1. Volume 136, Issue 3, Pages 978-989 (March 2009)
Adipose-Derived Mesenchymal Stem Cells Alleviate Experimental Colitis by Inhibiting Inflammatory and Autoimmune Responses 
Manuel A. González, Elena Gonzalez–Rey, Laura Rico, Dirk Büscher, Mario Delgado 
Gastroenterology 
Volume 136, Issue 3, Pages (March 2009)
DOI: /j.gastro
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2. Figure 1
Treatment with hASCs protects against colitis. (A and B) Colitis was induced by intracolonic administration of 3 mg TNBS (5 mg/mouse when indicated). Mice were treated intraperitoneally with 106 hASCs (0.3 × 106/mouse when indicated) 12 hours after injection of TNBS. Control mice received 50% ethanol or hASCs alone. (A) Clinical evolution was monitored by body weight changes, colitis score, survival, and macroscopic damage score. (B) Histopathologic analysis, inflammatory infiltration (number of CD4+ and CD11b+ cells per field), and MPO activity in the colon were determined at day 3. (C) hASC treatment abrogates established colitis. Colitis induced with TNBS (3 mg/mouse) at day 0 was treated from day 6 (arrow) with hASCs (106/mouse/day). (D) hASC treatment reduces disease recurrence. Colitis was induced by administration of TNBS (1.5 mg/mouse) at days 0 and 9 (arrows) and treated 12 hours after the first injection of TNBS with hASCs (106/mouse). Numbers in parentheses represent mortality after the second infusion of TNBS. n = 5–8 mice/group. *P < .001 vs TNBS-treated mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

3. Figure 2
Biodistribution of hASCs in colitic mice. (A) CFSE-labeled hASCs were injected intraperitoneally to naïve and TNBS-colitic mice and their presence in various organs determined by flow cytometry at the indicated times. Dashed lines in histograms set negative staining. Bottom panels represent the number of CFSE+HLA-ABC+CD11b− cells per 100 mg tissue. n = 3–4 mice/group. (B) The expression of various chemokine receptors (gray histograms) was analyzed by flow cytometry in hASCs. Black thin lines are the isotype controls. The numbers represents the percentage for a given chemokine receptor. Histograms are representative of 3 experiments. (C) Chemotaxis assay of hASCs against different chemokines was performed as described in Supplementary Methods (see supplemental material online at and expressed as percentage of cell migration against control medium (horizontal line). n = 4. *P < .01 vs medium.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
hASC treatment reduces systemic and colonic inflammatory responses in colitis. (A and B) TNBS-colitic mice were treated with hASCs and cytokine/chemokine contents in (A) colon and (B) sera determined at day 3. n = 5–6 mice/group. *P < .001 vs TNBS-treated mice. (C) Macrophages (Mφ, 5 × 104) isolated from spleen/MLNs of TNBS-colitic mice at the peak of disease were cocultured with hASCs (5 × 104) without (unstimulated) or with lipopolysaccharide (0.5 μg/mL) and cytokines determined after 24 hours. Anti–IL-10 and anti–TGF-β antibodies were added to neutralize secreted IL-10 and TGF-β and indomethacin was added to inhibit PGE2 production. TNF-α–stimulated hASC-conditioned medium was added to macrophage cultures. When indicated, macrophages and hASCs were separated by a semipermeable membrane in Transwells (n = 4). *P < .001 vs macrophages; ^P < .001 vs hASC-treated macrophages.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

5. Figure 4
hASC treatment down-regulates Th1 cytokine response. MLNs isolated from untreated or hASC-treated colitic mice were cultured without (unstimulated) or with PMA + con A (stimulated). (A) Proliferation and cytokine production were assayed after 96 hours and 48 hours, respectively. (B) Intracellular cytokine expression was analyzed by flow cytometry in gated CD4 cells. (C) Sera isolated at day 4 from untreated or hASC-treated colitic mice were assayed for anti-TNBS immunoglobulin G levels. n = 5 mice/group. *P < .001 vs TNBS-treated mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

6. Figure 5
hASCs deactivate colonic effector Th1 cells in vitro. MLNs isolated from TNBS-treated mice were cocultured with hASCs without (unstimulated) or with PMA + con A (stimulated). When indicated, anti–IL-10, anti–TGF-β antibodies, and indomethacin were added to cocultures, and MLNs and hASCs were separated in a Transwell system. TNF-α–stimulated hASC-conditioned medium was added to some MLN cultures. Proliferation and cytokine contents were determined after 96 hours and 48 hours, respectively. MLNs were analyzed for CD4 and intracellular cytokine expression (dot plots: numbers represent cell percentage in each quadrant). n = 6. *P < .001 vs MLNs. ^P < .001 vs hASC-treated MLNs. N.D., no data.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

7. Figure 6
hASCs induce Tr response in colitis. (A) Percentage of CD4+CD25+Foxp3+ cells was determined in MLNs isolated at different times after infusion of TNBS in untreated and hASC-treated colitic mice. n = 5 mice/group. *P < .001 vs TNBS-treated mice. (B) Effect of adoptive transfer of CD4+ and CD4+CD25− T cells isolated from MLNs of untreated (CD4control) or hASC-treated TNBS mice (CD4ASC) on the progression of colitis. n = 5 mice/group.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Syngeneic and allogeneic murine ASCs protects against TNBS-induced colitis. (A) Colitic BALB/c mice were treated with allogeneic (from C57Bl/6 mice) or syngeneic (from C/c mice) mASCs 12 hours after infusion of TNBS. Clinical evolution and severity were monitored by body weight changes and survival for different days. Histopathologic analysis, inflammatory infiltration, colitis score, and MPO activity in the colon were determined at day 3. (B) CFSE-labeled allogeneic mASCs were injected into colitic mice and their presence in various organs determined at different times. (C) Cytokine contents in colonic protein extracts (day 3) from untreated or allogeneic mASC-treated mice were determined by enzyme-linked immunosorbent assay. (D) MLN lymphocytes isolated at day 3 were analyzed for CD4 and CD25 and intracellular cytokine and FoxP3 expression by flow cytometry. Double staining for IFN-γ/IL-2, TGF-β/IL-10, and CD25/FoxP3 expression was performed in gated CD4 T cells. Numbers represent cell percentage in each quadrant. n = 4–6 mice/group. *P < .001 vs TNBS-treated mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions

9. Figure 8
mASCs induce T-suppressive responses. (A) MLNs (105) isolated from TNBS-colitic mice were cocultured with different numbers of allogeneic mASCs. Proliferation and cytokine contents were determined after 96 hours and 48 hours, respectively. (B) After 96-hour culture, 2 × 104 T cells (TASC) were isolated from the MLN/mASC cocultures, rested for 48 hours, and added to MLNs (105) isolated from TNBS-treated mice and activated with PMA + con A. MLN T cells (Tcontrol) from TNBS-treated mice were used as controls. When indicated, anti–IL-10 and/or anti–TGF-β antibodies or IL-2 was added to TASC/MLN cocultures, and TASC and responder MLNs were separated in a Transwell system. Proliferation was determined after 96 hours. n = 8. *P < .001 vs MLNs. ^P < .001 vs TASC-treated MLNs (none). (C) Disease evolution after the adoptive transfer of Tcontrol and TASC into colitic mice. n = 5–8 mice/group. *P < .001 vs TNBS-treated mice.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions