6B). At day 21 several pan-centromeric-positive cells did not express pan-cytokeratin, suggesting that some undifferentiated HLSCs were still present Metabolism inhibitor in liver parenchyma (Fig. 6B). In the GalN/LPS model of FLF, HLSC-CM showed a similar protective activity on liver function and morphology compared to HLSCs (Figs. 1C, 7A,B). The increased presence of PCNA-positive cells demonstrated liver regeneration in mice treated with HLSC-CM (Fig. 7C) and apoptosis was significantly reduced compared to mice treated with vehicle alone (Fig. 7D). In vitro studies confirmed that HLSC-CM at doses as low as 29 µg protein/mL protected human hepatocytes from
GalN-induced apoptosis (Fig. 7E). Moreover, increasing concentrations of HLSC-CM enhanced proliferation of human hepatocytes (Fig. 7F). Similar results were obtained with murine
hepatocytes isolated from both normal and FLF mice (Fig. 3S) As shown in Table 1 and Fig. 4S, HLSC-CM contained several growth factors/cytokines potentially involved in liver protection and regeneration, the most represented of which were interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and MSP, reconfirmed by single ELISA. The concentration of HGF in HLSC-CM was ∼50-fold higher than in MSC-CM. Treatment of HLSC-CM with neutralizing antihuman HGF antibody abrogated the protective effect of HLSC-CM (Fig. 1C), and the survival rate of mice treated with rhHGF was ∼40%, suggesting a relevant role of HGF in the hepatoprotective Selleckchem Trametinib effect of HLSC-CM (Fig. 1C). We also evaluated the amount of human and murine VEGF, IL-6, and HGF in SCID mice with FLF after intravenous injection with HLSCs. Human HGF and IL-6 levels in mouse serum peaked at 7 hours after HLSC injection (hIL6 = 596 ± 390 pg/mL; hHGF = 482 ± 370 pg/mL), decreased
after 24 hours (hIL6 = 46 ± 19 pg/mL; hHGF = 2 ± 1.6 pg/mL), and were undetectable after day 7. Human VEGF was undetectable. In mice injected with HLSCs, murine HGF was not significantly different from untreated controls (respectively 101 ± 49 pg/mL versus 107 ± 19 pg/mL). An increase of murine HGF (159 ± 106 pg/mL) was observed after 24 hours, decreasing thereafter. Levels of Amoxicillin murine VEGF and IL-6 did not increase at any time after HLSC administration. The results of our study can be summarized as follows: (1) HLSCs and HLSC-CM strikingly improved survival and reduced plasma levels of liver enzymes and ammonium in mice with FLF, and (2) the protective effect of HLSCs and HLSC-CM was due to reduced apoptosis, necrosis, and enhanced proliferation. Cell therapy based on hepatocyte transplantation is a potential treatment option in patients with acute liver failure.14 Transplanted hepatocytes may replace liver functions allowing endogenous regeneration. However, the availability of a suitable cellular source is a limiting factor, but stem cells could represent an ideal cell source for liver regeneration.