Differential localization patterns of pyruvate kinase isoforms in murine naïve, formative, and primed pluripotent states.

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of the pluripotency continuum, referred to as naïve and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways, including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naïve cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg Effect. Until recently, metabolism has been regarded as a by-product of cell fate, however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg Effect. Both isoforms catalyze the final, rate limiting step of glycolysis, generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naïve and primed pluripotency is not well understood. The primary objective of this study was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm, but also accumulate in differential subnuclear regions of mESC, mEpiLCs, and mEpiSCs as determined by a quantitative confocal microscopy employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 changes during the transition from mESCs, mEpiLCs, and mEpiSCs. Finally, we have comprehensively validated the appropriateness and power of the Pearson's correlation coefficient and Manders's overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 may assist 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.

3D Immunofluorescent Image Colocalization Quantification in Mouse Epiblast Stem Cells.

This chapter details 3D morphological topography of colony architecture optimization and nuclear protein localization by co-immunofluorescent confocal microscopy analysis. Colocalization assessment of nuclear and cytoplasmic cell regions is detailed to demonstrate nuclear and cytoplasmic localization in mEpiSCs by confocal microscopy and orthogonal colocalization assessment. Protein colocalization within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.

Modulation of PKM1/2 Levels by Steric Blocking Morpholinos Alters the Metabolic and Pluripotent State of Murine Pluripotent Stem Cells.

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described "formative" pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms 1/2 (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 messenger RNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the "formative" state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression toward a primed pluripotent state.

Flow Cytometric Characterization of Pluripotent Cell Protein Markers in Naïve, Formative, and Primed Pluripotent Stem Cells.

Here we describe methodologies to characterize, delineate, and quantify pluripotent cells between naïve, formative, and primed pluripotent state mouse embryonic stem cell (mESCs) populations using flow cytometric analysis. This methodology can validate pluripotent states, sort individual cells of interest, and determine the efficiency of transitioning naïve mESCs to a primed-like state as mouse epiblast-like cells (mEpiLCs) and onto fully primed mouse epiblast stem cells (mEpiSCs). Quantification of the cell surface markers; SSEA1(CD15) and CD24 introduces an effective method of distinguishing individual cells from a population by their respective positioning in the pluripotent spectrum. Additionally, this protocol can be used to demarcate and sort cells via fluorescently activated cell sorting for downstream applications. Flow cytometric analysis within mESCs, mEpiLCs, and mEpiSCs can be efficiently completed using these optimized protocols.