Therapeutic effect of hepatocyte growth factor-overexpressing bone marrow-derived mesenchymal stem cells on CCl4-induced hepatocirrhosis.

Hepatocirrhosis is one of the most severe complications of chronic hepatic disease in terms of medical intervention, and the available therapies are limited and not very successful. In this study, bone marrow-derived mesenchymal stem cells (BM-MSCs) from host rats were transduced with an adenoviral vector labelled with green fluorescent protein (EGFP) to overexpress hepatocyte growth factor (HGF). The therapeutic effect of these modified stem cells (HGF-BM-MSC group) transplanted intravenously into hepatocirrhosis model rats treated with CCl4 was evaluated using serological, biochemical and histological approaches. We compared the rats in the HGF-BM-MSC group with those in the other groups (rats treated with BM-MSCs, rats treated with HGF and untreated rats (Controls)) in detail. The localisation of EGFP-tagged BM-MSCs in the injured liver was evaluated using a microscope, and the cells co-expressed hepatocyte nuclear factor 4α, albumin and cytokeratin 18. After treatment for 4 weeks, the HGF-BM-MSC, BM-MSC and HGF groups exhibited increased protein and mRNA levels of hepatocyte nuclear factor 4α, albumin and cytokeratin 18, but decreased levels of aspartate aminotransferase, alanine aminotransferase and total bilirubin. These findings indicate that BM-MSC transplantation and HGF application have great potential for the treatment of hepatocirrhosis.

Mesenchymal Stem Cells with Enhanced Bcl-2 Expression Promote Liver Recovery in a Rat Model of Hepatic Cirrhosis.

BACKGROUND/AIMS: Mesenchymal stem cell (MSC) transplantation has emerged as an option for the treatment of chronic hepatic cirrhosis, while its therapeutic efficacy could be improved. The bcl-2 gene is anti-apoptotic and can help cell survival and proliferation. Therefore, we explored whether transplanted MSCs with enhanced bcl-2 expression may be beneficial in the treatment of experimental cirrhosis in rats. METHODS: MSCs were isolated from rat bone marrow, expanded in vitro and transfected with adeno-associated virus (AAV) engineered the bcl-2 gene (AAV-bcl-2). Rats with cirrhosis induced by carbon tetrachloride (CCl4) were treated with AAV-bcl-2 infected BMSCs-AAV-bcl-2, with the cells traced in vivo post transplantation. Liver pathology and function were evaluated 7, 14, 21, and 28 days post transplantation, respectively. RESULTS: On day 7 post transplantation, the infused AAV-bcl-2 had integrated into the hepatocyte-like cells (HLCs) that expressed albumin (ALB), Cytokeratin 18 (CK18), and hepatocytes nuclear factor 4a (HNF4a). On day 28 post transplantation, rats in the cirrhosis + BMSCs-AAV-bcl-2 group showed the most dense HLCs, highest mRNA and protein levels of ALB, CK18, and HNF4a, compared to the other groups. Their liver function recovered most rapidly in 4 week observation, while histological sign of cirrhosis remained at the end of this period. CONCLUSION: BMSCs over expressing bcl-2 gene showed better survival, and enhanced the differentiation into hepatocytes-like cells, and appeared to promote the recovery of liver function in rats with experimental cirrhosis.

Stem cell transplantation for the treatment of liver diseases: A systematic review and meta-analysis.

BACKGROUND/AIMS: The therapeutic efficacy of stem cell transplantation in liver diseases has not yet been determined. The objective of this study was to conduct a meta-analysis to evaluate changes in liver function and clinical outcome following stem cell transplantation in patients with liver disease. MATERIALS AND METHODS: A literature review of NCBI, Cochrane Library, and MEDLINE was performed. Eligible studies reported liver function indices and prothrombin time (PT) before and after transplantation. The weighted mean difference (WMD) was defined by the distinction before and after stem cell transplantation. Either a fixed-effects model or random-effects model was used to analyze the data. RESULTS: A total of 17 publications involving 21 original studies were included. We found that the levels of serum albumin significantly increased at 4, 8, 12, 24, and 48 weeks after stem cell transplantation compared with that at baseline. Serum alanine aminotransferase levels notably decreased at 1, 3, 4, 12, 24, and 48 weeks after stem cell transplantation. Aspartate aminotransferase levels significantly decreased at 4, 8, 12, and 48 weeks after transplantation. Total bilirubin levels significantly decreased at 4, 12, 24, and 48 weeks after transplantation. PT decreased at 4, 12, 24, and 48 weeks after transplantation. The MELD score significantly decreased at 24 weeks after transplantation. Stem cell infusion through the hepatic artery had better biochemical outcomes than an injection through the portal vein. CONCLUSION: Our meta-analysis verified that there are clinical and biochemical improvements in patients who suffered from liver diseases after stem cell transplantation, suggesting that stem cell transplantation may be a viable clinical solution for treating such patients.

Ex vivo-expanded bone marrow stem cells home to the liver and ameliorate functional recovery in a mouse model of acute hepatic injury.

BACKGROUND: Stem cell transplantation provides a theoretical approach for liver regeneration medicine; it may promote liver regeneration and self-repair. However, the transplantation of bone marrow-mesenchymal stem cells expanded ex vivo as a therapy for liver disease has rarely been investigated. This study aimed to explore whether bone marrow stem cells expanded ex vivo home to the liver and foster hepatic recovery after CCl4 injury. METHODS: Bone marrow cells from BALB/c mice were expanded ex vivo by multiple-passage cultivation, characterized by cytoflow immunofluorescence, and pre-labeled with PKH26 before intravenous infusion into animals treated with CCl4. The integration of bone marrow cells into the liver was examined microscopically, and plasma hepatic enzymes were determined biochemically. RESULTS: Cultured bone marrow cells exhibited antigenic profiles comparable to those of primary medullary stem cells. Double immunofluorescence showed colocalization of these cells with proliferative activity and albumin expression in the liver of CCl4-treated mice. Densitometry showed increased in situ cell proliferation (50+/-14 vs 20+/-3 cells/high-power field, P<0.05) and albumin expression (149+/-25 vs 20+/-5 cells/high-power field, P<0.05) in the liver, as well as reduced serum aminotransferase levels (P<0.05) and better survival rates (P<0.05) in animals receiving cultured bone marrow cells relative to controls. CONCLUSIONS: Ex vivo-expanded bone marrow cells are capable of relocating to and proliferating in the chemically-injured liver. Transplantation of these pluripotent stem cells appears to improve serum indices of liver function and survival rate in mice after CCl4-induced hepatic damage.

STAT3 and beta-catenin signaling pathway may affect GSK-3beta expression in hepatocellular carcinoma.

BACKGROUND/AIMS: To study the correlation and significance of beta-catenin, STAT3 and GSK-3beta signaling pathway in hepatocellular carcinoma (HCC). METHODOLOGY: The HCC cell line HepG2 was transfected with small interfering RNA (siRNA) directed against 8-catenin or STAT3. After 72 and 96h, protein was extracted and the protein expression of beta-catenin, STAT3, and GSK-3beta was detected by Western blot analysis. RESULTS: After siRNA directed against beta-catenin was transfected into HepG2 cells, beta-catenin protein expression was decreased at 72 and 96h, GSK-3beta and p-GSK-3beta protein expression increased gradually at 72 and 96h, and STAT3 protein expression showed no change following transfection. After siRNA directed against STAT3 was transfected into HepG2 cells, STAT3 protein expression was decreased at 72 and 96h and beta-catenin, GSK-3beta and p-GSK-3beta protein expression all increased at 72h and decreased at 96 h after transfection. CONCLUSION: In HCC, the beta-catenin signaling pathway may regulate GSK-3beta protein expression and the STAT3 signaling pathway may regulate beta-catenin and GSK-3beta protein expression, thereby playing key roles during HCC genesis and development.

Hepatocyte growth factor promotes liver regeneration induced by transfusion of bone marrow mononuclear cells in a murine acute liver failure model.

BACKGROUND/PURPOSE: Bone marrow mononuclear cell (BMMC) transplantation has been shown to facilitate tissue and organ regeneration and repair. BMMC transplantation may be a potential therapy for acute liver failure, and its effect might be further improved. Hepatocyte growth factor (HGF) plays an important role in liver cell development, and may ameliorate hepatic fibrosis or cirrhosis in animal models. We therefore explored a potential synergistic effect of the co-application of HGF and BMMCs in liver regeneration following carbon tetrachloride (CCl(4))-induced acute hepatic injury. METHODS: We established a murine acute liver failure model induced by CCl(4) administration, and studied the effect of BMMC transplantation in combination with HGF. We used 4 groups of animals, one group was transfused with PKH26-labeled BMMCs (5 × 10(6)) and HGF [50 ng/(kg days) × 7 days] (BMMCs + HGF group), one group received BMMCs only, one group received HGF only, and one group received saline solution (0.9% NaCl) alone. The effects were examined by biochemical measurements of liver enzymes and quantitative image analysis for PKH26 labeling, and by determining proliferating cell nuclear antigen (PCNA) and albumin expression 4 weeks after the BMMC transplantation. RESULTS: PKH26-labeled BMMCs were detected in transplanted mouse livers, most of which expressed PCNA. PCNA and albumin expressions were increased significantly in the BMMCs + HGF group compared with the expressions of these parameters in the other 3 groups. Liver function, reflected by serum aminotransferase activity, was also improved in the BMMCs + HGF group relative to that in the other groups. CONCLUSIONS: Data from the present study appear to suggest that BMMC transplantation combined with HGF administration exhibits a synergistic beneficial effect on improving both functional and histological liver recovery in a mouse model of acute liver failure.

Granulocyte colony-stimulating factor enhances bone marrow mononuclear cell homing to the liver in a mouse model of acute hepatic injury.

BACKGROUND: Experiments have reported that granulocyte colony stimulating factor (G-CSF) can mobilize stem cells. However, few studies have examined the effect of G-CSF on bone marrow mononuclear cell (BMMC) mobilization, in particular regarding their capability to home to acutely injured liver. AIMS: The aim of this study was to evaluate the effort of G-CSF on BMMC homing to the liver following chemically-induced hepatic failure. METHODS: BMMC were isolated from mice, pre-labeled with PKH26 and infused into the mice in which hepatic injury had been induced followed by administration of G-CSF or vehicle. Livers were studied by fluorescent microscopy after transplantation of pre-labeled BMMC. RESULTS: PKH26 labeled cells were found in liver tissue at 102 ± 10 cells/high power field in the BMMC+G-CSF group and 30 ± 5 cells/high power field in the BMMC group, but none in the G-CSF group and the control group (P < 0.05). In the former two groups the majority of PKH26 labeled cells colocalized with proliferative cell nuclear antigen (PCNA). The number of PCNA positive cells in the BMMC+G-CSF group was 20 ± 4 cells/high power field, while in the BMMC group it was 14 ± 2 cells/high power field, in the G-CSF group 12 ± 2 cells/high power field, and 8 ± 1 cells/high power field in the control group. Moreover, albumin expression was increased in the BMMC+G-CSF treated group (149 ± 7/high power field) relative to the BMMC group (48 ± 6/high power field), the G-CSF group (44 ± 5/high power field) and the vehicle group (30 ± 6/high power field), with the former three groups showing elevated levels as compared to vehicle control (30 ± 6) (P < 0.05). CONCLUSION: Transplanted BMMC may home to injured liver, which appears to be enhanced by G-CSF administration.

Stromal cell derived factor-1 enhances bone marrow mononuclear cell migration in mice with acute liver failure.

AIM: To evaluate the number of bone marrow mononuclear cells (BMMC) that are migrated to the liver following transplantation of murine BMMC into mice with acute liver injury. METHODS: BMMC were isolated from the bone marrow of mice in a lymphocyte separation medium and then labeled with PKH26. The labeled cells were subsequently infused into the caudal veins of BALB/c mice with hepatic injury induced by carbon tetrachloride and 2-acetylaminofluorene. Mice in experimental group were treated with stromal cell-derived factor-1 (SDF-1) which was injected intraperitoneally after transplantation of BMMC. Mice in control group were injected intraperitoneally with 0.1 mL of saline (0.9% NaCl) after transplantation of BMMC. After 2 wk, migration of the cells in experimental group was studied by fluorescence microscopy. The expression of proliferating cell nuclear antigen and albumin was quantified with manual methods in both groups. The serum transaminase levels at different time points were compared between the two groups. RESULTS: The labeled "cells" were found in the portal region and central veins of hepatic lobules. The PKH26-labeled cells appeared at an average frequency of 108 +/- 8/high power field in the experiment group and 65 +/- 8/high power field in the control group (P < 0.05). The total number of positive cells was 29 +/- 7/high power field in the experimental group and 13 +/- 2/high power field in the control group. The albumin expression level was also higher in the experimental group than in the control group (29 +/- 7 vs 13 +/- 2, P < 0.05). The total number of crossing points was 156 +/- 5/high power field in the experimental group and 53 +/- 5/high power field in the control group (P < 0.05). The serum alanine aminotransferase levels in experimental and control groups were measured at different time points (120 +/- 40 vs 118.50 +/- 1.75, P > 0.05; 80.60 +/- 6.50 vs 101.08 +/- 5.67, P < 0.05; 50.74 +/- 5.38 vs 80.47 +/- 4.62, P < 0.05; 30.54 +/- 2.70 vs 60.72 +/- 4.37, P < 0.05; 30.77 +/- 5.36 vs 40.47 +/- 6.50, P < 0.05). At the same time, the serum aspartate aminotransferase levels were measured in experimental and control groups at different time points (122.55 +/- 1.46 vs 120.70 +/- 4.22, P > 0.05; 54.26 +/- 6.50 vs 98.70 +/- 8.20, P < 0.05; 39.47 +/- 5.39 vs 78.34 +/- 4.50, P < 0.05; 28.94 +/- 2.70 vs 56.44 +/- 4.28, P < 0.05; 30.77 +/- 5.45 vs 42.50 +/- 6.28, P < 0.05). CONCLUSION: SDF-1 can promote the migration of BMMC to the liver of mice with acute liver failure.