Bioenergetic Changes Underline Plasticity of Murine Embryonic Stem Cells

Abstract

Murine embryonic stem cells (mESCs) are endowed by a time-dependent window of plasticity during their early commitment steps. Indeed, while mESCs deprived of leukemia inhibitory factor (LIF) for 24 hours revert to their naive pluripotent state after subsequent LIF readdition, cells deprived of LIF for 48 hours are no longer efficient in reverting, upon LIF addition, and undergo irreversible differentiation. We investigated undisclosed bioenergetic profiles of early mESCderived committed cells versus their undifferentiated states in order to reveal specific bioenergetic changes associated with mESC plasticity. Multiparametric bioenergetic analysis revealed that pluripotent (+LIF) and reversibly committed cells (−LIF24h) are energetically flexible, depending on both oxidative phosphorylation (OXPHOS) and glycolysis. They exhibit high mitochondrial respiration in the presence of the main energetic substrates and can also rely on glycolysis in the presence of OXPHOS inhibitor. Inhibition of the glycolysis or mitochondrial respiration does not change drastically the expression of pluripotency genes, which remain well expressed. In addition, cells treated with these inhibitors keep their capacity to differentiate efficiently upon embryoid bodies formation. Transition from metabolically active mESCs to irreversibly committed cells is associated with a clear change in mitochondrial network morphology, to an increase of adenosine triphosphate (ATP) produced from glycolysis and a decline of ATP turnover and of the mitochondrial activity without change in the mitochondrial mass. Our study pointed that plasticity window of mESCs is associated with the bivalent energetic metabolism and potency to shift to glycolysis or OXPHOS on demand. LIF removal provokes glycolytic metabolic orientation and consecutive loss of the LIFdependent reversion of cells to the pluripotent state. STEM CELLS 2019;37:463-475 SIGNIFICANCE STATEMENTMurine embryonic stem cells rely on leukemia inhibitory factor (LIF) cytokine to maintain their naïve pluripotent state. They undergo differentiation by simple LIF removal with a 48-hour period of plasticity window in which cells can maintain a pluripotent behavior upon LIF readdition. The study has established key point of metabolic changes, by multiparametric bioenergetic analysis, during this period of transition from naïve pluripotent toward committed cell states. Gene expression profiles and functional tests allow to conclude that cells maintained with LIF or depleted of LIF for 24 hours are energetically flexible, depending on both oxidative phosphorylation and glycolysis, whereas after 48 hours of LIF withdrawal, they are mainly glycolytic.

Normal Hematopoetic Stem and Progenitor Cells Can Exhibit Metabolic Flexibility Similar to Cancer Cells

“…Apoptosis was detected with an Annexin V-FITC kit (Beckman Coulter, Carlsbad, CA, USA) according to the manufacturer's protocol. Briefly, 10 5 cells from each of the experimental conditions were stained with Annexin V-FITC solution (AnnV) and propidium iodide (PI, 250 µg/mL) for 15 min at 4 • C in the dark, washed in phosphate buffer saline (PBS), and analyzed with a flow cytometer (BD Bioscience, FACS Canto II) (17). This technique allow to detect: unlabelled viable cell subpopulation (AnnV − / PI − ); early apoptotic cell subpopulation that have bound only AnnV (Ann + /PI − ); necrotic cell subpopulation (representing the cells in post-apoptosis necrosis or late apoptosis) that have both bound AnnV and have been labeled with PI (Ann + /PI + ).…”

“…Cells were then fixed with 900 µl of 70% cold ethanol (added dropwise), and incubated for 1 h at 4 • C. Cells were washed and resuspended in PBS with 0.2 mg/mL of bovine RNase A and incubated for 1 h at 37 • C in the dark. PI (10 µg/mL) was added, and the analysis was performed using flow cytometry (Bioscience, FACS Canto II) (17). The percentage of cells in each of the different cell cycle phases (G0/G1, S, and G2/M) was calculated from the linear graph on which cell counts were plotted relative to cell DNA content.…”

“…Mitochondria were detected using mitochondrial-selective fluorescent labeling with MitoTracker Green, according to the manufacturer's instructions (Molecular Probes). A total of 10 4 cells were treated with Mitotracker green (100 nM) for 20 min at 37 • C in the dark, then washed in PBS and analyzed using flow cytometry as previously published (17).…”

Redox Homeostasis and Regulation in Pluripotent Stem Cells: Uniqueness or Versatility?

“…Since mitochondrial enzymes of ETC are one of the main sources of intracellular ROS, highlighting the peculiarities of PSC energy metabolism led to the concept that the redox homeostasis in PSCs significantly differs from that of their differentiated progeny cells and that PSCs can limit intracellular ROS production to minimize ROS-induced oxidative damage. However, recent studies have shown that PSCs have not only phenotypic but also metabolic plasticity and, therefore, are able to adjust their redox metabolism to the conditions of their microenvironment [26,27]. These cells a priori have the capacity to exist not only in hypoxic conditions but also in normoxia (21% O 2 ) [26] and even hyperoxia (>21% O 2 ) [28].…”