MON-471 Establishment of Adrenal Cortex Like Cells From Human Induced Pluripotent Stem Cells That Produce Adrenal Hormones

Abstract Disclosure: J. Thyagraj: None. M.M. Laronda: None. Adrenal insufficiency (AI) is a rare and chronic condition characterized by the adrenal cortex’s inability to secrete adequate amounts of steroid hormones. Since the adrenal cortex and the gonads originate from a common progenitor cell, adrenal morbidities can occur co-occur with differences in sex development (DSD/intersex), primary ovarian insufficiency or infertility. Disruptions in reproductive functions associated with adrenal co-morbidities can be difficult to diagnose until during or after puberty. Our lab is interested in studying pathways involved in adrenal hormone production within patients with disrupted reproductive functions caused by specific gene variants. We aimed to optimize a culturing system that generates human adrenal cortex like cells (hACLC) from human induced pluripotent stem cells (hiPSCs) and to establish the baseline hormone outputs of functional hACLC aggregates. Using the protocol ligand-based protocol by Sakata et al., hiPSCs were differentiated into adrenogonadal primordium-like cells then hACLCs. 14.06% of cells differentiated hACLC aggregates expressed the adrenal cortex cell surface protein DLK1. We cultured the aggregates in submerged culture (sub) with the published air-liquid interface (ALI) culture to choose a culture that facilitated maximum hormone production. The levels of cortisol were significantly lower in the ALI than in sub across all time intervals (20.442+/-1.84, 26.61+/-3.66), DHEA was significantly reduced over time in Sub (8.007+/-1.05, 7.349+/-1.14) but not ALI (5.380 +/- 0.5, 2.735 +/- 0.38) and there was no significant difference in estradiol production in either condition (217+/-29.7, 118+/-923). Neither culture condition produced detectable levels of aldosterone. As submerged culture system performed better and maintained their aggregate form, we continued experiments with this method and measured their baseline hormone ranges over a week. The hACLC aggregates produced the most DHEA (14.20 ng/ml) on day 22 which significantly decreased by day 25 and 28 (7.930-14.20 ng/ml). The differentiated hACLCs also produced cortisol in the range of 20.44-26.62 ng/ml. Overall, the levels of cortisol remained consistent in all time intervals. Estradiol production also peaks at day 22 (638.2 pg/ml) followed by a significant decline on day 25 and day 28. On average, a hACLC aggregate produced up to 14.20 ng/ml DHEA, 26.62 ng/ml cortisol and 638.2 pg/ml estradiol. Our findings support a previously published protocol for generating hACLCs from hiPSCs with adjustments to increase secretion of cortisol, DHEA and estradiol. Ongoing research is advancing these tools to understand how patient-specific gene variants can influence adrenal cortex development and hormone production and may act as a predictive measure of pubertal-onset adrenal insufficiency. Presentation: Monday, July 14, 2025