Metabolic plasticity during transition to naïve-like pluripotency in canine embryo-derived stem cells

Pluripotent stem cells (PSCs) have been described in naïve or primed pluripotent states. Domestic dogs are useful translational models in regenerative medicine, but their embryonic stem cells (cESCs) remain narrowly investigated. Primed-like cESCs expanded in the presence of leukemia inhibitory factor and fibroblast growth factor 2 (LIF-FGF2) acquire features of naïve pluripotency when exposed to chemical inhibitors and LIF (2iL). However, proliferation of cESCs is influenced by the pluripotent state and is comparatively slower than human or mouse PSCs. We propose that different metabolic pathway activities support ATP generation and biomass accumulation necessary for LIF-FGF2 and 2iL cESC proliferation. We found that 2iL cESCs have greater respiratory capacity, altered mitochondrial chain complex stoichiometry and elevated mitochondrial polarization state. Yet, 2iL-enriched cESCs exhibited immature ultrastructure, including previously unrecognized changes to cristae organization. Enhanced ATP level in 2iL cESCs is associated with altered retrograde signalling, whereas LIF-FGF2 cESCs exhibit a lipogenic phenotype. Inhibition of oxidative phosphorylation impaired proliferation and ATP production in 2iL cESCs but not LIF-FGF2 cESCs, which remained sensitive to glycolysis inhibition. Our study reveals distinct bioenergetic mechanisms contributing to steady-state expansion of distinct canine pluripotent states that can be exploited to improve derivation and culture of canine PSCs.