Regiospecific Repopulation of the Liver Figure 3. HPLC profile of bilirubin species excreted in bile. (A) The bile of untreated Gunn rats contained some unconjugated bilirubin, but no bilirubin glucuronides. (B) Bile of the wildtype Wistar-RHA donors contained predominantly bilirubin diglucuronide and monoglucuronide and a very small amount of unconjugated bilirubin. (C) Three months after hepatocyte transplantation following HIR to 1/3 of liver, the pigment profile of bile in transplant-recipient Gunn rats became similar to that of the normal Wistar rats, except that there was still a significant amount of unconjugated bilirubin in the bile. (D) Three months after hepatocyte transplantation following HIR to 2/3 of liver, the pigment profile of bile in transplant-recipient Gunn rats became similar to that of Gunn rats received HIR to 1/3 of liver. doi:10.1371/journal.pone.0046775.g003 replaced by DPPIV+ donor hepatocytes, indicating massive proliferation of the donor cells. In contrast, the non-irradiated half of the lobe contained only individual DPPIV+ donor hepatocytes, indicating lack of significant donor cell proliferation (Figure 1). Serum bilirubin levels in Gunn rat recipients In order to demonstrate the functionality of the repopulated donor hepatocytes in irradiated host liver, we transplanted congeneic ugt1a1-proficient Wistar hepatocytes in jaundiced ugt1a1-deficient Gunn rats, following regiospecific preparative HIR. The effect of liver replacement in Gunn rat recipients by transplanted hepatocytes is shown in Figure 2. In the group receiving HIR of the median lobe (30% of the liver mass) only, mean serum bilirubin levels declined from pretransplantation values of 5.1760.78 mg/dl to 0.9660.30 mg/dl (mean 6 SD) in 8 weeks after hepatocyte transplantation (p,0.0001), and remained close to that level throughout the study. In the group of Gunn rats that received median and left lobes irradiation (60% of the liver mass), serum bilirubin levels declined more rapidly than in the group receiving HIR of the median lobe only, so that the mean values were significantly lower in this group at 4-weeks. However, serum bilirubin concentrations in both groups stabilized at similar levels (1.1260.22 mg/dl) in 8 weeks. In both HIR groups, the serum bilirubin levels were significantly lower than in the group receiving laparotomy and Ad-HGF, but no HIR, at all time points beyond 4 weeks after transplantation (p,0.0001). Analysis of bilirubin species excreted in bile Bile duct cannulae were placed, as previously described [16], at the end of the observation period in all Gunn rat recipients in order to examine the bilirubin species in the bile of recipient animals. Representative HPLC analyses of pigments excreted in the bile collected from untreated Gunn rats (Fig. 3A), congeneic wildtype Wistar-RHA rats (Fig. 3B) and Gunn rats receiving the various treatments are shown in Figure 3. As expected, the bile of untreated Gunn rats contained only unconjugated bilirubin, no bilirubin glucuronides (Figure 3A), whereas bile of the Wistar- PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e46775