Endocrine Abstracts

Background: Biological sex differences arise from the interaction between genetic and hormonal factors. These differences extend from external genitalia to the developing brain. Neurons exhibit sex-dependent transcriptional profiles that are associated with variations in neurodevelopmental processes and disease susceptibility. Steroid hormone receptors, including the androgen receptor (AR) and the estrogen receptors (ESR1 and ESR2), are present at low levels in certain cell types in the developing human brain. This suggests that neuronal differentiation may be sensitive to variations in hormone levels. Gonadotropin-releasing hormone (GnRH) neurons are of particular interest because they are part of the hypothalamic-pituitary-gonadal axis, which links brain development with steroid hormone production and feedback. Complete androgen insensitivity syndrome (CAIS) is a condition in which hormone signaling is altered. CAIS-derived human induced pluripotent stem cell (hiPSC) lines provide a human model to investigate neurodevelopment influenced by hormones.

Methods: Fully characterized CAIS-derived hiPSC lines were used to establish a 30-day differentiation protocol for GnRH neurons. To enable controlled, mechanistic studies of steroid hormone signaling, hiPSC lines derived from donors with intact steroid hormone receptors serve as the basis for CRISPR/Cas9-mediated genome editing. Loss-of-function mutations are introduced into the AR, ESR1, and ESR2 genes in both 46,XX and 46,XY genetic backgrounds to generate isogenic cell models.

Results: The differentiation protocol reliably produces GnRH neurons and provides a platform to compare neuronal populations derived from control and CAIS hiPSC lines. Preliminary analysis reveals transcriptional differences between 46,XY control and CAIS neuronal populations, indicating altered transcriptional programs associated with impaired androgen signaling. The CRISPR/Cas9-generated receptor knockout lines will enable receptor-specific perturbation while minimizing the impact of genetic variability between cell lines.

Conclusion: The combination of wild-type and genome-edited isogenic hiPSC lines establishes a human in vitro platform to investigate how steroid hormone signaling influences neuronal development. Together with long-read genomics approaches, the investigation of receptor-specific effects on transcriptional programs and transcript architecture during GnRH neuron differentiation will be allowed.