Altered Response of FOP Connective Tissue Stem Cells to Inflammatory Cytokines

1. Insult causing Inflammation Macrophage Activation Lymphocyte Activation TNF-α IFN-γ SHED Cells 6 Hours of TNFα + IFNγ Activated Mesenchymal Stem Cells FOP: TGF-ß FOP Lysate Normal Lysate Normal: TGF-ß Anti-TGFß1 44kD Less immunosuppression Immense Inflammation Heterotopic Ossification Immunosuppression Resolves Inflammation Altered Response of FOP Connective Tissue Stem Cells to Inflammatory Cytokines Discussion Transforming Growth Factor – Beta = immunosuppressive FOP flare ups are characterized by immense inflammation Mesenchymal Stem Cells are immunosuppressive when costimulated by Interferon gamma and Tumor Necrosis Factor Alpha Hypothesis: SHED cells from FOP patients will have lower exogenous amounts of the immunosuppressive TGF-B after stimulation with both IFN-gamma and TNF-alpha, allowing for immense inflammation during a flare up Ren, Guangwen, Liying Zhang, Xin Zhao, Guangwu Xu, Yingyu Zhang, Arthur I. Roberts, Robert C. Zhao, and Yufang Shi. "Mesenchymal Stem Cell-Mediated Immunosuppression Occurs via Concerted Action of Chemokines and Nitric Oxide." Cell Stem Cell 2.2 (2008): 141-50. Print. Lyons, Rusette M., Larry E. Gentry, A. F. Purchio, and Harold L. Moses. "Mechanism of Activation of Latent Recombinant Transforming Growth Factor Beta 1 by Plasmin." The Journal of Cell Biology 110.4 (1990): 1361-367. Print. References SHED Cells do not secrete exogenous TGF-B Longer Stimulation A greater concentration of cytokines Use a monoclonal primary antibody instead of a polyclonal Genetically sequence the 55KD TGF-B like band to determine it’s identifty Future Directions Introduction Methods SHED cells = Tie2+ stem cells which are of endothelial origin (makes them biologically relevant) ~Less invasive to obtain, capacity to undergo endochondral ossification Results Culture FOP and Wild Type Shed Cells, stimulate with 20ng/mL TNF-alpha, 20ng/mL IFN-gamma, or 20ng/mL of both TNF-alpha and IFN-gamma for 6 hrs, 24 hrs, or 48 hrs. Collect media and lyse cells. Western Blot and probe for TGF-B. Objectives Brendan Everett, Robert Pignolo, Frederick Kaplan Fibrodysplasia Ossificans Progressiva (FOP) is a rare genetic disorder characterized by progressive, episodic heterotopic endochondral ossification of muscles, tendons, and ligaments. Heterotopic ossification is commonly triggered by soft tissue injury followed by an immense inflammatory response. The cells which undergo ossification are originally vascular endothelial cells which have dedifferentiated into MSC’s before differentiating into bone. A growing body of literature has shown MSC’s to have immunosuppressive properties once stimulated by IFN-gamma and either IL-1 or TNF-alpha. TGF-B mouse knock-outs are characteruzed by immense inflammation, showing the immunosuppressive properties of TGF-B. We hypothesize that the immense inflammation which characterize FOP flare ups may be due to decreased TGF-B secreted from MSC which were stimulated by TNF-alpha and IFN-gamma. Robert Wood Johnson Medical School, UMDNJ and Perelman School of Medicine, University of Pennsylvania Media 24 Hours of TNF-α N Y N Y Pure TGF-ß Normal Normal FOP FOP Anti-TGF-ß1 12.5KD