SAT-185 - Ligand-Based Differentiation of Induced Pluripotent Stem Cells into Granulosa-Like Cells

Polycystic ovary syndrome (PCOS) is a common reproductive and metabolic disorder in women. The etiology of PCOS is still unknown, but a role for anti-Müllerian hormone (AMH) signaling has been implicated. AMH is predominantly expressed by granulosa cells (GCs) of ovarian preantral and small antral follicles, where it signals through the specific AMH type II receptor (AMHR2) and shared type I receptors, thereby exerting its gatekeeper function in ovarian folliculogenesis. In PCOS, AMH expression is increased and prolonged. This disrupted expression pattern may contribute to follicular arrest. To gain more insight into the molecular mechanisms induced by AMH in PCOS, we aim to employ human induced pluripotent stem cells (iPSCs)-derived granulosa-like cells to determine the downstream transcriptional effect of altered AMH signaling.

To differentiate iPSC into granulosa-like cells, we used a ligand-induced differentiation protocol. The assessment for correct lineage commitment and GC markers (FOXL2, CYP19A1, HSD17B1, AMH, AMHR2, FSHR) was performed by gene expression analysis. Additionally, transcription factors associated to AMH expression (GATA4, RUNX1, RUNX2 and SF1) are assessed. AMHR2 functionality was assessed by measuring AMH-induced Smad1/5 phosphorylation using Western blotting.

Our results indicate that the ligand-induced differentiation protocol suppresses expression of pluripotency markers. Expression of the early- (FOXL2) and late-follicular stage marker (CYP19A1) remained low. HSD17B1 expression increased 5-10 fold. Also AMH expression increased 3-5 fold, but its relative expression remained low. Nevertheless, transcription factors that have been implemented in the regulation of AMH transcription showed a substantial increase in expression peaking at 4000 fold induction for GATA4, 10-15 fold induction for RUNX1, 100-400 fold induction for RUNX2, and 20 fold induction for SF1. We observed a 2500 fold increase in FSHR expression during differentiation and a 400-600 fold increase in AMHR2 expression at endpoint of differentiation (day 14). Next, we assess whether these iPSC-derived granulosa-like cells were responsive to AMH. Despite a relatively high baseline Smad1/5 phosphorylation level, stimulation with exogenous AMH for 1 hr resulted in a mild increase (~1.4 fold) in Smad1/5 phosphorylation. Studies to determine FSH responsiveness of these iPSC-derived granulosa-like cells are ongoing.

Our findings demonstrate that iPSCs can be differentiated into granulosa-like cells that are responsive to AMH. While further optimization of the differentiation protocol is needed to enhance AMH expression, this differentiation protocol can be used to compare AMH signaling in iPSCs derived from women with PCOS and normo-ovulatory women.

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