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Supplementary Materials and Methods

MSC culture and expansion

For all sources, mesenchymal stem cells (MSCs) were isolated using the classical

adhesion method. When subconfluency (80–90%) was achieved, adherent cells were

detached with 0.25% Trypsin-EDTA (Gibico) and expanded by replating at lower

density (1000 cells/cm2). MSCs were generally evaluated after two passages. To

confirm the mesenchymal nature of cells, clone-forming unit fibroblast (CFU-F) assay,

phenotype and differentiation assays were performed as described.1

MSC were characterized according to the International Society for Cellular Therapy

(ISCT) criteria and as previously described.2 Briefly, adherent cells after 3-4 weeks of

culture were characterized by flow cytometry using the commercial Human

Mesenchymal Stem Cell Marker Antibody Panel by following the manufacturer's

instructions. Data were acquired and analyzed on a Kaluza® flow cytometry analysis

software from Beckman Coulter.

Multipotency of hUC-MSCs was tested for differentiation along the osteogenic, and

adipogenic lineages using the Mesenchymal Stem Cell Functional Identification

Kit (R&D Systems, Minneapolis, MN), as per manufacturer's instructions. In other

experiments, hUC-MSCs were primed with Pam3CSK4 (1μg/ml, TLR2), poly (I:C)

(1μg/ml, TLR3), lipopolysaccharide (LPS) (10ng/ml, TLR4) (Sigma–Aldrich), Tumor

Necrosis Factor (TNF)-α (50 ng/ml) and/or interferon (IFN)-γ (103 U/ml)

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(R&D Systems, Minneapolis, MN) and subsequently analyzed for cell surface

markers by flow cytometry and cytokine secretion by enzyme-linked immunosorbent

assay (ELISA).

For PGE2 synthesis inhibition experiments, MSCs were resuspended in complete

medium in the presence or absence of PGE2 inhibitors NS-389 (5μM; Cayman

Chemicals, Ann Arbor, MI) or indomethacin (5μM; ICN Chemicals, Irvine, CA) for

48 h, and coculture experiments were then carried out as described in the presence of

the inhibitors.

Assessment of TLR and cytokine transcription by real-time polymerase chain

reaction (RT-PCR)

Total mRNA was isolated from hUC-MSC using the TRIzol® Reagent (Life

Technologies). cDNA was obtained by reverse transcription of 1 μg mRNA using

PrimeScript RT Master Mix (Takara Bio Company, Japan) for 5 min at 25 ºC, 30 min

at 42 ºC, and 5 min at 85 ºC. RT-PCR was performed on an iQ5 (Bio-Rad) Sequence

Detection System with One Step PrimeScript™ RT-PCR Kit (Takara Bio Company,

Japan). Primers specific for human cytokine were detailed in Supplementary Table 1

(5’-3’ forward and reverse, respectively).

We analyzed the data using the comparative threshold cycle method based on the

relative expression of the target gene mRNA vs. glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) levels as a reference.

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MSC treatment

TLR3 and TLR4 ligation

To assess the influence of TLR3 and TLR4 ligation, MSC were activated for 1 h by

poly(I:C) at 1 μg/ml (Sigma–Aldrich) and lipopolysaccharide (LPS) at 10 ng/ml

(Sigma–Aldrich), respectively.3

TLR3 inhibition

MSCs were pre-treated with 10 ng/ml poly (I:C) for 48 h to activate TLR3 signaling.

TLR3 signaling in MSCs was inhibited by downregulating TLR3 expression with

small RNA interference (siRNA). Specific sequence for human TLR3 siRNA was

designed and synthesized by Ribobio (Guangzhou, China). Three suitable sequences

were selected as follows:

TLR3–siRNA#1, 5’ GAAGCUAUGUUUGGAAUUA dTdT 3’,

3’ dTdT CUUCGAUACAAACCUUAAU 5’;

TLR3–siRNA#2, 5’ AUAGGUGCCUUUCGUCAUA dTdT 3’,

3’ dTdT UAUCCACGGAAAGCAGUAU 5’;

TLR3–siRNA#3, 5’ GGAGCACCUUAACAUGGAA dTdT 3’,

3’ dTdT CCUCGUGGAAUUGUACCUU 5’

and mixed as siRNA for the following experiments. MSCs were cultured to 40%

confluency and transfected with the siRNA duplex and negative control siRNA using

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Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the

manufacturer’s recommended protocol. 50 nM siRNA was used for 6-well plates.

After 24 hours of transfection, the cells were stimulated with 1 μg/ml poly (I:C) for

another 1 hour and harvested for reverse transcription-PCR and western blotting.

Lentiviral vector-mediated gene knockdown in MSC

The Jagged-1 and Notch-1 knockdown constructs expressing siRNA targeting

endogenous Jagged-1 and Notch-1 (siRNA Jagged-1 and siRNA Notch-1) were

encoded into a lentivirus based siRNA vector pGLV3/H1/GFP (LV3). Target

sequences were designed and selected with software Designer 3.0 provided by

GenePharma. Additionally, LV3 containing nonspecific siRNA (LV3-GFP) was used

as a negative control.

MSC were seeded and cultured in six-well plates for 24 h. The lentiviral vectors

(carrying LV3-GFP or LV3- GFP siRNA Jagged-1 and Notch-1) were then added to

the wells at a multiplicity of infection (MOI) value of 100:1 and cultured with MSC

for 24 h. After 24 h, the culture medium was changed.

The sequences were as follows:Notch-1

5’-GGGACATCACGGATCATATGG-3’

5’-GCATGCATCACGACATCGTGA-3’

5’-CGAACCAATACAACCCTCTGC-3’

Jagged-1

5’-GCACCTCTGACTCCTATTACC-3’

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5’-GGGCCTACTGTGAAACCAATA-3’

5’-GCAACACAGGCCCTGACAAAT-3’

NC

5’-TTCTCCGAACGTGTCACGT-3’

Induction of Colitis and Study Design

Both colitis mice model were induced using the classical method as described

previously.4, 5 Briefly, trinitrobenzene sulfonic acid (TNBS, 3 or 5 mg; Sigma, St

Louis, MO) in 50% (v/v) ethanol (100 μl) was administered intra-rectally in BALB/c

mice. Control mice received 50% ethanol alone. Animals were treated

intraperitoneally with medium or with different numbers (106 cells/mouse) of

hUC-MSCs, TLR3-activated hUC-MSCs, or syngeneic mASCs (isolated from

BALB/c mice) 2 hours after instillation of TNBS.

Animals were monitored for the appearance of diarrhea, body weight loss, and

survival. At day 4, blood samples and various segments of the colon were collected.

Tissue segments were immediately frozen in liquid nitrogen for histologic studies,

protein extraction, cytokine determination, and measurement of myeloperoxidase

(MPO) activity. In vitro models were divided into seven groups (Table 2).

Clinical scoring of colitis

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The clinical scoring of a disease activity index (DAI) for TNBS-induced colitis was

based on weight loss, stool consistency, and bleeding, as described previously by

Murthy et al (Supplementary Table 3).6 The DAI was scored 0–4 for each parameter

and then averaged for each mouse and each group.

Macroscopic and Microscopic (Histological) Assessment

Colonic weight and length were measured as gross indicators of colitis. Colons were

also examined under a dissecting microscope and graded for macroscopic lesions.

Macroscopic assessment of the colitis severity was scored according to a previously

established scoring system (Supplementary Table 4).7 For evaluation of macroscopic

damage, colons were graded for macroscopic lesions (scale 0–10) based on criteria

reflecting inflammation (ie, hyperemia, bowel thickening, and extent of ulceration).8

For histological analysis, colons were fixed, sectioned, and stained with hematoxylin

and eosin. Histological changes were graded semiquantitatively from 0 to 4 according

to previously described criteria as follows (Neurath et al., 1995, Supplementary

Table 5). For histopathologic analysis, the number of CD4+ immunostained cells and

inflammation score were determined in colon cross sections.

MPO Assay

Neutrophil infiltration in the colon was monitored by measuring MPO activity as per

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manufacturer’s instructions (MPO Detection Kit; Nanjing Jiancheng Bioengineering

Institute, Nanjing, Jiangsu Province, China). MPO activity per gram of wet tissue was

calculated as MPO activity (U/g wet tissue) =A460/ 11.3* tissue weight (g) where A460

is the change in the absorbance of 460-nm light from 1 to 3 minutes after the initiation

of the reaction.

Cytokine Measurements

Cytokine levels were determined in colon mucosa. Colon homogenates were obtained

using a Potter-Elvehjem glass homogenizer at 4ºC in four volumes of Greenberger

lysis buffer (150 mM NaCl, 15 mM Tris, pH 7.4, 1 mM MgCl2, and 1% Triton X-100)

supplemented with protease inhibitors (Roche, Almere, The Netherlands). Samples

were centrifuged at 30,000g for 10 minutes at 4ºC and stored at -80ºC until cytokine

determination. Protein content was determined using the BCA Protein Assay (Thermo

Scientific Pierce, Etten-Leur, the Netherlands).

The levels of IFN -γ, interleukin (IL)-6, IL-23, IL-12/23p40, IL-4, IL-10, INF-γ and

TNF-α, IL-21 and IL-23, TGF-β and IL-17 proteins in the colon extracts and sera at

on day 4 after colitis induction were determined by ELISA using a custom

Luminex-based multiplex immunoassay kits (Procarta Cytokine Assay Kit:

Affymetrix, Santa Clara, CA). Briefly, the colon extracts and sera were incubated

with polystyrene beads coated with antibodies corresponding to the different

cytokines, and developed according to the manufacturer’s instructions. Bead size and

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fluorescence were measured on a Luminex 200 and data was analyzed using the

Masterplex QT software (Miraibio).

PGE2 and TGF-β concentrations were measured using a commercial ELISA kit

(eBioscience, San Diego, CA) according to the manufacturer’s protocol. For detection

of PGE2 from mouse serum and colon, serum or protein lysates were purified by a

multiple affinity removal kit (Agilent Technologies, Santa Clara, CA) before

measuring with an ELISA kit (Cayman chemical company, Ann Arbor, MI).

Furthermore, the mRNA expression of mice cytokines was detected by RT-PCR.

Specific primers were detailed in Supplementary Table 6 (5’-3’ forward and reverse,

respectively).

In Vivo Imaging

Fluc-hUC-MSCs (a kind gift from Peng Xiang, Center for Stem Cell Biology and

Tissue Engineering, Sun Yat-Sen University) were tracked using a whole-body cooled

charge-coupled device (CCD) camera system (IVIS 200 Series Imaging System;

Xenogen, Alameda, CA). The distribution and fluorescence intensity of the

fluc-hUC-MSCs were monitored at days 1, 2 and 5 post-injection. Mice were

continually anesthetized with chloral hydrate during the experimental procedure.

Biodistribution of Transplanted hUC-MSCs

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Five mice from both the syngeneic and allogeneic hUC-MSCs groups were injected

with cell tracker (CM-DiI)-dyed hUC-MSCs or 5, 6-carboxy fluorescein succinimidyl

ester (CFSE)-labeled (red fluorescence) hUC-MSCs for the cell biodistribution study.

The presence of CFSE-labeled cells was examined in various tissues, including the

spleen, mesenteric lymph node, and intestine via flow cytometry. To visualize the

localization of labeled MSCs based on CM-DiI dye (red) fluorescence, fluorescent

images of cryosectioned tissue were collected using a confocal scanning laser system

(Olympus FluoView 1000; Olympus Corp., Lake Success, NY, USA).

Transwell migration assay

The migratory ability of hUC-MSCs was evaluated using 8-μm pore filters (Corning

Costar, Tewksbury, USA). In brief, hUC-MSCs were suspended in serum-free

medium and seeded into the upper well, and 1μg/ml poly (I:C) containing medium

was placed in the lower well. Following incubation for 12 h at 37 °C, cells that had

not migrated from the upper side of the filter were scraped off with a cotton swab, and

filters were stained with crystal violet. The number of cells migrated to the lower side

of the filter was counted under a light microscope at 10× magnification in five

randomly selected fields.

hUC-MSCs / MLN Proliferation Assay

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Single-cell suspensions (106/mL) obtained from MLNs were incubated in medium

(RPMI 1640 supplemented with 100 U/mL penicillin/ streptomycin, 2 mmol/L

L-glutamine, 50 μmol/L 2-mercaptoethanol, and 10% fetal calf serum) in the absence

or presence of anti-CD3/CD28. Cultured human hUC-MSCs were plated in 96-well

round-bottom plates (Corning, Life Sciences) and allowed to adhere overnight. MLNs

cells were labeled with CFSE (10 μM for 106 T cells; Molecular Probes, Eugene, OR),

resuspended at a concentration of 106 cells/ml. Primed and unprimed MSCs were

thoroughly washed after stimulation, either mitomycin C (10 mg/ml) treated or

untreated, and used for co-culture with MLNs in lymphoproliferation experiments.

T-cell proliferation was determined 3 days later by flow cytometry analysis of CFSE

fluorescence intensity.

To determine the cell-contact dependence of the suppressive response, we placed

MLN cells (105) in the upper insert of a transwell system (0.4-μm pore; Millipore,

Billerica, MA) and hUC-MSCs (5 × 104) in the lower well, and MLN proliferation in

the upper compartment was determined after 96 hours. Supernatants were collected at

days 5 of the MSC and MLNs co-cultures for measurement of IL-10 and PGE2 levels.

Western Blot

The cells or colon segments were harvested and lysed in a buffer supplemented with a

complete protease inhibitor cocktail (Roche) and 2 mmol/L dithiothreitol. Lysates

were resolved via 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis,

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transferred to nitrocellulose membranes, and immunoblotted with primary antibodies

such as TLR3, Jagged-1, Jagged-2 and GAPDH (all from Abcam Inc., Cambridge,

MA, USA). After immunoblotting with secondary antibodies, the reacted protein

bands were detected using enhanced chemiluminescence (ECL) detection (GE

Healthcare, USA) using an ImageQuant LAS 4000 mini system (GE Healthcare,

USA).

Statistical Analysis

Values among different groups were expressed as mean + SD. All of the statistical

comparisons were made by one-way analysis of variance followed by a Bonferroni

post hoc test for multigroup comparisons. GraphPad Prism software (version 5.01;

GraphPad Software, San Diego, CA) were used to perform all statistical analyses. For

all tests, statistical significance was set at P < 0.05.

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Reference

1. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, concepts, and assays. Cell stem cell 2008;2:313-319.

2. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315-7.

3. Waterman RS, Tomchuck SL, Henkle SL, Betancourt AM. A new mesenchymal stem cell (MSC) paradigm: polarization into a pro-inflammatory MSC1 or an Immunosuppressive MSC2 phenotype. PLoS One 2010;5:e10088.

4. Wirtz S, Neufert C, Weigmann B, Neurath MF. Chemically induced mouse models of intestinal inflammation. Nat Protoc 2007;2:541-6.

5. Gonzalez MA, Gonzalez-Rey E, Rico L, Buscher D, Delgado M. Adipose-derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology 2009;136:978-89.

6. Murthy S, Cooper HS, Shim H, Shah RS, Ibrahim SA, Sedergran DJ. Treatment of dextran sulfate sodium-induced murine colitis by intracolonic cyclosporin. Digestive diseases and sciences 1993;38:1722-1734.

7. Wallace JL, Keenan CM. An orally active inhibitor of leukotriene synthesis accelerates healing in a rat model of colitis. American Journal of Physiology-Gastrointestinal and Liver Physiology 1990;258:G527-G534.

8. Wallace JL, Braquet P, Ibbotson GC, MacNaughton WK, Cirino G. Assessment of the role of platelet-activating factor in an animal model of inflammatory bowel disease. J Lipid Mediat 1989;1:13-23.

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Supplementary Tables Table 1. Compiled list of human primers used in this study gene target sequence (5′-3′)

GAPDH-F CTCTCTGCTCCTCCTGTTCGAC

GAPDH-R TGAGCGATGTGGCTCGGCT

TLR3-F AGGAAAGGCTAGCAGTCATCC

TLR3-R AGCAACTTCATGGCTAACAGTG

TLR4-F AAGCCGAAAGGTGATTGTTG

TLR4-R CTGAGCAGGGTCTTCTCCAC

IL-6-F CCAGTACCCCCAGGAGAAGAT

IL-6-R TTGCCTTTTTCTGCAGGAAC

Cox-2-F ACTCTGGCTAGACAGCGTAA

Cox-2-R ACCGTAGATGCTCAGGGAC

IFN-γ-F TCGGTAACTGACTTGAATGTCCA

IFN-γ-R TCCTTTTTCGCTTCCCTGTTTT

IDO1-F AGACTGCTGGTGGAGGACATG

IDO1-R AAAGGACAAACTCACGGACTG

NOTCH-1-F 5'-AGCTCGTCCCCGCATTCCAA-3'

NOTCH-1-R 5'-AGGCAGGTGATGCTGGTGGA-3'

CXCL-10-F 5'-CAAGCCAATTTTGTCCACGT-3'

CXCL-10-R 5'-GTAGGGAAGTGATGGGAGAG-3'

Notch-1-F CAATGTGGATGCCGCAGT TGTG

Notch-1-R CAGCACCTTGGCGGTCTCGTA

Jagged-1-F CGGGATTTGGTTAATGGTTATC

Jagged-2-R ATAGTCACTGGCACGGTTGTAGCAC

Jagged-2-F ACCAGGTGGACGGCTTTG

Jagged-2-R CCGCGACAGTCGTTGA

Delta-1-F CCTACTGCACAGAGCCGATCT

Delta-1-R ACAGCCTGGATAGCGGATACAC

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Table 2 Grouping of mice used in vivo experiment

Group (mice) TNBS

treatment MSC treatment Indo

Control – – –

TNBS + – –

MSC + MSCs –

poly (I:C)-MSC + poly (I:C) -primed MSCs –

LPS-MSC + LPS -primed MSCs –

IFN-γ-MSC + IFN-γ-primed MSCs –

TNF-α-MSC + TNF-α-primed MSCs –

TLR3 siRNA MSC + TLR3 siRNA-transfected MSCs –

Notch-1 siRNA MSC + Notch-1 siRNA-transfected MSCs –

Jagged-1 siRNA MSC + Jagged-1 siRNA-transfected MSCs –

DAPT+ poly (I:C) MSC + DAPT+ poly (I:C) MSC –

Indo- + poly (I:C) –

Indo+ + poly (I:C) 5μM

Abbreviations: TNBS, trinitrobenzene sulfonic acid; TLR3, Toll-like receptor 3; MSC,

mesenchymal stem cell; siRNA, small RNA interference; Indo, indomethacin, a COX inhibitor. .

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Table 3 Disease activity index (DAI)

weight-loss stool scores bleeding scores scores

0 normal no bleeding 0

1~5 + Guiac occult blood test (minimal color change to green) 1

6~10 loose + Guiac occult blood test (maximal color change to blue) 2

11~15 blood visibly present in the stool and no clotting on the anus 3

>15 diarrhea gross bleeding from the anus with clotting present 4

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Table 4 Macroscopic score

Score Description

0 no ulcer and no inflammation;

1 local hyperemia without ulceration;

2 ulceration without hyperemia;

3 ulceration and inflammation at one site only;

4 two or more sites of ulceration and inflammation;

5 ulceration extending more than 2 cm

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Table 5 Histological score

Score Description

0 No evidence of inflammation

1 Low level of inflammation with scattered infiltrating mononuclear cells (1–2 foci)

2 Moderate inflammation with multiple foci

3 High level of inflammation with increased vascular density and marked wall thickening

4 Maximal severity of inflammation with transmural leukocyte infiltration and loss of goblet

cells

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Table 6. Compiled list of mice primers used in this study T subsets gene target sequence (5′-3′)

β-actin-F GTGGGCCGCCCTAGGCACCA

β-actin-R CTCTTTGATGTCACGCACGATT

Th17 RORγt-F CCGCTGAGAGGGCTTCAC

RORγt-R TGCAGGAGTAGGCCACATTACA

IL-17-F CTCCAGAAGGCCCTCAGACTAC

IL-17-R AGCTTTCCCTCCGCATTGACACAG

Th1 STAT-4-F CCTTCTCCCCATGTCTCCAAGT

STAT-4-R CCGTTTGCACCGTCATTCA

IFN-γ-F GATGCATTCATGAGTATTGCCAAGT

IFN-γ-R GTGGACCACTCGGATGAGCTC

IL-6-F CCAGAAACCGCTATGAAGTTCC

IL-6-R TTGTCACCAGCATCAGTCCC

TNF-α-F ACAGAAAGCATGATCCGCG

TNF-α-R GCCCCCCATCTTTTGGG

Th2 IL-10-F CCCTTTGCTATGGTGTCCTT

IL-10-R TGGTTTCTCTTCCCAAGACC

IL-4-F CTTGAGAGAGATCATCGGCATTT

IL-4-R CTCACTCTCTGTGGTGTTCTTCGTT

STAT-6-F GATCATGAACAACACGGTGCC

STAT-6-R CGCTCACAGCGCTTTATTTTCT

Regulatory T IL-10-F CCCTTTGCTATGGTGTCCTT

IL-10-R TGGTTTCTCTTCCCAAGACC

Foxp3-F GGCCCTTCTCCAGGACAGA

Foxp3-R GCTGATCATGGCTGGGTTGT

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Supplementary Figures 1

Supplementary Figure 1. The effects of TLR3 signaling on hUC-MSC growth and

differentiation. (A) The grow curve of hUC-MSCs with(out) poly (I:C) priming. (B)

Differentiation capacity of poly (I:C)-MSC (upper panels) and naïve hUC-MSCs

(lower panels) into adipocytes and osteoblasts. Osteogenic differentiation was

detected using Alizarin Red S to reveal alkaline phosphatase activity. Lipid droplets

in the cell cytoplasm were stained with oil red O.

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Supplementary Figure 2

Supplementary Figure 2. The effect of TLR3 signaling on the phenotype of the

hUC-MSCs. Expressions of phenotypic surface antigens on hUC-MSCs were detected

via flow cytometry analysis.

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Supplementary Figure 3

Supplementary Figure 3. The siRNA itself has a negligible effect on the viability

with Annexin V- PI staining.

Note: By staining cells with Annexin V-FITC and PI, FACS was used to distinguish and

quantitatively determine the percentage of dead, viable, apoptotic and necrotic cells after siRNA

transfection.

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Supplementary Figure 4

Supplementary Figure 4. Poly (I:C)-induced PGE2 is responsible for the

anti-inflammatory activity of hUC-MSCs. Ligation of TLR3 on hUC-MSCs

enhance their suppressive effect on T-cell proliferation by increasing their Jagged-1

expression and, therefore, further activating its signaling to Notch receptors expressed

on T cells. Notch signaling regulates the immune-suppression of MSCs through the

production of PGE2. PGE2 is an important mediator of the MSC-mediated suppression

of T-cell proliferation and polarization.