SUPPLEMENTARY INFORMATION
SUPPLEMENTARY METHODS
Mice
The following strains of mice were used: C57BL/6J (H-2b), BALB/c (H-2d), OT2 TCR transgenic mice (Ly5.1+) specific for peptide Ovalbumin 323-339 (OVA323-339). They were purchased from Charles River Laboratories (Italy). All mice used as primary cell donors or recipients were between 8 and 12 wk of age. Procedures involving animals and their care conformed to institutional guidelines in compliance with national (4D.L. N.116, G.U., suppl. 40, 18-2-1992) and international (EEC Council Directive 86/609, OJ L 358,1,12-12-1987; National Institutes of Health Guide for the Care and Use of Laboratory Animals) law and policies. The protocol was approved by the Italian Ministry of Health on June 18th, 2007 and modified by Protocol 162/2011-B. All efforts were made to minimize the number of animals used and their suffering.
Isolation of murine MSCs (MSCs)
mMSCs were isolated by flushing the femurs and tibias from 8 week-old, C57Bl/6 female mice and cultured in 25 cm2 tissue culture flasks at a concentration of 2X106 cells/cm2 using complete Dulbecco modified Eagle medium low glucose (DMEM, Lonza, Braine-L’Alleud, Belgium) supplemented with 20% heat inactivated fetal bovine serum (Biosera, Ringmer, United Kingdom), 2mM glutamine (Lonza), 100 U/ml penicillin/streptomycin (Lonza) and 100 μg/ml gentamicin (Euroclone, Pero, Italy). Cells were incubated at 37°C in a 5% CO2 atmosphere. After 48 h, the non-adherent cells were removed. After reaching 70–80% confluence, the adherent cells were trypsinized (0.05% trypsin at 37°C for 3 minutes), harvested and expanded in larger flasks. mMSCs at passage 10 were screened by flow cytometry for the expression of CD106, CD45, CD117, CD73, CD105, MHC-I, SCA-1 and CD11b and used to perform experiments (BD Pharmingen, Oxford, UK).
Differentiation culture of mMSCs for adipogenic and osteogenic mesenchymal lineages
Adipogenic differentiation was induced by culturing confluent mMSCs in complete DMEM (high glucose) 20% FBS, supplemented with 1 µM dexamethazone, 0.5 mM 3-isobutyl-1-methylxanthine and 0.1 mM indomethacine (Sigma-Aldrich). Osteogenic differentiation was achieved by culturing mMSCs in complete DMEM (low glucose) 20% FBS, supplemented with 1 nM dexamethazone, 50 µM ascorbic acid, and 20 mM β-glycerophosphate (Sigma-Aldrich).
Each specific differentiation medium was changed every 2–3 days. Confirmation of differentiation of the cells to adipocytes and osteocytes were performed by staining with oil red O and Alizarin Red, respectively.
Flow cytometry analysis
Cells were analyzed on a CantoII (BD Biosciences), and post-analysis of flow cytometry data was performed with FlowJo software (Tree Star Inc.). The following antibodies were used in experiments and were purchased from BD Biosciences or eBioscience: anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-CD11c (clone HL3), anti CD45.1 (clone A20). FoxP3 staining was performed according to manufacture instructions (e-Bioscience, San Diego, CA).
Quantitative RT-PCR
Total RNA was isolated from untreated and encapsulated mMSCs using RNeasy Mini Kit (Quiagen, Venlo, The Netherlands) according to the manufacturer's instructions.
RNA was reverse-transcribed using the High-Capacity cDNA reverse transcription Kit, (Applied Biosystems, Carlsbad, CA). Quantitative PCR analysis was performed with TaqMan Gene Expression MasterMix (Applied Biosystems). The following probes for Taqman Gene Expression Assay (Applied Biosystems) were used: 18S (Hs99999901_s1), FABP4 (Mm00445880_m1), Col2a1 (Mm01309565_m1), OC (Bglap1;Bglap-rs1;Bglap2) (Mm03413826_mH). qRT-PCR data were expressed as target gene expression level (normalized AU) of three cell samples.
Cell encapsulation in alginate microcapsules
The capsules were prepared as described 1. Briefly the mMSC were thoroughly mixed with 1.6% alginate solution at a final concentration of 8x106 cells/ml of alginate. The suspension was extruded through a microdroplet generator combining air shears with mechanical pressure. The capsules diameter is about 600 µm. The alginate droplet were collected in 1.2% Bacl2 (Sigma Aldrich, Milano, Italy), immediately turning into gel microbeads. The final microcapsule preparation was incubated for additional 6-24 hours for sterility and viability checking as described above. Where needed, cells were retrieved from capsules by incubation in EDTA 5mM for 10 minutes at 37°C.
Viability of E-MSC
Immediately before, after capsule preparation and at different time points, MSCs viability was assessed by staining the preparations with ethidium bromide (EB; Sigma-Aldrich) and fluorescein diacetate (FDA; Sigma-Aldrich) that stain dead and live cells in red and green respectively, as previously described 2. Cells were visualized by fluorescence microscopy (BX51, Olympus)
In vivo immunological studies
CD4+ T cells were isolated from the spleen and lymph nodes (LNs) of Ly5.1 OT2 mice using the CD4+ Isolation Kit (Miltenyi Biotec), the cells were labeled with 1 μM CFSE for 10 minutes at 37°C and 106 were transferred i.v. into C57BL/6J female (sex-matched) mice.
The following day mice were given s.c. injections into the footpads with 30 μl of an emulsion containing 100ng OVA323-339 peptide (ISQAVHAAHAEINEAGR; AnaSpec, Fremont, CA) and 1mg/ml Mycobacterium tuberculosis in CFA (Sigma-Aldrich).
After 24 hours 0.4 x 106 MSCs were injected i.v in the tail vein or 50 ml of capsules entrapping MSCs, resuspended in 400ml of HBSS, were injected s.c. in the back of the mouse.
Immunized mice were sacrificed 2 days later and the popliteal draining LNs (dLNs) were collected and treated with 1.5 mg/ml collagenase IV (Sigma-Aldrich) and 0.2 mg/ml DNAse (Roche) at 37°C for 40 min. Cells were washed in PBS, counted and stained with specific antibodies.
GVHD experimental model
BALB/c mice received myeloablative total-body irradiation (700 cGy, 137Cs source, RadGil, Gilardoni, Italia) in two doses with a 2 hours interval, followed by i.v. infusion of 1 x 107 donor B6 BM cells and 1.5 x 107 B6 spleen cells as a source of allogeneic T cells. Syngeneic transplant was performed infusing the same concentration of Balb/c BM and spleen cells. BM suspensions were prepared by flushing femurs with RPMI 1640 medium supplemented with 10 % FCS. Splenocytes were obtained by gently crushing the spleen in complete medium to release the cells. After erythrocyte lysis cells were filtered to remove debris and washed twice in PBS before injection. To prevent infections induced by conditioning regiments, irradiated mice received gentamicin (70 mg/L) in their drinking water beginning 7 days before transplantation and continuing until 15 days after transplant. Every two days after transplant mice were monitored for survival and clinically evaluated by a standard scoring system that generates a composite GVHD score comprised of individual scores for weight loss, posture (hunching), activity, fur texture, skin integrity, diarrhea, occult blood in feces. Each parameter was graded 0 to 2 according to occurrence and degree of intensity. A clinical index was subsequently generated by summation of scores of the seven criteria (maximum index =14), as previously described 3.
ELISA
CCL8 and sTNFr1 protein levels were measured by ELISA (R&D Systems) following the manufacturer’s instructions.
Histopathology
The liver were fixed in buffered formaldehyde (4% in PBS) for 24 h and routinely processed for paraffin embedding. Sections 4 μm-thick were obtained and stained with haematoxylin and eosin. For CD3 staining, the sections were deparaffined, the antigen retrieval was performed with Citrate buffer (Biocare Medical, Concord, CA). Endogenous peroxidase activity and the specific sites were blocked. Primary antibody used was anti-CD3 (Dako) and the appropriate secondary antibody was used. Immunoreactivity was visualized with 3,3-diaminobenzidine (DAB, Sigma). Sections were counterstained with haematoxylin and mounted in Eukitt. Liver was examined under an optical microscope (Olympus BX51, Japan) using a 10x, 20x and 40x objective. The images were captured under the microscope with a resolution of 4080 x 3072 pixels (Olympus DP71 camera) and evaluated using the Olympus CellA program.
Specimens of liver were provided for histopathologic assessment of GVHD using a semiquantitative scoring system for abnormalities known to be associated with GVHD 4. Eleven parameters were scored for liver (portal tract expansion by an inflammatory cell infiltrate, lymphocytic infiltrate of bile ducts, bile duct epithelial cell apoptosis, bile duct epithelial cell sloughing, vascular endothelialitis, parenchymal apoptosis, parenchymal microabscesses, parenchymal mitotic figures, hepatocellular cholestasis, hepatocellular steatosis and lobular inflammation). The scoring system for each parameter was as follows: 0 indicates normal; 0.5, focal and rare; 1, focal and mild; 2, diffuse and mild; 3, diffuse and moderate; and 4, diffuse and severe.
Histopathological scores were performed in samples obtained from mice at days 6 after the induction.
Statistical analysis
Log-rank test was used for survival analysis. Differences between two groups were compared using t test or Mann Whitney. Values of P less than .05 were considered significant.
REFERENCES
1. Luca G, Calvitti M, Nastruzzi C, Bilancetti L, Becchetti E, Angeletti G, et al. Encapsulation, in vitro characterization, and in vivo biocompatibility of Sertoli cells in alginate-based microcapsules. Tissue Eng 2007;13(3):641-8.
2. Miyamoto M, Morimoto Y, Nozawa Y, Balamurugan AN, Xu B, Inoue K. Establishment of fluorescein diacetate and ethidium bromide (FDAEB) assay for quality assessment of isolated islets. Cell transplantation 2000;9(5):681-6.
3. Castor MG, Rezende B, Resende CB, Alessandri AL, Fagundes CT, Sousa LP, et al. The CCL3/macrophage inflammatory protein-1alpha-binding protein evasin-1 protects from graft-versus-host disease but does not modify graft-versus-leukemia in mice. J Immunol 2010;184(5):2646-54.
4. Zhao F, Zhang Y, Wang H, Jin M, He S, Shi Y, et al. Blockade of osteopontin reduces alloreactive CD8+ T cell-mediated graft-versus-host disease. Blood 2011;117(5):1723-33.
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