NMR ANALYSIS OF BIOARTIFICAL LIVER FROM PROGENITORS

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The long-term goal of this project is to develop a liver lobule model, or bioreactor, to study all aspects of liver biology. Initial investigations will use the human hepatic cell-line, HepG2, under two growth conditions, promoting (1) growth versus (2) differentiation. If successful, the experiments will be repeated with normal hepatic progenitors isolated from human livers using multiparametric fluorescence activated cell sorting (FACS) and a known antigenic profile. The extent of differentiation will be characterized using molecular biology techniques and correlated with NMRS-visible biomarkers of metabolism. HepG2 cells will be grown in a novel coaxial bioreactor that is the same dimension as the liver lobule, and coated with extracellular matrices described by the two culture conditions. The morphology and immunohistochemistry of microtomed bioreactor sections will be analyzed at different stages of growth and compared to the normal liver lobule. In situ hybridization assays of HepG2 cells in the microtomed sections will determine the localization of mRNAs transcribing extracellular matrix proteins. Normal liver lobule metabolism will be assessed in situ by NMRS. 31P NMRS will be used to monitor cell growth. P-450 activity will be measured in the hepatocytes by monitoring p-triflouromethylanisole metabolism by 19F NMRS. Gluconeogenesis/glycogen catabolism and glycolysis/glyconeogenesis, and glutathione levels and the glycine cycle will be measured by monitoring metabolism of l- 13C- glucose/3- 13C-pyruvate and 2- 13C-glycine, respectively, using 1H [13C]-HMQC (heteronuclear multiple-quantum coherence) NMRS. Ureogenesis and glutamine synthetase activity will be measured by 1H[15N]-HMQC NMRS detection of 1 5NH4 metabolism.