The absence of advanced technologies such as genomic modification and artificial reproductive technologies (ART) limits marsupial research and conservation. We are working on filling this gap by generating stem cells lines, focusing on spermatogonial stem cells (SSCs), the undifferentiated progenitors of sperm. As SSCs have well-defined applications in genetic modification and in vitro gametogenesis, they provide a logical and valuable avenue to rapidly develop these technologies in marsupials. However, the lack of established markers for marsupial SSCs prevents the application of traditional enrichment techniques.
In this study, the fat-tailed dunnart (Smithopsis crassicaudata) seminiferous epithelial cycle was defined using histology, and key steps of SSC development were identified to optimise their enrichment. The timing of gonocyte migration to the basement membrane was defined using immunohistology targeting UCHL1 (PGP9.5), a marker of A-single/paired undifferentiated spermatogonia. To enrich for live dunnart SSCs, testis tissue was enzymatically digested followed by differential plating, and fluorescence-activated cell sorting using custom dunnart antibodies specific for spermatogonia and germ cells (TSPAN8 and KIT). Quantitative real-time PCR was used to analyse the expression of stem cells markers (GFRA1, POU5F1), differentiating germ cells markers (ESCO2) and somatic cells (GATA6, SOX9, NR2F2, CYP11A1) within sorted populations.
UCHL1 staining demonstrated that dunnart SSC differentiation has occurred by day 110 in the dunnart. Therefore, to maximise SSC enrichment efficiency, dunnarts younger than 110 days old should be used. Increased germ cell markers and decreased somatic cell markers were observed in the non-adherent fraction of differentially plated single cell suspensions. Consistent with this, KIT-specific antibodies enriched for differentiating germ cells. All these findings will enable an increase in dunnart SSC enrichment. This will allow us to determine culture conditions and increase the efficiency of downstream applications such as SSC-based marsupial genomic editing, as well as ART techniques to enhance marsupial conservation efforts.