P04 - Modeling X-linked hypopituitarism using iPSC-derived hypothalamic-pituitary organoids

Introduction SOX3 is an X-linked transcription factor involved in early development of the pituitary gland and central nervous system. Mutations in SOX3, including copy number variants (CNVs) such as duplications and deletions, are associated with diverse clinical phenotypes, including intellectual disability with growth hormone (GH) deficiency, panhypopituitarism, and XX male sex reversal. However, SOX3 gene dosage alone cannot explain how similar CNVs can lead to unrelated phenotypes. Recent studies suggest that genomic duplications may disrupt the three-dimensional chromatin architecture, particularly affecting topologically associated domains (TADs). Structural variations (SVs) involving TADs may result in gene misexpression by exposing promoters to ectopic regulatory elements. This mechanism, termed “TADopathy,” has been implicated in several rare diseases. In this study, we aim to model SOX3 CNVs associated with hypopituitarism using iPSC-derived hypothalamic-pituitary organoids to investigate the pathogenic role of this gene in a rare and inaccessible tissue. Methods To model representative SOX3 duplications and deletions, we used two wild-type induced pluripotent stem cell (iPSC) lines to generate isogenic lines via CRISVar, a genome editing approach that employs paired sgRNAs to induce targeted SVs spanning several kilobases. Edited iPSCs were genotyped by breakpoint PCR and subcloned to obtain pure monoclonal lines. CNVs were validated by droplet digital PCR (ddPCR) assays. The iPSCs were then differentiated into hypothalamic-pituitary organoids using an established 3D culture protocol. Results We successfully established the 3D differentiation protocol in our lab using wild-type iPSCs. Expression of pituitary and hypothalamic markers (e.g. HESX1, RX, LHX3, PITX1, TBX19, ACTH) was confirmed by qPCR and immunofluorescence at multiple time points (d6, d18, d30, d48, d74, d105). In parallel, we performed CRISVar on control iPSCs and generated polyclonal and oligoclonal lines positive for representative SOX3 intra-TAD and inter-TAD duplications and deletions. Multiple oligoclonal lines are currently being genotyped and subcloned to obtain monoclonal lines. Additionally, we already obtained a monoclonal iPSC line carrying a SOX3 deletion validated by ddPCR. Conclusions There remains a significant gap in understanding the pathogenic mechanisms underlying SOX3 CNVs. This human in vitro model has the potential to be a useful tool to study SOX3-related disorders and explore future therapeutic strategies.