Hepatocyte transplantation is being developed as a minimally invasive alternative to liver transplantation for patients with liver failure or inherite metabolic disorders. Major hurdles to the broad application of this promising therapeutic modality are the scarcity of high quality donor organs and the need for prolonged immunosuppression to prevent allograft rejection. This research proposal is to test the hypothesis that induced pluripotent stem cells (iPSC) derived from the skin of patients with Crigler-Najjar syndrome type 1 (CN1), who have severe lifelong jaundice due to uridinediphosphoglucuronate glucuronosltransferase-1 (UGT1A1) deficiency can be corrected genetically, and then manipulated in culture to generate hepatocytes expressing active UGT1A1. We further hypothesize that appropriate host preparation will permit sufficient repopulation of the livers of the Gunn rat model of CN1 to ameliorate hyperbilirubinemia.
The specific aim 1 is (a) Generating and characterizing iPS cells from skin fibroblasts from normal subjects and CN1 patients, (b) correcting the genetic defect by homologous recombination at a genomic "safe harbor", using zinc-finger nuclease or TALE-nuclease technology, and (c) differentiating the genetically corrected iPSCs into hepatocyte-like cells (iHep) in culture. The gene expression profile and metabolic function of the derived hepatocytes will be compared with those of adult and fetal human hepatocytes.
Specific aim 2 is (a) Transplanting the iHeps into the liver of Gunn rats following appropriate host preparation, and evaluating the metabolic effect of the transplantation. Gunn rats will be treated with a tacrolimus-based immunosuppressive regimen that has been shown to prevent the rejection of xenografted human hepatocytes in our laboratory. The recipients will be subjected to preparative hepatic irradiation and mitotic stimulation by a single injection of an adenovector, expressing hepatocyte growth factor to induce repopulation of the liver with the transplanted hepatocytes. Serum bilirubin levels will be followed, the extent of liver repopulation will be determined and biliary excretion of bilirubin glucuronides will be analyzed by high-pressure liquid chromatography. (b) Finally, a group of recipient rats and immunodeficient mice will be observed life-long to evaluate the tumorigenic potential of the transplanted iPSC-derived hepatocytes. Successful completion of this project will provide a renewable source of autologous hepatocytes that, after additional modification and scale up, could be translated into cell transplantation-based treatment of CN1 and many other inherited liver diseases.
To develop liver cell transplantation as a minimally invasive alternative to liver transplantation for patients with inherited liver diseases, we will generate stem cells (iPSC) from the skin, correct the genetic defect, convert them to liver cells and then transplant them into a rat model of the disease.