Laurent Dollé

A role for autophagy during hepatic stellate cell activation

BACKGROUND & AIMS Autophagy is a metabolic process that degrades and recycles intracellular organelles and proteins with many connections to human disease and physiology. We studied the role of autophagy during hepatic stellate cell (HSC) activation, a key event in liver fibrogenesis. METHODS Analysis of the autophagic flux during in vitro activation of primary mouse HSCs was performed using a DsRed-GFP-LC3B encoding plasmid. The effect of autophagy inhibition by bafilomycin A1 on the in vitro activation process of human and mouse HSCs was examined by measuring proliferation, presence of activation markers by RT-qPCR, immunofluorescence, and Western blotting. Analysis of lipid droplet and microtubule-associated protein light chain 3 beta (LC3B) colocalization in the presence of PDGF-BB was investigated by immunocytochemistry. RESULTS A significant increased autophagic flux was observed during culture induced mouse HSC activation. Treatment of mouse HSCs and human HSCs with autophagy inhibitor bafilomycin A1 results in a significant decreased proliferation and expression of activation markers. In addition, lipid droplets and LC3B colocalization was increased after PDGF-BB treatment in quiescent HSCs. CONCLUSIONS During HSC activation, autophagic flux is increased. The demonstration of partly inhibition of in vitro HSC activation after treatment with an autophagy inhibitor unveils a potential new therapeutic strategy for liver fibrosis.

Osteopontin neutralisation abrogates the liver progenitor cell response and fibrogenesis in mice

Background Chronic liver injury triggers a progenitor cell repair response, and liver fibrosis occurs when repair becomes deregulated. Previously, we reported that reactivation of the hedgehog pathway promotes fibrogenic liver repair. Osteopontin (OPN) is a hedgehog-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesised that OPN may modulate liver progenitor cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralisation on murine liver fibrosis. Methods Liver progenitors (603B and bipotential mouse oval liver) were treated with OPN-neutralising aptamers in the presence or absence of transforming growth factor (TGF)-β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralisation (using OPN-aptamers or OPN-neutralising antibodies) on liver progenitor cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3,5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by quantitative real time (qRT) PCR, Sirius-Red staining, hydroxyproline assay, and semiquantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. Results OPN is overexpressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound healing by modulating TGF-β signalling. In vivo, OPN-neutralisation attenuates the liver progenitor cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. Conclusions OPN upregulation during liver injury is a conserved repair response, and influences liver progenitor cell function. OPN-neutralisation abrogates the liver progenitor cell response and fibrogenesis in mouse models of liver fibrosis.

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

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. (HEPATOLOGY 2012)

EpCAM and the biology of hepatic stem/progenitor cells.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein, which is frequently and highly expressed on carcinomas, tumor-initiating cells, selected tissue progenitors, and embryonic and adult stem cells. During liver development, EpCAM demonstrates a dynamic expression, since it can be detected in fetal liver, including cells of the parenchyma, whereas mature hepatocytes are devoid of EpCAM. Liver regeneration is associated with a population of EpCAM-positive cells within ductular reactions, which gradually lose the expression of EpCAM along with maturation into hepatocytes. EpCAM can be switched on and off through a wide panel of strategies to fine-tune EpCAM-dependent functional and differentiative traits. EpCAM-associated functions relate to cell–cell adhesion, proliferation, maintenance of a pluripotent state, regulation of differentiation, migration, and invasion. These functions can be conferred by the full-length protein and/or EpCAM-derived fragments, which are generated upon regulated intramembrane proteolysis. Control by EpCAM therefore not only depends on the presence of full-length EpCAM at cellular membranes but also on varying rates of the formation of EpCAM-derived fragments that have their own regulatory properties and on changes in the association of EpCAM with interaction partners. Thus spatiotemporal localization of EpCAM in immature liver progenitors, transit-amplifying cells, and mature liver cells will decisively impact the regulation of EpCAM functions and might be one of the triggers that contributes to the adaptive processes in stem/progenitor cell lineages. This review will summarize EpCAM-related molecular events and how they relate to hepatobiliary differentiation and regeneration.

Role of liver progenitors in acute liver injury.

Acute liver failure (ALF) results from the acute and rapid loss of hepatocyte function and frequently exhibits a fulminant course, characterized by high mortality in the absence of immediate state-of-the-art intensive care and/or emergency liver transplantation (ELT). The role of hepatocyte-mediated liver regeneration during acute and chronic liver injury has been extensively investigated, and recent studies suggest that hepatocytes are not exclusively responsible for the regeneration of the injured liver during fulminant liver injury. Liver progenitor cells (LPC) (or resident liver stem cells) are quiescent in the healthy liver, but may be activated under conditions where the regenerative capacity of mature hepatocytes is severely impaired. This review aims to provide an overview of the role of the LPC population during ALF, and the role of putative cytokines, growth factors, mitogens, and hormones in the LPC response. We will highlight the potential interaction among cellular compartments during ALF, and discuss the possible prognostic value of the LPC response on ALF outcomes.

Alteration in N‐glycomics during mouse aging: a role for FUT8

We recently reported that N‐glycosylation changes during human aging. To further investigate the molecular basis determining these alterations, the aging process in mice was studied. N‐glycan profiling of mouse serum glycoproteins in different age groups of healthy C57BL/6 mice showed substantial age‐related changes in three major N‐glycan structures: under‐galactosylated biantennary (NGA2F), biantennary (NA2), and core α‐1,6‐fucosylated ‐β‐galactosylated biantennary structures (NA2F). Mice defective in klotho gene expression (kl/kl), which have a shortened lifespan, displayed a similar but accelerated trend. Interestingly, the opposite trend was observed in slow‐aging Snell Dwarf mice (dw/dw) and in mice fed a calorically restricted diet. We also discovered that increased expression and activity of α‐1,6‐fucosyltransferase (FUT8) in the liver are strongly linked to the age‐related changes in glycosylation and that this increased FUT8 and fucosylation influence IGF‐1 signaling. These data demonstrate that the glycosylation machinery in liver cells is significantly affected during aging and that age‐related increased FUT8 activity could influence the aging process by altering the sensitivity of the IGF‐1R signaling pathway.

High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

Role of liver progenitors in liver regeneration.

During massive liver injury and hepatocyte loss, the intrinsic regenerative capacity of the liver by replication of resident hepatocytes is overwhelmed. Treatment of this condition depends on the cause of liver injury, though in many cases liver transplantation (LT) remains the only curative option. LT for end stage chronic and acute liver diseases is hampered by shortage of donor organs and requires immunosuppression. Hepatocyte transplantation is limited by yet unresolved technical difficulties. Since currently no treatment is available to facilitate liver regeneration directly, therapies involving the use of resident liver stem or progenitor cells (LPCs) or non-liver stem cells are coming to fore. LPCs are quiescent in the healthy liver, but may be activated under conditions where the regenerative capacity of mature hepatocytes is severely impaired. Non-liver stem cells include embryonic stem cells (ES cells) and mesenchymal stem cells (MSCs). In the first section, we aim to provide an overview of the role of putative cytokines, growth factors, mitogens and hormones in regulating LPC response and briefly discuss the prognostic value of the LPC response in clinical practice. In the latter section, we will highlight the role of other (non-liver) stem cells in transplantation and discuss advantages and disadvantages of ES cells, induced pluripotent stem cells (iPS), as well as MSCs.

Low hepatocyte repopulation from stem cells: a matter of hepatobiliary linkage not massive production.

Figure 1. A, Label (BrdrU) retaining peribiliary hepatocytes (black nuclei) in murine acute acetaminophen toxicity represent the majority of label retaining cells in this injury model and probably derive from the smallest cholangiocytes of the canals of Hering (CoH; DAB stained pan-keratin, brown) to which they are connected (stain: hematoxylin counterstain; original magnification: 40). B, Laminin staining in human liver is continuous around all ducts and ductules, but is interupted at the CoH: hepatocyte interface (arrow), a distinctive discontinuity that may relate to a specialized repopulating function of the CoH as a stem cell niche and be responsible for the unique ductular reactions that are seen in liver, but no other organ (DAB;stain: hematoxylin counterstain; original magnification: 40). C, Laminin staining of human pancreas shows continuous basement membrane around all duct, acinar, and islet structures (*; DAB; stain: hematoxylin counterstain; original magnification: 20). D, Laminin staining of human colon shows continuous basement membrane around all intestinal crypts (DAB; stain: hematoxylin counterstain; original magnification: 10). (Laminin staining images provided by Christine Sempoux, Université Catholique de Louvain, Belgium.) Low Hepatocyte Repopulation From Stem Cells: A Matter of Hepatobiliary Linkage Not Massive Production

Advances in hepatic stem/progenitor cell biology.

The liver is famous for its strong regenerative capacity, employing different modes of regeneration according to type and extent of injury. Mature liver cells are able to proliferate in order to replace the damaged tissue allowing the recovery of the parenchymal function. In more severe scenarios hepatocytes are believed to arise also from a facultative liver progenitor cell compartment. In human, severe acute liver failure and liver cirrhosis are also both important clinical targets in which regeneration is impaired, where the role of this stem cell compartment seems more convincing. In animal models, the current state of ambiguity regarding the identity and role of liver progenitor cells in liver physiology dampens the enthusiasm for the potential use of these cells in regenerative medicine. The aim of this review is to give the basics of liver progenitor cell biology and discuss recent results vis-à-vis their identity and contribution to liver regeneration.