Successful isolation of liver progenitor cells by aldehyde dehydrogenase activity in naïve mice.

UNLABELLED: The role of progenitor cells in liver repair and fibrosis has been extensively described, but their purification remains a challenge, hampering their characterization and use in regenerative medicine. To address this issue, we developed an easy and reproducible liver progenitor cell (LPC) isolation strategy based on aldehyde dehydrogenase (ALDH) activity, a common feature shared by many progenitor cells. We demonstrate that a subset of nonparenchymal mouse liver cells displays high levels of ALDH activity, allowing the isolation of these cells by fluorescence-activated cell sorting. Immunocytochemistry and qPCR analyses on freshly isolated ALDH(+) cells reveal an enrichment in cells expressing liver stem cell markers such as EpCAM, CK19, CD133, and Sox9. In culture, the ALDH(+) population can give rise to functional hepatocyte-like cells as illustrated by albumin and urea secretion and cytochrome P450 activity. ALDH1A1 expression can be detected in canals of Hering and bile duct epithelial cells and is increased on liver injury. Finally, we showed that the isolation and differentiation toward hepatocyte-like cells of LPCs with high ALDH activity is also successfully applicable to human liver samples. CONCLUSION: High ALDH activity is a feature of LPCs that can be taken advantage of to isolate these cells from untreated mouse as well as human liver tissues. This novel protocol is practically relevant, because it provides an easy and nontoxic method to isolate liver stem cells from normal tissue for potential therapeutic purposes.

Distinct roles for non-muscle myosin II isoforms in mouse hepatic stellate cells.

BACKGROUND & AIMS: Upon liver injury, hepatic stellate cells (HSCs) undergo dramatic morphological and functional changes including migration and contraction. In the present study, we investigated the role of myosin II isoforms in the development of the contractile phenotype of mouse HSCs, which are considered therapeutic targets to decrease portal hypertension and fibrosis. METHODS: We characterized the expression of myosin IIA and IIB in primary mouse HSCs and addressed their function by gene knock-down using isoform-specific siRNAs. RESULTS: We found that myosin IIA and IIB are differentially expressed and localized and have clearly different functions in HSCs. Myosin IIA is mainly located in the subcortical area of quiescent HSCs and at α-SMA-containing stress fibres after activation, while myosin IIB is located in the cytoplasm and at the edge of protrusions of quiescent HSCs, at stress fibres of activated cells, and at the leading edge of lamellipodia. Knock-down of myosin IIA in HSCs influences cell size and shape, results in the disruption of stress fibres and in a decrease of focal adhesions, and inhibits contractility and intra-cellular Ca(2+) release but increases cell migration. Myosin IIB contributes to the extension of lamellipodia and cell spreading but has no direct role in stress fibres and focal adhesion formation, contraction, or intra-cellular Ca(2+) signalling. CONCLUSIONS: In mouse HSCs, myosin IIA and IIB clearly fulfil distinct roles. Our results provide an insight into the contractile machinery of HSCs, that could be important in the search for new molecules to treat portal hypertension.

Blebbistatin inhibits contraction and accelerates migration in mouse hepatic stellate cells.

BACKGROUND AND PURPOSE: Blebbistatin, an inhibitor of myosin-II-specific ATPase, has been used to inhibit contraction of invertebrate and mammalian muscle preparations containing non-muscle myosin. Activated hepatic stellate cells have contractile properties and play an important role in the pathophysiology of liver fibrosis and portal hypertension. Therefore, hepatic stellate cells are considered as therapeutic target cells. In the present study, we studied the effect of blebbistatin during the transition of mouse hepatic stellate cells into contractile myofibroblasts. EXPERIMENTAL APPROACH: Effects of blebbistatin on cell morphology were evaluated by phase contrast microscopy. Cell stress fibres and focal adhesions were investigated by dual immunofluorescence staining and visualized using fluorescence microscopy. Contractile force generation was examined by silicone wrinkle formation assays and collagen gel contraction assays. Intracellular Ca(2+) release in response to endothelin-1 was measured by using Fluo-4. Cell migration was measured by wound healing experiments. KEY RESULTS: In culture-activated hepatic stellate cells, blebbistatin was found to change both cell morphology and function. In the presence of blebbistatin, stellate cells became smaller, acquired a dendritic morphology and had less myosin IIA-containing stress fibres and vinculin-containing focal adhesions. Moreover, blebbistatin impaired silicone wrinkle formation, reduced collagen gel contraction and blocked endothelin-1-induced intracellular Ca(2+) release. Finally, it promoted wound-induced cell migration. CONCLUSIONS AND IMPLICATIONS: By inhibiting myosin II ATPase, blebbistatin has profound effects on the morphology and function of activated hepatic stellate cells. Our data suggest that myosin II could be a therapeutic target in the treatment of liver fibrosis and portal hypertension.

Vinculin and cellular retinol-binding protein-1 are markers for quiescent and activated hepatic stellate cells in formalin-fixed paraffin embedded human liver.

Hepatic stellate cells (HSCs) have important roles in the pathogenesis of liver fibrosis and cirrhosis. As response to chronic injury HSCs are activated and change from quiescent into myofibroblast-like cells. Several HSC-specific markers have been described in rat or mouse models. The aim of our work was to identify the best marker(s) for human HSCs. To this end we used the automated high throughput NexES IHC staining device (Ventana Medical Systems) to incubate sections under standardized conditions. Formalin fixed paraffin embedded (FFPE) normal and diseased human livers were studied. With immunohistochemistry we examined the expression of synemin, desmin, vimentin, vinculin, neurotrophin-3 (NT-3), alpha-smooth muscle actin (alpha-SMA), cellular retinol-binding protein-1 (CRBP-1), glial fibrillary acidic protein (GFAP), cysteine- and glycine-rich protein 2 (CRP2), and cytoglobin/stellate cell activation-associated protein (cygb/STAP). This is the first study in which a series of HSC markers is compared on serial FFPE human tissues. CRBP-1 clearly stains lobular HSCs without reacting with smooth muscle cells (SMCs) and shows variable cholangiocyte positivity. Vinculin has a similar staining pattern as CRBP-1 but additionally stains SMCs, and (myo)fibroblasts. In conclusion, we therefore propose to use CRBP-1 and/or vinculin to stain HSCs in human liver tissues.

Expression of somatostatin receptors in splanchnic blood vessels of normal and cirrhotic rats.

BACKGROUND/AIMS: Somatostatin has been used for over two decades to treat acute variceal bleeding. Although it is assumed that somatostatin lowers portal pressure by constriction of the splanchnic arteries, little is known about the expression of somatostatin receptors (SSTR) in splanchnic blood vessels. In this study we investigated SSTR expression in splanchnic blood vessels from normal and cirrhotic rats. METHODS/RESULTS: Cirrhosis was induced by intraperitoneal injection of 50 mg thioacetamide twice a week for 14 weeks. In portal vein, mesenteric artery and aorta of normal and cirrhotic rats, mRNA for the five known SSTR was measured by quantitative reverse transcriptase-polymerase chain reaction. SSTR subtypes 1, 2, 3 and 4 were expressed, but subtype 5 was undetectable. In the portal vein of cirrhotic animals, SSTR1 was significantly down-regulated as compared with controls. Otherwise, no major differences in receptor expression between normal and cirrhotic animals were observed. Using immunohistochemistry, we identified all five receptors, although the staining of receptor 5 was very weak. CONCLUSION: All five SSTR are expressed in splanchnic blood vessels. Our results suggest that cirrhosis reduces expression of SSTR1 in portal vein. In other vessels, no major differences between the normal and cirrhotic state were noted.

Trichostatin a enhances gap junctional intercellular communication in primary cultures of adult rat hepatocytes.

The effects of histone deacetylase inhibitor Trichostatin A (TSA) on connexin (Cx) expression and gap junctional intercellular communication (GJIC) were investigated in primary cultures of adult rat hepatocytes. GJIC was monitored by using the scrape-loading/dye transfer method. Immunoblotting and immunocytochemistry were used to investigate Cx protein levels and localization. Cx gene expression was studied by means of quantitative reverse transcriptase-polymerase chain reaction. TSA increased Cx32 protein levels and affected negatively the Cx26 protein levels. The latter was preferentially located in the cytosol of cultured cells. TSA also promoted the appearance of Cx43 in the nuclear compartment of primary cultured hepatocytes. Overall, this resulted in enhanced GJIC activity. It is important to note that the time of onset of TSA treatment was crucial for the extent of its outcome and that the effects of TSA on Cx protein levels occurred independently of transcriptional changes. TSA differentially affects Cx proteins in primary rat hepatocyte cultures, suggesting distinct regulation and/or distinct roles of the different Cx species in the control of hepatic homeostasis. TSA enhances GJIC between primary cultured rat hepatocytes, an interesting finding supporting its use to further optimize liver-based in vitro models for pharmacotoxicological purposes.