Human Liver Fibroblasts Research Articles

Generation of An Induced Pluripotent Stem Cell Line from Human Liver Fibroblasts from A Patient with Combined Hepatocellular-Cholangiocarcinoma.

Objective Combined hepatocellular-cholangiocarcinoma (cHCC-CC) is a rare type of primary liver cancer with characteristics of both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). The pathogenesis of cHCC- CC is poorly understood due to a shortage of suitable in vitro models. Due to scarce availability of human liver tissue, induced pluripotent stem cells (iPSCs) are a useful alternative source to produce renewable liver cells. For use in the development of liver pathology models, here we successfully developed and evaluated iPSCs from liver fibroblasts of a patient with cHCC-CC. Materials and Methods In this experimental study, human liver fibroblasts (HLFs) were obtained from the liver biopsy of a 69-year-old male patient with cHCC-CC and transduced with a retroviral cocktail that included four factors - OCT4, SOX2, KLF4, and c-MYC (OSKM). Pluripotency of the iPSCs was determined by alkaline phosphatase (AP) staining, quantitative real-time polymerase chain reaction (PCR), and immunofluorescence. We induced in vitro embryoid body (EB) formation and performed an in vivo teratoma assay to confirm their differentiation capacity into the three germ layers. Results HLF iPSCs derived from the cHCC-CC patient displayed typical iPSC-like morphology and pluripotency marker expression. The proficiency of the iPSCs to differentiate into three germ layers was assessed both in vitro and in vivo. Compared to normal control iPSCs, differentiated HLF iPSCs showed increased expressions of HCC markers alpha-fetoprotein (AFP) and Dickkopf-1 (DKK1) and the CC marker cytokeratin 7 (CK7), and a decreased expression of the CC tumour suppressor SRY-related HMG-box 17 (SOX17). ConclusionWe established HLF iPSCs using liver fibroblasts from a patient with cHCC-CC for the first time. The HLF iPSCs maintained marker expression in the patient when differentiated into EBs. Therefore, HLF iPSCs may be a sustainable cell source for modelling cHCC-CC and beneficial for understanding liver cancer pathology and developing therapies for cHCC-CC treatment.

Disrupted Murine Gut-to-Human Liver Signaling Alters Bile Acid Homeostasis in Humanized Mouse Liver Models.

The humanized liver mouse model is being exploited increasingly for human drug metabolism studies. However, its model stability, intercommunication between human hepatocytes and mouse nonparenchymal cells in liver and murine intestine, and changes in extrahepatic transporter and enzyme expressions have not been investigated. We examined these issues in FRGN [fumarylacetoacetate hydrolase (Fah-/-), recombination activating gene 2 (Rag2-/-), and interleukin 2 receptor subunit gamma (IL-2rg -/-) triple knockout] on nonobese diabetic (NOD) background] and chimeric mice: mFRGN and hFRGN (repopulated with mouse or human hepatocytes, respectively). hFRGN mice showed markedly higher levels of liver cholesterol, biliary bilirubin, and bile acids (liver, bile, and plasma; mainly human forms, but also murine bile acids) but lower transforming growth factor beta receptor 2 (TGFBR2) mRNA expression levels (10%) in human hepatocytes and other proliferative markers in mouse nonparenchymal cells (Tgf-β1) and cholangiocytes [plasma membrane-bound, G protein-coupled receptor for bile acids (Tgr5)], suggestive of irregular regeneration processes in hFRGN livers. Changes in gene expression in murine intestine, kidney, and brain of hFRGN mice, in particular, induction of intestinal farnesoid X receptor (Fxr) genes: fibroblast growth factor 15 (Fgf15), mouse ileal bile acid binding protein (Ibabp), small heterodimer partner (Shp), and the organic solute transporter alpha (Ostα), were observed. Proteomics revealed persistence of remnant murine proteins (cyotchrome P450 7α-hydroxylase (Cyp7a1) and other enzymes and transporters) in hFRGN livers and suggest the likelihood of mouse activity. When compared with normal human liver tissue, hFRGN livers showed lower SHP mRNA and higher CYP7A1 (300%) protein expression, consequences of tβ- and tα-muricholic acid-mediated inhibition of the FXR-SHP cascade and miscommunication between intestinal Fgf15 and human liver fibroblast growth factor receptor 4 (FGFR4), as confirmed by the unchanged hepatic pERK/total ERK ratio. Dysregulation of hepatocyte proliferation and bile acid homeostasis in hFRGN livers led to hepatotoxicity, gallbladder distension, liver deformity, and other extrahepatic changes, making questionable the use of the preparation for drug metabolism studies.

547 CHOLANGIOCARCINOMA IN ITALY: A NATIONAL SURVEY ON DEMOGRAPHIC AND CLINICAL CHARACTERISTICS, DIAGNOSTIC MODALITIES AND TREATMENT

547 CHOLANGIOCARCINOMA IN ITALY: A NATIONAL SURVEY ON DEMOGRAPHIC AND CLINICAL CHARACTERISTICS, DIAGNOSTIC MODALITIES AND TREATMENT

Establishment and characterization of atypical fibroblasts from human adult liver contributing to hepatocyte cord‐like arrangement

We report the phenotypic and functional characterization of fibroblasts established in culture from the non-parenchymal epithelial cell populations of adult human livers. Human liver fibroblasts (hLF) expressed mesenchymal antigens vimentin, alpha-smooth muscle actin, collagen, fibronectin, CD73, CD90, CD105, and CD166 together with non-mesenchymal antigens cytokeratins 8 and 18, glial fibrillary acidic protein, and nestin. Mixed cell lineage-specific protein expression was not associated with stem-like cell properties. Coculturing hepatocytes onto confluent hLF showed that they survived and maintained metabolic activity such as albumin, glycogen, and urea production. Moreover, hepatocytes formed cord-like arrangements resembling those established in vivo. Hepatocyte arrangement depended on cell-to-cell contact and the tissue origin of fibroblasts. Time-lapse video imaging of cocultured cells showed that hepatocyte arrangement was coordinated by the stretching and shortening of underneath hLF. Our data suggest that hLF may represent resident fibroblasts of the adult human liver, which could assume guiding functions for hepatic epithelial cells.

Cytokinesis-block micronucleus assay in primary human liver fibroblasts exposed to griseofulvin and mitomycin C.

Primary liver fibroblasts were applied in a cytokinesis-block micronucleus assay in combination with fluorescence in situ hybridization (FISH) using two protocols. In protocol A (Prot. A), cytochalasin B (Cyt B) was added at the end of the treatment time directly to the medium containing the standard compounds, whereas in protocol B (Prot. B) the chemical-containing medium was removed and fresh medium with Cyt B was added. The study was performed using the aneugen griseofulvin (GF) and the clastogen mitomycin C (MMC) as standard compounds. With both protocols GF induced a significant increase in MN frequency over controls in a dose-related manner at the lower concentrations tested (7.5 and 15 microg/ml). At the highest dose (30 microg/ml) the aneugen effect was substantially reduced. MN induction obtained with Prot. A was significantly higher ( approximately 3-fold) than with Prot. B at the most effective concentration. The aneugen effect induced by GF did not change when different cell densities were used, but again with Prot. A we obtained the highest effect. MN induced by MMC showed a dose- and time-dependent increase in both protocols. In contrast to GF, the greater clastogenic response induced by MMC in human liver fibroblasts was obtained with Prot. B, approximately 3-fold higher than Prot. A at the most effective concentration and approximately 2-fold with 24 h treatment at 0.17 microg/ml MMC. With GF, the FISH data in human liver fibroblasts (80% C+MN) were fairly consistent with those obtained in the rodent cell lines. In human whole blood cultures, the same dose used in our experiment produced a relatively higher percentage of C+MN. FISH analysis showed that MMC induced mainly MN containing acentric fragments rather than whole chromosomes. In conclusion we have demostrated that chemically induced genetic effects are strongly dependent on the cell culture employed, treatment schedule and intra- and post-treatment experimental conditions.

Subcellular Distribution of Glucocorticoid Receptor in Cultured Rat and Human Liver–Derived Cells and Cell Lines: Influence of Dexamethasone

Glucocorticoid receptor (GR) distribution in isolated rat hepatocytes and nonparenchymal hepatic stellate cells, Kupffer cells, and liver fibroblasts with and without dexamethasone treatment was investigated by immunostaining and confocal laser scanning microscopy. In addition, human liver fibroblasts, Hep3B and HepG2 cells were investigated. Subcellular distribution of GR immunostaining was assessed semiquantitatively by digital image analysis. Short–term incubation of rat liver cells with dexamethasone resulted in an increase of nuclear staining. The same was true for human liver fibroblasts. In contrast, predominant nuclear staining could be observed in untreated as well as in dexamethasone–treated Hep3B and HepG2 cells. By means of reverse–transcription polymerase chain reaction, it could be shown that messenger RNA of both known human GR isoforms, hGR α and nonhormone–binding hGR β, are present in human cells. Furthermore, dexamethasone binding indicates that hGR α protein is expressed in all human cells investigated. The data of this study show that GR is present in all cells investigated. Rat liver cells and human liver fibroblasts contain a translocating GR, suggesting that glucocorticoid action is receptor mediated in these cells. Nuclear localization of unliganded GR in Hep3B and HepG2 indicates that factors other than glucocorticoids may direct subcellular GR distribution.

Subcellular distribution of glucocorticoid receptor in cultured rat and human liver-derived cells and cell lines: Influence of dexamethasone

Glucocorticoid receptor (GR) distribution in isolated rat hepatocytes and nonparenchymal hepatic stellate cells, Kupffer cells, and liver fibroblasts with and without dexamethasone treatment was investigated by immunostaining and confocal laser scanning microscopy. In addition, human liver fibroblasts, Hep3B and HepG2 cells were investigated. Subcellular distribution of GR immunostaining was assessed semiquantitatively by digital image analysis. Short-term incubation of rat liver cells with dexamethasone resulted in an increase of nuclear staining. The same was true for human liver fibroblasts. In contrast, predominant nuclear staining could be observed in untreated as well as in dexamethasone-treated Hep3B and HepG2 cells. By means of reverse-transcription polymerase chain reaction, it could be shown that messenger RNA of both known human GR isoforms, hGR alpha and nonhormone-binding hGR beta, are present in human cells. Furthermore, dexamethasone binding indicates that hGR alpha protein is expressed in all human cells investigated. The data of this study show that GR is present in all cells investigated. Rat liver cells and human liver fibroblasts contain a translocating GR, suggesting that glucocorticoid action is receptor mediated in these cells. Nuclear localization of unliganded GR in Hep3B and HepG2 indicates that factors other than glucocorticoids may direct subcellular GR distribution. (Hepatology 1996 Oct;24(4):928-33)

Correlation between complementation group for immortality and DNA synthesis inhibitors

Previous studies had demonstrated that a DNA synthesis inhibitor(s) was produced by senescent but not young human diploid fibroblasts (HDF). Analysis of immortal human cell lines led to the finding that SUSM-1, carcinogen-treated immortal human liver fibroblast cells, expressed a potent inhibitor of DNA synthesis that was active in proliferation-competent young HDF but did not affect the SUSM-1 cell line itself. To determine whether one mechanism of escape from senescence to the immortal phenotype involved the loss of response to such DNA synthesis inhibitors, we initiated the present study analyzing a larger number of immortal human cell lines representative of the four complementation groups for indefinite division identified to date. We have found a correlation between the assignment of a cell line to Complementation Group D and the production of DNA synthesis inhibitors coupled with inability to respond to the inhibitory factors. We have also observed a correlation between the ability of immortal cell lines to respond to such DNA synthesis inhibitory factors and assignment to Complementation Group B. These data suggest DNA synthesis inhibitors are involved in the limited lifespan of normal cells and that the immortalization process may involve alterations in the activity of or response to such inhibitors.

Hepatocytes as feeder-layers for in vitro cultivation of plasmodium falciparum blood-stages

To improve the in vitro growth of Plasmodium falciparum we attempted to cultivate its erythrocytic stages on monolayers of functionally active hepatocytes. Hepatocytes from Swiss Albino mice were isolated by perfusing the liver with a collagenase solution and were co-cultured with a liver epithelial cell type in RPMI 1640 medium supplemented with 10% human umbilical cord serum. The results show that the presence of hepatocytes improves both the multiplication rates of three strains of P. falciparum already in cultivation and the proliferation of freshly isolated strains. Of nine primary isolates tested, only three could be adapted in the standard conditions, whereas all grew readily in the presence of hepatocytes. After two to three weeks of culture with feeder cells, all the strains could be maintained continuously in standard conditions. Similar results were obtained using hepatocytes from another rodent species. Growth was also improved using the supernatant from hepatocyte cultures. No improvement resulted from the use of two human hepatoma cell lines, one rat hepatoma, human embryonic lung fibroblasts, human liver fibroblasts and rat liver epithelial cells as feeder layers. From these results it appears that better culture media can be designed and that the effect of hepatocytes is probably related to the specific functions exhibited by these cells. Hepatocytes may act either by removing toxic substances, particularly lactic acid in the Krebs and Cori cycles, and/or supplying nutrients essential to the parasite.