Figure 4. Immunofluorescent staining for ugt1a1 in different liver lobes. Three months after hepatocyte transplantation following HIR to median lobe (ML), 20% of the irradiated median lobe was replaced by donor hepatocytes, whereas the unirradiated left (LL), right (RL) and caudate (CL) lobes contained only small ugt1a1+ hepatocyte clusters. doi:10.1371/journal.pone.0046775.g004 RHA rats contained predominantly bilirubin diglucuronide and monoglucuronide, and only a small amount of unconjugated bilirubin (Figure 3B). In the groups receiving hepatocyte trans- plantation after preparative HIR targeted to the median lobe (Figure 3C) or the median plus left lobe (Figure 3D) of the liver, the pigment profile of bile became similar to that of the wildtype Wistar-RHA donor rats, except that there was still a significant amount of unconjugated bilirubin in the recipient Gunn rat bile. Immunofluorescent staining for ugt1a1 in different liver lobes Liver repopulation was examined three months after hepatocyte transplantation and conformal HIR to the median liver lobe, by immunofluorescent staining for ugt1a1. As expected, transplanted hepatocytes selectively repopulated the median lobe. Based on examination of 20 fields at 2006 magnification, approximately 20% (range 18–22%) of the irradiated lobe was replaced by ugt1a1+ donor hepatocytes, whereas the unirradiated left, and right lobes contained only small occasional ugt1a1+ hepatocyte clusters. When both the median and the left lobe were irradiated, 18–30% hepatocytes in each lobe were repopulated by the transplanted cells. The caudate lobe, which receives the least scattered radiation because of its anatomical location, contained only individual engrafted donor hepatocytes (Figure 4). Ugt1a1 content of the different lobes Three months after hepatocyte transplantation and preparative HIR to the median and left lobe, 52-kd ugt1a1-immunoreactive bands were observed in all four lobes of Gunn rat livers. However, the ugt1a1 content in the irradiated median and left lobe was much higher than that of unirradiated right and caudate lobe (Figure 5). Ugt1a1 activity toward bilirubin Following transplantation, ugt1a1 activity in the liver homog- enates was assayed with bilirubin as the acceptor substrate. In Gunn rat livers that received regiospecific HIR prior to hepatocyte transplantation, ugt1a1 activity in irradiated median and left lobes Regiospecific Repopulation of the Liver Figure 5. Western blot for ugt1a1 in the Gunn rat liver following HIR to 2/3 of liver and hepatocyte transplantation. Three months after hepatocyte transplantation, ugt1a1 protein was demonstrated in the irradiated and unirradiated liver lobes of Gunn rats by western blot analysis. However, the ugt1a1 content in irradiated lobes was much higher than that of unirradiated lobes. RHA, Wistar- RHA rat liver; ML, median lobe; LL, left lobe;, RL, right lobe; CL, caudate lobe. doi:10.1371/journal.pone.0046775.g005 was about 25–40% of the activity found in control Wistar-RHA rat livers (2.660.14 mmol/g liver per hour). These values were significantly higher than in the unirradiated lobes of Gunn rat livers, which were less than 5% of the activity in livers of Wistar- RHA rats (P,0.01). Similarly, ugt1a1 activity in the median lobe of Gunn rats that had received preparative HIR to that lobe only was about 21–24% of the activity in wildtype RHA rats, and was significantly higher than the activity in the unirradiated left, right, and caudate lobes. The ugt1a1 activity was undetectable in untreated Gunn rats (Figure 6). Discussion Crigler-Najjar syndrome type 1 (CN1) is a potentially lethal autosomal recessive disorder characterized by life long non- hemolytic unconjugated hyperbilirubinemia. The disorder results from a deficiency of ugt1a1 activity, which is critical for glucuronidation and excretion of bilirubin [27]. Although phototherapy and plasmapheresis treatment have extended the life expectancy of patients with this disorder, liver transplantation is still the only definitive treatment [27]. As liver transplantation is a formidable surgical procedure, hepatocyte transplantation is being explored as a minimally invasive alternative to whole organ transplantation in the management of several liver-based meta- bolic disorders and liver failure [1–7,27]. Because a fraction of the normal hepatic enzyme content is often sufficient to support metabolic function, we and others have proposed that transplan- tation of isolated hepatocytes might cure several inherited liver- based metabolic disorders. After extensive preclinical research in animal models, we performed hepatocyte transplantation in a patient with CN1, which resulted in a significant reduction of serum bilirubin levels [1]. However, the levels were not low enough to obviate the need for phototherapy. Following our report, several groups confirmed that hepatocyte transplantation successfully, but partially, ameliorated hyperbilirubinemia in CN1 patients [1,4,28,29]. Thus, it was clear that the number of hepatocytes that could be safely engrafted into the liver of CN1 patients was not sufficient to achieve complete cure of the disease. We then performed further preclinical studies to achieve proliferation of the engrafted hepatocytes within the liver. Host hepatocytes in CN1 patients, as in most other liver-based inherited metabolic disorders, have normal proliferative capacity. As the liver to body weight ratio is maintained within a tight limit by strong physiological mechanisms, the engrafted wildtype cells can proliferate only by replacing the host hepatocytes. Massive hepatic repopulation has been achieved by genetic, chemical and physical means to limit the mitotic capacity of the host [12,14,30,31]. Under these circumstances, mitotic stimuli after hepatocyte PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e46775