8053 A Ligand-Based Differentiation Protocol for Human Induced Pluripotent Stem Cells Recapitulates Granulosa Cell Development and Induces Steroidogenic Pathway Genes

Abstract

Abstract Disclosure: H. Kubo: None. M.M. Laronda: None. Sex determination is a developmental process that defines the gonad as a testis or ovary, and influences sex hormone production. While key signaling events are well conserved across mammals, there can be differences between species. Therefore, there is a need to develop a human model to fully understand this critical process in humans. We have developed a ligand-based differentiation protocol for human induced pluripotent stem cells (hiPSCs) to generate granulosa-like cells (GLCs). Additionally, hiPSC-derived GLCs could become the foundation for a cell-based hormone replacement therapy that recapitulates cyclicity, physiologic levels of hormones, and production of peptide hormones that are not included in currently available hormone replacement therapies. We aim to gain further understanding of the key factors and culture conditions necessary for functional GLCs that produce ovarian hormones, which includes maintained expression of FOXL2. We used our monolayer differentiation protocol that includes WNT-agonist CHIR99021 treatment for 2 days, followed by a combination treatment of BMP4, FST, and DKK1-inhibitor Gallocyanine for 3 days. We have tested our protocol on three different hiPSC lines: IISH2i-BM9s, STiPS-A3, and STiPS-A6. Expression of pluripotency marker POU5F1 is reduced during differentiation, while expression of the intermediate mesoderm markers PAX2, LHX1, and WT1 and bipotential gonad marker GATA4 peak at day 2. Peak expression of the bipotential gonad marker NR5A1 occurred at day 2 in the STiPS-A6 cells, but at day 5 in IISH2i-BM9 and STiPS-A3 cells. Interestingly, FOXL2 expression peaks at day 2 in all three lines and drops by day 5. In the IISH2i-BM9s, FOXL2 protein peaks at day 2 by Jess Western while there is the highest percentage of FOXL2-positive cells at day 5 by immunofluorescence. In murine granulosa cells, FOXL2 maintenance depends on NR5A2. NR5A2 expression drops in the GLCs, which suggests that NR5A2 loss may be preventing FOXL2 maintenance. Additionally, as FOXL2 is required for expression of aromatase and HSD17B1, we are using this model to interrogate NR5A2 upstream of FOXL2 and control hormone production. CYP19A1 and HSD17B1 along with AMH are induced during this differentiation protocol, suggesting these GLCs can be steroidogenic and produce peptide hormones. Additionally, scRNA-seq analysis of the GLCs is underway to identify types of granulosa cells produced by this protocol. Future directions include optimizing GLC culture conditions to generate hormones and to test if NR5A2 inhibitors alter FOXL2 expression. Put together, the data suggests that cells differentiated with our protocol show promise as a human model of ovarian development and a foundation for a personalized cell-based hormone replacement therapy. This work is supported by the NIH/NICHD R01HD104683 (MML), HuBMAP U01HD110336 (MML), and Gesualdo Family Research Scholar (MML). Presentation: 6/2/2024