Autophagy-Modulated Human Bone Marrow-Derived Mesenchymal Stem Cells Accelerate Liver Restoration in Mouse Models of Acute Liver Failure

Abstract

Background: Mesenchymal stem cells (MSCs) have been recently received increasing attention for cell-based therapy, especially in regenerative medicine. However, the low survival rate of these cells restricts their therapeutic applications. It is hypothesized that autophagy might play an important role in cellular homeostasis and survival. This study aims to investigate the regenerative potentials of autophagy-modulated MSCs for the treatment of acute liver failure (ALF) in mice.

Methods: ALF was induced in mice by intraperitoneal injection of 1.5 ml/kg carbon tetrachloride. Mice were intravenously infused with MSCs, which were suppressed in their autophagy pathway. Blood and liver samples were collected at different intervals (24, 48 and 72 h) after the transplantation of MSCs. Both the liver enzymes and tissue necrosis levels were evaluated using biochemical and histopathological assessments. The survival rate of the transplanted mice was also recorded during one week.

Results: Biochemical and pathological results indicated that 1.5 ml/kg carbon tetrachloride induces ALF in mice. A significant reduction of liver enzymes and necrosis score were observed in autophagy-modulated MSC-transplanted mice compared to sham (with no cell therapy) after 24 h. After 72 h, liver enzymes reached their normal levels in mice transplanted with autophagy-suppressed MSCs. Interestingly, normal histology without necrosis was also observed.

Conclusion: Autophagy suppression in MSCs ameliorates their liver regeneration potentials due to paracrine effects and might be suggested as a new strategy for the improvement of cell therapy in ALF.

Keywords: Acute liver failure; Autophagy; Mesenchymal stem cells.

Fig. 1

Confirmation of acute liver failure (ALF) induction in mouse models using different concentrations of CCl4. (A) Histo/histopatological assay. Liver samples were collected 24 h after treatment with 0.5, 1, 1.5, 2 and 2.5 ml/kg CCl₄ in olive oil (three mice for each dose). Mice without any injection and mice injected with olive oil were considered as control. Multiple central lobular necroses (long narrow arrows) was induced using 1 ml/kg CCl₄. The injection of 1.5 ml/kg CCl₄ induced interalobular necrotic bridge (short thick arrow) and diffuse necrosis (medium narrow arrows). Severe diffuse necrosis and complete destruction of lobules (short arrows) were seen after the injection of higher CCl₄ doses (H&E stain, magnification 40×, scale bar: 500 µm). (B) Biochemical assay of ALF-induced mice (three mice for each dose). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were evaluated 24 h after the injection of 0.5, 1, 1.5, 2 and 2.5 ml/kg CCl₄ in olive oil. ALT and AST serum levels were significantly higher in CCl₄-received mice in comparison to the control groups (those without any injection and those that received olive oil alone). (mean±SD, *P≤0.05, **P≤0.01, ***P≤0.001). (C) The quantification of necrosis score in at least 30 microscopic fields. Administration of 1 and 1.5 ml/kg CCl₄ induced type two necrosis but 2 and 2.5 ml/kg CCl₄ led to type three necrosis. (D) The survival rate of ALF-induced mice. The survival rates of 10 ALF-induced mice were recorded during seven days after CCl₄ injection. Also, >50% death was recorded in 2 and 2.5 ml/kg CCl₄-treated groups. Altogether, 1.5 ml/kg CCl₄ was chosen as the optimized ALF-inducing dose. Cont, control

Fig. 2

Therapeutic effects of different mesenchymal stem cells (MSCs) in ALF-induced mice 24 h after cell therapy. Normal MSCs and autophagy-modulated MSCs were transplanted in acute liver failure (ALF)-induced mice, and normal mice that received normal MSCs were considered as control. Their therapeutic potentialities were evaluated after 24 h. (A) Histo/histopathological assay. Accelerated repairing was observed in ALF-MSC-shRNA 3 mice received autophagy-modulated MSCs. Mild necrosis (narrow arrows) without interalobular necrotic bridge vs. persistent interalobular necrotic bridge (thick arrow) in mice that received no MSCs (sham group) and multiple diffuse necrosis in ALF-MSC that received MSCs without any autophagy modulation (H&E stain, magnification ×100, scale bar: 500 µm). (B) Biochemical assay. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum levels were reduced significantly in mice received autophagy-modulated MSCs (ALF-MSC-shRNA 3) and MSCs without any autophagy modulation (ALF-MSC) compared with sham. (mean±SD, *P≤0.05 and **P≤0.01). (C) Necrosis score quantification. Reduction of necrosis score from two to one was occurred after the administration of MSC-shRNA 3. There was no alteration of necrosis score in other groups

Fig. 3

Therapeutic effects of different mesenchymal stem cells (MSCs) in acute liver failure (ALF)-induced mice 48 h after cell therapy. Normal MSCs and autophagy-modulated MSCs were transplanted in ALF-induced mice. Normal mice received normal MSCs and were considered as control. Their therapeutic potentialities were evaluated after 48 h. (A) Histo/histopathological assay. There was no significant necrosis in ALF-MSC-shRNA 3, and inflammatory cells (narrow arrows) contributed to a progressive regeneration rate. Persistent diffuse necrosis and interalobular necrotic bridge (thick arrow) were observed in sham group (H&E staining, magnification 100×, scale bar: 500 µm). (B) Biochemical assay. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were decreased continually. Their serum levels were significantly lower in ALF-MSC-shRNA 3 and ALF-MSC compared with sham (mean±SD, *P≤0.05, **P≤0.01, ***P≤0.001). (C) Necrosis score quantification. As shown in the figure, necrosis score was reduced from one to zero in MSC-shRNA 3 and from two to one in ALF-MSC. However, the necrosis score in sham group was two

Fig. 4

Therapeutic effects of different mesenchymal stem cells (MSCs) in acute liver failure (ALF)-induced mice 72 h after cell therapy. Normal MSCs and autophagy-modulated MSCs were transplanted in ALF-induced and normal mice that received normal MSCs were considered as control. Their therapeutic potentialities were evaluated after 72 h. (A) Histo/histopathological assay. A decreased number of inflammatory cells (narrow arrows) and the presence of repaired cells (thick arrow) were observed in ALF-MSC-shRNA 3. ALF-MSC (received MSCs without any manipulation) revealed increased inflammation and mild necrosis. Necrosis was observed in sham group (H&E stain, magnification 100×, scale bar: 500 µm). (B) Biochemical assay. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels approximately returned to the normal level in ALF-shRNA 3 compared with sham (mean±SD, ***P≤0.01). There was no significant difference between ALT and AST of ALF-MSC and sham. (C) Necrosis score quantification. Necrosis score was zero in MSC-shRNA 3 but the score was one in ALF-MSC and two in the sham group

Fig. 5

The survival rate of different transplanted mice. The survival rate of understudied mice was recorded during seven days of mesenchymal stem cells (MSCs) post transplantation. Approximately all ALF-induced mice transplanted with MSC-shRNA 3 (ALF-MSC-shRNA 3) stayed alive during this time period