1. Volume 145, Issue 6, Pages 1392-1403.e8 (December 2013)
Human Umbilical Cord Blood Mesenchymal Stem Cells Reduce Colitis in Mice by Activating NOD2 Signaling to COX2 
Hyung–Sik Kim, Tae–Hoon Shin, Byung–Chul Lee, Kyung–Rok Yu, Yoojin Seo, Seunghee Lee, Min–Soo Seo, In–Sun Hong, Soon Won Choi, Kwang–Won Seo, Gabriel Núñez, Jong–Hwan Park, Kyung–Sun Kang 
Gastroenterology 
Volume 145, Issue 6, Pages e8 (December 2013)
DOI: /j.gastro
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2. Figure 1
Administration with NOD2-activated hUCB-MSCs enhanced the protective effects against DSS-induced colitis in mice. (A–C) Clinical progression in DSS-induced colitic mice was monitored. Numbers of mice were as follows: naive mice, 10–14; PBS mice, 12–20; fibroblast mice, 6–10; MSC mice, 12–20; MSC+MDP mice, 12–20; and MSC-siNOD2+MDP mice, 12–29. (A) Mantel–Cox analysis of survival rate, (B) percentage of body weight loss, (C) disease activity index for colitis severity, and (D and E) on day 10, animals were killed for further evaluation. (D) Colon length measurement. (E) Histopathologic analysis of colon. Bar, 500 μm. Six mice per group were used. (F) Enlargement of mesenteric lymph nodes was evaluated. Five mice per group were used. *P < .05, **P < .01, ***P < Results are shown as mean ± SD.
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3. Figure 2
NOD2-activated hUCB-MSCs reduce colonic inflammation in mice. (A) DSS-induced colitic mice were injected intraperitoneally with hUCB-MSCs and IL-6, IFN-γ, TNF-α, and IL-10 levels in colon were determined on day 5 by enzyme-linked immunosorbent assay. (B) Neutrophil infiltration was determined by colonic MPO activity assay. (C and D) Inflammatory T lymphocyte and phagocyte infiltration were measured by counting cells per microscopic field on immunostained colon sections. (C) CD4+ cell counts. (D) CD11b+ cell counts. (E) Colonic infiltration of CD4+CD25+FoxP3+ cells were determined by flow cytometry on day 5. *P < .05, **P < .01, ***P < Three to 6 mice per group were used. Results are shown as mean ± SD.
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4. Figure 3
NOD2 is crucial for the protective ability of hUCB-MSCs against TNBS-induced colitis. (A–C) Gross and histologic observations in TNBS-induced colitic mice were performed. Numbers of mice were as follows: naive mice, 7; EtOH mice, 8; PBS mice, 13; fibroblast mice, 15; MSC mice, 18; MSC + MDP mice, 18; and MSC-siNOD2 + MDP mice, 13. (A) Survival rate and body weight loss. (B) Measurement of colon length. (C) Histopathologic evaluation of colon sections. Five mice per group were used, Bar, 500 μm. (D) NOD2-deficient hUCB-MSCs without MDP stimulation were injected intraperitoneally into colitic mice. Percentage of survival rate and body weight loss were measured. Numbers of mice were as follows: EtOH mice, 8; PBS mice, 13; MSC-siCTL mice, 18; and MSC-siNOD2 mice, 13. (E) Nine days after colitis induction and hUCB-MSC administration, a second dose of TNBS was inoculated and survival rate was analyzed. Numbers of mice were as follows: EtOH mice, 9; PBS mice, 8; MSC mice, 10; and MSC+MDP mice, 10. Numbers in parentheses represent the percentage of mice that were dead. *P < .05, **P < .01, ***P < Results are shown as mean ± SD.
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5. Figure 4
MDP enhanced the immunosuppressive effect of hUCB-MSCs. (A) After treatment with ligands, hUCB-MSCs were co-cultured with hUCB-MNCs and MNC proliferation was determined by the bromodeoxyuridine kit. (B and C) hUCB-MNCs were cultured with the culture media of hUCB-MSCs (UCM). hUCB-MNC proliferation was determined by the bromodeoxyuridine kit. UCM 618 and 620; UCM from 618 and 620 hUCB-MSCs. (D) Human Jurkat cells and (E) mouse splenocytes were cultured in the presence of UCM and their proliferation was determined. *P < .05, ***P < Results show 1 representative experiment of at least 3. Results are shown as mean ± SD.
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6. Figure 5
MDP-induced PGE2 is responsible for the anti-inflammatory activity of hUCB-MSCs. (A and B) hUCB-MSCs were treated with each indicated ligand. (A) The PGE2 concentration was measured from culture supernatant by enzyme-linked immunosorbent assay. (B) Cellular COX-2 expression was determined by Western blot analysis. (C) PGE2 concentration in UCM was detected. (D) hMNCs and (E) mouse splenocytes were cultured at the presence of various doses of PGE2 and their proliferation was determined by the bromodeoxyuridine kit. (F) hUCB-MSCs were treated with MDP alone or MDP + indomethacin and UCM was collected. hUCB-MNCs were cultured in the presence of each UCM and MNC proliferation was determined. *P < .05, **P < .01, ***P < Results show 1 representative experiment of at least 3. Results are shown as mean ± SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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7. Figure 6
MDP enhanced the immunosuppressive effect of hUCB-MSCs through a NOD2-RIP2–dependent pathway. (A) hUCB-MSCs were transfected with siRNAs, and then were treated with MDP. Protein levels of COX-2 were examined by Western blot analysis. (B) UCM was harvested from cells treated with MDP after siRNA transfection. PGE2 concentration was measured in UCM by enzyme-linked immunosorbent assay. (C) siRNA-transfected hUCB-MSCs without MDP stimulation were determined for COX-2 expression on the protein level. (D) hUCB-MSCs were treated with siRNA without MDP stimulation, and PGE2 secretion was detected from culture supernatant using an enzyme-linked immunosorbent assay kit. In addition, PGE2 concentration in UCM was measured. (E) hUCB-MNCs were cultured in UCM and IL-10 production was measured in the culture supernatant. (F) hUCB-MNCs cultured in UCM were analyzed for regulatory T-cell population by flow cytometry. Results are 1 representative experiment of 2 or 3 or the cumulative of 3 independent experiments. *P < .05, **P < .01, ***P < Results are shown as mean ± SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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8. Figure 7
NOD2-mediated PGE2 production and ensuing IL-10 induction are crucial for the attenuation of colitis. (A) siRNA for COX-2 was transfected into hUCB-MSCs. Cells were stimulated with MDP and injected intraperitoneally into DSS-induced colitic mice. Survival rate, disease activity index, and histopathologic score were analyzed. Numbers of mice were as follows: naive mice, 10; PBS mice, 12; MSC-siControl (CTL) mice, 12; MSC-siCOX2+MDP mice, 12; and MSC-siCTL+MDP mice, 10. (B) COX-2–inhibited hUCB-MSCs were administered into TNBS-induced colitic mice and disease progress was monitored. Numbers of mice were as follows: EtOH mice, 8; PBS mice, 15; MSC-siCTL+MDP mice, 20; and MSC-siCOX2+MDP mice, 10. (C) PGE2 concentration was measured in both serum and colon of hUCB-MSCs transplanted colitic mice at days 3, 5, and 7. (D) IL-10–neutralizing antibody was injected intraperitoneally daily from day 0 to day 5 into hUCB-MSC–administered colitic mice. Survival rate, body weight loss, and histologic score were evaluated. Numbers of mice were as follows: naive mice, 10; PBS mice, 10; MSC+MDP mice, 10; and MSC+ MDP+IL-10 neutralizing antibody (NA) mice, 10. *P < .05, **P < .01, ***P < Results are shown as mean ± SD.
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9. Supplementary Figure 1
LPS pretreatment did not enhance the protective effects of hUCB-MSCs. (A–D) Colitis was induced by the addition of 3% DSS in drinking water for 7 days. Mice were injected intraperitoneally with LPS or MDP pretreated hUCB-MSCs (2 × 106) 24 hours after DSS addition. Disease severity was evaluated by gross and histologic analyses. (A) Survival rate analysis. (B) Body weight loss. Ten mice per group were used. (C) Measurement of reduction in colon length. (D) Histologic scoring of colon. Four to 5 mice per group were used. *P < .05, **P < .01, ***P < Results are shown as mean ± SD.
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10. Supplementary Figure 2
NOD2-activated hUCB-MSCs reduce colonic infiltration of CD4+ and CD11b+ cells. (A and B) hUCB-MSCs were injected intraperitoneally into colitic mice. On day 5, colons were digested for single-cell suspensions and incubated with CD4 and CD11b antibodies. (A) Percentage of CD4+ cells, and (B) Percentage of CD11b+ cells were determined by flow cytometry. Five to 10 mice per group were used. *P < .05, **P < .01, ***P < Results are shown as mean ± SD.
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11. Supplementary Figure 3
NOD2-activated hUCB-MSCs increased colonic infiltration of Foxp3+ cells. (A) Frozen colon sections were immunostained for Foxp3 (Green). Bar, 100 μm. (B) Protein levels of Foxp3 in colon segment lysates were detected by Western blot analysis. (C) Quantification of FoxP3 protein levels. *P < .05, ***P < Results are shown as mean ± SD.
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12. Supplementary Figure 4
NOD2 activation did not modulate the migratory ability of hUCB-MSCs. (A and B) CFSE-labeled hUCB-MSCs were injected intraperitoneally into DSS-induced colitic mice and at 1, 3, and 7 days after injection, the colon sections were examined for green fluorescent cells with a confocal microscope. (A) CFSE-labeled cells in frozen colon sections were detected with fluorescent microscopy. Bar, 100 μm. (B) The number of green fluorescent cells per microscopic field was counted. Three mice per group were used. (C and D) CFSE-labeled cells were injected intraperitoneally into colitic mice. At different time points, colons, MLNs, and spleens were digested for single-cell suspensions and CFSE-positive cells were determined by flow cytometry. (C) CFSE-positive cells in single-cell suspensions isolated from inflamed colons were detected on days 1, 3, 5, 7, and 10. The number of CFSE-positive cells in 1 × 105 colonic cells were determined by flow cytometry. (D) CFSE-positive cells in single-cell suspensions isolated from spleens and mesenteric lymph nodes were detected on days 1 and 3. Three to 4 mice per group were used. Results are shown as mean ± SD.
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13. Supplementary Figure 5
Failure of induction in IDO-1 activation and NO production by Toll-like receptor (TLR) and NOD-like receptor (NLR) ligands stimulation. (A–C) hUCB-MSCs were treated with Pam3CSK4, LPS, Tri-DAP, and MDP for 24 hours. (A) The protein level of IDO-1 was determined using Western blot analysis. (B) The supernatant of hUCB-MSCs treated with TLR and NLR ligands were collected and NO production was examined by the Griess reaction. LPS-treated RAW264.7 cells were used as positive control. (C) Protein expression level of inducible NO synthase (iNOS) was detected by immunoblotting. Results show 1 representative experiment of 3.
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14. Supplementary Figure 6
Effect of specific siRNAs for NOD2 and Rip2. (A) hUCB-MSCs were transfected with specific siRNAs for NOD2 and Rip2 for 48 hours, and the levels of NOD2 and Rip2 were analyzed using Western blot analysis. (B) Enhancement of COX-2 expression in hUCB-MSCs by MDP stimulation was not affected by control siRNA transfection. Results show 1 representative experiment of 3.
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15. Supplementary Figure 7
NOD2 and Rip2 regulate the suppressive activity of hUCB-MSCs on hMNC proliferation. (A) hUCB-MNCs treated with Concanavalin A (ConA) were cultured for 5 days in the presence of UCM harvested from siRNA transfected hUCB-MSCs. hUCB-MNC proliferation was determined by the bromodeoxyuridine (BrdU) kit. (B) MLR using NOD2 and RIP2 siRNA-transfected UCM was performed and hMNC proliferation was determined. **P < .01, ***P < Results show 1 representative experiment of 2 or 3. Results are shown as mean ± SD.
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16. Supplementary Figure 8
MDP promoted the hUCB-MSCs–mediated induction of regulatory T cells in hMNCs through the NOD2-RIP2 pathway. hUCB-MNCs cultured in UCM for 5 days were analyzed for CD4+ CD25+ FoxP3+ cell population by flow cytometry. Dot plot images for Figure 6F.
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17. Supplementary Figure 9
Intracellular COX-2 expression level of hUCB-MSCs detected in mice colon was increased by MDP stimulation. hUCB-MSCs and MDP-MSCS were injected intraperitoneally into colitic mice. On day 3, colons were digested for single-cell suspensions and incubated with human CD73, permeabilized, and then incubated with human COX-2 antibodies. Intensity of human COX-2 expression among human CD73-positive cells was determined by flow cytometry.
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18. Supplementary Figure 10
COX-2 inhibition decreased colonic IL-10 level by MDP-MSC transplantation. (A) DSS-induced colitic mice were injected intraperitoneally with MDP-MSCs or COX-2–inhibited MDP-MSCS and mouse IL-10 levels in colon were determined on day 5 by enzyme-linked immunosorbent assay. **P < .01.
Gastroenterology  , e8DOI: ( /j.gastro )
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