Morphological and functional studies on heterogeneous cell population derived from human ovarian follicular liquid

The origin of oocytes and primary follicles in ovaries of adult mammalian females is still a matter of dispute. The components of new primary follicles, primitive granulose and germ cells, differentiate sequentially and de novo from mesenchymal progenitor cell residing in the ovarian tunica albuginea (TA). It appears that mesenchymal progenitor cells contribute to the generation of epithelial cells similar to granulosa cells (GCs). The aim of this work is to study the morphological and functional characteristics of cell populations collected from human ovaries and to analyse particularly the growth of mesenchymal-like GCs in vitro in medium without any growth factor (such as leukemia-inhibiting factor) beyond 20 days. The interest in this study is to use waste biological liquid picked-up during human assisted reproduction techniques as a new possible source of mesenchymal stem cells (MSCs) to use in in vitro oocytes maturation techniques. Cells obtained from human follicular fluid grow in vitro in minimal medium condition, without any kind of growth factor. Morphological analysis revealed an heterogeneous cell population, with cells displaying a fibroblast-like, epithelial-like and also neuron-like shapes. The characteristics of fibroblast-like cells were similar to mesenchymal stem cells, while keeping also some aspects of ovary granulosa cells. Using immunocytochemistry and flow cytometry these cells have been shown to be positive for several mesenchymal stemness markers, including CD90, CD73, CD44, CD105, but not cytokeratins (expressed in epithelial cells) and they are also negative to CD34, CD45 and other aspecific markers. To isolate CD44+ cells of ovary-derived stem cell population, in this study we have performed experiments using immunomagnetic procedure. Parallel experiments were done on MSC cells from human bone marrow that served as positive control. Cell proliferation activity at different times in minimal culture conditions and colony forming unit capacity were evaluated too. After 15-20 days in culture, BrdU incorporation show about 20% proliferating cells less then after 1 week. Colony formation was observed after 13 days in culture. The multipotency of the subset of CD44+ cells was also established by in vitro differentiation into other cell types, such as osteoblasts. We have demonstrated the possibility that cells with mesenchymal plasticity can be isolated simply collecting waste follicular fluid, avoiding scraping of human ovaries. We also demonstrated the presence of a small subpopulation of neural-like cells in culture derived from the waste ovarian follicular fluid.