Regiospecific Repopulation of the Liver Figure 1. Regiospecific liver repopulation in DPPIV2 recipients by donor DPPIV+ hepatocytes following conformal HIR of one half of the median lobe. All liver lobes, except one half of the median lobe were shielded from HIR. Three months after hepatocyte transplantation, liver sections were stained histochemically for DPPIV activity. A region at the junction of the irradiated and non-irradiated regions of the median lobe is shown. Panel a. Low power view showing clusters of DPPIV-positive donor hepatocytes (red, white arrow) in the upper part of the section that had received preparative HIR, and the absence of such clusters in the lower part of the section that had been shielded from HIR. Panel b. High power view showing a donor hepatocyte cluster. The black bars indicate 100 mm. doi:10.1371/journal.pone.0046775.g001 difluoride membranes (Bio-Rad). The membranes were blocked with 5% fat-free milk powder in PBS followed by incubation with the rabbit antiserum directed against the rat ugt1a1 amino- terminal domain. After washing with PBS, the membranes were incubated with a horseradish peroxidase–conjugated goat anti- rabbit IgG (Sigma) and developed using an enhanced chemilumi- nescence reagent (ECL Plus; Amersham, Arlington Heights, IL). Measurement of serum bilirubin levels Approximately 0.4 ml of blood was collected from the rat tail vein before any experimental procedure (pretransplant) and at various time points after transplantation under isoflurane anes- thesia, and serum bilirubin levels were determined using an automated clinical microchemistry system (Bayer Chem One; Bayer Corp., Tarrytown, NY). Analysis of bilirubin species excreted in bile Bile samples were collected from the bile ducts of the Gunn rats after completion of serial blood collection, and were analyzed by reverse-phase high-performance liquid chromatography as de- scribed [16]. Assay for ugt1a1 activity toward bilirubin Ugt1a1 enzyme activity in the rat livers was analyzed as described previously [26]. Statistical analysis Repopulation of donor cells was assessed by counting the number of ugt1a1-stained donor hepatocytes in host liver lobes after ugt1a1 immunohistochemistry. Sampling regions were chosen at random for digital acquisition for data quantitation in a blinded fashion. A total of 20 fields at 2006 magnification, each from irradiated and non-irradiated liver lobes of three rats per treatment group were used for each data point. The serum bilirubin values were expressed as the mean 6 standard deviation (SD) and the ugt1a1 enzyme activity was expressed as percent of ugt1a1 activity in the wildtype Wistar RHA rat liver in order to estimate the extent of repopulation of host liver by donor hepatocytes. Statistical differences between two groups were determined by a two-sided Student t test. P values less than 0.05 were considered statistically significant. Results Targeted HIR enabled regiospecific liver repopulation in DPPIV2 recipients To determine the effect of targeted HIR to the liver, DPPIV+ F344 hepatocytes were transplanted into congeneic DPPIV2 rat livers that had been subjected to preparative HIR of one half of the median lobe only. Three months after transplantation, 30– 60% of the irradiated region of the median lobe was selectively Figure 2. Conformal HIR followed by hepatocyte transplanta- tion ameliorates bilirubin levels in a hyperbilirubinemia model. The control group of Gunn rats received Ad-HGF followed by hepatocyte transplantation without HIR (triangles, n = 4). One experi- mental group received Ad-HGF and preparative median lobe HIR (1/3 of liver, diamonds, n = 6) before hepatocyte transplantation. Another experimental group received Ad-HGF and preparative HIR of the median plus left lobe (2/3 of liver, circles, n = 8) before hepatocytes transplantation. doi:10.1371/journal.pone.0046775.g002 PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e46775