Isolation, Purification, and Culture of Primary Murine Hepatic Stellate Cells: An Update.

In the healthy liver, quiescent hepatic stellate cells (HSCs) are found in the perisinusoidal space (i.e., the space of Dissé) in close proximity to endothelial cells and hepatocytes. HSCs represent 5-8% of the total number of liver cells and are characterized by numerous fat vacuoles that store vitamin A in the form of retinyl esters. Upon liver injury caused by different etiologies, HSCs become activated and acquire a myofibroblast (MFB) phenotype in a process called transdifferentiation. In contrast to quiescent HSC, MFB become highly proliferative and are characterized by an imbalance in extracellular matrix (ECM) homeostasis, by producing an excess of collagen and blocking its turnover by synthesis of protease inhibitors. This leads to a net accumulation of ECM during fibrosis. In addition to HSC, there are fibroblasts in the portal fields (pF), which also have the potency to acquire a myofibroblastic phenotype (pMF). The contributions of these two fibrogenic cell types (i.e., MFB and pMF) vary based on the etiology of liver damage (parenchymal vs. cholestatic). Based on their importance to hepatic fibrosis, the isolation and purification protocols of these primary cells are in great demand. Moreover, established cell lines may offer only limited information about the in vivo behavior of HSC/MFB and pF/pMF.Here we describe a method for high-purity isolation of HSC from mice. In the first step, the liver is digested with pronase and collagenase, and the cells are dissociated from the tissue. In the second step, HSCs are enriched by density gradient centrifugation of the crude cell suspension using a Nycodenz gradient. The resulting cell fraction can be further optionally purified by flow cytometric enrichment to generate ultrapure HSC.

Phalloidin Staining for F-Actin in Hepatic Stellate Cells.

During the development of liver fibrosis, hepatic stellate cells undergo a transition from a quiescent phenotype into a proliferative, fibrogenic, and contractile, α-smooth muscle actin-positive myofibroblast. These cells acquire properties that are strongly associated with the reorganization of the actin cytoskeleton. Actin possesses a unique ability to polymerize into filamentous actin (F-actin) form its monomeric globular state (G-actin). F-actin can form robust actin bundles and cytoskeletal networks by interacting with a number of actin-binding proteins that provide important mechanical and structural support for a multitude of cellular processes including intracellular transport, cell motility, polarity, cell shape, gene regulation, and signal transduction. Therefore, stains with actin-specific antibodies and phalloidin conjugates for actin staining are widely used to visualize actin structures in myofibroblasts. Here we present an optimized protocol for F-actin staining for hepatic stellate cells using a fluorescent phalloidin.

Induction of Obstructive Cholestasis in Mice.

Experimental bile duct ligation (BDL) in rodents causes cholestatic liver injury characterized by structural and functional alterations that include periportal biliary fibrosis. These changes are time-dependent and based on excess accumulation of bile acids in the liver. This in turn causes damage of hepatocytes and functional loss, leading to recruitment of inflammatory cells. Liver resident pro-fibrogenic cells facilitate extracellular matrix synthesis and remodeling. The proliferation of bile duct epithelial cells provokes a ductular reaction characterized by bile duct hyperplasia. Experimental BDL surgery is technically simple and quick to perform and reliably generates progressive liver damage with a predictable kinetics. The cellular, structural, and functional alterations induced in this model are similar to that in humans suffering from diverse forms of cholestasis including primary biliary cirrhosis (PBC) or primary sclerosing cholangitis (PSC). Therefore, this extrahepatic biliary obstruction model is used in many laboratories worldwide. Nevertheless, BDL can result in significant variations and high mortality rates when surgery is carried out by untrained or inexperienced personnel. Here we present a detailed protocol to achieve a robust experimental obstructive cholestasis in mice.

Immunohistochemical Detection of Estrogen Receptor-Beta (ERß) with PPZ0506 Antibody in Murine Tissue: From Pitfalls to Optimization.

The estrogen receptor beta (ERß) is physiologically essential for reproductive biology and is implicated in various diseases. However, despite more than 20 years of intensive research on ERß, there are still uncertainties about its distribution in tissues and cellular expression. Several studies show contrasts between mRNA and protein levels, and the use of knockout strategies revealed that many commercially available antibodies gave false-positive expression results. Recently, a specific monoclonal antibody against human ERß (PPZ0506) showed cross-reactivity with rodents and was optimized for the detection of rat ERß. Herein, we established an immunohistochemical detection protocol for ERß protein in mouse tissue. Staining was optimized on murine ovaries, as granulosa cells are known to strongly express ERß. The staining results were confirmed by western blot analysis and RT-PCR. To obtain accurate and reliable staining results, different staining conditions were tested in paraffin-embedded tissues. Different pitfalls were encountered in immunohistochemical detection. Strong heat-induced epitope retrieval (HIER) and appropriate antibody dilution were required to visualize specific nuclear expression of ERß. Finally, the specificity of the antibody was confirmed by using ovaries from Esr2-depleted mice. However, in some animals, strong (non-specific) background staining appeared. These signals could not be significantly alleviated with commercially available additional blocking solutions and are most likely due to estrus-dependent expression of endogenous immunoglobulins. In summary, our study showed that the antibody PPZ0506, originally directed against human ERß, is also suitable for reliable detection of murine ERß. An established staining protocol mitigated ambiguities regarding the expression and distribution of ERß in different tissues and will contribute to an improved understanding of its role and functions in murine tissues in the future.

Isolation of Hepatocytes from Liver Tissue by a Novel, Semi-Automated Perfusion Technology.

Primary hepatocytes are a major tool in biomedical research. However, obtaining high yields of variable hepatocytes is technically challenging. Most protocols rely on the two-step collagenase perfusion protocol introduced by Per Ottar Seglen in 1976. In this procedure, the liver is perfused in situ with a recirculating, constant volume of calcium-free buffer, which is maintained at 37 °C and continuously oxygenated. In a second step, the liver is removed from the carcass and perfused with a collagenase solution in order to dissociate the extracellular matrix of the liver and liberate individual cells. Finally, the dissected hepatocytes are further purified and concentrated by density-based centrifugation. However, failure in proper cannulation, incomplete enzymatic digestion or over-digestion can result in low cell yield and viability. Here we present a novel semi-automated perfusion device, which allows gentle, rapid and efficient generation of a single-cell suspension from rodent livers. In combination with prefabricated buffers, the system allows reliable and highly reproducible isolation of primary hepatocytes.

Isolation and Culture of Primary Murine Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) are found in the perisinusoidal space of the liver (i.e., the space of Dissé). They represent 5-8% of the total number of liver cells. In normal liver, these cells have a quiescent phenotype and are characterized by numerous fat vacuoles that store vitamin A in a form of retinyl ester. In injured liver, these cells transdifferentiate into a myofibroblast phenotype, become highly proliferative and are responsible for excess collagen synthesis and deposition during fibrosis. Due to their exceptional pathophysiological relevance, several isolation and purification protocols of primary HSCs have been established that provide the basis for studying HSC biology in vitro. We here describe a method for high-purity isolation of HSCs from mice. This protocol includes the enzymatic digestion of the liver tissue by pronase and collagenase, cellular enrichment by centrifugation of the crude cell suspension through a Nycodenz density gradient, and a final (optional) flow cytometric enrichment that allows generating ultrapure HSC fractions.

Isolation of Mature (Peritoneum-Derived) Mast Cells and Immature (Bone Marrow-Derived) Mast Cell Precursors from Mice.

Mast cells (MCs) are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC) or mucosal (MMC) type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT) and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs) and immature MC precursors from the bone marrow (BM). The latter are differentiated in vitro to yield BM-derived MCs (BMMC). These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research.

Isolation and time lapse microscopy of highly pure hepatic stellate cells.

Hepatic stellate cells (HSC) are the main effector cells for liver fibrosis. We aimed at optimizing HSC isolation by an additional step of fluorescence-activated cell sorting (FACS) via a UV laser. HSC were isolated from livers of healthy mice and animals subjected to experimental fibrosis. HSC isolation by iohexol- (Nycodenz) based density centrifugation was compared to a method with subsequent FACS-based sorting. We assessed cellular purity, viability, morphology, and functional properties like proliferation, migration, activation marker, and collagen expression. FACS-augmented isolation resulted in a significantly increased purity of stellate cells (>99%) compared to iohexol-based density centrifugation (60-95%), primarily by excluding doublets of HSC and Kupffer cells (KC). Importantly, this method is also applicable to young animals and mice with liver fibrosis. Viability, migratory properties, and HSC transdifferentiation in vitro were preserved upon FACS-based isolation, as assessed using time lapse microscopy. During maturation of HSC in culture, we did not observe HSC cell division using time lapse microscopy. Strikingly, FACS-isolated, differentiated HSC showed very limited molecular and functional responses to LPS stimulation. In conclusion, isolating HSC from mouse liver by additional FACS significantly increases cell purity by removing contaminations from other cell populations especially KC, without affecting HSC viability, migration, or differentiation.

Molecular response of liver sinusoidal endothelial cells on hydrogels.

There is a high demand for the isolation of primary endothelial cells for biomaterial endotheliazation studies, tissue engineering, and artificial organ development. Further, biomarkers for monitoring the response of endothelial cells in biomaterials science are required. We systematically compared two strategies for isolating liver sinusoidal endothelial cells (LSEC) from mouse liver. We demonstrate that fluorescence-activated cell sorting results in a considerably higher purity (~97%) compared to magnetic-assisted cell sorting (~80%), but is associated with a lower yield and recovery rate. Cell repellent polyethylene glycol (PEG) substrates affected the morphology of primary LSEC in culture and significantly downregulated the intracellular adhesion molecule (ICAM) and upregulated the vascular cell adhesion molecule (VCAM). This molecular response could partially be reverted by further modification with arginylglycylaspartic acid (RGD). Thus, usage of PEGylated materials may reduce, while applying RGD may support endotheliazation of materials, and we could relate LSEC attachment to their expression of ICAM and VCAM mRNA, suggesting their usage as biomarkers for endothelialization.

Bile duct ligation in mice: induction of inflammatory liver injury and fibrosis by obstructive cholestasis.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Primary cultures of glomerular parietal epithelial cells or podocytes with proven origin.

Parietal epithelial cells (PECs) are crucially involved in the pathogenesis of rapidly progressive glomerulonephritis (RPGN) as well as in focal and segmental glomerulosclerosis (FSGS). In this study, transgenic mouse lines were used to isolate pure, genetically tagged primary cultures of PECs or podocytes using FACsorting. By this approach, the morphology of primary glomerular epithelial cells in culture could be resolved: Primary podocytes formed either large cells with intracytoplasmatic extensions or smaller spindle shaped cells, depending on specific culture conditions. Primary PECs were small and exhibited a spindle-shaped or polygonal morphology. In the very early phases of primary culture, rapid changes in gene expression (e.g. of WT-1 and Pax-2) were observed. However, after prolonged culture primary PECs and podocytes still segregated clearly in a transcriptome analysis--demonstrating that the origin of primary cell cultures is important. Of the classical markers, synaptopodin and podoplanin expression were differentially regulated the most in primary PEC and podocyte cultures. However, no expression of any endogenous gene allowed to differentiate between the two cell types in culture. Finally, we show that the transcription factor WT1 is also expressed by PECs. In summary, genetic tagging of PECs and podocytes is a novel and necessary tool to derive pure primary cultures with proven origin. These cultures will be a powerful tool for the emerging field of parietal epithelial cell biology.

Secreted cysteine-rich FGF receptor derives from posttranslational processing by furin-like prohormone convertases.

Cysteine-rich FGF receptor (CFR) was originally identified as a FGF2 receptor and found to be identical to Golgi complex-localized glycoprotein-1 (GLG1), also known as MG-160, and to a murine E-selectin ligand-1 (ESL-1). Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the cis-medial Golgi complex, a substantial proportion of CFR is secreted but the underlying mechanism is unknown. CFR contains several possible furin-like proprotein convertase (PC) and matrix metalloproteinase cleavage sites. Cells expressing CFR were treated with the furin protease inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (decCMK) or the MMP-inhibitor GM6001. In the presence of furin-like PC inhibitor, secretion of CFR was almost completely inhibited. Secretion was not affected by the GM6001 inhibitor. The secreted forms were further characterized by creating different mutant CFR proteins with N-terminal and C-terminal tags. Immunoblot analysis and immunofluorescence indicated, that successive endoproteolytical processing of CFR which takes place in the Golgi complex is essential for secretion. Secreted CFR bound to heparan sulphate proteoglycan (HSPG) could trap FGFs and thereby directly competing with tyrosine kinase receptors for FGF binding.

Expression of E-selectin ligand-1 (CFR/ESL-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis.

Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.

The Marburg I variant (G534E) of the factor VII-activating protease determines liver fibrosis in hepatitis C infection by reduced proteolysis of platelet-derived growth factor BB.

UNLABELLED: Genetic risk factors play an important role for the progression of liver fibrosis in chronic hepatitis C virus (HCV) infection, but functional data on specific alleles and their related proteins are limited. Platelet-derived growth factor BB (PDGF-BB) is one of the strongest mitogens for hepatic stellate cells and is considered as a critical soluble mediator of liver fibrosis in vitro and in vivo. The biological activity of PDGF-BB is dependent on its degradation by the factor VII-activating protease (FSAP). Here, we demonstrate that a coding polymorphism (G534E) in the gene for FSAP is significantly associated with severe HCV-induced liver fibrosis (odds ratio, 2.59; P = 0.017), which is independent of age, gender, and presence of diabetes in multivariate analysis. These genetic findings were replicated in a cohort of patients with liver transplantation due to HCV-induced cirrhosis (OR, 2.56; P = 0.011). Functional dissection of the association demonstrates that the single amino acid change encoded by G534E in the FSAP protein does not influence PDGFbeta receptor or alpha-smooth muscle actin expression but completely abrogates FSAP-mediated inhibition of PDGF-BB-induced proliferation of primary stellate cells in vitro. CONCLUSION: The G534E variant of FSAP is a risk locus for HCV-induced liver fibrosis and cirrhosis by determining PDGF-BB-mediated hepatic stellate cell proliferation through a single amino acid substitution in FSAP. FSAP G534E might be useful for risk stratification in patients with HCV infection.

Maternal factor V Leiden mutation is associated with HELLP syndrome in Caucasian women.

OBJECTIVE: There is growing evidence that hypertensive pregnancy complications and other adverse pregnancy outcomes are associated with the presence of inherited or acquired thrombophilias. As hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome is one of the most severe forms of pre-eclampsia we aimed to assess the prevalence of the factor V Leiden, the prothrombin 20210G >A mutation and the methylenetetrahydrofolate reductase (MTHFR) 677C >T polymorphism in women with HELLP syndrome and in their fetuses from the same index pregnancy. DESIGN: The study was performed retrospectively in a case-control design. SAMPLE: Seventy-one mother-child pairs with HELLP syndrome and 79 control mother-child pairs with uncomplicated pregnancies were included in the study. METHODS: Genotyping of the three thrombophilic mutations was performed using the LightCycler technology. The chi-squared test was used for statistical analysis. Main outcome measures were maternal and fetal genotypes and their correlation with clinical parameters. RESULTS: Maternal heterozygosity for factor V Leiden was significantly more prevalent in the HELLP group than in controls (OR 4.45, 95% CI 1.31-15.31). No significant association was observed for maternal prothrombin mutation or MTHFR polymorphism (p=0.894, p=0.189, respectively). The fetal genotype was not associated with HELLP syndrome for any of the three mutations investigated. Analysis of gene-gene interactions and genotype-phenotype correlation with respect to clinical parameters and perinatal outcome revealed no further differences. CONCLUSIONS: Our study confirms that women heterozygous for factor V Leiden have an increased risk of developing HELLP syndrome, while the most frequent mutations of the prothrombin and MTHFR gene do not play a major role in the pathogenesis of HELLP syndrome.

Evaluation of a novel reverse-hybridization StripAssay for typing DNA variants useful in diagnosis of adult-type hypolactasia.

BACKGROUND: Adult-type hypolactasia is a genetically determined inability to digest lactose after weaning. Two single-nucleotide polymorphisms (C-13910T, G-22018A) located upstream of the lactase gene (LCT) within the gene MCM6 are associated with the lactase persistence/non-persistence trait in patients of European descent. Therefore, the genotyping of these SNPs has been established as a diagnostic tool for adult-type hypolactasia. We have recently shown that several novel allelic variants located in close proximity to the C-13910T SNP interfere with the diagnostic accuracy of real-time PCR-based genotyping methods. METHODS: We describe here the validation of a comprehensive reverse-hybridization teststrip-based assay for the detection of common and novel LCT SNPs (C-13907G, C-13910T, T-13913C, G-13914A, T-13915G, and G-22018A). This assay is based on multiplex DNA amplification and ready-to-use membrane teststrips containing variant-specific oligonucleotide probes immobilized as an array of parallel lines. RESULTS: We evaluated the novel reverse-hybridization StripAssay on 125 DNA samples in comparison to LightCycler analysis and sequencing. The outcome of StripAssay genotyping was found to be completely concordant with that obtained by sequencing. CONCLUSIONS: The StripAssay represents an accurate and robust screening tool to identify multiple LCT/MCM6 variants in a rapid manner. It overcomes diagnostic pitfalls that were reported and allows the simultaneous genotyping of closely spaced LCT variant sites in a single-step diagnostic approach.

Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury.

Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.

Pitfalls in LightCycler diagnosis of the single-nucleotide polymorphism 13.9 kb upstream of the lactase gene that is associated with adult-type hypolactasia.

BACKGROUND: Patients presenting with symptoms of lactose intolerance are in some centres routinely tested for a single-nucleotide polymorphism C-13910T, which is located upstream of the lactase gene (LCT) and is tightly associated with genetically determined lactase persistence/non-persistence. Typing of this polymorphism enables differential diagnosis for genetic versus secondary causes of lactose intolerance. Several PCR-based methods have been established as tests for this SNP. In particular, automated genotyping assays conducted on LightCycler platforms provide a rapid, labour-saving means for routine high-throughput analysis of this variant. Recently, several novel allelic variants have been identified in non-European populations. Three of these variants occur in close proximity to C-13910T, but their effect on the genetic test is unknown. METHODS: Here we analyse whether the occurrence of C-13907G, T-13913C, and T-13915G, affect the diagnostic accuracy of C-13910T typings obtained using the LightCycler MutaREAL lactase real-time PCR kit. RESULTS: Genotyping of DNA samples harbouring respective variants or combinations thereof significantly influenced the LightCycler analysis. Some allelic combinations generated melting profiles that prevented the correct assignment of C-13910T. CONCLUSIONS: We conclude that genotyping of the C-13910T variant with the MutaREAL lactase real-time PCR kit in non-Europeans is prone to error and should be omitted.