Doctor of Philosophy
Physiology and Pharmacology
Betts, Dean H.
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from large mammals reproduce few characteristics displayed by rodent or human counterparts. This is complicated by the inherent plasticity of mammalian ESC/iPSC cultures that resemble a variety of developmental stages including the naïve and primed pluripotent states. Defining the extrinsic signals that modulate the developmental identity of canine ESC/iPSC (i.e. primed versus naïve) will improve knowledge integration with more sophisticated rodent and primate research. In this thesis, I sought to determine if manipulation of the culture environment can promote nuclear and metabolic reprogramming of canine cell lines towards a more primitive state of pluripotency. Using growth factors and kinase inhibitors to modulate pluripotency of canine ESCs (cESCs), I demonstrated that cESCs exhibit the plasticity to adopt multiple developmental identities. I observed that lineage-specific differentiation of cESCs is influenced by pluripotent state modulation, coincident with changes to colony architecture, transcriptional and epigenetic markers of developmental maturity. I found that primed- and naïve-like cESCs exhibit pluripotent-state specific mitochondrial structure and function, including differential reliance on glucose oxidation pathways for steady-state proliferation in vitro. Lastly, using comparative sequence analysis and biochemical assays, I observed that evolutionary differences in genomic CpG density at pluripotency-associated promoters correlate with functionally relevant cytosine modifications to the induction and maintenance of pluripotency. I showed the utility of physiological metabolites that regulate 5-methylcytosine oxidation in promoting cellular and transcriptional features associated with early nuclear reprogramming of canine fibroblasts (e.g. mesenchymal-to-epithelial transition). Taken together, my work establishes a binary cESC model of primed- and naïve-pluripotent states, providing insight into shared and divergent features of pluripotency progression and acquisition in placental mammals.
Tobias, Ian C., "Phenotypic and Metabolic Plasticity In Canine Cellular Reprogramming" (2019). Electronic Thesis and Dissertation Repository. 6224.