IntroductionFibrodysplasia Ossificans Progressiva (FOP) is a rare genetic

disorder characterized by progressive, episodic heterotopic endochondral ossification of muscles, tendons, and ligaments. The mutation results in unregulated BMP signaling through a TGF-B signaling pathway.The heterotopic ossification in FOP is commonly triggered by soft tissue injury followed by an immense inflammatory response, muscle death, fibroproliferation, angiogenesis, chondrogenesis, and ultimately osteogenesis. Some of 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. Similarly, TGF-B mouse knock-outs experience immense inflammation because they have lost the immunosuppressive properties of TGF-B. We hypothesize that the immense inflammation characteristic to FOP flare ups may be due to decreased TGF-B secreted from MSC which have been stimulated by TNF-alpha and IFN-gamma.

Objectives

• 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

Methods

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.

Results

Discussion

Lymphocyte Activation

Insult causing Inflammation

IFN-γ

Macrophage Activation

TNF-α

Activated Mesenchymal

Stem Cells

FOP: TGF-ß

Less immunosuppression

Immense Inflammation

Normal: TGF-ß

Immunosuppression

Resolves inflammation

Future Directions

• Use a new primary antibody (preferably monoclonal)

• Sequence the 55KD TGF-B like band to determine it’s identity

References

• 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.