SUN-123 - Experimental Validation of DHX37 Dynamics in Human Gonadogenesis using iPSC-derived Models and Single-Cell Transcriptomics

Pathogenic variants in the RNA helicase DHX37 are established causes of 46,XY Differences in Sexual Development (DSD). While previous computational analyses from our group have mapped the DHX37 transcriptomic signature in fetal gonads, experimental models are essential to elucidate its functional role during early human gonadal cell specification. This study aims to experimentally validate the temporal dynamics and cellular localization of DHX37 using a human induced pluripotent stem cell (iPSC) model of primordial germ cell-like cells (PGCLCs) and somatic gonadal cells. We employed an optimized in vitro differentiation protocol to drive human iPSCs toward germline and somatic lineages. DHX37 protein expression and subcellular distribution were quantified via flow cytometry and high-resolution immunofluorescence at key developmental milestones in the differentiation protocols (Days 0, 1, 3, 5, 15, and 18). These findings were integrated with refined single-cell RNA sequencing (scRNA-seq) clusters from human fetal gonadal tissues. Our experimental model successfully recapitulated the DHX37 expression trajectory predicted by scRNA-seq. We observed a robust upregulation of DHX37 specifically during the transition from pluripotency to the PGCLC stage. Quantitative flow cytometry revealed that the percentage of DHX37+ cells peaks at Day 5, coinciding with the expression of the germline markers EPCAM, TFAP2C, and CXCR4. Immunofluorescence confirmed that DHX37 maintains a strict nuclear localization within the specifying primordial germ cells, supporting its role in nuclear RNA processing during lineage commitment. This study provides experimental evidence of DHX37’s specific involvement in the early stages of human gonadal cell development. By bridging the gap between transcriptomic predictions and in vitro functional modeling, we demonstrate that DHX37 is a critical player in early gonadogenesis, offering a powerful platform to investigate the molecular mechanisms of DSD-associated mutations.

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