Sena Yildiz1, Jac Steenbergen2, Anke McLuskey-Dankbar3, Hana Kubo 4, Monica Laronda5, Jenny Visser6
(1) Erasmus MC/Department of Internal Medicine, Rotterdam, Netherlands. (2) Erasmus MC/Department of Internal Medicine, Rotterdam, Netherlands. (3) Erasmus MC/Department of Internal Medicine, Rotterdam, Netherlands. (4) Northwestern University, Feinberg School of Medicine/Department of pediatrics, Chicago, USA. (5) Northwestern University, Feinberg School of Medicine/Department of Obstetrics and Gynecology, Chicago , USA. (6) Erasmus MC/Department of Internal Medicine, Rotterdam, Netherlands.
Introduction:
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) has been implicated. AMH is predominantly expressed by granulosa cells (GCs) of ovarian preantral and small antral follicles and acts as a gatekeeper in follicular differentiation. In PCOS, AMH expression is increased and also its window of expression is prolonged. Yet, the mechanisms regulating AMH expression in general and in PCOS remain unknown. Therefore, we aim to study the transcriptional regulation of AMH using human induced pluripotent stem cells (iPSCs).
Methods:
To differentiate iPSC into granulosa-like cells, we applied several ligand-induced differentiation protocols which we analyzed in combination with two types of base medium. Assessment for correct lineage commitment and GC markers (FOXL2, CYP19A1, AMH, AMHR2, FSHR) was performed by gene expression analysis.
Results:
Our preliminary results indicated that all protocol combinations suppress expression of the pluripotency markers. However, differential expression of early and late stage GC markers was observed. In human embryonic stem cell (hES) medium expression of the early GC marker FOXL2 increased by 3-10 fold and expression of the late follicular stage marker CYP19A1 increased by ~100 fold. Despite a 3-5 fold increase in AMH expression, expression remained low. In contrast, a strong induction in AMHR2 expression (200-300 fold) was observed, but only when iPSCs were differentiated in RPMI medium.
Conclusion:
Our results indicate that iPSCs can be differentiated into granulosa-like cells. The differential expression of GC markers under the different protocols may reflect the expression pattern at different follicular stages. RNA sequencing analysis is currently conducted for in depth transcriptomic analysis. Furthermore, studies are ongoing to determine whether addition of oocyte growth factors to the differentiation protocols increases AMH expression.