Abstract
The earth faces unprecedented extinction levels, termed "the sixth extinction". Advanced assisted reproductive technology such as in vitro gametogenesis and cryobanking emerged as pivotal strategies in safeguarding the genetic diversity in mammalian species. However, oocyte scarcity, harvesting and cryopreservation challenges persist due to the absence of species-specific protocols. To save the genetics that could be forever lost, the use of somatic cells instead of reproductive tissues to generate offspring has been investigated. The most commonly investigated source of somatic cells are fibroblasts, but we will establish protocols for keratinocytes and mesenchymal stem cells which are expected to yield higher reprogramming efficiency and to anticipate situations where no fibroblasts are available. The goal is to provide biobanks with a variety of options depending on the available banked samples. While the ability to generate induced pluripotent stem cells (iPSCs) from different endangered mammalian species has been shown, the struggle to acquire a true endogenous pluripotent state of the reprogrammed cells remains. We propose a plasmid based transfection to provide a solid reprogramming. The generation of primordial germ cell-like cells from iPSCs and the creation of putative follicle structures is still in its infancy. By developing a co-culture system between primordial germ cell-like cells and ovarian somatic stem cells, we will develop a 3D ovaroid which will be able to create putative follicle structures. This project aims to bridge the gap between mice and wildlife applications by using the pig as a relevant animal model species. By enhancing our understanding of reproductive biology in this kind of model, this proposal will contribute to reversing biodiversity loss and aid wildlife population management.
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