Modelling ovarian development using human pluripotent stem cells

Gonads originate from the intermediate mesoderm and start out as a paired structure that has the capacity to further differentiate into both testes and ovaries depending on the sex chromosomes of an individual. In the past, it was largely assumed that ovarian development was a passive process that occurred due to the lack of male determining factors. However, research in recent years has shown that ovarian development is an intricate process that requires the involvement and interplay of many different genes and pathways. Mutations in genes involved in ovarian development can lead to a variety of gonadal disorders, which can result in infertility. Unfortunately, despite increasing research efforts over the last few years, ovarian development is not well understood and the function of many genes involved in early ovarian development remain unknown for the most part.

The NR5A1 gene is thought to be a crucial factor for normal gonadal development and function. Additionally, it is also an important regulator of steroidogenesis. Previous studies done by our group have elucidated the role of NR5A1 in male gonadal differentiation as being necessary for the generation of more mature male somatic gonadal cells. However, its role in the maturation of female bipotential gonadal-like cells has not been studied yet. Another important gene, FOXL2, encodes a transcription factor that is well known for its role in female sex determination and ovarian development, function and maintenance. It has also been identified as one of the earliest markers of ovarian development. However, its role in the formation of granulosa cell precursors is unknown.

Human pluripotent stem cells provide a valuable method to model human gonadal development as they can be differentiated into derivatives of all three germ layers, namely endoderm, mesoderm and ectoderm. Although there are protocols available by which stem cells can be differentiated to gonadal cells, in most cases the resulting cells are immature and the protocols itself do not recapitulate gonadal development completely. Previously, our laboratory developed a protocol for gonadal differentiation that accurately replicates gonadal development up to the bipotential gonadal cell stage.

In this thesis work, I aimed to elucidate the role of NR5A1 and FOXL2 in early human ovarian development. To do so, a female human embryonic stem cell line (H9) was genome-edited using the CRISPR/Cas9 technology to induce either NR5A1 or FOXL2. The dual-inducible activation lines were then subjected to the previously established gonadal differentiation protocol and either NR5A1 or FOXL2 was activated at day 4 of differentiation.

Initial RT-qPCR findings combined with bulk RNA-sequencing results showed that NR5A1 induction did not upregulate many bipotential or female gonadal genes aside from INHA, but instead significantly increased the expression of several steroidogenic genes. Additionally, several pathways relating to adrenal or ovarian steroidogenesis seemed to be upregulated. Therefore, it seems that in contrast to its role in male gonadal development, NR5A1 steers the differentiating cells towards a potential steroidogenic cell fate instead of a more mature gonadal cell fate in a female background. Similar experiments showed that FOXL2 on the other hand was found to not merely be a marker of early ovarian development, but a driver in the formation of early supporting gonadal cells, which are the precursors of granulosa cells. Moreover, the combination of FOXL2 activation and the gonadal differentiation protocol allowed us to replicate the early weeks of ovarian development in vitro.

Altogether, this thesis work provides valuable insight in the functional role of NR5A1 and FOXL2 in early human ovarian development. This information in turn contributes to our understanding of pathological gonadal development, which will hopefully allow for the establishment of better therapies and personalised patient care for individuals suffering from gonadal disorders and infertility.

This thesis is based on the following publications:

I. Danti L, Lundin K, Sepponen K, Yohannes DA, Kere J, Tuuri T, Tapanainen JS. CRISPR/Cas9-mediated activation of NR5A1 steers female human embryonic stem cell-derived bipotential gonadal-like cells towards a steroidogenic cell fate. J Ovarian Res. 2023 Sep 20;16(1):194. https://doi.org/10.1186/s13048-023-01264-5

II. Danti L, Lundin K, Nedeczey-Ruzsák P, Tuuri T, Tapanainen JS. FOXL2 drives the differentiation of supporting gonadal cells in early ovarian development. Reprod Biol Endocrinol. 2025 Mar 18;23(1):44. https://doi.org/10.1186/s12958-025-01377-0