Role of Royal Jelly Treated Adipose-Derived Stem Cell-Extracellular Vesicles on Fibroblast Proliferation, Migration, and Collagen Production

Figure 2

Morphological characterization of extracellular vesicles (EVs) prepared from ADSCs or ADSCs treated with royal jelly. (a) Particle distribution of ADSC-EVs, nRJ-ADSC-EVs, and pRJ-ADSC-EVs measured by nanoparticle tracking analysis. (b) Particle concentration of ADSC-EVs, nRJ-ADSC-EVs, and pRJ-ADSC-EVs was detected by NTA (n = 5). (c) Proliferation of each RJ-treated ADSC was measured by WST-1 assay. ADSCs were seeded in 96-well plates and were allowed to attach for 24 h. ADSCs were then treated with the indicated concentrations of each RJ sample for 48 h (n = 12). (d) Subcellular localization and size characterization of EVs in ADSCs treated with RJ examined by TEM. Each lower panel indicates enlarged area that is outlined in each upper panel. Multiple nanovesicles are encapsulated in multivesicular bodies. Arrowhead identifies multivesicular bodies or exosomes, whereas the arrow denotes the endoplasmic reticulum. (e) Representative SEM and TEM micrographs of prepared EVs. (f) Full-length western blot images for the exosomal protein (Alix, CD9) and non-exosomal protein makers (GM130): (A) ADSCs; (F) nRJ-ADSCs; (E) pRJ-ADSCs. (g) Uptake of each EV by NHDFs. NHDFs were exposed to each EV labeled with PKH67 (green) for 24 h. Arrows indicated PKH67-labeled EVs taken into NHDFs. Blue: nucleus stained with Hoechst33342. Red: cell membrane stained with CellROX™ Deep Red. Data are presented as means ± SE. Statistical analysis was performed using the Tukey–Kramer test, and differences were considered significant at .

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