Isolation and characterization of mesenchymal stromal cells derived from equine ovarian follicular aspirates

The use of mesenchymal stromal cells (MSCs) in equine reproduction is increasing its interest in the treatment of specific pathologies. MSCs have been isolated from follicular aspirates obtained during transvaginal oocyte aspiration in women, offering a novel source for autologous therapies in reproductive treatments. However, this approach has not been tested in mares despite the common use of transvaginal oocyte aspiration for oocyte collection to produce equine embryos in vitro. Our study aimed to investigate the feasibility of isolating MSCs from equine ovarian follicular aspirates obtained by ovum pick-up (OPU). Follicular aspirates from six mares were processed to establish adherent cell cultures. Our results showed that these cells exhibited the spindle-like morphology associated with MSCs. Isolated cells were positive for CD29 and CD90 (characteristic MSCs surface markers), and negative for CD45 and CD19 (hematopoietic markers), as well as major histocompatibility complex class II (MHC-II) as asessed by flow cytometry. Gene expression analysis by RT-qPCR revealed significant upregulation of the MSC-related genes THY1 and FGF2 in cultured cells compared to the native follicular aspirates, indicating the enrichment of a MSCs population during in vitro expansion. Trilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages was successfully demonstrated by morphological differentiation and specific staining. These findings demonstrate that equine ovarian follicular aspirates contain a viable population of MSCs that can be efficiently isolated, expanded, and differentiated in vitro. This work offers a novel and practical alternative for obtaining equine MSCs, with potential applications in the development of cell-based therapies within the reproductive and regenerative contexts.