Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Differential Oxygen Tension And Environmental Inflammatory Conditions Optimize Mesenchymal Stem Cell Fate And Immunosuppressive Potential
Rohini L. Kadle, BA, Marc A. Soares, MD, Nakul Rao, MD, April Duckworth, MD, Chin Park, MD, Joshua David, BS, Christopher Lopez, BS, Anna Zhou, BA, Nicholas Brownstone, BA, Rita Abigail Sartor, BS, Camille Kim, BS, Piul S. Rabbani, PhD, Daniel J. Ceradini, MD.
NYU, New York, NY, USA.

PURPOSE:
Mesenchymal stem cells (MSCs) are known to have powerful immunosuppressive properties and promote allograft tolerance, as previously demonstrated by our lab. Determining the environmental oxygen tension and inflammatory conditions under which MSCs are supremely primed for this immunosuppressive function is essential to their utilization in promoting graft tolerance. We hypothesize that priming MSCs by growing them under hypoxic conditions or exposing them to inflammatory cytokines will increase their immunosuppressive potential.
METHODS:
MSCs derived from Lewis rat bone marrow were expanded in either normoxia or hypoxia (5% O2 versus 0.5% O2). During expansion, MSCs were exposed to the inflammatory cytokines IFNγ and/or TNFα. Markers for immunosuppressive function, stemness and differentiation were assessed using quantitative real-time PCR and flow cytometry. Proliferation and migration were also assessed via MTT and Transwell assays.
RESULTS:
Growth in hypoxia at 5% O2 increased cyclinD2, a marker of cell cycle progression (1.7-fold, p<0.05) and showed no change in p21, a marker of replicative senescence, indicating that hypoxia promotes proliferation. MSCs grown in hypoxia demonstrated preservation of stemness, as measured by flow cytometry (97.9% CD90+, CD29+ MSCs in hypoxia versus 63.9% in normoxia). There was no significant change in markers of differentiation or immunosuppression in hypoxia versus normoxia. Growth at 0.5% O2 resulted in a decrease in all markers assessed, including those for immunomodulation, replication, and differentiation.
Exposure to cytokines increased the expression of immunosuppressive markers CXCL9 and CXCL10 (>300-fold and >700-fold respectively, p<0.0001) as well as the immunosuppressive molecule iNOS (>30-fold, p<0.05). Inflammatory cytokines also decreased markers of osteogenic differentiation (alkaline phosphatase, >4-fold, p<0.01) and adipogenic differentiation (lipoprotein lipase, >4-fold, p<0.05), indicating that exposure to cytokines promotes the undifferentiated state of MSCs. Exposure to cytokines led to increased migration of MSCs, as determined by a Transwell assay (p<0.01), but also leads to decreased proliferation as seen by an MTT assay (p<0.01) and a slight increase in replicative senescence (p21, 1.5-fold, p<0.05).
CONCLUSION:
MSCs grown under hypoxic conditions show increased proliferation and maintain their stemness. However, an extremely low oxygen tension negatively affects MSC stemness and MSC immunosuppressive properties. Priming MSCs with cytokines significantly increases the expression of immunosuppressive markers while also increasing senescence and decreasing proliferation. Here we have shown the potential in priming MSCs with hypoxic conditions and/or cytokines to optimize their immunomodulation.


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