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theses

Mesenchymal stromal cells (MSCs) hold great potential as a cellular therapy due in part to their tissue protective and regenerative abilities, achieved via the secretion of many bioactive molecules and induced by microenvironmental cues including soluble factors. However, translation of MSCs into the clinic, where variability exists amongst both MSC donors and recipients, has not resulted in dramatic success. Efforts to identify activation inputs that promote specific MSC phenotypes have been inconsistent and lack systematic optimization. The goal of our work is to address these hurdles using a combination of approaches. We aimed to (1) develop a systematic approach for optimizing activation parameters (type and/or combinations of activating molecule, concentration, and duration of exposure) that enhance MSC immunomodulation, (2) characterize MSCs treated with the resulting pre-activation protocol, and (3) test the performance of the pre-activation protocol using multiple MSC donors. In a high throughput in vitro screening assay designed using fractional factorial design of experiments, we identified interleukin (IL)-1β and lipopolysaccharide (LPS), individually and in combination, as optimal activators of MSC attenuation of pro-inflammatory macrophage tumor necrosis factor (TNF)-α production via prostaglandin E2 (PGE2) secretion. Further optimization of dose and duration of pre-activation, however, revealed that brief (1 hour) exposure of to 1 ng/mL IL-1β alone resulted in the highest sustained upregulation of PGE2 secretion post-activation. This pre-activation protocol generated MSCs whose PGE2 secretion was more sensitive to induction by secondary inflammatory molecules than MSCs with no pre-activation. IL-1β pre-activation led to enhanced MSC-mediated attenuation of macrophage TNF-α in a co-culture assay. This protocol was also successful in enhancing these functions for multiple MSC donors, although variability was noted in the absolute secretion levels of PGE2, the level of improvement in macrophage modulation, and the correlation of MSC PGE2 and macrophage TNF-α. Using the previous assays, we found that alginate encapsulated MSCs retained the ability to respond to activation factors. Statistical analysis revealed that macrophage TNF-α level was more significantly a function of PGE2 level, MSC activation factor, and macrophage donor rather than MSC culture format (monolayer versus encapsulated).

ETD_6762

Ph.D.

Includes bibliographical references

by Andrea Gray

doi:10.7282/T3154K14

ETD doctoral

The author owns the copyright to this work.