Australian marsupial populations are in significant decline. While traditional conservation approaches (e.g., habitat protection and breeding programs) have proven useful, next-generation technologies—such as genetic modification to enhance population fitness—are required to stem continuing losses. This study generated induced pluripotent stem cells (iPSCs) from the fat-tailed dunnart to provide critical starting material for such technologies. Pluripotency of the iPSCs was validated using transcriptomic and epigenomic analyses, embryoid body formation, differentiation into primordial germ cell-like cells (PGCLCs), and injection into mouse embryos.
Dunnart iPSCs strongly activated transcriptomic and epigenomic networks associated with pluripotency in other species; however, unique patterns of activation and inhibition were also observed. The dunnart iPSCs readily formed embryoid bodies expressing markers associated with differentiation into all three germ layers, as well as extraembryonic lineages. Furthermore, injection of dunnart iPSCs into mouse morulae or blastocysts demonstrated their capacity to contribute to both the inner cell mass and the trophoblast lineage. Applying established methods for differentiating mouse or human iPSCs into PGCLCs resulted in the robust induction of a similar PGCLC transcriptional network in dunnart iPSCs.
Altogether, this research has established reliable methods for generating dunnart iPSCs that exhibit characteristics of totipotency and a functional capacity to contribute to early embryonic development in vitro. However, validation in longer-term mouse or dunnart embryo cultures and further molecular analysis is essential for definitive classification. Genome-wide analysis of pluripotency gene activation and inhibition revealed a unique pattern, potentially reflecting a marsupial-specific pluripotency network currently under investigation. Fully elucidating this network will provide valuable insight into the most effective applications of marsupial iPSCs in next-generation conservation technologies such as de-extinction and programmed genetic fitness.