Human pluripotency acquisition and maintenance: Examining the relationship between lactate, cell state, and cell fate
Abstract
Human pluripotent stem cells (hPSCs) have transformed the field of regenerative medicine by advancing drug development, disease modelling, and cell replacement therapy. However, low reprogramming efficiency and high phenotypic variability hinder their translation and commercialization. To address these issues, understanding the mechanisms regulating hPSC state and fate is critical to further optimizing maintenance and reprogramming methodologies. Metabolism has long been considered a product of cell fate changes but is now recognized as a driver. Indeed, studies have shown that promoting a metabolic shift from oxidative phosphorylation to glycolysis is essential to reprogramming oxidative somatic cells into induced pluripotent stem cells (iPSCs), which are glycolytic. More than a product of glycolysis, the metabolite lactate is an essential cellular fuel source, signalling molecule, and substrate for the posttranslational and epigenetic modification, lysine lactylation. However, the relationship between lactate and pluripotency needs further investigation. In this thesis, I demonstrated that restricting human fibroblast cells to lactate as a fuel source, followed by recovery in glucose-containing medium (lactate preconditioning), primes fibroblasts to switch from oxidative phosphorylation to glycolysis, in part, through reactive oxygen species-mediated hypoxia inducible factor 1-alpha stabilization. Further, lactate preconditioning increases the transcript abundance of critical facilitators of early somatic cell reprogramming. Applying this lactate preconditioning strategy during early somatic cell reprogramming, I showed that lactate exposure appears to increase the percent of prospective hiPSC colonies that evade death or differentiation following colony picking to successfully establish hiPSC lines. Finally, I explored the relationship between metabolism, lactylation, lactate transport, and pluripotency gene expression in naïve-like and primed human embryonic stem cells (hESCs). I found that histone lactylation and acetylation levels were higher in hESCs than in somatic cells. Further, exogenous lactate increased lactylation, acetylation, and essential pluripotency gene transcripts in hESCs. I also showed that naïve-like hESC colonies exhibit distinct spatial distribution of mitochondrial activity and proteins involved in lactate transport and production. Indeed, naïve-like hESC colonies exhibit higher lactylation levels peripherally, coinciding with elevated levels of SOX2, a core pluripotency marker. Together, these findings suggest that lactate and associated histone lysine lactylation may act as novel regulators of human pluripotency.