Ultrafiltration Segregates Tissue Regenerative Stimuli Harboured Within and Independent of Extracellular Vesicles

Cooper, T. T.; Sherman, S. E.; Dayarathna, T.; Bell, G. I.; Ma, J.; McRae, D. M.; Lagugne-Labarthet, F.; Pasternak, S. H.; Lajoie, G. A.; Hess, D. A.

doi:10.1101/2020.01.28.923037

bioRxiv · 10.1101/2020.01.28.923037

Ultrafiltration Segregates Tissue Regenerative Stimuli Harboured Within and Independent of Extracellular Vesicles

Cooper, T. T.·Sherman, S. E.·Dayarathna, T.·Bell, G. I.·Ma, J.·McRae, D. M.·Lagugne-Labarthet, F.·Pasternak, S. H.·Lajoie, G. A.·Hess, D. A.

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Abstract

The release of extracellular vesicles (EVs) from human multipotent stromal cells (MSC) has been proposed as a mechanism by which MSC mediate regenerative functions in vivo. Our recent work has characterized MSC derived from human pancreatic tissues (Panc-MSC) that generated a tissue regenerative secretome. Despite these advancements, it remains unknown whether regenerative stimuli are released independent or within extracellular vesicles. Herein, this study demonstrates ultrafiltration is a simple method to enrich for EVs which can be injected in murine models of tissue regeneration. The enrichment of EVs from Panc-MSC conditioned media (CM) was validated using nanoscale flow cytometry and atomic force microscopy; in addition to the exclusive detection of classical EV-markers CD9, CD81, CD63 using label-free mass spectrometry. Additionally, we identified several pro-regenerative stimuli, such as WNT5A or ANGPT1, exclusive to EV-enriched CM. Endothelial cell tubule formation was enhanced in response to both Panc-MSC CM fractions in vitro yet only intramuscular injection of EV-enriched CM demonstrated vascular regenerative functions in NOD/SCID mice with unilateral hind-limb ischemia (*<p<0.05). Furthermore, both EV-depleted and EV-enriched CM reduced hyperglycemia following intrapancreatic injection in hyperglycemic mice (**p<0.01). Collectively, understanding the functional synergy between compartments of the secretome is required to advance cell-free biotherapeutics into applications of regenerative medicine.

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Cooper, T. T., Sherman, S. E., Dayarathna, T., Bell, G. I., Ma, J., McRae, D. M., Lagugne-Labarthet, F., Pasternak, S. H., Lajoie, G. A., Hess, D. A.. 2020-01-29. Ultrafiltration Segregates Tissue Regenerative Stimuli Harboured Within and Independent of Extracellular Vesicles. https://doi.org/10.1101/2020.01.28.923037

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