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. 2022 May 2;13:878194. doi: 10.3389/fimmu.2022.878194

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Copyright © 2022 Kang, Huang, Gajendrareddy, Lu, Shirazi, Ravindran and Cooper

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Effect of TNFα EVs on MSC osteogenic differentiation. (A) Representative confocal images of fluorescently labeled control and TNFα EVs (green) endocytosed by MSCs. Scale bar=20μm in all images. The red staining in the images indicate actin counter stain and blue staining shows the nuclei (DAPI). (B) Dose-dependent and saturable endocytosis of fluorescently labeled control and TNFα EVs by MSCs. (C) Fold change in the expression of osteoinductive marker genes in MSCs in the presence of control and TNFα EVs (72hrs post treatment) in the presence of growth medium. Data represents fold change with respect to no EV controls. Note that TNFα preconditioning does not generate osteoinductive property in MSC EVs. (D) ALP activity in MSCs cultured in osteogenic differentiation medium in the presence/absence of control or TNFα EVs. Note that while EVs increase ALP activity in general, TNFα preconditioning does not increase osteoinductive potential. (E) Representative images of alizarin red stained MSC cultures after 14 days of culture in osteogenic medium in the presence/absence of EVs. Note that while EV presence increased calcium deposition, no change was observed between control and TNFα preconditioned groups. *: statistical significance (P < 0.05) with respect to untreated control, #: statistical significance (P < 0.05) between control and TNFα EV groups as calculated by Tukey’s test post ANOVA.