Extracellular vesicles derived from differentiated granulosa-like cells restore the ovarian function of rats with premature ovarian insufficiency

Specifically differentiated cells exhibit greater therapeutic efficacy than mesenchymal stem cells (MSCs), and extracellular vesicles (EVs) present therapeutic benefits similar to those of parental cells and fewer safety issues. Ovarian granulosa cells (OGCs) play a critical role in the pathogenesis of premature ovarian insufficiency (POI), a common gynecological disease that can cause infertility and has no effective treatment. Here, we investigated whether umbilical cord mesenchymal stem cells (UCMSCs) can differentiate into ovarian granulosa-like cells (GLCs) and whether GLC-EVs are more effective in restoring ovarian function than UCMSC-EVs are in POI model rats. Here, we differentiated rat UCMSCs (rUCMSCs) into GLCs in vitro using cytokines and hormones and isolated GLC-EVs. We then used chemotherapy-induced POI model rats to verify the ability of GLC-EVs to repair ovarian function. We found that GLCs/GLCs-EVs expressed granulosa cell markers (FOXL2 and FSHR). We demonstrated that GLC-EVs outperformed rUCMSC-EVs by restoring the estrous cycle and ovarian morphology, increasing the number of follicles, regulating serum hormone levels, and restoring fertility in POI model rats. Mechanistically, GLC-EVs showed enhanced ovarian tropism. Proteomic analysis identified PLAU as a key component of GLC-EVs, and subsequent antibody blockade experiments demonstrated that PLAU contributes to primordial follicle activation through promoting FOXO3A phosphorylation (pFOXO3A). This study provides the first proof that EVs derived from differentiated cells enhance therapeutic precision for POI, improve the tissue targeting of EV therapy, and provide a generalized strategy for clinical cell-free therapy.