Adhesion of human haematopoietic (CD34+) stem cells to human liver compartments is integrin and CD44 dependent and modulated by CXCR3 and CXCR4.

BACKGROUND/AIMS: Haematopoietic stem cells (HSC) have previously been shown in some studies to migrate to damaged and diseased liver where a small proportion will engraft. Such cells can promote liver repair in rodent models of liver injury and lead to improved liver function in uncontrolled clinical studies. In order to maximize the engraftment of cells for clinical applications it is necessary to understand the molecular mechanisms that regulate stem cell recruitment and retention. Our aim therefore was to determine which factors where involved in adhesion of circulating HSC to liver endothelium and sequestration around epithelial cells within the liver. METHODS: We examined the ability of CD34+ populations from peripheral and mobilized blood and the CD34-expressing cell line KG1a to bind to human hepatic sinusoidal endothelial (HSEC) and biliary epithelial cells (BEC) in vitro. RESULTS: We report that all CD34(+) populations express alpha4beta1, beta2 integrins and CD44. Liver tissue sections and primary liver cells expressed the corresponding ligands VCAM-1/fibronectin, ICAM-1 and CD44. Pertussis toxin was shown to decrease binding of CD34(+) cells and the cells migrated to CXCR3 and CXCR4 ligands. CONCLUSIONS: CD34(+) populations use alpha4beta1, beta2 integrins and CD44 receptors to bind to the ligands VCAM-1/fibronectin, ICAM-1, and hyaluronic acid expressed on sinusoidal vessels in tissue sections and to primary human HSEC. Binding to BEC was mediated by the interaction of beta1 and beta2 integrins with VCAM-1 and ICAM-1 respectively. A role for chemokines is supported by our finding that pertussis toxin inhibits CD34(+) cell adhesion to BEC and HSEC and by the ability of CD34(+) cells to migrate to CXCR3 and CXCR4 ligands.

A novel cell-surface marker found on human embryonic hepatoblasts and a subpopulation of hepatic biliary epithelial cells.

The nature of the cells that contribute to the repopulation of the liver after hepatic necrosis or cirrhosis remains uncertain, in part because we lack specific markers to facilitate identification and prospective isolation of progenitor cells. The monoclonal antibody GCTM-5 reacts with a minority subpopulation of cells in spontaneously differentiating cultures of pluripotent human embryonal carcinoma or embryonic stem cells. The epitope recognized by GCTM-5 is found on a 50-kDa protein present on the surface of these cells. In tissue sections of first-trimester human embryos, GCTM-5 specifically stained hepatoblasts and no other cell type examined. In normal pediatric or adult liver, GCTM-5 reacted with a minority population of luminal bile duct cells. In diseased livers, the numbers of GCTM-5-positive cells were increased compared with normal liver; antibody staining was restricted to a subpopulation of ductular reactive cells, and among this subpopulation we observed GCTM-5-positive cells that did not express cytokeratin 19 or N-CAM, classical makers of ductular reactive cells. Live GCTM-5-positive cells could be isolated from diseased livers by immunomagnetic sorting. These results suggest that GCTM-5 will be a useful reagent for defining cell lineage relationships between putative progenitor populations in embryonic liver and in the biliary epithelium during tissue repair.

The role of Notch receptor expression in bile duct development and disease.

Mutations in the Jagged1 gene, a ligand for the Notch signalling pathway, have been implicated in the pathogenesis of Alagille syndrome (AGS), resulting in bile duct paucity. Recently, a mouse model for AGS suggested that abnormalities of the Notch2 receptor, as well as of Jagged1, may be present. Expression patterns of Notch receptors have not been described in the developing human liver or in paediatric liver. The expression of Notch receptors and ligands was examined in fetal, paediatric normal, and diseased human liver by RT-PCR and immunohistochemistry. RT-PCR showed Notch1-4 mRNA expression to be present. In fetal liver, Notch3 protein was expressed on mesenchymal cells, closely adjacent to ductal plate cells that expressed Jagged1. In paediatric normal liver, Notch1 and Notch2 were present on mature bile duct cells. Notch expression was altered in disease, with distinct differences in AGS from extrahepatic biliary atresia (EHBA) and alpha1-anti-trypsin deficiency (alpha1AT). In AGS, where extensive ductular reaction was present, Jagged1 was expressed on ductular reactive cells (DRCs), along with marked Notch2 and Notch3 staining. Where there was ductular paucity, Notch2 and Notch3 were not expressed on remaining biliary epithelial cells. In EHBA and alpha1AT, Notch receptor expression was not seen on DRCs. Instead, Notch2 and Notch3 were expressed by stromal cells. In all diseases, Notch3 was expressed on neovessels in portal tracts and cirrhotic fibrous septa. In conclusion, Notch3 is expressed in close proximity to Jagged1 at the time of ductal plate formation, suggesting that Notch3 is important for bile duct development. The expression of both Notch2 and Notch3 in AGS on DRCs confirms that these receptors may be important in the pathogenesis of this disease. Further studies are required to investigate the presence of Notch2 and Notch3 at other periods in liver development and to clarify the role of Notch signalling in paediatric cholestases.

Nomenclature of the finer branches of the biliary tree: canals, ductules, and ductular reactions in human livers.

The work of liver stem cell biologists, largely carried out in rodent models, has now started to manifest in human investigations and applications. We can now recognize complex regenerative processes in tissue specimens that had only been suspected for decades, but we also struggle to describe what we see in human tissues in a way that takes into account the findings from the animal investigations, using a language derived from species not, in fact, so much like our own. This international group of liver pathologists and hepatologists, most of whom are actively engaged in both clinical work and scientific research, seeks to arrive at a consensus on nomenclature for normal human livers and human reactive lesions that can facilitate more rapid advancement of our field.

Human liver-derived stem cells.

The search for human oval cells or bi-potential stem cells in the human liver is the subject of intensive investigation. Fetal hepatocytes (hepatoblasts) have some proliferative and bipotential capacity, but access to sufficient numbers of cells remains limiting. Candidate stem cells in the adult normal and diseased human liver have been identified using markers such as OV6, CD34, c-kit and NCAM. Lack of stem cell marker specificity however, remains a problem and further, more specific markers are required. The molecular signaling molecules and transcription factors that control proliferation and cell differentiation pathways into hepatocyte or biliary phenotype are beginning to emerge. However, whether any of the current liver stem cell approaches will be converted into effective clinical cell transplantation or gene therapy treatments has yet to be proven.

Progenitor cells of the biliary epithelial cell lineage.

Stem-like cells have been identified in liver that are able to differentiate in vivo and in culture to biliary epithelial cells (BEC), hepatocytes and oval cells. The growth factors/cytokines and signal pathways required for the differentiation processes are beginning to be evaluated. There is increasing evidence to suggest that these stem-like cells may originate from both the bone marrow population or from a precursor remnant from liver embryogenesis, as they share many of the same markers (CD34, c-kit, CD45). Most recently, it has been shown that a population of progenitor cells can copurify with mesenchymal bone marrow cells and differentiate under specific culture conditions to form both hepatic epithelial and also endothelial cells. The interaction of haemopoietic and mesenchymal stem cells needs further evaluation. The close association of ductular reactive cells and neovessels in end-stage cholestatic liver diseases and the relation to Jagged/Notch signalling pathway may be important in the regulation of stem cells to form both biliary epithelial and endothelial cells.

Altered Notch ligand expression in human liver disease: further evidence for a role of the Notch signaling pathway in hepatic neovascularization and biliary ductular defects.

The Jagged and Delta family of transmembrane proteins are ligands for Notch receptors, which control the proliferation and/or differentiation of many cell lineages. Expression and localization of these ligands in the adult human liver has not been fully elucidated, nor whether dysregulation of these proteins contributes to liver disease processes. We have examined expression of the five known Notch ligands in human liver. Expression of Jagged-1 and Delta-4 mRNA was seen in normal and diseased liver tissue, whereas Jagged-2, Delta-1, and Delta-3 mRNA was undetectable. In primary liver cell isolates, Jagged-1 expression was found in all cell types, whereas Delta-4 was present in biliary epithelial and liver endothelial cells, but absent in hepatocytes. Interestingly, Jagged-1 mRNA expression was significantly up-regulated in diseased liver tissue. By immunohistochemistry, Jagged-1 expression was present on most structures in normal tissue. However in disease, strikingly strong Jagged-1 immunoreactivity was observed on many small neovessels and bile ductules. The expression of downstream modulators and effectors of Notch signaling was also detectable in purified cell isolates. This, together with aberrant Jagged-1 expression suggests that the Notch signaling pathway may play a role in the neovascularization and biliary defects observed in the liver during the development of cirrhosis.