Endocrine Abstracts

JOINT3135

Background: Sex-determination (SD) is defined as differentiation of the bipotential anlage into either testes or ovaries. The gene networks and mechanisms involved in the cell fate decision that direct the precursor somatic cells to commit to either Sertoli (testis) or granulosa cells (ovary) in the human are poorly understood, in part, due to a lack of evolutionary conservation and the absence of a suitable in-vitro model.

Material and methods: Using only defined culture media, we have developed robust protocols to sequentially differentiate human XX iPSCs to granulosa-like cells (iGLCs) and primordial germ cell-like cells (PGCLCs).

Results: During differentiation lineage appropriate markers are expressed, with terminally differentiated cells expressing those for primary follicle (e.g. FOXL2, RSPO1) even in prolonged culture (>2 months), suggesting that they are bona fide ovarian somatic cell precursors. We also derived primordial germ cell-like cells (PGCLCs) from iPSCs, defined by expression of SOX17, BLIMP1, TFAP2C and NANOS3. To generate 3D ovarian organoids (ovaroids) we have co-cultured in-vitro derived PGCLCs with iGLCs to evaluate their ability to reconstitute follicular structures and development associated with primordial germ cell differentiation towards oogonia. Using genome editing, we are using this system to introduce variants we previously defined as causing SRY-negative 46,XX testicular or ovotesticular Disorders/Differences in Sex Development (e.g. NR5A1, NR2F2).

Conclusion: Ovaroids provide an infinite and powerful supply of material to define the mechanisms involved in directing and maintaining cellular identity in human gonadal cells. This system will lead to a better understanding of factors and mechanisms involved in human ovary formation and a wide range of ovarian pathologies, including infertility.