Exosomes derived from Polygonum multiflorum-treated human dental pulp stem cells (hDPSCs): New approach in regenerative medicine

Abstract

Extracellular vesicles (EVs) are currently being extensively utilized in clinical trials. Our previous study demonstrated that the conditioned medium of human dental pulp stem cells (hDPSCs) treated by 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (THSG) exhibits biological activity and anti-inflammatory responses, which are believed to be regulated by EVs. To date, no study has thoroughly investigated the potential of the exosomes released by THSG-treated hDPSCs. In this study, we utilized the conditioned medium (CM) of hDPSCs with/without THSG stimulation to compare the differences in exosome extraction using ultracentrifugation (UC), tangential flow filtration (TFF), and TFF combined with size-exclusion chromatography (TFF + SEC) methods, aiming to establish an effective purification method. Subsequently, we analyzed the contents of the exosomes using proteomic strategy. The results showed that TFF + SEC was more effective in reducing protein contamination compared to CM and TFF alone. TFF effectively concentrated large-scale CM and maintained exosome particle sizes consistently around 85–100 nm. Flow cytometry analysis revealed that the presence of THSG increased exosome secretion from hDPSCs, concurrently leading to the identification of 219 unique proteins through proteomic analysis, including BMP-1, VEGF, and members of the MMP family. Compared to non-lyophilized exosomes, lyophilized exosomes showed no change in protein activity, indicating that lyophilization was an effective preservation method. Collectively, these findings present a combined extraction strategy for dental stem cell exosomes and reveal the high purity and proteome composition of THSG-exosomes, thereby highlighting their potential applications in regenerative therapy.