Construction of human pluripotent stem cell-derived testicular organoids and their use as humanized testis models for evaluating the effects of semaglutide

<b>Background:</b> The generation of human testicular organoids from human induced pluripotent stem cells (hiPSCs) presents exciting opportunities for gonadal developmental biology, and reproductive disease modeling. However, creating organoids that closely mimic the tissue structure of testes remains challenging. <b>Methods:</b> In this study, we established a method for generating testicular organoids (TOs) from hiPSCs using a stepwise differentiation approach and a combination of hanging drop and rotational culture systems. The capability of hiPSC-derived precursor testicular cells to self-assemble into organoids was confirmed by detection of morphology, single-cell RNA-sequencing, and protein profiles. The reliability of testicular organoids as a drug evaluation model was assessed by the measurements of transcriptome signatures and functional features, including hormone responsiveness and blood-testis barrier (BTB) formation, and drug sensitivity assessment by recording cell viability and BTB integrity in organoids exposed to reproductive toxicants. Finally, we applied testicular organoids to evaluate the effects of semaglutide, a glucagon-like peptide-1 receptor agonist (GLP-1 RA), on testicular function, thereby underscoring their utility as a model for drug evaluation. <b>Results:</b> These organoids exhibited testicular cord-like structures and BTB function. RNA sequencing and functional assays confirmed that testicular organoids possess gene expression profiles and endocrine functions regulated by gonadotropins, closely resembling those of testicular tissue. Notably, these organoids displayed sensitivity to semaglutide. Treatment with semaglutide resulted in reduced testosterone levels and downregulation of <i>INHBB</i> expression, aligning with previous clinical observations. <b>Conclusions:</b> These findings introduced a method for generating testicular organoids from human pluripotent stem cells, highlighting their potential as valuable models for studying testicular function, drug toxicity, and the effects of compounds like semaglutide on testicular health.