TSG-6 Released from Intradermally Injected Mesenchymal Stem Cells Accelerates Wound Healing and Reduces Tissue Fibrosis in Murine Full-Thickness Skin Wounds  Yu Qi, Dongsheng Jiang, Anca Sindrilaru, Agatha Stegemann, Susanne Schatz, Nicolai Treiber, Markus Rojewski, Hubert Schrezenmeier, Seppe Vander Beken, Meinhard Wlaschek, Markus Böhm, Andreas Seitz, Natalie Scholz, Lutz Dürselen, Jürgen Brinckmann, Anita Ignatius, Karin Scharffetter-Kochanek  Journal of Investigative Dermatology  Volume 134, Issue 2, Pages 526-537 (February 2014) DOI: 10.1038/jid.2013.328 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Mesenchymal stem cells (MSCs) accelerate anti-fibrogenic healing with suppression of macrophage-derived tumor necrosis factor (TNF)-α. (a) Representative wounds injected with phosphate-buffered saline (PBS; Co) or MSCs. (b) Statistical analysis of 20 wounds/group expressed as percentage of day-0 wound size. Results given as mean±SD (n=5), *P<0.05, **P<0.01, and ***P<0.001 by Mann–Whitney test. (c) Representative photomicrographs of Masson Trichrome– and Picrosirius Red–stained sections from day-15 wounds injected with PBS (Co) or MSCs. Bars=200 μm. (d) TNF-α-specific enzyme-linked immunosorbent assay of day-2 wound Mφ lysates from PBS (Co) or MSC-injected wounds. (e) Immunostaining for TNF-α and F4/80 in day-2 PBS (Co) or MSC-injected wounds. Bars=25 μm. (f) Quantification of total and TNF-α+F4/80+ Mφ in PBS and MSC-injected wounds. Sections from eight wounds per group were analyzed in 15 high-power fields (HPF) per section. Results given as mean±SD (n=4), ***P<0.001 by Student’s t-test. DAPI, 4,6-diamidino-2-phenylindole. Journal of Investigative Dermatology 2014 134, 526-537DOI: (10.1038/jid.2013.328) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Mesenchymal stem cell (MSC)-derived TNF-α-induced protein 6 (TSG-6) suppresses macrophage activation in vitro. (a) Reverse transcriptase–PCR for TSG-6 in non-activated bone marrow (BM)-MSCs, tumor necrosis factor (TNF)-α-treated BM-MSCs, and BM-MSC-activated Mφ co-cultures. Results given as mean TSG-6/GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression±SD (n=3), *P<0.05, ***P<0.001 by Student’s t-test. (b) Western blot of TSG-6 expression in lysates from non-activated BM-MSCs, TNF-α-treated BM-MSCs, BM-MSCs+non-activated Mφ co-cultures, and BM-MSCs+activated Mφ co-cultures. Semiquantitative densitometric analysis of digitized blots given as mean TSG-6/β-actin signal±SD (n=3), *P<0.05 by Student’s t-test. (c) TNF-α enzyme-linked immunosorbent assay with supernatants from non-activated or activated Mφ stimulated with recombinant human TSG-6 (rhTSG-6). Results presented as mean±SD (n=3), *P<0.05 by Student’s t-test. (d) TNF-α concentrations measured in supernatants from non-activated Mφ, activated Mφ, co-cultures of activated Mφ with BM-MSCs, TSG-6 small interfering RNA (siRNA)-silenced BM-MSCs, or scrambled (scr) siRNA-transfected BM-MSCs. Data given as mean±SD (n=3), *P<0.05, **P<0.01 by Student’s t-test. Journal of Investigative Dermatology 2014 134, 526-537DOI: (10.1038/jid.2013.328) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Mesenchymal stem cell (MSC)-derived TNF-α-induced protein 6 (TSG-6) reduces tumor necrosis factor (TNF)-α and improves wound closure in vivo. (a) Real-time reverse transcriptase–PCR for human TSG-6 in MSC-injected mouse wounds at indicated time points. Results given as mean human TSG-6/GAPDH (glyceraldehyde 3-phosphate dehydrogenase) expression±SD (n=5). (b) Representative photomicrographs of day-2 wounds injected with phosphate-buffered saline (PBS; Co) or MSCs 24 h post wounding stained for human-specific β2M (green) and TSG-6 (red) with overlay (yellow) indicating TSG-6-producing MSCs. Nuclear staining with DAPI (4,6-diamidino-2-phenylindole), bars=25 μm. (c) Western blot with lysates from day-3 wounds injected with recombinant human TSG-6 (rhTSG-6) or PBS (Co). Semiquantitative densitometric analysis of digitized blots expressed as mean TNF-α/β-actin signal±SD (n=5), *P<0.05 by Student’s t-test. (d) TNF-α-specific enzyme-linked immunosorbent assay of day-5 wound lysates from PBS (Co), MSCs, TSG-6-silenced MSCs, or srambled (scr) small interfering RNA (siRNA)-transfected MSC-injected wounds. *P<0.05 by Student's t-test. (e) Statistical analysis of 20 wounds/group expressed as percentage of day-0 wound size for PBS-injected wounds, MSC-injected wounds, scr siRNA-transfected MSC-injected wounds, and TSG-6-silenced MSC-injected wounds. Results presented as mean±SD (n=5), *P<0.05 by Student's t-test. **P<0.01, ***P<0.001 by Mann–Whitney test. Journal of Investigative Dermatology 2014 134, 526-537DOI: (10.1038/jid.2013.328) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Mesenchymal stem cells (MSCs) reduce granulation tissue formation. (a) Western blot analysis of α-smooth muscle actin (α-SMA) from wound lysates of day-5, -7, and -10 wounds injected either with phosphate-buffered saline (PBS; Co), MSCs, TNF-α-induced protein 6 (TSG-6)-silenced MSCs, or srambled (scr) small interfering RNA (siRNA)-transfected MSCs. (b) Semiquantitative densitometric analysis of digitized blots given as mean α-SMA/β-actin signal±SD (n=5), *P<0.05, ***P<0.001 by Student’s t-test; NS, not significant. (c) Immunostaining for the classical myofibroblast-specific marker α-SMA of wound sections derived from day-10 wounds injected with PBS (Co) or MSCs 24 h after wounding. Bars=100 μm. DAPI, 4,6-diamidino-2-phenylindole. Journal of Investigative Dermatology 2014 134, 526-537DOI: (10.1038/jid.2013.328) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Mesenchymal stem cell (MSC)-derived TNF-α-induced protein 6 (TSG-6) contributes to reduced tissue fibrosis. Representative photomicrographs of Masson Trichrome–stained sections from day-15 wounds injected with (a) phosphate-buffered saline (PBS; Co), (b) MSCs, or (c) TSG-6-silenced MSCs. Collagen fibrils stained blue. Epidermis and hair follicles stained brownish-red. White arrows indicate fibrotic tissue depths. The white rectangles indicate the area of magnification as shown in the outer right panels. Bars=100 μm. (d) The fibrotic tissue depths were quantified in serial sections in the center of day-15 wounds treated with PBS (Co), MSCs, or TSG-6-silenced MSCs. Assessment of fibrotic tissue depths was done in a blinded fashion. Results presented as mean±SD (n=5). ***P<0.001 by Student’s t-test. Reverse transcriptase–PCR of (e) colα1(I), (f) colα2(I), and (g) colα1(III) expression levels for day-2, -5, -10, -21, and -42 PBS (Co) or MSC-injected wounds. Results expressed as mean±SD (n=5), *P<0.05, **P<0.01, ***P<0.001 by Student’s t-test. (h) Following pepsin digestion, equal protein concentration of lysates for PBS (Co) or MSC-injected wounds were subjected to gel electrophoresis and subsequent Coomassie blue staining. (i) Breaking strength was measured as the ratio of wounds versus corresponding normal skin from PBS (Co) or MSC-treated mice at days 21and 42 post wounding. Results are expressed as mean±SD (n=5), *P<0.05, by Student’s t-test. Journal of Investigative Dermatology 2014 134, 526-537DOI: (10.1038/jid.2013.328) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Mesenchymal stem cell (MSC)-derived TNF-α-induced protein 6 (TSG-6) contributes to reduced tissue fibrosis. Representative photomicrographs of Masson Trichrome–stained sections from day-15 wounds injected with (a) phosphate-buffered saline (PBS; Co), (b) MSCs, or (c) TSG-6-silenced MSCs. Collagen fibrils stained blue. Epidermis and hair follicles stained brownish-red. White arrows indicate fibrotic tissue depths. The white rectangles indicate the area of magnification as shown in the outer right panels. Bars=100 μm. (d) The fibrotic tissue depths were quantified in serial sections in the center of day-15 wounds treated with PBS (Co), MSCs, or TSG-6-silenced MSCs. Assessment of fibrotic tissue depths was done in a blinded fashion. Results presented as mean±SD (n=5). ***P<0.001 by Student’s t-test. Reverse transcriptase–PCR of (e) colα1(I), (f) colα2(I), and (g) colα1(III) expression levels for day-2, -5, -10, -21, and -42 PBS (Co) or MSC-injected wounds. Results expressed as mean±SD (n=5), *P<0.05, **P<0.01, ***P<0.001 by Student’s t-test. (h) Following pepsin digestion, equal protein concentration of lysates for PBS (Co) or MSC-injected wounds were subjected to gel electrophoresis and subsequent Coomassie blue staining. (i) Breaking strength was measured as the ratio of wounds versus corresponding normal skin from PBS (Co) or MSC-treated mice at days 21and 42 post wounding. Results are expressed as mean±SD (n=5), *P<0.05, by Student’s t-test. Journal of Investigative Dermatology 2014 134, 526-537DOI: (10.1038/jid.2013.328) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 TNF-α-induced protein 6 (TSG-6) leads to a low anti-fibrogenic transforming growth factor (TGF)-β1/TGF-β3 ratio. (a) Active TGF-β1- and TGF-β3-specific enzyme-linked immunosorbent assays (ELISAs) were performed on day-2, -5, -7, and -10 wound lysates from phosphate-buffered saline (PBS)-injected or mesenchymal stem cell (MSC)-injected wounds. *P<0.05 and ***P<0.001 by one-tailed Student’s t-test. (b) Active TGF-β1- and TGF-β3-specific ELISAs were performed on day-5 wound lysates wounds injected with PBS or MSCs or TSG-6-silenced MSCs. The ratios of active TGF-β1 to TGF-β3 protein concentrations of the different experimental groups are shown (outer right panel). (c) Active TGF-β1- and TGF-β3-specific ELISAs were performed on day-3 wound lysates from PBS or recombinant human TSG-6 (rhTSG-6)-injected wounds. The ratio of active TGF-β1 to TGF-β3 protein is shown (outer right panel). Results are presented as mean±SD (n=3), *P<0.05, **P<0.01, and ***P<0.001 by Student’s t-test. Journal of Investigative Dermatology 2014 134, 526-537DOI: (10.1038/jid.2013.328) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions