Volume 26, Issue 1, Pages 162-172 (January 2018) Mesenchymal Stromal Cells Inhibit Inflammatory Lymphangiogenesis in the Cornea by Suppressing Macrophage in a TSG-6-Dependent Manner  Hyun Beom Song, Se Yeon Park, Jung Hwa Ko, Jong Woo Park, Chang Ho Yoon, Dong Hyun Kim, Jeong Hun Kim, Mee Kum Kim, Ryang Hwa Lee, Darwin J. Prockop, Joo Youn Oh  Molecular Therapy  Volume 26, Issue 1, Pages 162-172 (January 2018) DOI: 10.1016/j.ymthe.2017.09.026 Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 1 MSCs Inhibit Corneal NV and Inflammation (A) Experimental scheme and clinical scores for corneal NV. Right after suture placement to induce corneal NV, either MSCs (1 × 106 cells in 100 μL of Hank’s balanced salt solution [BSS]) or the same volume of BSS was injected into tail vein. The corneas were assayed 7 days later. Representative corneal photographs and H&E staining of corneal sections are shown (scale bars, 50 μm). (B) Immunostaining of the corneal whole mounts for CD31, LYVE-1, and CD11b. Asterisks depict the suture locations. (C) Clinical scoring of corneal new vessel ingrowth and quantitation of LYVE-1-stained area in corneal flat mounts. Dot indicates a single animal, and the bar indicates the mean ± SD. (D–F) Real-time RT-PCR analysis for endothelial (D), pro-inflammatory (E), anti-inflammatory, and anti-angiogenic molecules (F). Shown are the relative values of mRNA levels to the levels in normal corneas. Data are presented as mean ±/+ SD from three independent experiments, each with five mice per group. *p < 0.05; **p < 0.01; ****p < 0.0001. Molecular Therapy 2018 26, 162-172DOI: (10.1016/j.ymthe.2017.09.026) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 2 Correlation between Clinical Corneal NV Scores and Transcript Levels of Molecular Markers (A) Scheme of experiments. The sutures (0, 1, 2, or 3) were applied to the corneal stroma to induce a gradient of NV at day 0. At day 7, the corneas were clinically scored for NV and assessed for expression of lymphatic vessel and pro-angiogenic macrophage markers by real-time RT PCR. (B) The correlation was shown between clinical NV scores and mRNA levels of markers expressed on vessels (CD31 and LYVE-1). (C) The correlation was shown between clinical NV scores and mRNA levels of vascular growth factors (VEGF-A, -B, and -C). (D) The correlation was shown between clinical NV scores and mRNA levels of markers expressed on pro-angiogenic macrophages (Tek, MRC1, and MRC2). Dot indicates a single animal, and R represents Pearson R coefficient. Molecular Therapy 2018 26, 162-172DOI: (10.1016/j.ymthe.2017.09.026) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 3 The Effects of MSCs in Inhibiting Corneal NV and Inflammation Were Dependent on TSG-6 Expression (A) Experimental scheme. Corneal NV was induced at day 0, immediately after which 1 × 106 MSCs transfected with TSG-6 siRNA (TSG-6 siRNA MSC) or the cells transfected with control scrambled siRNA (SCR siRNA MSC) were injected. BSS (Hank’s BSS) was injected in positive controls. (B) The plasma concentration of human TSG-6 (hTSG-6) as assayed by ELISA. (C) Representative corneal photographs and corneal whole-mount immunostaining for CD31 and LYVE-1. (D and E) Corneal NVs were clinically graded under slit-lamp biomicroscopy (D), and the LYVE-1-stained area was quantitatively measured in corneal flat mounts at day 7 (E). Dot represents a single animal, and the bar the mean ± SD. (F and G) Real-time RT-PCR analysis of the cornea for endothelial markers (F) and vascular growth factors (G). (H) ELISA for the protein levels of vascular growth factors (VEGF-C, -D, -A) in the cornea. (I and J) Real-time RT-PCR analysis of the cornea for proangiogenic macrophage markers (I) and inflammatory cytokines (J). Shown are the relative values of mRNA levels in sutured corneas to the levels in normal corneas. Data are presented as mean ±/+ SD from four independent experiments, each with five mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, not significant. Molecular Therapy 2018 26, 162-172DOI: (10.1016/j.ymthe.2017.09.026) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 4 MSCs Reduce the Number of Circulating and Cornea-Infiltrating Pro-angiogenic Monocytes/Macrophages in a TSG-6-Dependent Manner (A) Representative flow cytometry plots for CD11b+Ly6C+ cells in the blood, spleen, and cervical draining lymph nodes (CLNs) at day 1 after corneal NV induction. (B) Quantitative flow cytometry results for CD11b+Ly6C+ cells in the blood, spleen, and CLN at day 1. Dot indicates a single animal, and the bar indicates the mean ± SD. (C) Immunostaining of the corneal whole mounts for CD11b, Ly6G, and VEGFR-3 at day 1. Asterisks depict the corneal suture locations. (D) Counting of VEGFR-3+CD11b+ cells in the corneal whole mounts. (E) Real-time RT-PCR analysis for VEGFR-3 in the cornea. The mRNA level in sutured corneas relative to the levels in normal corneas is presented in mean + SD from four independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001. ns, not significant. Molecular Therapy 2018 26, 162-172DOI: (10.1016/j.ymthe.2017.09.026) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

Figure 5 Systemic Depletion of CD11b+Ly6C+ Monocytes at the Time of Suture Application Inhibits Corneal NV (A) Experimental scheme. Corneal NV was induced at day 0 in mice depleted of CD11b+Ly6C+ cells by injection of blocking antibody (RB6-8C5). Isotype IgG was injected into control mice. (B) Flow cytometric confirmation for depletion of CD11b+Ly6C+ cells in the blood at day 1. Dot indicates a single animal, and the bar indicates the mean ± SD. (C) Clinical corneal NV scoring at day 7. Dot indicates a single animal, and the bar indicates the mean ± SD. (D–G) Real-time RT-PCR analysis of the corneas for endothelial markers (D), vascular growth factors (E), proangiogenic macrophage markers (F), and inflammatory cytokine (G). Data are presented as mean + SD from three independent experiments, each with at least three mice per group. (H) Schematic representation of the MSC effects in corneal NV. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns, not significant. Molecular Therapy 2018 26, 162-172DOI: (10.1016/j.ymthe.2017.09.026) Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions