The emergence of multiple testicular cell lineages in human stem cell-derived testis-like organoids [RNA-Seq]

Reproductive development is a complex process orchestrated by precise gene expression and cellular interactions. Disruption to this process can result in Differences of Sex Development (DSDs) which occur in approximately 1-2% of live births. We have previously developed a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into testis-like organoids as a model to study the molecular mechanisms underlying reproductive development and DSD. Here, we performed bulk and single-cell RNA sequencing on testis-like organoids to investigate their transcriptional landscape. Transcriptomic analysis revealed six cell clusters expressing markers associated with bipotential, early Sertoli, and testicular interstitial cells. To address the limited emergence of mature cell types, we generated a doxycycline-inducible NR5A1/SF1 hiPSC line, which successfully triggered the expression of Leydig cell markers upon overexpression. Our findings provide a comprehensive transcriptional profile of hiPSC-derived testis-like organoids, offering insights into human fetal gonadal development and a novel disease modelling system for DSDs.