Marsupial research and conservation are limited by the absence of advanced technologies such as artificial reproductive technologies (ART). We are working on filling this gap by developing stem cell lines for marsupials. Our research focuses on spermatogonial stem cells (SSCs), the undifferentiated progenitors of sperm. SSCs provide a logical and valuable means to develop ART in marsupials. However, the lack of established cell markers for marsupial SSCs prevents the use of traditional enrichment techniques.
In our study, fat-tailed dunnart SSCs were enriched by targeting the highly conserved retinoic acid (RA) synthesis pathway which is vital for the initiation of spermatogonial differentiation. Dunnart testes were digested to single cells and stained with a fluorescent dye (ALDEFLUORTM) that allowed measurements of aldehyde dehydrogenase (ALDH) activity, a surrogate marker of RA synthesis which is low in SSCs. Cells were subsequently fluorescence-activated cell sorted based on their ALDH activity. Quantitative PCR was used to analyse the expression of stem cell (POU5F1, GFRA1, ETV5) and somatic cell markers (CYPA11A1, NR2F2, GATA6, SOX9) within sorted populations. Culture of sorted populations was performed to assess SSC enrichment, viability, and proliferative ability.
Increased SSC markers and reduced somatic cell markers were found within the ALDH low population as predicted. Similarly, cultured populations demonstrated a reduced somatic cell presence within the ALDH low population when compared to the ALDH high population. These results suggest that marsupial SSC enrichment can be achieved though sorting cells by ALDH expression. This provides us with the ability to define culture conditions for SSCs and identify novel markers for their future use in ART, genomic editing, conservation, and research applications.