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A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening
Published on: May 12, 2017
Metabolic characterization of a paused-like pluripotent state
Maria Inês Sousa1, Bibiana Correia2, Ana Sofia Rodrigues2
1CNC-Center for Neuroscience and Cell Biology, CIBB, Azinhaga de Santa Comba, Polo 3, University of Coimbra, Coimbra, Portugal; University of Coimbra, Department of Life Sciences, Calçada Martim de Freitas, 3000-456 Coimbra, Portugal.
Abstract:
Embryonic diapause is a conserved reproductive strategy in which development arrests at the blastocyst phase. Recently mammalian target of rapamycin (mTOR) inhibition was shown to induce diapause on mouse blastocysts and a paused-like state on mouse embryonic stem cells (mESCs). In this work, we aimed to further characterize this new paused-pluripotent state, focusing on its glycolytic and oxidative metabolic function. We therefore exposed mESCs, to the mTOR inhibitor INK-128 and evaluated proliferation, pluripotency status and energy-related metabolism, as well as the mTOR inhibition status and translational function. Unexpectedly, in our hands INK-128 did not inhibit the phosphorylation of mTOR or its downstream targets after 48 h. Accordingly, no alterations on protein translational function were observed. Nonetheless, INK-128 could still successfully induce a paused-like state in naïve mESCs regardless of their culturing conditions, by greatly slowing proliferation without affecting pluripotency status. This effect was more prevalent in 2i cultured cells. Interestingly, in this paused-like state, mESCs present a glucose-related hypometabolic profile, which is a hallmark of diapaused blastocysts, with decreased glycolytic and oxidative metabolism and decreased nutrient uptake. Despite the lack of mTOR inhibition and translational suppression, INK-128 still induced a paused-like pluripotent state through cell cycle and metabolic modulation, rather than by translational suppression, suggesting more than one avenue for this type of pluripotent phenotype.
Insights
Mammalian target of rapamycin (mTOR) inhibition can induce a paused-like state in mouse embryonic stem cells (mESCs). This study found INK-128 slowed mESC proliferation and induced hypometabolism, mimicking embryonic diapause, independent of mTOR inhibition.
Area of Science:
- * Developmental Biology
- * Stem Cell Biology
- * Reproductive Biology
Background:
- * Embryonic diapause is a state of developmental arrest at the blastocyst stage, conserved across many species.
- * Mammalian target of rapamycin (mTOR) inhibition has been linked to inducing diapause in mouse blastocysts and a paused-like state in mouse embryonic stem cells (mESCs).
- * The metabolic characteristics of this paused-pluripotent state, particularly its reliance on glycolysis and oxidative metabolism, require further investigation.
Purpose of the Study:
- * To characterize the glycolytic and oxidative metabolic functions of the paused-pluripotent state induced by mTOR inhibition in mESCs.
- * To evaluate the effects of the mTOR inhibitor INK-128 on mESC proliferation, pluripotency, and energy metabolism.
- * To investigate the mechanism by which INK-128 induces a paused-like state, specifically whether it involves mTOR inhibition and translational suppression.
Main Methods:
- * Mouse embryonic stem cells (mESCs) were cultured and treated with the mTOR inhibitor INK-128.
- * Proliferation rates, pluripotency markers, and energy metabolism (glycolytic and oxidative) were assessed.
- * mTOR inhibition status, downstream target phosphorylation, and protein translation were evaluated.
Main Results:
- * INK-128 treatment did not inhibit mTOR phosphorylation or downstream targets after 48 hours, nor did it alter protein translation.
- * Despite the lack of mTOR inhibition, INK-128 successfully induced a paused-like state in mESCs, characterized by significantly slowed proliferation without affecting pluripotency.
- * mESCs in the paused-like state exhibited a hypometabolic profile, with decreased glucose uptake, glycolysis, and oxidative metabolism, mirroring diapaused blastocysts.
Conclusions:
- * INK-128 can induce a paused-pluripotent state in mESCs through cell cycle and metabolic modulation, independent of direct mTOR inhibition and translational suppression.
- * The observed hypometabolic profile in paused mESCs is a key characteristic shared with diapaused blastocysts.
- * This suggests that multiple pathways may lead to the induction of a pluripotent paused state, offering new insights into developmental arrest and stem cell regulation.
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