Investigation of the protective effect of human adipose mesenchymal stem cells-derived exosomes and nanovesicles against cyclophosphamide-induced damage on human (HGRC1) and rat (SİGC) granulosa cell lines

Aim: In this study, it is aimed to investigate the antiapoptotic and therapeutic effects of exosomes (EXO) and nanovesicles (NV) derived from ADMSCs by inducing apoptosis with 4-hydroperoxy cyclophosphamide (4-HC) in both human (HGrC1) and rat (SIGC) granulosa cell lines under in vitro conditions. Material and method: EXOs and NVs were isolated from ADMSCs and their characterization was performed using flow cytometry method. EXOs and NV's were cocultured with HGrC1 and SIGC cell lines both alone and together with 4-HC and cell viability was determined by using the MTT method. After all applications, both protein and mRNA expression levels of apoptosis (Caspase 3, Bax and Bcl-2) and cell proliferation (Ki-67) markers were analyzed by indirect immunocytochemistry and qRT-PCR methods. Moreover, the cell cycle analyzes in all groups were performed by using flow cytometry method. Results: It was determined that EXOs and NVs derived from ADMSC induced granulosa cell proliferation after coculturing with HGrC1 and SIGC cell lines. In both cell lines it was found that Caspase 3 and Bax expressions decreased and Bcl-2 and Ki-67 expressions increased in the groups which cocultured EXOs and NVs together with 4-HC compared to the only 4-HC group. In addition, it was observed that the percentage of cells kept in S phase decreased and the percentage of cells in the G2/M phase increased in the groups cocultured with EXOs and NVs. Conclusions: It was found that EXOs and NVs had no cytostatic or cytotoxic effects on both granulosa cell lines, they had protective capacities against the apoptotic effect of 4-HC and they also induced granulosa cell proliferation. It was found that EXO and NV do not have cytostatic or cytotoxic effects on both granulosa cell lines, they have protective capacities against the apoptotic effect of 4-HC and they also induced granulosa cell proliferation. With this study, it was shown for the first time in the literature that artificially generated NVs from ADMSCs have similar therapeutic effects like EXOs. These results suggest that EXO and NV's can be an effective and new method in the prevention and treatment of POI.