Background: Human gonads initially develop in a bipotential form that subsequently differentiates into either testes or ovaries. Disruptions to gonad development often result in Disorders of Sex Development (DSD) in humans. Genomic analysis of these patients has identified variants in both known and novel DSD genes. However functional analysis of these variants is severely hampered by the lack of a human fetal gonad cell line. Recent studies have differentiated human pluripotent stem cells (hPSCs) into renal lineages. Aggregates of these cells, termed kidney organoids, grown in vitro recapitulate features of functional embryonic kidneys.
Aim: Given the shared developmental origin of gonad and kidney, we aim to optimise protocols to allow differentiation of hPSCs into male gonad cells, to create “testes in a dish”.
Methods: Starting with male hPSCs, ten different growth factor conditions were tested for their ability to induce gonadal fate after seven days treatment. We also undertook a comprehensive analysis of RNA-Seq data to determine markers of the bipotential gonad and fetal testis. Differentiated cells were screened using qRT-PCR and immunofluorescence.
Results: Differentiation studies showed high induction of bipotential gonad markers when hPSCs are treated for four days with CHIR99021 followed by three days with FGF9/BMP4. Furthermore, up-regulation of the testis marker SOX9 indicated these cells are differentiating towards a testis fate.
Conclusions: These data provide evidence for the induction of gonadal lineages from hPSCs. We are now aggregating these differentiated cells to create testis organoids.
Funding Source: Murdoch Children’s Research Institute