Fig 1.
Characterization of commercial murine mesenchymal stem cells.
C57BL/6 MSCs (Invitrogen, # S1502-100) were cultured as recommended by the manufacturer and utilized below passage 18. (A) Morphology of MSCs in culture observed with ×40 magnification on an inverted phase-contrast microscope. MSC cells present with a characteristic spindle-shape morphology. Scale bar, 100 μm. (B) Proper stem cell markers expression in cultured MSC. On passage 18, MSC were immunotypified for CD34, CD45, Sca-1, CD44 and CD29. MSC showed negative for hematopoietic markers CD34 and CD45 and positive for Sca-1, CD44 and CD29. Histograms for isotype controls are shown in grey. (C) Invitrogen MSCs are immunosuppressive. CD4 T-cells were isolated from healthy C57BL/6 mice using CD4+ untouched kit (Invitrogen) and labeled with Cell Trace Violet® (CTV; Invitrogen) for proliferation analysis. 300,000 CTV-labeled CD4+ T-cells were stimulated with anti-CD3/anti-CD28 in the presence of indicated amounts of MSC and T-cell proliferation was assessed by flow cytometry 60 h later, as described in Methods. Left Panel, representative histograms from T-cell cultures showing dilution patterns of CTV signal in CD4+-gated cells. To localize the pool of non-proliferating cells with undiluted CTV, control cultures were kept not stimulated (basal). Proliferation indexes were calculated from cell events at each dilution peak using described algorithms. MSC cause a dose-dependent decrease in T-cell proliferation. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 3).
Fig 2.
IL-1β pre-treatment increases immunosuppressive properties of MSCs.
MSC were seeded at 4,800 cells/cm2 and grew for 24 h. MSC cultures were then supplemented with 20 ng/mL IL-1β, 25 ng/mL IFN-γ, or the combination of both cytokines as described in Methods and MSC cells were grew for additional 48 h. The ending day for the cytokine treatment, splenic TCD4+ lymphocytes were isolated from healthy C57BL/6 mice, labeled with CTV for proliferation assessment and stimulated with anti-CD3/anti-CD28 for 4 h. MSC were then added to CTV-labeled TCD4+ cells (ratio 1:10 MSC:TCD4+) and co-cultures were continued for additional 60 h as described in Methods. Proliferation controls included the determination of background and maximal proliferation for the T-cell preparation (basal, No MSC and anti-CD3/anti-CD28, respectively), and basal MSC-mediated immunosuppression was determined using untreated MSC (MSC Ctrl). Priming with IL-1β increases immunosuppressive properties of MSC. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 3). Right Panel, representative histograms showing gating strategy and CTV dilution peaks for each treatment.
Fig 3.
GAD-67 gene expression regulation by pro-inflammatory cytokines IL-1β and IFN-γ in MSCs.
MSC were seeded at 4,800 cells/cm2 and grew for 24 h. MSC cultures were then supplemented with 20 ng/mL IL-1β, 25 ng/mL IFN-γ, or the combination of both cytokines, and MSC cells were grew for indicated times. Control cultures were identically treated but cytokines were not added. (A) GAD-67, GAD-65 and ABAT mRNA levels were determined 48 h after cytokine treatment by RT-qPCR. IL-1β selectively increases GAD-67 mRNA levels in MSCs and IFN-γ potentiates IL-1β effects. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 3). (B) Sister cultures of experiments as in A were set and protein lysates prepared at 24 or 48 h after cytokine treatment. Proteins (30 μg) were subjected to 8% SDS-PAGE and GAD-67 protein levels were determined by Western blot using β-actin as a loading control. 15 μg of total brain lysate was used as a positive control. Lower panel, densitometric analysis of Western blot results. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 11).
Fig 4.
Increased GABA secretion with GAD-65/GAD-67 co-expression.
MSCs were seeded at 10,000 cells/cm2 in 6 well plates and grew overnight. Cells were then transfected with Fugene6 as described in Methods using 1 μg total plasmid, and cells were recovered for 24 h. Co-expression with GAD-65 and GAD-67 used equivalent amounts of each plasmid. (A) Conditioned media was pre-cleared by centrifugation and secreted GABA levels were enzymatically determined using a fluorescence-coupled assay. Basal levels were 2.48 ± 0.83 μM and results were expressed as fold change to GAD-65 transfected (GAD-65) cells. Maximal secretion of GABA was detected using GADs co-expression. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 5). (B) 15 μg (GAD-65, β-actin) or 30 μg (GAD-67) proteins were separated by 8% SDS-PAGE, proteins transferred to PVDF membranes and GAD-65, GAD-67 or β-actin immunodetected by Western blotting. Note that GAD levels in co-transfected cells were approximately half the levels of GADs expressed individually.
Fig 5.
GAD-65/GAD-67 co-expression increases MSC immunosuppression.
MSCs were seeded and transfected as indicated in Fig 2. Where indicated, IL-1β treatment was initiated right after addition of DNA complexes. 24 h after treatment, MSC:TCD4+ co-cultures were assembled at MSC:TCD4+ ratios of (A) 1:30; (B) 1:15; or (C) 1:10, and cell proliferation was assessed by FACS 60 h later as before. GADs co-expression increases immunosuppressive properties of MSC. *, p<0.05 (Kruskal-Wallis test with Dunn’s correction, N = 3). (D) Dose-dependent inhibition of T-cell proliferation by MSCs transfected with GFP or GADs. Where indicated, MSC were transfected with single or double amount of coding plasmids, or co-treated with IL-1β. Controls for T-cell proliferation include 1, basal, non-stimulated cultures; and 2, maximal stimulation (no MSC treatment). MSC doses in co-culture experiments were 50,000 (3,8,13,18), 40,000 (4,9,14,19), 30,000 (5,10,15,20), 20,000 (6,11,16,21) or 10,000 cells (7,12,17,22) and TCD4+ cells were kept constant at 300,000 cells per well. Similar immunosuppression was obtained using GADs expression or IL-1β priming. Image is a representative experiment out of 2 independent assays.
Fig 6.
IL-1β priming increases NO levels and immunosuppressive properties of MSC.
MSC were seeded and treated with IL-1β for 24 h as described before. The ending day for the cytokine treatment, total splenocytes were isolated from healthy C57BL/6 mice, labeled with CTV for proliferation assessment and stimulated with anti-CD3/anti-CD28 for 4 h. MSC were then added to pre-seeded splenocytes at indicated doses, and co-cultures were developed for additional 60 h. Then, conditioned media was recovered for nitric oxide determinations (A) and cells were resuspended for splenic T-cell proliferation assessment by FACS (B). Priming with IL-1β increased NO levels in cell culture and potentiates immunosuppressive properties of MSC. *, p<0.05; **, p<0.01 (Mann-Whitney t-test, N = 5).
Fig 7.
GADs expression increases NO levels and immunosuppressive properties of MSC.
MSC were seeded, transfected with GADs or GFP, and recovered for 24 h as before. Total splenocytes were isolated from healthy C57BL/6 mice, and cells were labeled with CTV for proliferation assessment as described in Fig 6. MSC were then added to CTV-labeled splenocytes cells at indicated doses, and co-cultures were developed for additional 60 h. Then, conditioned media was recovered for nitric oxide determinations (A) cells were gently resuspended and splenic T-cell proliferation assessed by FACS (B). Expression of GADs caused an increase of NO levels in cell culture and potentiates immunosuppressive properties of MSC. *, p<0.05 (Mann-Whitney t-test, N = 4–8).
Fig 8.
GADs expression does not increase nitric oxide levels per se in MSC.
MSCs were seeded and transfected as indicated in Fig 4 or treated with cytokines as in S2 Fig. After 48 h, conditioned media was collected and assayed for NO levels as described in Methods. Plated media without cells was also included as control (media). GAD expression does not increase steady state levels of secreted nitric oxide. Results are from 4 independent experiments with determinations performed in triplicate. ***, p<0.001 (Kruskal-Wallis test with Dunn’s correction). NS, not statistically significant.