Sertoli cells play a crucial role in the process of germ line development and are essential for spermatogenesis. However, studying the early germ line in human development is challenging and is lacking accurate model systems to recapitulate early primordial germ cell (PGC) and gonadal development. Current models rely and focus on ectopic grafting and xenoorganoid approaches. Xenoorganoids, in which PGC-like cells (hPGCLCs) derived from human pluripotent stem cell (hPSCs) are co-cultured with somatic cells derived from embryonic gonads or neonatal testis from rodents, offer a great tool to gain insights into germ line development and maturation due to its availability and reproducibility. However, these approaches always require the use of animals and do not represent accurately human physiology, especially in the context of the germ line.

Here, we established a human gonad in a dish derived from human pluripotent stem cells that can recapitulate early gonadal and germ line development. Somatic cells of the male gonad, mainly Sertoli cell-like cells (SCLCs), as well as hPGCLCs were derived from hPSCs and co-cultured together in a two-dimensional (2D) system. The cellular identity of SCLCs was confirmed by immunohistochemistry with the markers SOX9, GATA4, WT1, AMH and Claudin-11 that are associated with Sertoli cell development and maturation. Their transcriptomic profile was analysed by RNA-Seq and compared with a data set from embryonic gonads covering different developmental stages from 8 weeks post conception (pcw). Additionally, SCLCs showed a cellular response when stimulated with Follicle stimulating hormone (FSH) indicated by elevated intracellular cyclic AMP (cAMP). Co-localization, migration and morphology of hPGCLCs towards SCLCs in the co-cultures was assessed by various imaging approaches. hPGCLCs survived for up to four weeks in our system and showed co-localization and cluster formation along cord-like structures formed by SCLCs. After an extended culture period, we observed a transcriptional transition and downregulation of early germ line gene BLIMP1 while hPGCLCs maintained strong expression of TFAP2C.

We foresee that this system is useful for disease modelling of infertility, developmental studies and drug screening on human germ cells. Additionally, long term cultures may provide options for generating and expanding human male germ cells from induced pluripotent stem cells.