Stem cells from follicular fluid present multilineage differentiation capacity and form oocyte-like structures under the influence of FSH hormone

Abstract

Background & Aim Recent studies highlight the existence of putative stem cells derived from human ovarian follicular liquid collected after routine procedures for in vitrofertilization techniques (Riva F et al, 2009; Polimeni M et al, 2012).The aim of this study was to isolate, characterize and evaluate the pluripotency of this heterogeneous cell population of stem cells isolated from follicular fluid. For this purpose human ovarian follicular fluids were collected from women during the IVF procedure at the Embryoclinic Assisted Reproduction Unit (Thessaloniki, Greece). Methods, Results & Conclusion The collection was performed during the retrieval process of oocytes by transvaginal ultrasound-guided aspiration needle. After removal of the cumulus oophorus-oocyte complex the remained follicular fluid was centrifuged and the pellet was analyzed by flow cytometry for total immunophenotypic characterization. Cells were seeded on plastic surfaces in DMEM medium supplemented with fetal bovine serum, penicillin/streptomycin and gentamycin for incubation at 37οC/5% CO2. After 10-day cultivation the cells were analyzed for the expression of mesenchymal and pluripotent stem cell markers with flow cytometry. The induced differentiation capacity of isolated cells towards bone, adipose and endothelium in specific culture mediums was estimated via alizarin red, oil red staining and flow cytometry respectively. Cultures of follicular stem cells were checked for their ability to form oocyte like structures with the addition of FSH hormone in different concentrations. Real time PCR was performed for primordial germ stem cells markers Sox-2 Nanog and OCT4. A population of CD34-/CD45+/CD90+/CD105+/CD133-/SSEA4-/OCT4-/CD44+ stem cells were isolated. Osteogenic, adipogenic and endothelial multilineage differentiation capacity of cells derived from follicular liquid after culture was successfully confirmed. Oocyte like structure were observed in different FSH concentrations in the culture medium. After the presence of FSH hormone, cells were positive for the primordial stem cells markers Sox-2, Oct4 and Nanog while expression was gradually decreased in larger FSH concentrations. Conclusively, stem cells from follicular fluid show multilineage potential capacity and express primordial stem cell markers in specific conditions. Follicular fluid could be used as an alternative source of pluripotent stem cells and it might be a hopeful cure to women suffer from premature ovarian failure.