Australian marsupial populations are in significant decline. While traditional approaches to conservation such as habitat protection and breeding programmes have proven useful in specific cases, next generation technologies are required to stem the tide of population losses. Such technologies include the generation of primordial germ cells (PGCs) from induced pluripotent stem cells (iPSCs), that may serve as starting material for in vitro gametogenesis, in vitro fertilisation, and testis transplantations.
This study generated iPSCs from primary dunnart fibroblasts using a transposase-based approach and dox-inducible OSKLM factors. Pluripotency of iPSCs was validated using transcriptomic analysis and embryoid body formation. Subsequently, PGC-differentiation protocols established in mouse or human iPSCs were applied. The extent of PGC differentiation was measured using qPCR for genes associated with pluripotency and PGC specification (POU5F1, NANOG, KLF4, PRDM1, PRDM14, KIT), as well as flow cytometry to assess cell surface expression of ITGA6.
Dunnart iPSCs demonstrated morphological features characteristic of a pluripotent state, and strongly activated transcript networks associated with pluripotency in other species. Embryoid bodies were readily formed, and expressed markers associated with differentiation into multiple germ layers, validating the functional pluripotency of dunnart iPSCs. Applying methods for differentiating mouse or human iPSCs into PGCs resulted in robust induction of PGC-specific genes in multiple dunnart iPSC lines. Consistent with results in human iPSCs, PGC differentiation increased the expression of ITGA6.
Altogether this study has established a method to reliably generate marsupial iPSC lines that exhibit critical hallmarks of pluripotency. These cells exhibit features of germline competence as evidenced by expression of PGC-specific markers in response to factors involved in mouse and human PGC differentiation. These findings provide an invaluable tool for development of next-generation conservation technologies that can be used to bolster population numbers and genetic diversity, as well as in marsupial de-extinction efforts.