Abstract
BackgroundAdvances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro. Here we extend the model to human first-trimester embryonic gonadal tissue.ResultsTesticular cell suspensions reorganised into testis-like organoids with distinct seminiferous-like cords situated within an interstitial environment after 7 days. In contrast, tissue reorganisation failed to occur when mesonephros, which promotes testicular development in vivo, was included in the tissue digest. Organoids generated from dissociated female gonad cell suspensions formed loosely organised cords after 7 days. In addition to displaying testis-specific architecture, testis-like organoids demonstrated evidence of somatic cell differentiation. Within the 3-LGS, we observed the onset of AMH expression in the cytoplasm of SOX9-positive Sertoli cells within reorganised testicular cords. Leydig cell differentiation and onset of steroidogenic capacity was also revealed in the 3-LGS through the expression of key steroidogenic enzymes StAR and CYP17A1 within the interstitial compartment. While the 3-LGS generates a somatic cell environment capable of supporting germ cell survival in ovarian organoids germ cell loss was observed in testicular organoids.ConclusionThe 3-LGS can be used to generate organised whole gonadal organoids within 7 days. The 3-LGS brings a new opportunity to explore gonadal organogenesis and contributes to the development of more complex in vitro models in the field of developmental and regenerative medicine.
Highlights
Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro
Gonocytes in the human embryonic testis are characterised by the expression of pluripotency-associated factors (e.g. POU5F1) which are gradually downregulated from approximately 12 wpc with a reciprocal upregulation of proteins associated with germ cell progression (e.g. MAGE-A4) marking the transition from gonocyte to pre-spermatogonium [4, 7, 8]
Dissociated testis tissue comprising small cell aggregates and single cells (n = 3; 8, 8.5, and 8.5–9 wpc) reaggregated (Fig. 1B) and reorganised into testis-like organoids (TO) with distinct seminiferous-like cords situated within an interstitial environment similar to morphological structures observed in age-matched controls within 7 days (Fig. 1C, D)
Summary
Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Human gonad development begins between 3.5 and 4.5 weeks post conception (wpc) during which the primordial germ cells (PGCs) migrate to the primordial gonadal ridge [1]. At the same time as Sertoli cell fate is established, organisation of the seminiferous cords is initiated. Gonocytes in the human embryonic testis are characterised by the expression of pluripotency-associated factors (e.g. POU5F1) which are gradually downregulated from approximately 12 wpc with a reciprocal upregulation of proteins associated with germ cell progression (e.g. MAGE-A4) marking the transition from gonocyte to pre-spermatogonium [4, 7, 8]. Adjacent to the developing gonadal ridge, the mesonephros develops into the Wolffian duct, generating various external urogenital structures as well as contributing endothelial cells to the developing testis [11]
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