augments the initial engraftment of the donor cells by transiently disrupting the endothelial barrier [11]. In addition, HIR allows the engrafted cells to respond more efficiently to mitotic stimuli, such as partial hepatectomy or expression of hepatic growth factor (HGF), than the host hepatocytes. This leads to extensive repopulation of the liver with complete correction of the metabolic defect in the uridinediphosphoglucuronate glucuronosyltransferase 1A1 (ugt1a1)-deficient Gunn rat model of Crigler-Najjar syn- drome type 1 (CN1) [16] and a mouse model of primary hyperoxaluria type 1 (PH1) [17]. Despite the encouraging results in the rodent models, whole liver HIR has not been tested as a preparative regimen in clinic trials because the risk of RILD may be greater in primates, including humans. However, recent clinical studies using confor- mal HIR have shown that partial irradiation of the liver markedly reduces the occurrence of RILD and allows far greater doses of radiation to be delivered safely [18,19]. Therefore, we wanted to test the hypothesis that preparative HIR of one or two selected liver lobes should result in a sufficient wild-type donor cell mass for correction of an inherited metabolic liver disease. Initially, for the convenience of identification of the donor cells, we transplanted hepatocytes from F344 rats into congeneic DPPIV2 recipients by injection into the splenic pulp [15]. To verify the regiospecificity of liver repopulation, we irradiated one half of the median lobe, followed by mitotic stimulation by injecting an adenovector expressing HGF (Ad-HGF) intravenously [20]. To determine whether repopulation of one or two of the five liver lobes is sufficient to correct hyperbilirubinemia in Gunn rats, wildtype hepatocytes from congeneic Wistar rats (Roman High Avoidance, RHA strain) were transplanted after regional HIR to the median lobe (30% of liver mass) or median plus left anterior lobes (60% of liver mass). Mitotic stimulation was provided by injection of Ad-HGF. Our results demonstrate for the first time that repopulating a single liver lobe by regional HIR is sufficient to cure hyperbilirubinemia in Gunn rats. Combined with mitotic stimulation, conformal HIR permits marked liver repopulation by transplanted hepatocytes, thereby correcting hyperbilirubinemia, as seen after whole liver HIR. Materials and Methods Animals Male, 250–300 g wildtype Fisher 344 (F344) rats were purchased from Taconic Farms (German Town, NY). Wildtype Wistar-RHA rats, congeneic Gunn rats and DPPIV- deficient F344 rats were bred and maintained on normal rat chow diet and the climate-controlled facility of the Institute for Animal Studies at the Albert Einstein College of Medicine in a 12-hr light/dark cycle. This study was approved by the Institutional Animial Care and Use Committee of the Albert Einstein College of Medicine. Viral Vector An adenoviral vector expressing human HGF was generated by the Cell Culture and Genetic Engineering Core of the Marion Bessin Liver Research Center of the Albert Einstein College of Medicine [21]. Regional Hepatic irradiation Regional HIR in rats was performed as reported with some modifications [15]. Briefly, the liver was exposed by midline incision and all liver lobes and other abdominal organs were covered by 2 mm-thick lead shields, except the region targeted to be irradiated. We have previously conducted a dose escalation (15–50 Gy) study of preparative HIR and demonstrated 50 Gy Regiospecific Repopulation of the Liver was optimal in achieving .80% repopulation of Gunn rat livers by transplanted hepatocytes [16]. We, therefore, administered a HIR dose of 50 Gy selectively to the exposed half of the median lobe in DPPIV2 rats (n = 3), and the whole median lobe (n = 6) or median and left lobe (n = 8) in Gunn rats, using a Philips orthovoltage unit operating at 320 kVP, 5 mA, and 0.5 mm copper filtration at 200cGy/min. Control Gunn rats (n = 4) underwent laparotomy, but no HIR was delivered. In our study we had to deliver intra- operative HIR to rat livers in order to spatially confine the irradiation dose to specific liver lobes because of the use of orthovoltage animal irradiator and the small volume of liver in rats. However clinical translation of our preparative HIR technique could be easily achieved in humans via external beam RT using modern 3D conformal techniques. Indeed, fractionated external beam RT has been used as a preparative regimen for hepatocyte transplantation in F344 rats using a clinical linear accelerator [22,23]. Hepatocyte isolation and transplantation Donor hepatocytes were isolated from DPPIV+ wildtype F344 rats or ugt1a1+ wildtype Wistar-RHA rats by an in situ two-step collagenase perfusion procedure [24]. Male DPPIV2 Fisher rats and Gunn rats (180–200 g body weight) that had received regional HIR served as recipients for congeneic normal F344 and Wistar- RHA rat hepatocytes, respectively. Twenty-four hours after conformal HIR, 107 hepatocytes were transplanted by intrasplenic injection as described [16]. Ad-HGF (1011 particles) was injected via the tail vein 24 hours after hepatocyte transplantation [20]. Control rats received sham surgery (no HIR), Ad-HGF injection and hepatocyte transplantation. Histochemical staining for DPPIV activity The median lobe subjected to conformal HIR was separated from other liver lobes and snap-frozen in pre-cooled 2-methylbu- tane at 280uC. Five-micron thick cryostat liver sections were fixed for 5 min in cold 95% ethanol/5% glacial acetic acid (99:1vol/vol) on ice and followed by a 5 min wash in cold 95% ethanol on ice. The sections were then air-dried and stained for DPPIV histochemistry as previously described [25]. Immunofluorescent staining for ugt1a1 expression Five-micron frozen sections of liver tissues were fixed with ice- cold acetone/methanol for 45 minutes. Then the sections were incubated for 60 minutes at room temperature with the primary antibody (rabbit polyclonal antibody raised against a 31-mer polypeptide corresponding to the unique amino-terminal domain of rat ugt1a1) at 1:50 dilution after blocking with donkey serum. The sections were washed thrice with phosphate-buffered saline (PBS) and incubated for 45 minutes at room temperature with the secondary antibody, Cy3-conjugated donkey anti-rabbit IgG (H+L) at 1:200 dilution (Jackson ImmunoResearch Laboratories, Inc.). After extensive washing, the cover slips were mounted onto the slides with Vectashield (Vector Laboratories) and imaged using a Nikon Eclipse TE 2000-S fluorescent microscope. Measurement of ugt1a1 protein content in different lobes Liver fragments (100–150 mg) from each lobe were homoge- nized in a glass-Teflon motorized homogenizer and protein concentrations were determined (Bio-Rad protein assay). The proteins (100 mg/lane) were subjected to electrophoresis on 5%– 7.5% polyacrylamide gradient SDS/glycine gel. Following elec- trophoresis, proteins were transferred onto a polyvinylidene PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46775