No animal received any medical support (e.g., infusions, drugs) during the entire experimental period. The transplantation procedures were all performed by the same B-ultrasound expert with 5 years of extensive
experience. Animals were evaluated for up to 6 months after transplantation. As biochemical markers of liver metabolism, coagulation and hepatocyte damage, alanine aminotransferase (ALT), prothrombin time, total bilirubin, ammonia, blood urea nitrogen, and creatinine levels were analyzed find more prior to hBMSC transplantation (baseline data) and then on days 1, 2, and 3 and weeks 1, 2, 3, 5, and 8 after transplantation. Survival was analyzed using a Kaplan-Meier plot and log-rank analysis. The data are expressed as the mean ± SD and were evaluated via Student t test and one-way analysis of variance with SPSS software version 16.0 (SPSS, Chicago, IL). The significance for all statistical analyses was defined as P < 0.05. To determine the effect of the transplanted hBMSCs on liver regeneration, hBMSC-derived hepatocytes engrafted in liver tissues were tracked using immunohistochemistry with the human hepatocyte-specific marker ALB (Bethyl, Montgomery, TX) and a hepatocyte-specific antigen antibody (HSA) (Abcam, Cambridge, UK). Liver tissues were harvested after the pigs died of FHF in the control and PVT groups. In
the IPT group, because many Erlotinib unforeseen risks exist in FHF animals that undergo several partial hepatectomies and to ensure an adequate number of surviving animals for follow-up, liver tissue was
harvested from five animals. Three liver sections were harvested from each of the left, middle, and right lobes (10-20 g, each sample) via small partial hepatectomy under sterile conditions on weeks 2, 3, MCE 5, 10, 15, and 20 after transplantation. Immunohistochemical analyses of ALB and HSA were performed using serial sections. The hepatectomy procedure was performed by a surgeon with 10 years of experience in liver transplantation. Each liver tissue specimen was analyzed by hematoxylin and eosin (H&E) staining. For H&E staining, each liver tissue section (4-μm-thick) was heat-fixed at 60°C for 1 hour and stained with H&E as described.16 For immunohistochemistry, serial tissue sections were applied to poly-L-lysine-coated slides. After the sections were dewaxed, rehydrated, and washed, endogenous peroxidases were inactivated with 3% H2O2 for 10 minutes at room temperature. The sections were incubated overnight with primary anti-human antibodies (ALB, 1:10,000, and HSA 1:1,000) with no cross-reactivity to pig tissues. The sections were washed with phosphate-buffered saline three times and incubated with the appropriate secondary antibodies at 37°C for 1 hour.