Regiospecific Repopulation of the Liver Figure 6. Ugt1a1 activity toward bilirubin in Gunn rats that received conformal HIR before hepatocyte transplantation. Ugt1a1 activities in liver homogenates of different recipient groups as in Figure 2 are shown (means6SD) as percent of the activity in the liver of Wistar-RHA rats (2.6 mmol/g liver per hour). doi:10.1371/journal.pone.0046775.g006 transplantation can cause massive preferential proliferation of the host cells, which competitively repopulate the host liver [13]. As the chemical methods used to inhibit host hepatocyte proliferation are not readily translatable to clinical use, we investigated preparative HIR for achieving liver repopulation by transplanted hepatocytes. These studies showed that whole liver HIR in combination with a mitotic stimulus, such as partial hepatectomy or HGF expression, results in massive liver repop- ulation and complete correction of the metabolic defect in Gunn rats [16], as well as in a mouse model of PH1 (hepatic alanine:glyoxylate aminotransferase deficiency) [17,20]. In addi- tion, we have found that preparative HIR enhances engraftment in non-human primates [11]. A major hurdle to the application of this promising technology to clinical hepatocyte transplantation is concern about inducing RILD. Whole liver irradiation in primates, including humans, is associated with RILD that differs in severity and pathological characteristics from HIR-induced RILD in rodents. Therefore, it is critical to determine the tolerable dose of HIR in humans, and, if possible devise strategies to prevent clinical RILD. In humans, irradiation targeted to parts of the liver is tolerated much better than whole liver irradiation. It has been reported that therapeutic irradiation of one third of the liver volume in humans is tolerated up to doses as high as 90 Gy without manifestation of RILD [18,19]. In fact, when radiation was targeted to ,25% of the liver volume, RILD did not occur at any irradiation dose tested. In contrast to the treatment of the liver by drugs or chemicals, modern conformal irradiation technology permits HIR delivery to precisely targeted regions. This should permit repopulation of specific regions of the liver, keeping the remaining liver unperturbed. To take advantage of this unique characteristic of irradiation, it would be important to estimate the volume of the liver that must be repopulated to achieve a cure of a metabolic liver disease. Our results in the DPPIV2 recipient rats demon- strate that the area of the liver to be repopulated can be precisely mapped by targeting preparative HIR to that area. Percent repopulation of the liver was somewhat lower in the Gunn rat recipients than in the DPPIV- rats, which could have resulted from the strain difference. Nevertheless, we show that as little as 20% repopulation of 30% of the liver volume in the Gunn rat recipients was sufficient to correct hyperbilirubinemia. Repopulation of twice that volume reduced serum bilirubin levels more rapidly, but ultimately the long-term effect was similar with both HIR groups. Excretion of bilirubin glucuronides in the bile confirmed the function of the repopulating donor cells in vivo. In addition, there was a significant unconjugated bilirubin peak in the bile of the transplant recipients. A similar pattern is also found in human subjects with Gilbert syndrome, who have a partial deficiency of bilirubin glucuronidation [32]. This finding is consistent with the fact that the ugt1A1 deficiency was partially, but not completely normalized. In terms of potential clinical application, it should be noted that the therapeutic objective in patients with CN1 is to reduce serum bilirubin concentrations to safe levels, which can be clearly achieved by partial restitution of hepatic bilirubin glucuronidation. Although we used an adenoviral vector to express HGF, for clinical application the mitotic stimulation will need to be achieved by alternative methods. Humanized activating antibodies to c-met, which have a much longer half-life in the circulation are being evaluated toward this goal in preclinical studies. In conclusion, we have shown for the first time that targeted irradiation of liver lobes can be used to precisely delineate a fraction of the liver volume to be repopulated by transplanted hepatocytes. Partial repopulation of one third of the liver volume using this method cured hyperbilirubinemia in the Gunn rat model of CN1. Our finding is highly relevant to potential clinical application of preparative HIR for hepatocyte transplantation because irradiation of a limited liver volume is tolerated much better than whole liver irradiation in the clinic. Modern conformal irradiation techniques can precisely delineate regions of the liver to PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46775