups of animals we observed the emergence of cells with scant cytoplasm and oval‐shaped nuclei, but this response was more dramatic in the KO. We utilized the oval cell marker A6 to compare the response between the two groups. Contrary to short‐term exposure (<4wk), KO liver harbored significantly more A6 positive cells than WT. This coincided with more fibrosis in the KO than observed in WT liver. We also observed nodular formation upon macroscopic examination of the KO liver. These likely indicate continued expansion and lack of differentiation of oval cells in this model. In sum, these findings indicate diverse roles for Wnt/β ‐catenin signaling during oval cell response in mice.

Insights into the polymorphism of the diatomPhaeodactylum tricornutumBohlin

AbstractPhaeodactylum tricornutumis a pleiomorphic diatom that exists in three different forms depending on environmental conditions, viz. oval, fusiform or triradiate. In spite of numerous observations of the three morphotypes, very little information is available about the process of transformation. In this review, we summarize results of previous studies and add new observations on cell wall structure of the three morphotypes and the environmental conditions under which each form appears (for different strains ofPhaeodactylum). New data on the cellular mechanisms of transformation between the three morphotypes and on the differentiation of oval cells are also presented. The ultrastructural morphology of the three morphotypes and strain/genotype-dependent transformations of morphotypes are discussed.

Hepatic non-parenchymal cells and extracellular matrix participate in oval cell-mediated liver regeneration

To elucidate the interaction between non-parenchymal cells, extracellular matrix and oval cells during the restituting process of liver injury induced by partial hepatectomy (PH). We examined the localization of oval cells, non-parenchymal cells, and the extracellular matrix components using immunohistochemical and double immunofluorescent analysis during the proliferation and differentiation of oval cells in N-2-acetylaminofluorene (2-AAF)/PH rat model. By day 2 after PH, small oval cells began to proliferate around the portal area. Most of stellate cells and laminin were present along the hepatic sinusoids in the periportal area. Kupffer cells and fibronectin markedly increased in the whole hepatic lobule. From day 4 to 9, oval cells spread further into hepatic parenchyma, closely associated with stellate cells, fibronectin and laminin. Kupffer cells admixed with oval cells by day 6 and then decreased in the periportal zone. From day 12 to 15, most of hepatic stellate cells (HSCs), laminin and fibronectin located around the small hepatocyte nodus, and minority of them appeared in the nodus. Kupffer cells were mainly limited in the pericentral sinusoids. After day 18, the normal liver lobule structures began to recover. Local hepatic microenvironment may participate in the oval cell-mediated liver regeneration through the cell-cell and cell-matrix interactions.

Expression of matrix metalloproteinase-2 and -9 and of tissue inhibitor of matrix metalloproteinase-1 in liver regeneration from oval cells in rat

Oval cells participate in liver regeneration when hepatocyte replication is impaired. These precursor cells proliferate in periportal regions and organize in ductules. They are surrounded by a basement membrane, the degradation of which by matrix metalloproteinases (MMP) might trigger their terminal differentiation into hepatocytes. We studied the expression of MMP-2 and MMP-9 and that of one of their tissue inhibitors (TIMP-1) in a model of hepatic regeneration from precursor cells. Regeneration was induced by treating rats with 2-acetylaminofluorene followed by partial hepatectomy. MMP-2 and MMP-9 hepatic expression paralleled oval cell number with a peak at day 9–14 after hepatectomy. They were mainly detected in oval cells. TIMP-1 mRNA and oncostatin M receptor mRNA, a major regulator of TIMP-1 synthesis, markedly increased from day 1 after surgery until day 9 and then declined; they were mainly detected in interlobular bile duct cells and oval cells until day 14. In agreement with the in vivo data, the WB-F344 liver precursor cell line expressed MMP-2 and MMP-9, as well as TIMP-1 and oncostatin M receptor. These data suggest that (a) early increased TIMP-1 synthesis by biliary and oval cells favors basement membrane deposition around proliferating ductular structures through MMP inhibition, (b) delayed increased MMP expression, concomitant to decreased TIMP-1 synthesis, leads to basement membrane degradation, preceding oval cell differentiation, (c) the oncostatin M pathway might play a major role in increased TIMP-1 synthesis.

Beta‐catenin regulates proliferation and differentiation of oval cells in mouse liver

Atypical ductular proliferation (ADP) occurs in the liver following DDC‐induced liver injury. We noted blunted ADP in mice liver lacking β‐catenin. Also based on the similarities between the processes of ADP in mice and oval cell response in rats, and the role of Wnt/β‐catenin signaling in both events, we sought to examine whether presence of active‐β‐catenin would provide a growth or differentiation advantage to atypical ductular cells in mice. Liver‐specific β‐catenin over expressing mice (TG) with a stabilizing mutation at ser45, were fed the DDC diet. No significant differences in appearance and numbers of A6‐positive cells were observed. Intriguingly, increased numbers of A6‐positive cells with hepatocyte‐like morphology were observed in TG mice. Contrary to short‐term exposure (<4wk) in β‐catenin knockout mice, long‐term exposure to DDC (5 months) led to a significant increase in A6‐positive cells with bridging fibrosis indicating their continued expansion and lack of differentiation. In sum, these findings indicate diverse roles Wnt/β‐catenin signaling during oval cell response in mice.

Characterization of cytokeratin 19-positive hepatocyte foci in the regenerating rat liver after 2-AAF/CCl4 injury

Partial hepatectomy or carbon tetrachloride (CCl4) injury, following treatment of rats with 2-acetylaminofluorene (2-AAF) to inhibit proliferation of hepatocytes, induces proliferation of oval cells and possibly their differentiation into nodular foci of hepatocytes when higher doses of 2-AAF are used. Unfortunately, immunohistochemistry in previous studies failed to show oval cell markers in these foci, and thereby to demonstrate the precursor-product relationship between oval cells and hepatocytes. Immunohistochemistry on livers of rats treated with high dose 2-AAF/CCl4 was used. We found 7.6% of the hepatocyte foci were positive for an oval cell marker cytokeratin 19 (CK-19). These foci were positive for alpha-fetoprotein, less positive for carbamoylphosphate synthetase 1, and more positive for laminin in the basement membrane lining. Rarely present transitional foci had weaker expression of CK-19 and discontinuous laminin. Focal hepatocyte differentiation of oval cells was characterized by cell hypertrophy, membranous CK-19, and positive hepatocyte nuclear factor 4 (HNF-4). HNF-4+ small oval cells surrounding CK-19+ foci were frequently seen, suggesting that a paracrine mechanism(s) may be responsible for the enlargement of CK-19+ foci. In conclusions, oval cells appear to differentiate to CK-19+ foci and then to CK-19- foci in the high dose 2-AAF/CCl4 model.

Hepatic differentiation and transcriptional profile of the mouse liver epithelial progenitor cells (LEPCs) under the induction of sodium butyrate

The liver regenerates by progenitor cells when it is damaged in chronic liver diseases and extensive damage. The progenitor cells, also termed "oval cells" according to their morphological traits, can differentiate into hepatocytes and bile duct cells in vivo. To better understand the transcriptional pattern that accompanies the hepatic differentiation of oval cells, we applied cDNA microarray to analyze the oval cell-derived liver epithelial progenitor cells (LEPCs) during in vitro induced differentiation. Upon exposure to sodium butyrate, a histone deacetylase inhibitor, cultured LEPCs differentiate and express functional hepatocyte markers albumin, tryptophan 2, 3-dioxygenase and alcohol dehydrogenase. For expression profiling, cells were harvested at 6 h, 12 h, 1 d, 3 d and 7 d after exposure to sodium butyrate. After analyzing the microarray data by SOM clustering, total of 796 differentially regulated genes were grouped into 48 clusters. Consistent with the phenotype change of LEPCs after sodium butyrate treatment, many hepatocyte functional genes are revealed by analyzing the clusters containing genes up-regulated through all the time points. The clusters, containing down-regulated genes immediately after the induction, are also analyzed. The microarray data was validated by analyzing the expression of selected genes by quantitative real-time PCR. A set of genes expressed synergistically in these clusters may play a central role during the process of differentiation. Sodium butyrate decreases cyclin B1 and Cdk4 expression, which would be associated with LEPCs growth arrest shortly after treatment. Bmi1, a polycomb group protein, is also down-regulated immediately after treatment and remains at a low level during the induction. These findings highlight the key molecular mechanisms by which sodium butyrate, mediates its effects on cell growth arrest and induction of differentiation. In conclusion, our data reflect a global view of gene expression during hepatic differentiation of LEPCs induced by sodium butyrate.

Long-term Extensive Expansion of Mouse Hepatic Stem/Progenitor Cells in a Novel Serum-Free Culture System

The liver has high regenerative potential. We attempted to establish a novel culture system for extensive expansion of fetal mouse hepatic stem/progenitor cells and to characterize cultured cells. Hepatic spheroids collected from 6-day floating cultures were cultured on collagen-coated dishes in serum-free conditions in medium containing growth factors. Cultured cells were mainly characterized by immunocytochemistry and flow cytometry or transplanted into adult mice. Approximately 400 expanding hepatic spheroids were generated from every 1 x 10(6) fetal liver cells. Subsequently, highly replicative colonies were subcultured with maintaining colony formation on collagen-coated dishes. These colonies consisted of small immature alpha-fetoprotein-positive cells and hepatocytic and cholangiocytic lineage-committed cells. The immature alpha-fetoprotein-positive cells could be expanded in a reproducible manner at least 5 x 10(5)-fold (which involved at least 30 passages over >6 months) without losing differentiation potential. Flow cytometric analysis showed that all cultured cells expressed CD49f, but not CD34, Thy-1, c-kit, or CD45. Nearly 15% of the cells expressed Sca-1, and approximately 5%-20% of the cells were side population cells. Both sorted side population cells and Sca-1-positive cells (especially side population cells) produced a large number of alpha-fetoprotein-positive cells and lineage-committed cells. Expanded cells had bidirectional differentiation potential and improved serum albumin levels in mice with severe liver damage. Long-term extensive expansion of transplantable hepatic stem/progenitor cells was reproducibly achieved in a novel serum-free culture system. Moreover, this culture system yielded side population and Sca-1-positive cell populations that included hepatic stem/progenitor cells with differentiation and proliferation properties.

Oncostatin M Inhibits Proliferation of Rat Oval Cells, OC15-5, Inducing Differentiation into Hepatocytes

Oval cells of the liver participate in liver regeneration when hepatocytes are prevented from proliferating in response to liver damage. To clarify the role of oncostatin M (OSM) in the liver regeneration involving oval cells, we examined the expression of OSM and OSM-specific receptor (OSM-R) in the liver undergoing regeneration in the 2-acetylaminofluorene/partial hepatectomy model. Expression levels of OSM-R changed in correlation to the number of oval cells, and its expression was exclusively observed in oval cells. On the other hand, OSM was expressed in both oval cells and Kupffer cells. To examine the effect of OSM on the growth and differentiation of oval cells, rat oval cells (OC15-5) were incubated in conditioned medium of 293T cells expressing rat OSM cDNA. This resulted in suppression of growth, changes in morphology (microvilli and large cytoplasm with developed organelles), and expression of hepatocyte markers (albumin, tyrosine amino transferase, and tryptophan oxygenase). The effects of the conditioned medium with rat OSM were abrogated by introducing a small interfering RNA specifically targeting rat OSM-R into OC15-5 cells. These results indicate that OSM is a key mediator for inducing differentiation of OC15-5 cells into hepatocytes and suggest that the OSM/OSM-R system is pivotal in the differentiation of oval cells into hepatocytes, thereby promoting liver regeneration.

Expression of Stromal Cell-Derived Factor-1 and of Its Receptor CXCR4 in Liver Regeneration from Oval Cells in Rat

Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway.

Activation of NF-kappaB and STAT3 in rat oval cells during 2-acetylaminofluorene/partial hepatectomy-induced liver regeneration.

Proliferation and differentiation of hepatic stem cell progenies (i.e., oval cells) sustain liver regeneration when the replicative and functional capacity of hepatocytes is impaired. The signaling pathways that control stem cell activation remain poorly understood. In this study, we investigated the involvement of nuclear factor-kappa B (NF-kappaB) and signal transducer and activator of transcription 3 (STAT3) in oval cell-mediated liver regeneration induced by 2-acetylaminofluorene/partial hepatectomy (AAF/PH) protocol. Using OV1 as a marker for identification and sorting of oval cells, we established that both NF-kappaB and STAT3 were highly activated in the OV1(+) cell population. Three distinct subpopulations of oval cells were defined as OV1(low), OV1(medium), and OV1(high), based on the intensity of OV1 staining. Quantitative polymerase chain reaction analysis revealed that they represent different stages of oval cell differentiation along hepatocyte lineage. OV1(low) cells displayed the least differentiated phenotype as judged by high expression of c-kit and lack of hepatocytic differentiation markers, whereas OV1(high) cells lost c-kit expression, were more proliferative, and acquired more mature hepatocytic phenotype. Notably, NF-kappaB was activated uniformly in all three subpopulations of oval cells. In contrast, phosphorylation of STAT3 was detected only in OV1(high) cells. In conclusion, transcriptional activity supported by NF-kappaB and STAT3 is required for oval cell activation, expansion, and differentiation. The differential induction of NF-kappaB and STAT3 point to a distinct role for these transcription factors at different stages of hepatic stem cell differentiation.

2-acetylaminofluorene dose-dependent differentiation of rat oval cells into hepatocytes: Confocal and electron microscopic studies

The 2-acetylaminofluorene (AAF)/partial hepatectomy (PH) model is one of the most extensively studied experimental systems for oval cell proliferation and differentiation. We have previously described the oval cells as forming ductular structures surrounded by basement membrane, representing extensions of the canals of Hering. Herein we analyze the differentiation of oval cells into hepatocytes after varying degrees of liver damage induced by AAF. At a low dose of AAF, most oval cells synchronously differentiate into small hepatocytes by 6 days after the PH, resulting in complete restoration of the liver structure in 10 days. Higher doses of AAF delay the differentiation process and the new hepatocytes form foci, in contrast to what is observed at the low dose. Qualitatively, the differentiation process seems to be identical at the cellular level under both conditions. The transition from the expanding oval cell population into hepatocytes was correlated with the upregulation of hepatocyte nuclear factor 4 and the disappearance of the basement membrane. Also, the differentiation of oval cells into hepatocytes coincided with the loss of alpha-fetoprotein and OV-6 staining, and the replacement of the biliary cell-specific alpha6 integrin and connexin 43 with the hepatocyte-specific alpha1 integrin and connexin 32. In addition, bile canaliculi form between the new hepatocytes. In conclusion, these results indicate the rate of oval cell differentiation into hepatocytes is context dependent and suggest that, under favorable conditions, oval cells can complete this process much faster than previously appreciated.