Transplanted human amniotic membrane-derived mesenchymal stem cells ameliorate carbon tetrachloride-induced liver cirrhosis in mouse

Abstract

Background: Human amniotic membrane-derived mesenchymal stem cells (hAMCs) have the potential to reduce heart and lung fibrosis, but whether could reduce liver fibrosis remains largely unknown.

Methodology/principal findings: Hepatic cirrhosis model was established by infusion of CCl₄ (1 ml/kg body weight twice a week for 8 weeks) in immunocompetent C57Bl/6J mice. hAMCs, isolated from term delivered placenta, were infused into the spleen at 4 weeks after mice were challenged with CCl₄. Control mice received only saline infusion. Animals were sacrificed at 4 weeks post-transplantation. Blood analysis was performed to evaluate alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Histological analysis of the livers for fibrosis, hepatic stellate cells activation, hepatocyte apoptosis, proliferation and senescence were performed. The donor cell engraftment was assessed using immunofluorescence and polymerase chain reaction. The areas of hepatic fibrosis were reduced (6.2%±2.1 vs. control 9.6%±1.7, p<0.05) and liver function parameters (ALT 539.6±545.1 U/dl, AST 589.7±342.8 U/dl,vs. control ALT 139.1±138.3 U/dl, p<0.05 and AST 212.3±110.7 U/dl, p<0.01) were markedly ameliorated in the hAMCs group compared to control group. The transplantation of hAMCs into liver-fibrotic mice suppressed activation of hepatic stellate cells, decreased hepatocyte apoptosis and promoted liver regeneration. More interesting, hepatocyte senescence was depressed significantly in hAMCs group compared to control group. Immunofluorescence and polymerase chain reaction revealed that hAMCs engraftment into host livers and expressed the hepatocyte-specific markers, human albumin and α-fetoproteinran.

Conclusions/significance: The transplantation of hAMCs significantly decreased the fibrosis formation and progression of CCl₄-induced cirrhosis, providing a new approach for the treatment of fibrotic liver disease.

Figure 1. Characterization of human amniotic membrane-derived mesenchymal stem cells.

(A) Human amniotic membrane-derived mesenchymal stem cells (hAMCs) display fibroblastic morphology in cultures. (B) and (C) The differentiation of hAMCs into adipocytos and osteoblasts in cultures. Cells were incubated in adipogenic or osteogenic medium and then analyzed by cytochemical staining with Oil Red-O (B) and Alizarin red (C), respectively.

Figure 2. hAMCs transplantation reduced hepatic fibrosis.

(A) Micrographs of Masson's trichrome stained liver paraffin sections from control group and hAMCs group. (B) Fibrotic areas were quantified by computer assisted image analysis. Each value is the mean±SEM of determinations in 8 mice of each group. *p<0.05 compared with control group.

Figure 3. hAMCs transplantation reduced CCl₄-induced hepatic stellate cells activation.

(A) Micrographs of liver frozen sections from control group and hAMCs group stained with immunofluorescence for Desmin (left panel), DAPI (middle panel) and merged (right panel). (B) Micrographs of liver frozen sections from control group and hAMCs group stained with immunofluorescence forα-SMA (left panel), DAPI (middle panel) and merged (right panel). (C) Micrographs of liver frozen sections from control group and hAMCs group stained with immunofluorescence for GFAP (left panel), DAPI (middle panel) and merged (right panel). (D) Representative Western blots of liver extracts from control group and hAMCs group for expression of α-SMA. β-actin was used as an invariant control. (E) α-SMA protein levels relative to β-actin protein level were assessed by densitometric analysis. Each value is the mean±SEM of determinations in 5 mice of each group. ** p<0.01 compared with control group.

Figure 4. hAMCs transplantation suppressed hepatocyte apoptosis and promoted hepatocyte regeneration.

(A) Micrographs of liver paraffin sections from control group and hAMCs group stained immunohistochemically for TUNEL and PCNA. (B) Representative Western blots of liver extracts from two groups for expression of HGF and bcl-2. β-actin was used as an invariant control. HGF and bcl-2 protein levels relative to β-actin protein level were assessed by densitometric analysis. Each value is the mean±SEM of determinations in 8 mice of each group. *p<0.05 compared with control group.

Figure 5. hAMCs transplantation suppressed hepatocyte senescence.

(A) Micrographs of liver section stained histochemically for SA-β-Gal. (B) SA-β-Gal positive hepatocytes per field. (C) The levels of SOD activity in mouse liver homogenate from control group and hAMCs group. (D)Representative Western blots of liver extracts from control group and hAMCs group for expression of p16^INK4a, p21^Cipl, p27^Kipl, Sirt 1 and prdx I expression in livers from hAMCs group or control group. β-actin was used as an invariant control. (E) p16^INK4a, p21^Cipl, p27^Kipl, Sirt 1 and prdx I protein levels relative to β-actin protein level were assessed by densitometric analysis. Each value is the mean±SEM of determinations in 8 mice of each group. FOV = Field of view. *p<0.05 compared with control group.

Figure 6. hAMCs engraft in CCl₄ injured livers.

(A) Micrographs of liver frozen sections stained with immunofluorescence for DiI (left panel), α-fetoprotein (middle panel) and merged (right panel). (B) Micrographs of liver frozen sections stained with immunofluorescence for DiI (left panel), albumin (middle panel) and merged (right panel). Bars = 100 µm.