Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biology

Collaborative Specialization

Developmental Biology

Supervisor

Cumming, Robert C.

2nd Supervisor

Betts, Dean H.

Co-Supervisor

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.

Summary for Lay Audience

Human pluripotent stem cells (hPSCs) are cells that can become any cell type in the adult body. They can also divide to maintain their cell population indefinitely. These properties have led to significant advancements in regenerative medicine, an interdisciplinary field that seeks to develop and apply new treatments and technologies to heal and restore the function of damaged tissues and organs. However, the regenerative medicine applications of hPSCs are hindered by difficulties in obtaining and maintaining them. To address and develop solutions to these problems, the mechanisms that regulate how hPSCs obtain and maintain their pluripotent features must continue to be identified. Cellular metabolism encompasses the various processes by which cells turn nutrients into energy, use energy to grow, and expel waste. Metabolic nutrients can also alter biochemical and genetic events to alter cell identity and function. Manipulation of metabolic processes has been shown to direct cell identity and function changes. In this thesis, I explored the relationship between the metabolic nutrient lactate and the acquisition and maintenance of hPSCs. I demonstrated that temporarily depriving adult skin cells of sugar and instead providing them with lactate as a metabolic nutrient, changes the process by which adult skin cells turn nutrients into fuel. I called this strategy lactate preconditioning. I proceeded to use lactate preconditioning while generating hPSCs from adult skin cells. From this experiment, I observed that lactate preconditioning increased the likelihood of survival of newly generated prospective hPSCs, making it easier to obtain them from adult skin cells. Finally, I found that hPSCs have more gene markers caused by a lactate-dependent process called lactylation, than adult cells. Lactylation is a known modifier of gene expression and cell function. Further, lactylation, metabolic activity, and proteins involved in hPSC identity, lactate transport, and lactate production exhibited distinct spatial distribution within groups of hPSCs in a manner suggestive of a direct relationship between lactylation, lactate transport and pluripotent gene expression. These findings suggest that lactate metabolism and associated lactylation may act as new regulators of hPSC identity and function.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Available for download on Thursday, August 27, 2026

Included in

Cell Biology Commons

Share

COinS