Mesenchymal stem cells (MSCs) inhibit T-cell activation and proliferation but their effects on individual T-cell-effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD41 T cells toward the Th17 phenotype was examined. CD4 1 T cells exposed to Th17-skewing conditions exhibited reduced CD25 and IL-17A expression following MSC co-culture. Inhibition of IL-17A production persisted upon re-stimulation in the absence of MSCs. These effects were attenuated when cell-cell contact was prevented. Th17 cultures from highly purified naïve-and memoryphenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX-2 inhibitor. Media from MSC/Th17 co-cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC-mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation-induced IL-17A secretion by naturally occurring, effector-memory Th17 cells from a urinary obstruction model was also inhibited by MSC co-culture in a COX-dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T-cell precursors and inhibit naturally-occurring Th17 cells derived from a site of inflammation. Suppression entails cellcontact-dependent COX-2 induction resulting in direct Th17 inhibition by PGE2 via EP4.Key words: Immunosuppression . Mesenchymal stem cells . Stem cells . T helper cells . Th17 cells Supporting Information available online IntroductionThe immune suppressive properties of mesenchymal stem cells (MSCs) have garnered increasing attention over the past decade and constitute a central mechanism for MSC therapeutic benefits [1][2][3][4]. Specific modulatory effects of MSCs from human and experimental animal sources have been described for the differentiation, activation, proliferation and effector functions of multiple innate and adaptive immune cells [5][6][7][8][9][10][11]. Among these, MSC-mediated inhibition of primary T-cell activation and proliferation, suppression of DC maturation and promotion of regulatory phenotypes in 2840monocyte/macrophages and T cells have been most extensively characterised [7-9, 11, 12].In keeping with a paracrine or ''trophic'' model of MSC function in vivo [13], various MSC-produced soluble mediators have been implicated in these immunomodulatory effects including IL-10, IL-6, HGF, TGF-b, chemokine ligand-2 (CCL2), HLA-G, NO, tumor necrosis factor-inducible gene 6 protein (TSG-6), prostaglandin E2 (PGE2) and kyneurenine [1,2,7,9,12,[14][15][16]. For some such mediators, expression by MSCs may be dependent on pre-exposure to exogenous factors (e.g. IFN-g, TNF) or on contact-dependent MSC/target cell cross-talk [2,7,[16][17][18][19]. The potential for harnessing MSC immunomodulatory properties has been highlighted by results in pre-clinical models of autoimmunity, allotransplantation, sepsis and acu...

The use of multichannel polymer scaffolds in a complete spinal cord transection injury serves as a deconstructed model that allows for control of individual variables and direct observation of their effects on regeneration. In this study, scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] (OPF(+)) hydrogel were implanted into rat spinal cords following T9 complete transection. OPF(+) scaffold channels were loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats (eGFP-MSCs). Control scaffolds contained extracellular matrix only. The capacity of each scaffold type to influence the architecture of regenerated tissue after 4 weeks was examined by detailed immunohistochemistry and stereology. Astrocytosis was observed in a circumferential peripheral channel compartment. A structurally separate channel core contained scattered astrocytes, eGFP-MSCs, blood vessels, and regenerating axons. Cells double-staining with glial fibrillary acid protein (GFAP) and S-100 antibodies populated each scaffold type, demonstrating migration of an immature cell phenotype into the scaffold from the animal. eGFP-MSCs were distributed in close association with blood vessels. Axon regeneration was augmented by Schwann cell implantation, while eGFP-MSCs did not support axon growth. Methods of unbiased stereology provided physiologic estimates of blood vessel volume, length and surface area, mean vessel diameter, and cross-sectional area in each scaffold type. Schwann cell scaffolds had high numbers of small, densely packed vessels within the channels. eGFP-MSC scaffolds contained fewer, larger vessels. There was a positive linear correlation between axon counts and vessel length density, surface density, and volume fraction. Increased axon number also correlated with decreasing vessel diameter, implicating the importance of blood flow rate. Radial diffusion distances in vessels significantly correlated to axon number as a hyperbolic function, showing a need to engineer high numbers of small vessels in parallel to improving axonal densities. In conclusion, Schwann cells and eGFP-MSCs influenced the regenerating microenvironment with lasting effect on axonal and blood vessel growth. OPF(+) scaffolds in a complete transection model allowed for a detailed comparative, histologic analysis of the cellular architecture in response to each cell type and provided insight into physiologic characteristics that may support axon regeneration.

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