Histological organization in hepatocyte organoid cultures.

Hepatocytes and other cellular elements isolated by collagenase perfusion of the liver and maintained in defined culture conditions undergo a series of complex changes, including apoptosis and cell proliferation, to reconstruct tissue with specific architecture. Cultures in collagen-coated pleated surface roller bottles, with hepatocyte growth medium medium and in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF), form characteristic and reproducible tissue architecture composed of a superficial layer of biliary epithelial cells, an intermediate layer of connective tissue and hepatocytes, and a basal layer of endothelial cells. Dexamethasone, EGF, and HGF are required for the complete histological organization. Analysis of the structures formed demonstrates that the receptor tyrosine kinase ligands HGF and EGF are required for the presence, growth, and phenotypic maturation of the biliary epithelium on the surface of the cultures and for the formation of connective tissue in the cultures. Dexamethasone, in the presence of HGF and EGF, was required for the phenotypic maturation of hepatocytes. The results demonstrate the role of these molecules for the formation and phenotypic maturation of specific histological elements of the liver and suggest roles for these signaling molecules in the formation and structure of the in vivo hepatic architecture.

Changes in WNT/beta-catenin pathway during regulated growth in rat liver regeneration.

The wnt/beta-catenin pathway is important during embryogenesis and carcinogenesis. beta-Catenin interaction with E-cadherin has been shown to be crucial in cell-cell adhesion. We report novel findings in the wnt pathway during rat liver regeneration after 70% partial hepatectomy using Western blot analyses, immunoprecipitation studies, and immunofluorescence. We found wnt-1 and beta-catenin proteins to be predominantly localized in hepatocytes. Immediately following partial hepatectomy, we observed an initial increase in beta-catenin protein during the first 5 minutes with its translocation to the nucleus. We show this increase to be the result of decreased degradation of beta-catenin (decrease in serine phosphorylated beta-catenin) as seen by immunoprecipitation studies. We observed activation of beta-catenin degradation complex comprising of adenomatous polyposis coli gene product (APC) and serine-phosphorylated axin protein, beginning at 5 minutes after hepatectomy, leading to its decreased levels after this time. Quantitative changes observed in E-cadherin protein during liver regeneration are, in general, reverse to those seen in beta-catenin. In addition, using immunoprecipitation, we observe elevated levels of tyrosine-phosphorylated beta-catenin at 6 hours onward. Thus, changes in the wnt pathway during regulated growth seem to tightly regulate cytosolic beta-catenin levels and may be contributing to induce cell proliferation and target gene expression. Furthermore, these changes might also be intended to negatively regulate cell-cell adhesion for structural reorganization during the process of liver regeneration.

Direct retrovirus-mediated gene transfer to the synovium of the rabbit knee: implications for arthritis gene therapy.

We have investigated the feasibility of using high-titer murine leukemia virus-based retroviral vectors to deliver exogenous genes to naive and chronically inflamed knee joints of rabbits in vivo. Intraarticular injection of retrovirus encoding beta-galactosidase (beta-gal or lacZ) was found to transduce synoviocytes in both naive and inflamed joints, but a significantly higher number of lacZ+ cells were found in inflamed knees. Using a retrovirus encoding a secretable marker, human growth hormone (hGH), quantitative comparison of ex vivo and in vivo gene delivery methods demonstrated that transgene expression following in vivo gene transfer was at least equivalent to that of the ex vivo method in inflamed knees. In addition, hGH transgene expression was maintained for at least 4 weeks. These experiments suggest that high-titer retroviral vector could be used for efficient in vivo gene transfer to inflamed joints in patients with rheumatoid arthritis (RA).