An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development

Abstract

Currently an in vitro model that fully recapitulates the human embryonic gonad is lacking. Here we describe a fully defined feeder-free protocol to generate early testis-like cells with the ability to be cultured as an organoid, from human induced pluripotent stem cells. This stepwise approach uses small molecules to mimic embryonic development, with upregulation of bipotential gonad markers (LHX9, EMX2, GATA4, and WT1) at day 10 of culture, followed by induction of testis Sertoli cell markers (SOX9, WT1, and AMH) by day 15. Aggregation into 3D structures and extended culture on Transwell filters yielded organoids with defined tissue structures and distinct Sertoli cell marker expression. These studies provide insight into human gonadal development, suggesting that a population of precursor cells may originate from a more lateral region of the mesoderm. Our protocol represents a significant advance toward generating a muchneeded human gonad organoid for studying disorders/differences of sex development.

Single-Cell Atlas of the Chinese Tongue Sole (Cynoglossus semilaevis) Ovary Reveals Transcriptional Programs of Oogenesis in Fish

“…In contrast, krt18 , a marker gene of pre-granulosa cells and mural granulosa cells in human ovary ( Fan et al, 2019 ), was expressed in GC3. Additionally, GC3 showed specific high activity of TFs related to pre-granulosa cells, such as lhx9 , wt1, and irx3 ( Fu et al, 2020 ; Knarston et al, 2020 ) . Accordingly, GC3 might be pre-granulosa cells surrounding early follicles.…”

In-vitro cellular reprogramming to model gonad development and its disorders

“…However, they show a limited activation of endogenous Sertoli cell markers, with little to no activation of the key testis factor Sox9. An attractive route is the use of supplement enriched media to differentiate human pluripotent cells towards the IM and associated coelomic epithelium (Takasato and Little, 2016) and further towards granulosa-like (Lan et al, 2013) or Sertoli-like cells (Knarston et al, 2020;Rodriguez Gutierrez et al, 2018). However, the in-vitro derived Sertoli-like cells also lack robust and prolonged expression of key Sertoli-cell markers (SRY, SOX9, NR5A1, DMRT1) and their functionality was not unexplored.…”

“…Direct reprogramming of fibroblasts has also been attempted. However, the resultant populations lack appropriate and sustained expression of key testis-specific markers (Buganim et al, 2012;Knarston et al, 2020;Lan et al, 2013;Mae et al, 2013;Oeda et al, 2013;Rodriguez Gutierrez et al, 2018;Seol et al, 2018;Yoshino et al, 2021). No attempts were made to assess the entire transcription profiles, make comparisons to in-vivo counterparts, or, with the exception of Yoshino et al, (Yoshino et al, 2021) to determine the functional ability of the in-vitro derived gonadal cells.…”

Reprograming human fibroblasts into Sertoli cells: a tool for personalized medicine

“…Transient trans-differentiation of pluripotent stem cells (iPSCs) into Sertoli-like fate was achieved with specialized small molecules or biological factor supplementation (Knarston et al, 2020;Rodríguez Gutiérrez et al, 2018). A transgenic approach was able to produce potentially stable Sertoli-like cells using murine fibroblasts (Buganim et al, 2012) but this hadn't been reproducible with human cells.…”