Isolation, characterization and evaluation of growth kinetics and multilineage differentiation of ovine ovarian mesenchymal stem cells.

Mesenchymal stem cells (MSCs) derived from reproductive tissues are promising for regenerative and reproductive applications; however, ovarian MSCs from large animal models remain poorly characterized. The present study aimed to isolate and characterize the ovine ovarian MSCs and to evaluate passage-dependent changes in their morphology, clonogenicity, growth kinetics, immunophenotype, stemness-associated gene expression, and differentiation potential, while identifying an optimal passage for molecular analyses. This is the first report of isolation and characterization of MSCs from sheep ovary to the best of our knowledge. Plastic-adherent cells were isolated from sheep ovaries, cultured under standard conditions, and established as ovine ovarian mesenchymal stem cells. Based on morphological uniformity and proliferative stability, passage 3 (P3) cells were selected for molecular characterization using immunocytochemistry (ICC), flow cytometry, and quantitative RT-PCR (qRT-PCR). Clonogenicity was assessed by CFU-F assays, growth kinetics by population doubling time (PDT), and differentiation potential under adipogenic, osteogenic, and chondrogenic induction. Early passages (P1-P3) exhibited typical fibroblast-like morphology, high proliferative capacity, strong CFU-F efficiency, and short PDTs. Beyond passage 4, PDT increased, and clonogenicity declined. Molecular analyses of P3 cells confirmed strong expression of MSC markers (CD73, CD90, CD105, and CD44), pluripotency/stemness-associated genes (OCT4, SOX2, and NANOG), and absence of hematopoietic markers (CD34 and CD45). A transient CD34 transcript expression detected at early passages was completely lost by P3, indicating stabilization of the mesenchymal phenotype. The isolated MSCs showed efficient differentiation into adipogenic, osteogenic, and chondrogenic lineages. Prolonged culture induced morphological alterations and reduced proliferative and clonogenic potential. These findings establish the sheep ovary as a reliable source of MSCs and identify P3 as the optimal passage for molecular and functional studies, supporting the translational use of MSCs in reproductive and regenerative medicine.