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  • There is evidence from our study of

    2018-11-08

    There is evidence from our study, of extreme resilience in hepatocytes isolated from patients with metabolic liver disease and for recovery of hepatic function when the hepatocytes are removed from the donor. For example, ammonia metabolism improved in the PHO, CN, and CHF cases in cells cultured for 5days as compared to measurements made immediately after isolation. Conjugation of resorufin was completely absent in the cells from the CN cases, however, after 5days in culture the cells showed normal activity. Resorufin conjugation could be expected to recover, even in CN cases, because resorufin is conjugated by up to 9 different UGT enzymes, with UGT1A6 and 1A9 the most active (Tolando et al., 2012). While UGT1A1 (the form mutated in CN cases) remains inactive in the CN cells, data presented here indicate that the remaining UGTs recover activity in hepatocytes removed from the CN environment. The observations made from hepatocytes explanted from metabolic disease cases have provided clinical guidance for hepatocyte transplants. High CYP3A4 activities were noted in the OTC patients. When HTx was performed on these patients, because of the high CYP3A4 levels, normal doses of Tacrolimus were rapidly cleared resulting in insufficient immunosuppression until dosage could be adjusted. Significantly higher doses of Tacrolimus were required to maintain therapeutic levels. Two recent CN patients received HTx and whose immunosuppression post hepatocyte transplantation included Tacrolimus, prednisolone and mycophenolate mofetil (MMF). As we predicted from CN hepatocytes, conjugation was impaired, and a complete absence of mycophenolate glucuronide was noted in blood samples taken during the first post-transplant week. The patient registered extremely high mycophenolic Caspase-6, human recombinant protein levels when a normal dose of MMF was administered (533mg∗h/L; therapeutic range 30–60mg∗h/L). Their inability to metabolize the drug necessitated that MMF be discontinued. A subsequent patient was treated with a reduced dose of MMF. Samples taken during the first two post-transplant weeks showed a small, but gradually growing peak in the chromatograms at a retention time similar to mycophenolate glucuronide, suggesting that conjugation of mycophenolic acid is returning to normal over time, again, as predicted from experiments with the cultured cells. We are currently investigating drugs, diet and other factors that might influence these metabolic activities in such a disease-specific manner. Bhogal et al. recently reported the isolation of hepatocytes from diseased livers (Bhogal et al., 2011). Their disease selections were quite different from those in the current study, in that they focused on cirrhotic and fibrotic tissues from patients with alcoholic liver disease, primary biliary cirrhosis and primary sclerosing cholangitis. They reported an experience similar to ours in that the viability, total cell yield and the success rate with cirrhotic tissues were low. Like theirs, our experience suggests that cirrhotic tissues would not provide enough cells from a single donor for clinical transplantation. For the studies reported here with tissue from metabolic disease patients, we generally procured less than 1/10 of the total liver weight for isolation. Given the viability and cell yield reported here, even one lobe from many metabolic disease organs could provide billions of cells, enough for one or more clinical transplants. Transplants of MD cells to FRG immunodeficient mice support the hypothesis that these cells would perform well post transplantation as was evident from the robust growth and albumin secretion from cells from 5 metabolic donors in parallel with 3 “normal” ODs (Fig. 6A). Since each mg of human albumin correlates with 15–20% repopulation, these animals show 50–99% repopulation with human hepatocytes by 3months post transplant. Analysis of the liver of FRG mice transplanted with human hepatocytes isolated from metabolic diseased patients resulted in a rapid and efficient repopulation of the liver. There were no differences in the levels of repopulation of the liver with diseased cells or those isolated from normal organ donors (Figs. 6B, C).