Ameliorating liver fibrosis in an animal model using the secretome released from miR-122-transfected adipose-derived stem cells

Figure 1 Assessment of multilineage differentiation potential of miR-122-transfected adipose-derived stem cells. A: Flow cytometric analysis showing that miR-122 transfection did not alter the expression of surface markers of adipose-derived stem cells (ASCs). ASCs were negative for CD31 and CD45 (hematopoietic stem cell-associated markers) expression and positive for CD73 and CD105 (mesenchymal stem cell-associated markers) expression regardless of miR-122 transfection; B: Comparison of gross cell morphology between ASCs with/without miR-122 transfection. Cells appear to be identical regardless of miR-122 transfection; C, D: Photomicrographs showing successful differentiation of ASCs into adipocytes, osteocytes, and chondrocytes regardless of miR-122 transfection. The differentiated cells were identified using four distinct staining methods (Oil Red O, Alizarin Red, collagen type 1, and proteoglycan). Scale bars = 100 µm. Values are presented as mean ± standard deviation of three independent experiments. ^aP < 0.05. ASC: Adipose-derived stem cell; HSC: Hepatic stellate cell; PG: Proteoglycan.

Figure 2 In vitro experiments validating the effects of miR-122 transfection into Adipose-derived stem cells. A: Western blot analysis showing the expression of fibrotic and antifibrotic markers in miR-122-transfected adipose-derived stem cells (ASCs). miR-122-transfected ASCs showed decreased expression of fibrotic proteins (TGF β1, MMP2, and α-SMA) and increased expression of an antifibrotic protein (TIMP-1) than control ASCs. The graphs below microscopic figures show the relative densities of these markers; B, C: RT-PCR (left) and western blot analysis (right) of LX2 cells for the determination of the thioacetamide (TAA) concentration used for generating in vitro model of liver fibrosis. A TAA concentration of 2.5 mM was used for inducting LX2 cells into fibrosis; D: Effects of MCM in the in vitro model of liver fibrosis. The in vitro model of liver fibrosis was generated by treating human HSCs cells (LX2 cells) with a hepatotoxin (TAA). In western blot analysis (Left), MCM induced the lowest expression of fibrotic markers (MMP2, TGF-β1, and α-SMA) in the TAA-treated LX2 cells. Relative densities of fibrosis-related markers in each group (Right). Values are presented as mean ± standard deviation of three independent experiments. ^aP < 0.05 vs Ct. ^cP < 0.05 between NCM and MCM. α-SMA: Alpha-smooth muscle actin; COL1A1: Collagen type-1 alpha-1; Ct: Control; CM: The secretome obtained from ASCs after 48-h-incubation; MCM: The secretome released from miR-122-transfected ASCs; MMP-1: Metalloproteinases-1; TAA: Thioacetamide; TGF-β: Transforming growth factor-β; TIMP-1: Tissue inhibitor of metalloproteinases-1.

Figure 3 Determination of antifibrotic effects of MCM in the in vivo model of liver fibrosis. Control mice and thioacetamide (TAA)-treated mice (mouse model of liver fibrosis) were intravenously (using tail vein) infused with normal saline, CM, and MCM. A, B: Sirius red A and Masson’s trichrome B stains showing that MCM infusion significantly decreased the collagen content of the liver in the mouse model of liver fibrosis. Magnification × 400. Percentages of fibrotic areas were measured using NIH image J and expressed as relative values to those in normal livers; C: Western blot analysis of liver specimens. MCM infusion significantly increased the expression of PCNA (a proliferation marker), and significantly decreased the expression of α-SMA, TGF-β1, and MMP1 (fibrotic markers) and increased an antifibrotic marker (TIMP-1) in the mouse model of liver fibrosis. The relative densities of individual markers had been quantified using Image Lab 3.0 (Bio-Rad) software and then were normalized to that of β-actin in each group. Values are presented as mean ± standard deviation of three independent experiments. ^aP < 0.05 vs Ct (TAA). ^cP < 0.05 between TAA + NCM and TAA + MCM. α-SMA: Alpha-smooth muscle actin; Ct: Control; CM: The secretome obtained from ASCs after 48-h-incubation; MCM: The secretome released from miR-122-transfected ASCs; MMP-1: Metalloproteinases-1; PCNA: Proliferating cell nuclear antigen; TAA: Thioacetamide; TGF-β: Transforming growth factor-β; TIMP-1: Tissue inhibitor of metalloproteinases-1.

Figure 4 Immunohistochemical staining showing the effects of MCM on the expression of inflammatory and fibrotic markers in the livers. A, B: Upon comparing immunohistochemical staining patterns, MCM infusion led to higher expression of PCNA (an inflammatory marker), albumin, and TIMP-1 (an antifibrotic marker) A, and lower expression of α-SMA, TGF-β1, and MMP1 (fibrotic markers) B in the livers of TAA-treated mice. Percentages of immunoreactive areas were measured using NIH image J and expressed as relative values to those in normal livers. Magnification × 400. Values are presented as mean ± standard deviation of three independent experiments. ^aP < 0.05 vs Ct (TAA). ^cP < 0.05 between TAA + NCM and TAA + MCM. α-SMA: Alpha-smooth muscle actin; Ct: Control; CM: The secretome obtained from ASCs after 48-h-incubation; MCM: The secretome released from miR-122-transfected ASCs; MMP-1: Metalloproteinases-1; PCNA: Proliferating cell nuclear antigen; TAA: Thioacetamide; TGF-β: Transforming growth factor-β; TIMP-1: Tissue inhibitor of metalloproteinases-1.

Figure 5 Effects of MCM on the expression of antioxidant enzymes in the liver. Upon comparing immunohistochemical staining patterns, MCM infusion was observed to lead to a higher expression of SOD, catalase, and GPx in the livers of thioacetamide (TAA)-treated mice. The graphs below microscopic figures show the relative densities of these markers. Percentages of immunoreactive areas were measured using NIH image J and expressed as relative values to those in normal livers. Magnification × 400. Values are presented as mean ± standard deviation of three independent experiments. ^aP < 0.05 vs Ct (TAA). ^cP < 0.05 between TAA + NCM and TAA + MCM. Ct: Control; CM: The secretome obtained from ASCs after 48-h-incubation; GPx: Slutathione peroxidase; MCM: The secretome released from miR-122-transfected ASCs; SOD: Superoxide dismutase; TAA: Thioacetamide.

Figure 6 Determination of systemic effects of MCM and analysis of secretome components. A: Results of ELISA showing serum levels of inflammatory markers (IL-6 and TNF-α) in each group. MCM administration had the greatest effect on lowering the serum levels of IL-6 and TNF-α in thioacetamide (TAA)-treated mice; B: Serology tests of AST and ALT in the mouse model of liver fibrosis. MCM infusion had the greatest effect on decreasing the serum levels of AST and ALT; C: Heat map generated from label-free LC-MS for quantitative proteomics reflecting protein expression values of NCM and MCM. Samples are arranged in columns, proteins in rows. Red shading indicates increased expression in samples compared to control; green shading indicates reduced expression; and black shading indicates median expression. Each sample for LC-MS was pooled from three samples of the secretome. The components and concentrations of various essential proteins varied widely between NCM and MCM, validating the effects of miR-125 transfection. Specifically, MCM exhibited a significantly lower concentration of essential intermediates of TGF-β/Smad signaling, such as transgelin, PIN1, and Profilin-1, than NCM. Values are presented as mean ± standard deviation of three independent experiments. ^aP < 0.05 vs Ct (TAA). ^cP < 0.05 between TAA + NCM and TAA + MCM. ALT: Alanine transaminase; AST: Aspartate transaminase; TAA: Thioacetamide; TNF- α: Tumor necrosis factor-α.