In vitro characterization of granulosa progenitor cells isolated from bovine ovarian follicl

Ovarian granulosa is a highly available cell cluster that could be used as an alternative source of mesenchymal stem cells (MSCs) for veterinary medicine applications. To this, presumptive granulosa MSCs (gMSCs) were isolated by aspirating the follicular fluid from 2-3mm growing follicles from bovine ovaries collected at slaughterhouse. gMSCs were divided in three culture groups in basic medium supplemented with a different factor: group 1 was added with 2% fetal calf serum (FCS) only, group 2 with 10 ng/ml epidermal growth factor (EGF) and group 3 with 0.02% leukemia inhibitory factor (LIF). These cells were evaluated until passage (P) 5 for their proliferative potential and the expression of pluripotent (c-Myc and Oct-4), mesenchymal (CD29, CD44, CD73 and CD166), functional (FSHR and FST) and hematopoietic (CD34) markers by RT-PCR. Oct-4, CD73 and FSHR expression was also investigated by quantitative PCR (qPCR). Adipogenic and osteogenic differentiation potential was assessed at P3. Each culture condition supported the growth of cells that retained the capability to adhere to the plastic dish and expand exponentially with a low doubling time (1.21, 1.33, 2.13 days for FCS, EGF and LIF, respectively). All cells expressed pluripotent and mesenchymal markers but not hematopoietic markers until P5. A significant decrease in the FSHR expression and an increase in the CD73 and Oct4 expression were observed by qPCR at P3 compared to P0. A 100-fold decrease in the expression of FSHR, and a 10-fold and a 8 fold increase in the expression of CD73 and Oct-4 respectively, were detected in gMSC cultured in presence of LIF. The FST marker was expressed in all the tested conditions. Although the expression of mesenchymal and pluripotent markers was found in gMSCs, these cells did not differentiate into the adipogenic and osteogenic lineages. Our results differ from those reported for humans and gilts (Kossowska et al., 2009; Mattioli et al., 2012). We hypothesize that these differences might be attributable to the collection method and the follicular development stage. The present study was performed on cells collected by needle aspiration from growing follicles, whereas in human and gilts a scraping protocol from luteinizing ones was applied. Our findings demonstrate that the conditions we used allowed for the isolation of progenitor cells from the ovarian granulosa, although these cells did not show the differentiation capability probably due to epigenetic memory. This hypothesis was further confirmed by the expression of FST in each passage studied. In conclusion, the cells obtained in this study cannot be considered gMSCs and the question about the presence of isolable presumptive gMSCs from growing follicles remains open. Kossowska K. et al. The multipotency of luteinizing granulosa cells collected from mature ovarian follicles. Stem Cells 2009; 27(1): 210-219. Mattioli M et al. Osteo-regenerative potential of ovarian granulosa cells: an in vitro and in vivo study. Theriogenology 2012; 77(7): 1425-1437.