Markers of cellular reprogramming potential in fibroblast cell lines

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The biodiversity crisis is one of the most pressing environmental problems of our time, yet after >30 years of research, there has been limited progress on the implementation of assisted reproductive technologies for endangered mammals. Species-specific requirements for successful embryo development have impacted the advancement of in vitro-based embryo technologies resulting in a lack of effective protocols for key conservation species. The ability to preserve and access both female and male genetic material is of fundamental importance when developing strategies for the long-term sustainability of threatened wildlife species. As such, somatic cells are highly valued for their ease of collection, culture and cryopreservation from all animals, as well as their reprogramming capabilities and potential use in somatic cell-based technologies (somatic cell nuclear transfer, SCNT; induction of pluripotent stem cells, iPSCs). However, the production of living offspring from SCNT- and iPSC-derived embryos remains an inefficient and variable process. Our studies have resulted in the first-ever production of bison blastocysts by interspecies SCNT (iSCNT) in American (plains, wood) and European bison. Nevertheless, bison iSCNT embryos differed significantly from cattle SCNT controls in developmental potential, including cell number, apoptosis, and gene expression. Most importantly, multiple cell lines were assessed for SCNT with some cell lines being unable to produce blastocysts after repeated attempts. Recently, there has been a growing body of evidence indicating that the individual cell line is the greatest determinant of outcome following reprogramming for reasons beyond traditional cell health metrics. Metabolism plays a key role in cellular processes, and thus, in the regulation of the epigenome, a principal driver of reprogramming success. Our research lies in understanding the cellular and molecular factors influencing the pluripotency potential of somatic cells for the production of embryos by SCNT and iPSC techniques. The proposed research will focus on the characterization and modification of cellular metabolism in an effort to alter the epigenetic profile of somatic donor cells, and positively impact reprogramming and downstream embryo production events. The first phase of the study will characterize the metabolic profiles of bovine donor cells and identify metaboloepigenetic markers related to reprogramming outcomes. In the second phase, modification of the metaboloepigenetic status of the cells will be carried out in an effort to improve reprogramming capabilities. This study will increase our understanding of cellular determinants of reprogramming success, specifically the role of metabolic and epigenetic status of the donor cell, and provide us with potential methods for improving somatic cell-based technologies. This is a necessary step towards the application of SCNT and iPSC strategies in the recovery of threatened species.