Molecular Characterization By ddPCR Of In Vitro Differentiated Oocyte-like Cells From Oogonial Stem Cells

Claudia Felici
Erica Silvestris, Paola Cafforio, Stella D’Oronzo, Claudia Felici, Francesco Silvestris, Giuseppe Loverro
University of Bari Aldo Moro, Italy

Recent reports regarding the presence of OSCs in the ovaries of non-menopausal and menopausal women suggest that neo-oogenesis is inducible during ovarian senescence. However, there isn’t consensus on isolation methods of these cells, their spontaneous maturation in vitro, and the final differentiation state of the resulting putative oocytes. Ovarian cortex fragments from menopausal and non-menopausal women were processed by immuno-magnetic separation using a rabbit anti-human DDX4 antibody and cultured for up to 3 weeks. Large and small cells were individually isolated by DEPArray technology and early and late differentiation markers were measured by droplet digital PCR. The haploid versus diploid chromosomal content was investigated using fluorescence in situ hybridization (FISH). After immuno-magnetic enrichment, DDX4-positive OSCs from non-menopausal and menopausal women, under appropriate culture conditions, differentiate into large haploid oocyte-like cells expressing the major oocyte markers growth differentiation factor 9 (GDF-9) and synaptonemal complex protein 3 (SYCP3) and then enter meiosis. Moreover, in culture small DDX4 positive cells are also present which do not express differentiation markers. Therefore, we provide further evidence demonstrating the presence of stem-like cells with ovarian germ line properties within the otherwise exhausted oocyte reserve of menopausal human ovaries. These cells can be a source of oocytes that can be exploited to achieve fertility in women who are infertile or have an exhausted ovarian reserve.

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