The emergence of multiple testicular cell lineages in human stem cell-derived testis-like organoids.

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. In this study, we performed bulk and single-cell RNA-sequencing on these organoids to investigate their transcriptional landscape. Transcriptomic analysis revealed six distinct cell clusters expressing markers associated with bipotential, early Sertoli and testicular interstitial cells. These findings provide the first comprehensive transcriptional profile of hiPSC-derived testis-like organoids. Additionally, to address the limited emergence of mature cell types, we generated an inducible NR5A1/SF1 hiPSC line, which successfully triggered the upregulation of Leydig cell markers and additional Sertoli markers upon overexpression. Our findings show that our testis-like organoids are a valuable model system for studying DSDs in vitro.