Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Collaborative Specialization

Developmental Biology

Supervisor

Betts, Dean H.

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.

Summary for Lay Audience

Prior to implantation, an embryo is referred to as a blastocyst. The blastocyst contains a small pocket of cells called the inner cell mass. These cells can become all cell types of an individual, a characteristic coined ‘pluripotency’. Isolated inner cell mass cells can be grown in the lab to study how the embryo develops and how pluripotent stem cells function. Stem cells require sources of energy to maintain themselves and growth with the process of metabolism. Pluripotent stem cells progressively specialize over the timeline of the pluripotent continuum. The first stage of the continuum is called the naïve state, where a cell is pluripotent, but is not fully ready to turn into a new cell. First, a naïve cell must develop into a formative state cell, this state is an intermediate point needed to gain the ability to turn into any cell of the fetus, and the only state that a cell can turn into a germ cell. Before making the decision to leave pluripotency, the cell enters the final stage, the primed state, where it is ‘primed’ for cell lineage choices. This thesis examined two main proteins that are known to aid in cellular digestion of the building blocks needed to grow and generate energy, pyruvate kinase muscle isoforms 1 and 2 (PKM1/2). PKM1/2 affect the ability of pluripotent stem cells to stay as naïve cells or develop into becoming formative, or primed stem cells. This thesis utilized several improved methods to examine the effects of altering the levels and localization of PKM1/2 on pluripotent and metabolic state of the naïve, formative, and primed stages. This knowledge helps us to understand how embryonic stem cells stay pluripotent and specialize into other types of cells.

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