necrosis, 2 = ,30% necrosis, 3 = ,60% necrosis, and 4 = .60% necrosis. Immunohistochemical detection of LacZ-positive cells and apoptosis To detect injected LacZ-positive MSCs in the liver, the experimental rats were sacrificed 6 h after reperfusion. Thin frozen sections (10 mm) of the liver were fixed in 0.2% glutaraldehyde for 10 min at room temperature and incubated in a solution (X-gal) containing 1 mg/ml 5-bromo-4-chloro-3- indolyl b-D-galactopyranoside (X-gal; Sigma-Aldrich, USA), 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, and 2 mM MgCl2 in PBS at 37uC for 20 h. Paraffin sections of livers sacrificed 6 h after reflow were prepared. Apoptosis was determined by in situ detection of DNA fragmentation using terminal deoxynucleotidyl transferase-medi- ated 29-deoxyuridine 59-triphosphate nick-end labeling (TUNEL) assay. Quantification of TUNEL-positive hepatocyte nuclei was assessed by calculating the mean of the number of TUNEL- positive hepatocytes in 10 random 2006 fields per animal. References 1. Ben-Haim M, Emre S, Fishbein TM, Sheiner PA, Bodian CA, et al. (2001) Critical graft size in adult-to-adult living donor liver transplantation: impact of the recipient’s disease. Liver transpl 7: 948–953. 2. Azoulay D, Astarcioglu I, Bismuth H, Castaing D, Majno P, et al. (1996) Split- liver transplantation. The Paul Brousse policy. Ann Surg 224: 737–746. 3. Goss JA, Yersiz H, Shackleton CR, Seu P, Smith CV, et al. (1997) In situ splitting of the cadaveric liver for transplantation. Transplantation 64: 871–877. 4. Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, et al. (2008) IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury. Stem Cells 26: 2705–2712. 5. Tsai PC, Fu TW, Chen YM, Ko TL, Chen TH, et al. (2009) The therapeutic potential of human umbilical mesenchymal stem cells from Wharton’s jelly in the treatment of rat liver fibrosis. Liver transpl 15: 484–495. 6. Oyagi S, Hirose M, Kojima M, Okuyama M, Kawase M, et al. (2006) Therapeutic effect of transplanting HGF-treated bone marrow mesenchymal cells into CCl4-injured rats. J Hepatol 44: 742–748. 7. Wang Y, Sun Z, Qiu X, Li Y, Qin J, et al. (2009) Roles of Wnt/beta-catenin signaling in epithelial differentiation of mesenchymal stem cells. Biochem Biophys Res Commun 390: 1309–1314. 8. Forte A, Finicelli M, Mattia M, Berrino L, Rossi F, et al. (2008) Mesenchymal stem cells effectively reduce surgically induced stenosis in rat carotids. J Cell Physiol 217: 789–799. 9. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284: 143–147. 10. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, et al. (2006) Position Paper Minimal criteria for defining multipotent mesenchymal stromal cells. The international Society for Cellular Therapy position statement. Cytotherapy 8: 315–317. 11. Dahlke MH, Hoogduijn M, Eggenhofer E, Popp FC, Renner P, et al. (2009) Toward MSC in solid organ transplantation: 2008 position paper of the MISOT study group. Transplantation 88: 614–619. 12. van Poll D, Parekkadan B, Cho CH, Berthiaume F, Nahmias Y, et al. (2008) Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo. Hepatology 47: 1634–1643. 13. Parekkadan B, van Poll D, Suganuma K, Carter EA, Berthiaume F, et al. (2007) Mesenchymal Stem Cell-Derived Molecules Reverse Fulminant Hepatic Failure. PLoS ONE 2: e941. Impact of MSCs against Hepatic I/R Injury Liver regeneration rate To estimate the recovery rate of rat liver weight after partial hepatectomy, the resected liver was weighed at the time of partial hepatectomy, and when the rats were sacrificed the remnant liver was excised and weighed. The liver regeneration rate was expressed as remnant liver weight/estimated whole liver weight. The original whole liver weight was extrapolated by calculating the resected liver weight/0.663, based on the results of a pilot study that the resected liver weight in our model was equal to 66.3% of the original whole liver weight (66.365.5%, n = 6). Statistical analysis The results are given as the mean 6 SD. Statistical analysis was conducted using the student t-test for continuous data and the Mann-Whitney test for discontinuous data. A P value of ,0.05 was considered to be statistically significant. Author Contributions Conceived and designed the experiments: HK YF T. Teratani T. Tsuruyama SU. Performed the experiments: HK T. Teratani EK. Analyzed the data: HK. Contributed reagents/materials/analysis tools: EK JI NK KN. Wrote the paper: HK. 14. Lam SP, Luk JM, Man K, Ng KT, Cheung CK, et al. (2010) Activation of interleukin-6-induced glycoprotein 130/signal transducer and activator of transcription 3 pathway in mesenchymal stem cells enhances hepatic differentiation, proliferation, and liver regeneration. Liver transpl 16: 1195–1206. 15. Sato Y, Araki H, Kato J, Nakamura K, Kawano Y, et al. (2005) Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood 106: 756–763. 16. Baertschiger RM, Serre-Beinier V, Morel P, Bosco D, Peyrou M, et al. (2009) Fibrogenic Potential of Human Multipontent Mesenchymal Stromal Cells in Injured Liver. PLoS ONE 8: e6657. 17. Zhang D, Jiang M, Miao D (2011) Transplanted Human Amniotic Membrane- Derived Mesenchymal Stem Cells Ameliorate Carbon Tetrachloride-Induced Liver Cirrhosis in Mouse. PLoS ONE 2: e16789. 18. Hakamata Y, Murakami T, Kobayashi E (2006) ‘‘Firefly rats’’ as an organ/ cellular source for long-term in vivo bioluminescent imaging. Transplantation 81: 1179–1184. 19. Inoue H, Ohsawa I, Murakami T, Kimura A, Hakamata Y, et al. (2005) Development of new inbred transgenic strains of rats with LacZ or GFP. Biochem Biophys Res Commun 329: 288–295. 20. Meer C, Kley GA, Valkenburg PW (1976) Studies on the cause of death after permanent and temporary occlusion of the portal vein in rats. Circ Shock 3: 191. 21. Yadav SS, Gao W, Harland RC, Clavien PA (1998) A new and simple technique of total hepatic ischemia in the mouse. Transplantation 65: 1433–1436. 22. Banas A, Yamamoto Y, Teratani T, Ochiya T (2007) Stem cell plasticity: Learning from hepatogenic differentiation strategies. Dev Dyn 236: 3228–3241. 23. Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, et al. (2007) Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology 46: 219–228. 24. Inoue S, Hakamata Y, Kaneko M, Kobayashi E (2004) Gene therapy for organ grafts using rapid injection of naked DNA: application to the rat liver. Transplantation 77: 997–1003. 25. Fujishiro J, Takeda S, Takeno Y, Takeuchi K, Ogata Y, et al. (2005) Gene transfer to the rat kidney in vivo and ex vivo using an adenovirus vector: factors influencing transgene expression. Nephrol Dial Transplant 20: 1385–1391. 26. Suzuki S, Nakamura S, Koizumi T, Sakaguchi S, Baba S, et al. (1991) The beneficial effet of a prostaglandin I2 analog on ischemic rat liver. Transplan- tation 52: 979–983. PLoS ONE | www.plosone.org 8 April 2011 | Volume 6 | Issue 4 | e19195