Background & Aims-The ability to obtain unlimited numbers of human hepatocytes would improve development of cell-based therapies for liver diseases, facilitate the study of liver biology and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, can potentially differentiate into any cell type and could therefore be developed as a source of human hepatocytes.

On September 6 and 7, 2009 a meeting was held in London to identify and discuss what are perceived to be current roadblocks to effective hepatocyte transplantation as it is currently practiced in the clinics and, where possible, to offer suggestions to overcome the blocks and improve the outcomes for this cellular therapy. Present were representatives of most of the active clinical hepatocyte transplant programs along with other scientists who have contributed substantial basic research to this field. Over the 2-day sessions based on the experience of the participants, numerous roadblocks or challenges were identified, including the source of cells for the transplants and problems with tracking cells following transplantation. Much of the discussion was focused on methods to improve engraftment and proliferation of donor cells posttransplantation. The group concluded that, for now, parenchymal hepatocytes isolated from donor livers remain the best cell source for transplantation. It was reported that investigations with other cell sources, including stem cells, were at the preclinical and early clinical stages. Numerous methods to modulate the immune reaction and vascular changes that accompany hepatocyte transplantation were proposed. It was agreed that, to obtain sufficient levels of repopulation of liver with donor cells in patients with metabolic liver disease, some form of liver preconditioning would likely be required to enhance the engraftment and/or proliferation of donor cells. It was reported that clinical protocols for preconditioning by hepatic irradiation, portal vein embolization, and surgical resection had been developed and that clinical studies using these protocols would be initiated in the near future. Participants concluded that sharing information between the groups, including standard information concerning the quality and function of the transplanted cells prior to transplantation, clinical information on outcomes, and standard preconditioning protocols, would help move the field forward and was encouraged.

Mutations in the alanine-glyoxylate amino transferase gene (AGXT) are responsible for primary hyperoxaluria type I, a rare disease characterized by excessive hepatic oxalate production that leads to renal failure. We generated a null mutant mouse by targeted mutagenesis of the homologous gene, Agxt, in embryonic stem cells. Mutant mice developed normally, and they exhibited hyperoxaluria and crystalluria. Approximately half of the male mice in mixed genetic background developed calcium oxalate urinary stones. Severe nephrocalcinosis and renal failure developed after enhancement of oxalate production by ethylene glycol administration. Hepatic expression of human AGT1, the protein encoded by AGXT, by adenoviral vector-mediated gene transfer in Agxt ؊/؊ mice normalized urinary oxalate excretion and prevented oxalate crystalluria. Subcellular fractionation and immunofluorescence studies revealed that, as in the human liver, the expressed wild-type human AGT1 was predominantly localized in mouse hepatocellular peroxisomes, whereas the most common mutant form of AGT1 (G170R) was localized predominantly in the mitochondria.gene therapy ͉ knockout mouse ͉ oxalate ͉ urolithiasis ͉ nephrocalcinosis

Background Hepatocyte transplantation partially corrects genetic disorders and has been associated anecdotally with reversal of acute liver failure. Monitoring for graft function and rejection has been difficult, and has contributed to limited graft survival. Methods Preparative hepatic irradiation was examined in non-human primates as a strategy to improve engraftment of donor hepatocytes, and was then applied in human subjects. T-cell immune monitoring was also examined in human subjects to assess adequacy of immunosuppression. Results Porcine hepatocyte transplants engrafted and expanded to comprise up to 15% of irradiated segments in immunosuppressed monkeys preconditioned with 10Gy liver-directed irradiation. Two patients with urea cycle deficiencies had early graft loss following hepatocyte transplantation; retrospective immune monitoring suggested the need for additional immunosuppression. Preparative radiation, anti-lymphocyte induction, and frequent immune monitoring were instituted for hepatocyte transplantation in a 27-year old female with classical phenylketonuria. Post-transplant liver biopsies demonstrated multiple small clusters of transplanted cells, multiple mitoses, and Ki67+ hepatocytes. Mean peripheral blood phenylalanine (PHE) level fell from pre-transplant levels of 1343 ± 48μM (normal 30-119μM) to 854 ± 25μM (treatment goal ≤360μM) after transplant (36% decrease; p<0.0001), despite transplantation of only half the target number of donor hepatocytes. PHE levels remained below 900 μM during supervised follow-up, but graft loss occurred after follow-up became inconsistent. Conclusions Radiation preconditioning and serial rejection-risk assessment may produce better engraftment and long-term survival of transplanted hepatocytes. Hepatocyte xenografts engraft for a period of months in non-human primates and may provide effective therapy for patients with acute liver failure.

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