were crossed with SCID or RAG2/2 mice, as was previously described[11]. For G-CSF studies, 8 to 10 week old immune competent C57BL mice and C57BL hemophilic (C57BL Hem F8) mice were used. All mice were kept in small cages (up to five animals per cage) and fed sterile food. Pig embryos were obtained from the Lahav Institute of Animal Research (Kibbutz Lahav, Israel). Pregnant sows were operated on at embryonic day 28 (E28) for the harvest of liver, and at E42 for the harvest of pancreas and spleen, under general anesthesia. Warm ischemia time was less than 10 minutes and the embryos were transferred to cold PBS. Spleen, pancreas and liver precursors for transplantation were extracted under a light microscope and were kept in sterile conditions at 4uC in RPMI 1640 (Biological Industries, Beit HaEmek, Israel) prior to transplantation. Cold ischemia time until transplantation was less than 2 hours. Mouse embryos were obtained from C57BL/6 pregnant female mice. Pregnant mice were operated on at embryonic day 15 (E15), to remove fetal spleen tissue, and at day 16 (E16) for liver and pancreatic tissues, under general anesthesia. Warm ischemia time was less than 10 minutes, and the embryos were transferred to cold PBS. Spleen, pancreas and liver precursors for transplantation were extracted under a light microscope and were kept in sterile conditions at 4uC in RPMI 1640 (Biological Industries, Beit HaEmek, Israel) prior to transplantation. Cold ischemia time until transplantation was less than 2 hours. Transplantation Procedure Transplantation of pig precursors was completed as previously described[36]. Briefly, transplantation of the embryonic precursors was performed under general anesthesia (2.5% 2,2,2-Tribromo- ethanol in PBS, 10 ml/kg intraperitoneally). Host kidney was exposed through a left lateral incision. A 1.5-mm incision was made at the caudal end of the kidney capsule and donor precursors were grafted under the kidney capsule in fragments 1–2 mm in diameter. Morphometric Analysis Porcine E42 spleen and pancreatic grafts were formalin fixed and embedded in paraffin 3 months following transplantation. Consecutive 40 mm sections were cut and stained with pig specific anti ki67 antibody. The areas of interest were quantified using the Image Pro program (Media Cybernetics). ELISA Measurements of Pig Insulin and Albumin A porcine/human insulin kit (Catalog No. K6219, DAKO), in which the primary pig anti-insulin antibody does not cross-react with mouse insulin, was used, according to the manufacturer’s instructions, to follow pig insulin levels. Pig albumin in mouse serum was measured by a standard ELISA using primary affinity purified goat anti-pig albumin antibody (human, mouse, and bovine adsorbed), and secondary pig-specific horseradish-peroxi- dase conjugated antibody (Catalog Nos. A100–210A and A100– 210P, Bethyl). References Organ Size Control Histochemistry Histochemistry included hematoxylin/eosin (H&E) and periodic acid/Schiff (PAS) staining. For immunohistochemical labeling, the following antibodies were used: Goat anti-pig albumin antibody (Bethyl Laboratories, Montgomery, TX), rabbit anti-human glucagon (DAKO), guinea pig anti-rabbit insulin (DAKO), mouse anti porcine CD31 (Serotec, Enco Scientific Services Ltd Israel) mouse anti porcine vimentin (clone V9) (Dako) and mouse anti- human ki67 (clone MIB-1) (Dako). Paraffin sections (4 mM) were xylene deparaffinized and rehydrated. Endogenous peroxidase was blocked with 0.3% H2O2 in 70% methanol for 10 minutes. Antigen-retrieval procedures were performed according to the manufacturer’s (DAKO) instructions. After blocking, both paraffin sections and 6-mM cryosections were incubated with specific first antibody for 60 minutes. Detection of antibody binding was performed by using the following secondary reagents: DAKO peroxidase EnVision system for the detection of mouse and rabbit antibodies, and Sigma biotinylated anti-goat antibody (followed by extra avidin peroxidase reagent) for goat antibodies. In all cases, diaminobenzidine was used as a chromogen. Immunofluorescence protocols were applied using secondary antibodies: Donkey anti mouse Texas red (Jackson), and donkey anti rat conjugated CY2 or Texas red (Jackson). Treatment with Inhibitors Enoxaparine (Clexane 20 mg/0.2 ml, Rhone-poulenc, France) was used at a dosage of 200 mg/mouse, (dissolved in PBS) and 0.2 ml of the final solution was injected subcutaneously into each mouse once a day. Dabigatran etexilate (Boehringer Ingelheim Pharma KG, Biberach, Germany) was administered orally at a dosage of 30 mg/kg. A final volume of 0.3 ml dissolved in DDW was administered daily. PAR1 and PAR4 antagonists: The palmitoylated peptides: pal-RCLSSSAVANRS (PAR1 antagonist) and pal-SGRRYGHALR (PAR4 antagonist) were prepared by solid-phase peptide synthesis using in situ neutralization/HBTU by Hadar Biotec, Israel. The mice were treated by vehicle control, or with PAR1antagonist or PAR4 antagonist 0.5 mg/kg, intra- peritoneally on daily basis. Thrombin cleaved form of Osteopontin (tcOPN) was produced as previously described[37], administration of tcOPN was twice daily by orbital vein injection through all transplantation period. Statistical Analysis Comparisons between groups were evaluated by the Student’ test. Data were expressed as mean6SD, and were considered statistically significant at p values of 0.05 or less. Author Contributions Conceived and designed the experiments: AA DT GR YR. Performed the experiments: AA DT ES CR YK SC OT HK SEF. Analyzed the data: AA DT ES JJH GR YR. Contributed reagents/materials/analysis tools: UM JJH GR YR. 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(2005) Embryonic pig liver, pancreas, and lung as a source for transplantation: PLoS ONE | www.plosone.org 9 December 2009 | Volume 4 | Issue 12 | e8362