OR19-05 Human Induced Pluripotent Stem Cell-derived Granulosa-like Cells From Ligand-based Differentiation Express FOXL2 After Inhibition Of DKK1

Abstract

Abstract Disclosure: H. Kubo: None. M.M. Laronda: None. Premature ovarian insufficiency (POI) is characterized by the loss of ovarian function prior to an individual reaching the age of 40. POI can have serious ramifications on health, reducing average life expectancy, and can develop from iatrogenic treatments or genetic variants. POI patients can receive hormone replacement therapies (HRTs), but current methods are not dynamic, responsive, or comprehensive. Contrastingly, cell-based therapies have the potential to produce hormones other than estrogen or progesterone and respond dynamically to each patient. Using human induced pluripotent stem cells (hiPSCs) to generate such cell-based therapies may mitigate rejection upon transplantation and allow for personalized therapies for POI patients. Therefore, we propose to differentiate hiPSCs into granulosa like cells (GLCs) that are functionally the same as granulosa cells (GCs), which are responsible for aromatizing androgens into estrogens. One factor critical to steroidogenesis in GCs is FOXL2, which in mice is suppressed by DKK1, a WNT inhibitor. Therefore, we hypothesized that FOXL2 expression and subsequent steroidogenic functions in our GLCs will be influenced by DKK1. To generate GLCs, we treated the cells with WNT agonist CHIR99021 for two days which has been shown to induce the intermediate mesoderm, and then BMP4 and FST for three days to induce the ovarian somatic lineage. We analyzed our cultures for self-renewal (POU5F1), intermediate mesoderm (PAX2, LHX1, WT1), coelomic epithelium (EMX2), genital ridge (GATA4, NR5A1), and GC (FOXL2) markers using qPCR to assess correct lineage commitment. At day 2, we observed approximately 4000-fold induction of PAX2, 20-fold induction of LHX1, 35-fold increase of WT1, 3000-fold induction of GATA4, and 3.5-fold induction in FOXL2 transcript expression. At day 5, we observed 10-fold induction of NR5A1. We also observed a 7000-fold increase of DKK1 at day 2. Addition of Gallocyanine, a DKK1 inhibitor, from days 2 to 5 resulted in induction of FOXL2 protein expression at day 5, captured by immunofluorescence imaging. Analysis of the images showed that Gallocyanine-treated cells were 42.4% positive for FOXL2, while cells untreated were 0.325% positive for FOXL2. This data suggests that DKK1 does suppress FOXL2 expression in these hiPSC-derived GLCs and demonstrates the potential of these cells as a model to investigate mechanisms important to GC development and maintenance. Future steps include testing the steroidogenic abilities of the GLCs and identification of culture conditions needed to maximize steroidogenesis. This work is supported by the NIH/NICHD R01HD104683 (MML), HuBMAP U01HD110336 (MML), Warren and Eloise Batts Endowment (MML), a Debicki Foundation Grant (MML), the Burroughs Wellcome Fund Career Award at the Scientific Interface (MML), Gesualdo Family Research Scholar (MML), and NIH/NCI training grant T32 CA009560 (HK). Presentation Date: Saturday, June 17, 2023