5 × 106 (n = 5) or 0.2 × 106 HLSCs (n = 5); group 7, FLF mice intraperitoneally injected with concentrated HLSC-CM (n = 14); group 8, FLF mice intraperitoneally injected with concentrated MSC-CM (n = 5); group 9, FLF mice intraperitoneally injected with vehicle (PBS; n = 5); group 10, FLF mice intraperitoneally injected with concentrated HLSC-CM pretreated for 1 hour at 4°C with anti-HGF blocking antibody (100
mg/mouse; GeneTex, Aachen, Germany) (n = 5); group 11, FLF mice intraperitoneally injected with 25 ng/mouse of rhHGF (PreproTech, Rocky Hill, NJ,) (n = 5). The intravenous injection (120 mL) was performed in the vein of the tail within 30 seconds. In all experiments, cells cultured in T75 flasks until the 2-6 passage were detached by trypsin (0.5%
w/v), washed, and resuspended selleck in phosphate-buffered saline (PBS). Before in vivo injection, HLSCs were stained with the CellTrace CFSE Cell Proliferation Kit (Molecular Probes, Life Technologies, Paisley, UK).11 Full details are provided in the online Supporting Information. Full details are provided in the online Supporting Information. Full details are provided in the online Supporting Information. Data were analyzed using t tests, analysis of variance (ANOVA) with Erlotinib Newmann-Keuls’ or ANOVA with Dunnett’s multicomparison tests as appropriate. For survival experiments, a log-rank test was conducted. P < 0.05 was considered significant. The intraperitoneal administration of 600 mg/kg of GalN and 125 ng of LPS in SCID mice induced FLF with a 100% mortality rate within 8 hours (Fig. 1A). To evaluate whether HLSCs might rescue mice with FLF, the cells were administered 30 minutes after GalN/LPS-induced injury in different protocols: single intraperitoneal injection (3 × 107 cells); six repeated intravenous injections (3.3 × 105 cells,
total: 2 × 106 cells); LP injection at two concentrations (0.2 and 0.5 × 106). Survival rates were 77%, 100%, and 100% in the case of intraperitoneal, intravenous, and LP injections, see more respectively (Fig. 1A,B). An equal number of MSCs was intravenously injected six times (3.3 × 105 cells, total: 2 × 106 cells) using the same protocol. The survival rate was 33% (Fig. 1A). Cell injection timings were chosen on the basis of preliminary experiments performed at 30 minutes, 1 and 4 hours, which showed that cell administration after 30 minutes did not improve survival. As paracrine mechanisms of adult stem cells were related to their secreted factors, we investigated and compared the effects of HLSC-CM and MSC-CM to that of HLSCs. As shown in Fig. 1C, the intraperitoneal injection of HLSC-CM induced an 80% survival. Conversely, MSC-CM did not protect mice from FLF. All controls treated with vehicle alone (PBS) died within 8 hours, suggesting that the effect on survival did not depend on mice hydration (Fig. 1C). Histological analysis of mice treated with GalN/LPS showed extensive necrosis (Fig. 2A) and apoptosis (Fig.