Enhanced immunomodulatory effects of canine adipose tissue-derived mesenchymal stem cells in 3D culture

Figures

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  Figure 1. Characterization of cAT-MSC isolated from canine adipose tissue. (A) unstained cAT-MSC. (B) Differentiation into adipocyte, osteoblast, and chondrocyte was confirmed via Oil Red O, Alizarin Red, and Alcian Blue staining, respectively. (C) Flow cytometry analysis of cell surface markers. cAT-MSCs expressed CD29, CD44, CD 73 and CD90 and lacked CD45. (Blue: stained marker, red: unstained negative control).

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  Figure 2. Analysis of cAT-MSCs spheroid formation. (A) Images of cAT-MSCs spheroids taken at 12-h intervals over 48 h. (B) Analysis of spheroid area over 48 h using ImageJ. The size of cAT-MSCs spheroids significantly decreased within 48 h. (C) cAT-MSCs spheroids were harvested at 48 h, sectioned, and stained with H&E staining (scale bar = 0.020 mm). (D) Immunohistochemistry analysis of cAT-MSCs spheroids harvested at 48 h. Spheroids were stained with APC dye-conjugated IgG1 mouse monoclonal anti-pimonidazole antibody (red) and Hoechst 33342 (blue) (scale bar = 100 μm). *p < 0.05 and ***p < 0.001, as determined by one-way ANOVA.

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  Figure 3. Stemness-related protein expression in cAT-MSCs spheroids. (A) cAT-MSCs spheroids harvested 48 h after incubation and stained with anti-SOX2 antibody (green) and Hoechst 33342 (blue) were examined using a confocal microscope (scale bar = 50 μm). (B) cAT-MSCs spheroids harvested 48 h after incubation and stained with anti-OCT4 antibody (green) and Hoechst 33342 (blue) were examined using a confocal microscope (scale bar = 50 μm). (C) SOX2 and OCT4, and TSG6 were extracted from 2D-cultured cAT-MSCs and 3D-cultured cAT-MSCs spheroids and quantified using western blotting. (D) Relative protein expressions in 2D and 3D-cultured cAT-MSCs were analyzed using ImageJ. SOX2 and OCT4 expression was significantly increased in 3D-cultured cAT-MSCs spheroids compared to 2D-cultured cAT-MSCs. *p < 0.05 and ***p < 0.001, as determined by Student’s t-test.

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  Figure 4. Inflammation and angiogenesis factor expression in 2D-cultured cAT-MSCs and 3D-Cultured cAT-MSCs Spheroids. The expression level of factors were measured by RT-PCR and ELISA. The expression of inflammation-related factors (A; TGFβ1, HIF1-α, TSG6, COX2), cytokines (B; IL-1β, IL-6, IL-10) and Angiogenesis (C; VEGF-A, bFGF) were significantly increased in 3D-cultured cAT-MSCs spheroids compared to 2D-cultured cAT-MSCs. (D) PGE2 ELISA results in the culture media of 2D-cultured cAT-MSCs and 3D-cultured cAT-MSCs spheroids. The PGE2 concentration was significantly higher in cAT-MSCs spheroids than in 2D-cultured cAT-MSCs. *p < 0.05 and **p < 0.01, as determined by Student’s t-test.

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  Figure 5. Treatment of conditioned media of 3D-cultured cAT-MSCs reduces LPS-induced inflammation in DH82 cells. DH82 cells were treated with 0.1 uM dexamethasone, conditioned media (CM) of 2D-or 3D-cultured cAT-MSCs for 48 h after LPS treatment. Inflammatory response changes were then determined by flow cytometry and RT-PCR. (A) Scatter plots of DH82 cells. M1 was identified as CD206-CD86+ cells, and M2 was identified as CD206 + CD86-cells. (B) Percentages of M1 (CD206-CD86+) to M2 (CD206 + CD86-) determined by flow cytometry. M1 polarization was significantly reduced in DH82 cells treated with 3D-cultured cAT-MSCs CM compared to those treated with LPS alone. (C) Differences in mRNA expression of inflammatory cytokines in DH82 cells. The expression of TNFα, IL-1β, and IL-6 was significantly decreased in 3D-cultured cAT-MSCs CM-treated DH82 cells compared to those treated with LPS alone. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, as determined by one-way ANOVA.