Characterization and modulatory influence of pyruvate kinase muscle isoforms 1 and 2 within the murine pluripotent continuum
Abstract
Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of a pluripotency continuum, respectively referred to as naïve and primed pluripotent states. A third, recently discovered intermediate state has been described as the ‘formative state’. Metabolism has been traditionally regarded as a by-product of cell fate; however, recent evidence now supports metabolism as promoting stem cell fate. Pyruvate kinase muscle isoforms 1 and 2 (PKM1 and PKM2) catalyze the final, rate limiting step of glycolysis generating adenosine triphosphate (ATP) and pyruvate; however, the precise role(s) of these isozymes in naïve, formative, and primed pluripotency is unclear. Steric-blocking morpholino oligonucleotides were employed to modulate the levels of PKM1/2; this thesis characterized the cellular expression, localization patterns, and contributions of PKM1 and PKM2 in mESCs, chemically transitioned mouse epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting, flow cytometry, and confocal microscopy. My results indicate that PKM1 and PKM2 are not only localized to the cytoplasm, but also accumulate in distinct subnuclear regions of mESCs, mEpiLCs, and mEpiSCs as determined by a comprehensive and quantitative, confocal microscopy colocalization methodology.
In Chapters 2 and 3, I employed orthogonal projections, and airyscan processing to investigate the localization patterns of PKM1/2. I determined that the subnuclear localization of PKM1/2 shifts during the pluripotent development across mESCs, mEpiLCs, and mEpiSCs. The appropriateness and power of the Pearson’s Correlation Coefficient and Manders’ Overlap Coefficient for assessing nuclear and cytoplasmic protein colocalization in pluripotent stem cells (PSCs) by immunofluorescence confocal microscopy was validated and expanded upon. In Chapter 4, I describe a key research tool of this thesis using flow cytometry, this improved technique allows for the identification of formative pluripotency cells from naïve and primed populations using the cell surface markers SSEA1 and CD24. Additionally, I utilized this advanced methodology in Chapter 5 to assess the influence of PKM1/2 modulation on pluripotency state. Altering PKM1/2 levels affected the ability of naïve state cells to transition to the formative state, it also influenced the transition of formative cells to a primed-like state. In conclusion, the results suggest that nuclear PKM1/2 assists with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naïve, formative, and primed pluripotency.