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Translational and Clinical Research
Article first published online: 20 AUG 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 9, pages 2044–2053, September 2012
How to Cite
Giunti, D., Parodi, B., Usai, C., Vergani, L., Casazza, S., Bruzzone, S., Mancardi, G. and Uccelli, A. (2012), Mesenchymal Stem Cells Shape Microglia Effector Functions Through the Release of CX3CL1. STEM CELLS, 30: 2044–2053. doi: 10.1002/stem.1174
Author contributions: D.G. and A.U.: designed research; D.G., B.P., C.U., L.V., S.C., and S.B.: performed research; D.G., B.P., G.M., and A.U.: analyzed data; D.G. and A.U.: wrote the paper; A.U.: provided financial support to the study.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS July 20, 2012.
- Issue published online: 20 AUG 2012
- Article first published online: 20 AUG 2012
- Accepted manuscript online: 20 JUL 2012 10:50AM EST
- Manuscript Accepted: 19 JUN 2012
- Manuscript Revised: 6 JUN 2012
- Manuscript Received: 13 JAN 2012
- Italian Foundation for Multiple Sclerosis (FISM)
- Italian Ministry of Health (Ricerca Finalizzata)
- Regione Liguria, the Italian Ministry of University and Scientific Research (MIUR)
- Progetto Limonte, and the Fondazione CARIGE
Additional Supporting Information may be found in the online version of this article.
|SC-12-0047_sm_SupplFigure1.pdf||846K||Figure Supplementary 1: Efficiency of CX3CL1 silencing in MSC Efficiency of silencing of CX3CL1 at 24h is shown. We utilized 4 siRNA oligo-sequences targeting CX3CL1 (Qiagen). The oligo1 sequence 5′-TTGAAGTTGTTGATCCTTTA-3′, providing 80% efficiency of silencing, was chosen. Similar results were also obtained after 48h from transfection.|
|SC-12-0047_sm_SupplFigure2.pdf||64K||Figure Supplementary 2: MSC modulate the release of effector molecules by microglia The presence of MSC in culture with LPS-activated N9 cells (grey bars) significantly reduced the microglia production of the NO metabolites, nitrite and nitrate and TNF compared to control cells (N9+LPS, black bars), similar to levels produced by resting microglia (white bars) as measured by ELISA. MSC enhanced the secretion of IL1β and IGF1 by LPSactivated microglia (grey bars) compared to resting (white bars) and activated microglia in the absence of MSC (black bars). Results are depicted as mean ± SD of at least three independent experiments. *, p<0.05 by t test.|
|SC-12-0047_sm_SupplFigure3.pdf||1923K||Figure Supplementary 3: Effects on molecules expressed and released by primary microglia following activation in the presence of MSC LPS-activated microglia from primary cultures (grey bars) was cultured with MSC at 1:1 ratio (8×105 cells) and the expression of TNF, CX3CR1, NURR1, IGF1 and IL1β was quantified by Real Time PCR and compared to that one of LPS-activated microglia (black bars) and resting primary microglia (white bars). Results obtained for TNF and IL1β mRNA expression were also confirmed at protein level by ELISA. Results are shown as mean ± SD of at least three independent experiments. *, p<0.05 ; **, p<0.05 by t test.|
|SC-12-0047_sm_SupplFigure4.pdf||1598K||Figure Supplementary 4: MSC modulate the profile of genes expressed by microglia even when activated by IFNγ and CpG. Microglia activated with either LPS, IFNγ or CpG was cultured with MSC at 1:1 ratio (8x105 cells) (grey bars) and the expression of TNF, iNOS, HO1, IGF1, CX3CR1, NURR1 was quantified by Real Time PCR. Results were compared to gene expression levels of resting microglia (white bars) and activated microglia in the absence of MSC (black bars). Results obtained for IGF1 mRNA expression were also confirmed at protein level by ELISA. Results are shown as mean ± SD of at least three independent experiments. *, p<0.05; **, p<0.01 by t test.|
|SC-12-0047_sm_SupplFigure5.pdf||1230K||Figure Supplementary 5: MSC produce and express CX3CL1 but do not inhibit proliferation of microglia. A) The production of CX3CL1 was measured by ELISA on supernatants derived from MSC alone (white bar) or cultured in the presence of either 5ng/ml IFNγ (light grey bar), 1μg/ml LPS (dark grey bar) and in the presence of both stimuli (black bar). Quantitative gene expression of CX3CL1, in MSC activated as previously described, was analyzed by Real Time PCR. *, p<0.05 by t test. B) LPS-activated microglia proliferation was measured by intracellular incorporation of CFSE in the presence (right panel) or absence of MSC (middle panel), compared to activated microglia (left panel) at time 0 (T0) and 4 days post culture with or without MSC (T4). Results are shown as mean ± SD of at least three independent experiments.|
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