SUN-148 Successful Development of Gonadotropin-releasing Hormone Neurons from Human Pluripotent Stem Cells

Abstract Disclosure: M. Campbell: None. L. Plummer: None. S. Koli: None. R. Balasubramanian: None. S.B. Seminara: None. Background: Gonadotropin-releasing hormone (GnRH)-secreting neurons in the hypothalamus are central to the neuroendocrine regulation of human reproduction. Disruption of GnRH neuronal development and function is directly linked to both rare and common reproductive disorders. However, GnRH neurons are difficult to study because of their unique embryonic developmental origin, atypical structure, scattered distribution in the hypothalamus, and inaccessibility for human translational investigation. Hence, modeling human GnRH neuronal development and function will allow researchers to gain a better understanding of these neurons and eventually inform clinical translational research in patients with reproductive disorders. Methods: Following a previously published in vitro differentiation protocol (Keen et al., 2021), we optimized and differentiated well-characterized isogenic human induced pluripotent stem cells (hiPSC) into hypothalamic GnRH neurons and assessed their structure and function. Briefly, hiPSCs were subject to dual-SMAD inhibition for 10 days, and then FGF8 was added for an additional 25 days to direct their differentiation into GnRH neurons. A control experiment was performed where FGF8 was not included in the differentiation media. GnRH expression and morphology were assessed at days 10, 25 and 35 of the differentiation protocol. Results: Over the course of the differentiation, immunofluorescence imaging confirmed the presence of GnRH-expressing cells in both conditions. While GnRH1 expression increased significantly in response to FGF8, surprisingly, we observed some GnRH expression in control hiPSCs, albeit with significantly lower expression compared to FGF8-treated cells. Additional experiments will be needed to determine if there is an FGF8-independent GnRH neuronal differentiation pathway. Next, we utilized a context-based image processing platform (TissueGnostics-StrataQuest) to examine structural changes in cells from differentiation Day 25 to 35. Morphometric quantification measurements of soma size and shape showed that by day 35, GnRH neurons showed a decrease in size and elongation of the soma, indicating that GnRH maturation is complete by day 35 of the differentiation protocol. RNA-seq and GnRH secretion assays are currently underway. Conclusions: We have successfully developed hiPSC-generated GnRH neurons and demonstrate both FGF8-dependent and independent differentiation pathways. This unique resource will now allow us to define the critical molecular drivers of normal GnRH development and function; (2) enable the modeling of disorders of GnRH dysfunction such as Kallman syndrome and Idiopathic Hypogonadotropic Hypogonadism to identify the underlying disease pathomechanisms; and (3) develop new therapeutic strategies without relying on ethically complex access to human fetal tissue. Presentation: Sunday, July 13, 2025