S100A8/A9 Enhances Immunomodulatory and Tissue-Repairing Properties of Human Amniotic Mesenchymal Stem Cells in Myocardial Ischemia-Reperfusion Injury

Figures

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  Figure 1. The study flowchart.

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  Figure 2. The characteristics of hAMSCs. (A) Morphology of hAMSC at passage 7 under normal cultural condition. The morphology of hAMSCs show accurate spindle-shape under microscopic observation (Bar = 100 μm). (B) Cell proliferation test of hAMSC at passage 7. Measured the cell proliferation at day 0, 2, 4, 6, 8, and 10 after seeded approximately 4 × 104 of hAMSCs in 6-well plate through CCK-8 kit and ELISA determination (OD: 450 nm). (C) Flow cytometry analysis of hAMSCs surface marker expression. Flow cytometry results of hAMSCs at passage 7 represented the CD105, CD90, and CD73 positive. (D) Representative microscopic images of Alizarin Red S staining and Oil Red O staining to confirm osteogenic differentiation and adipogenic differentiation of plastic adherent hAMSCs in passage 7 after 14 days induced. Chondrogenic differentiation tests were confirmed by the formation of dense cell pellets and Alcian blue staining after 21 days induced. (Bar = 100μm (A,B); Bar = 200 μm (C)).

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  Figure 3. The comparison of expression levels of interested gene in hAMSCs with or without S100A8/A9 treatment. The significance among the groups was calculated using t test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

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  Figure 4. Analysis of the gene expressions in control cardiomyocyte group (Control CM) and H/R cardiomyocyte group (H/R CM) through qPCR. The significance among the groups was calculated using t test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 5. The immunomodulatory and cardioprotective effects of hAMSC-derived condition medium in the H/R model. (A) Cell viability tests of control, H/R CM, non-pretreated hAMSC (non-hAMSC), and pretreated hAMSC (pre-hAMSC) groups through trypan blue staining. (B) Microscopic images of each group after different treatment. (C) Gene expression levels in each group through qPCR. The significance among the groups was calculated using one-way ANOVA. * each group compared with H/R group. ** p < 0.01, *** p < 0.001, **** p < 0.0001. # pre-hAMSC group compared with non-hAMSC group. # p < 0.05, ### p < 0.001, #### p < 0.0001.

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  Figure 6. Cardiac function and fibrosis in each group during the 4-week period after the I/R injury. LVEFs (A) and FS (B) of control, sham, I/R, basal medium, non-hAMSC, and pre-hAMSC groups. (C–E) Representative bar graphs of left ventricular function in non-hAMSC and pre-hAMSC groups. (F) Masson’s Trichrome staining of histological transverse sections of hearts in each group 28 days after the I/R injury (Bar = 1 mm). (G) Quantification of heart fibrosis area in each group at 28 days after I/R surgery via Masson’s Trichrome staining. * Comparison with I/R group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. # Comparison with Basal Medium group. # p < 0.05, ## p < 0.01.