A rodent model to study toxicology and human viruses

DESCRIPTION (provided by applicant): A study of human hepatocytes is hindered by hepatocyte loss of liver-specific functions as de-differentiation occurs during cell culture. Hepaticus Inc has liscenced the technology to fill this need from Drs. George and Catherine Wu of University of CT Health Center. Injection of human hepatocytes into the peritoneum of embryonic rats results in a new born "tolerized" rat that does not mount an immune reaction to later transplantion with human hepatocytes. This unique system provides an inexpensive environment to study pathogenesis, immunology, natural history, and viral replication of Hepatitis B and C and other human liver diseases with immunologic component(s) including other forms of hepatitis, and autoimmune diseases. The Hepaticus system augments rodent cell studies with data from human cells that maintain human-specific functions. This is one step closer to testing a drug candidate in human patients without the risk. Multiple compounds can be studied at one time, helping to identify potential metabolic interactions. We will use several methods to show that human hepatocytes transplanted into the rat maintain human specific functions over time. We will show that Cytochrome P450 levels are preserved in the human hepatocytes using a Vivid Cytochrome P450 kit with specific substrates for human enzymes. The majority of drug-drug interactions are metabolism-based where two or more drugs compete for the same enzymes, usually members of the Cytochrome P450 family. We will also show that normal human hepatocytes continue to produce human albumin protein. The purpose of this proposal is to confirm that human hepatocytes harvested from a single transplant donor will propagate and maintain human-specific liver enzyme functions when transplanted into a tolerized rat. We will pre-label normal human hepatocytes prior to transplantation with a fluorescent dye, CFSE. Using enzymes the tissue will be dissocated and the isolated cells will be FACS sorted using flow cytometry methods to separate the human hepatocytes from rat cells. Molecular biological methods will be used to show human hepatocytes produce message for human albumin as well as humain albumin protein, thus further corroborating that hepatocytes maintain human-specific characteristics when grown in a liver from a tolerized rat.