Mouse embryonic stem (ES) glucose-6-phosphate (G6P) dehydrogenase-deleted cells ( G6pd delta), obtained by transient Cre recombinase expression in a G6pd -loxed cell line, are unable to produce G6P dehydrogenase (G6PD) protein (EC 1.1.1.42). These G6pd delta cells proliferate in vitro without special requirements but are extremely sensitive to oxidative stress. Under normal growth conditions, ES G6pd delta cells show a high ratio of NADPH to NADP(+) and a normal intracellular level of GSH. In the presence of the thiol scavenger oxidant, azodicarboxylic acid bis[dimethylamide], at concentrations lethal for G6pd delta but not for wild-type ES cells, NADPH and GSH in G6pd delta cells dramatically shift to their oxidized forms. In contrast, wild-type ES cells are able to increase rapidly and intensely the activity of the pentose-phosphate pathway in response to the oxidant. This process, mediated by the [NADPH]/[NADP(+)] ratio, does not occur in G6pd delta cells. G6PD has been generally considered essential for providing NADPH-reducing power. We now find that other reactions provide the cell with a large fraction of NADPH under non-stress conditions, whereas G6PD is the only NADPH-producing enzyme activated in response to oxidative stress, which can act as a guardian of the cell redox potential. Moreover, bacterial G6PD can substitute for the human enzyme, strongly suggesting that a relatively simple mechanism of enzyme kinetics underlies this phenomenon.

SummaryMetabolites are emerging as key mediators of crosstalk between metabolic flux, cellular signaling, and epigenetic regulation of cell fate. We found that the nonessential amino acid L-proline (L-Pro) acts as a signaling molecule that promotes the conversion of embryonic stem cells into mesenchymal-like, spindle-shaped, highly motile, invasive pluripotent stem cells. This embryonic-stem-cell-to-mesenchymal-like transition (esMT) is accompanied by a genome-wide remodeling of the H3K9 and H3K36 methylation status. Consistently, L-Pro-induced esMT is fully reversible either after L-Pro withdrawal or by addition of ascorbic acid (vitamin C), which in turn reduces H3K9 and H3K36 methylation, promoting a mesenchymal-like-to-embryonic-stem-cell transition (MesT). These findings suggest that L-Pro, which is produced by proteolytic remodeling of the extracellular matrix, may act as a microenvironmental cue to control stem cell behavior.

Signaling peptides of the extracellular environment regulate cell biological processes underlying embryonic development, tissue homeostasis, and pathophysiology. The heparan sulphate proteoglycans, glypicans, have evolved as essential modulators of key regulatory proteins such as Wnt, Bmp, Fgf, and Shh. By acting on signal spreading and receptor activation, glypicans can control signal read-out and fate in targeted cells. Genetic and embryological studies have highlighted that glypicans act in a temporal and spatially regulated manner to modulate distinct cellular events. However, alterations of glypican function underlie human congenital malformations and cancer. Recent reports are starting to reveal their mechanism of action and how they can ensure tight modulation of cell signaling.

Glucose-6-phosphate dehydrogenase-deleted embryonic stem (ES) cells (G6pdD) proliferate in vitro without special requirements, but when challenged with oxidants fail to sustain glutathione disulphide reconversion to reduced glutathione (GSH), entering a condition of oxidative stress. Here, we investigate the signalling events downstream of GSH oxidation in G6pdD and wild-type (wt) ES cells. We found that G6pdD ES cells are very sensitive to oxidants, activating an apoptotic pathway at oxidant concentrations otherwise sublethal for wt ES cells. We show that the apoptotic pathway activated by low oxidant concentrations is accompanied by mitochondria dysfunction, and it is therefore blocked by the overexpression of Bcl-X L . Bcl-X L does not inhibit the decrease in cellular GSH and reactive oxygen species formation following oxidant treatment. We also found that oxidant treatment in ES cells is followed by the activation of the MEK/ extracellular signal-regulated kinase (ERK) pathway. Interestingly, ERK activation has opposite outcomes in G6pdD ES cells compared to wt, which has a proapoptotic function in the first and a prosurvival function in the latter. We show that this phenomenon can be regulated by the cellular GSH level.

Increasing evidence indicates that metabolism is implicated in the control of stem cell identity. Here, we demonstrate that embryonic stem cell (ESC) behaviour relies on a feedback loop that involves the non-essential amino acid L-Proline (L-Pro) in the modulation of the Gcn2-Eif2α-Atf4 amino acid starvation response (AAR) pathway that in turn regulates L-Pro biosynthesis. This regulatory loop generates a highly specific intrinsic shortage of L-Pro that restricts proliferation of tightly packed domed-like ESC colonies and safeguards ESC identity. Indeed, alleviation of this nutrient stress condition by exogenously provided L-Pro induces proliferation and modifies the ESC phenotypic and molecular identity towards that of mesenchymal-like, invasive pluripotent stem cells. Either pharmacological inhibition of the prolyl-tRNA synthetase by halofuginone or forced expression of Atf4 antagonises the effects of exogenous L-Pro. Our data provide unprecedented evidence that L-Pro metabolism and the nutrient stress response are functionally integrated to maintain ESC identity.

ZFP57 is necessary for maintaining repressive epigenetic modifications at Imprinting control regions (ICRs). In mouse embryonic stem cells (ESCs), ZFP57 binds ICRs (ICRBS) and many other loci (non-ICRBS). To address the role of ZFP57 on all its target sites, we performed high-throughput and multi-locus analyses of inbred and hybrid mouse ESC lines carrying different gene knockouts. By using an allele-specific RNA-seq approach, we demonstrate that ZFP57 loss results in derepression of the imprinted allele of multiple genes in the imprinted clusters. We also find marked epigenetic differences between ICRBS and non-ICRBS suggesting that different cis-acting regulatory functions are repressed by ZFP57 at these two classes of target loci. Overall, these data demonstrate that ZFP57 is pivotal to maintain the allele-specific epigenetic modifications of ICRs that in turn are necessary for maintaining the imprinted expression over long distances. At non-ICRBS, ZFP57 inactivation results in acquisition of epigenetic features that are characteristic of poised enhancers, suggesting that another function of ZFP57 in early embryogenesis is to repress cis-acting regulatory elements whose activity is not yet required.

Biotechnologies such as high-throughput screening (HTS) enable evaluation of large compound libraries for their biological activity and toxic properties. In the field of drug development, embryonic stem (ES) cells have been instrumental in HTS for testing the effect of new compounds. We report an innovative method in one step to differentiate ES cells in neurons and glial cells. The four different neuronal subtypes, gamma-aminobutyric acid (GABA)-ergic, dopaminergic, serotonergic, and motor neurons, are formed in culture. This protocol is adaptable to small wells and is highly reproducible, as indicated by the Z-factor value. Moreover, by using either leukemia inhibitory factor (LIF) or recombinant Cripto protein in our culture conditions, we provide evidence that this protocol is suitable for testing the effect of different molecules on neuronal differentiation of ES cells. Finally, thanks to the simplicity in carrying out the experiment, this method provides the possibility of following the morphological evolution of the in vitro differentiating neuronal cells by timelapse videomicroscopy. Our experimental system provides a powerful tool for testing the effect of different substances on survival and/or differentiation of neuronal and glial cells in an HTS-based approach. Furthermore, using genetically modified ES cells, it would be possible to screen for drugs that have a therapeutic effect on specific neuronal pathologies or to follow, by time-lapse videomicroscopy, their ability to in vitro differentiate.

SummaryMetabolites and cofactors are emerging as key regulators of cell plasticity and reprogramming, and their role in the control of pluripotency is just being discovered. Here we provide unprecedented evidence that embryonic stem cell (ESC) pluripotency relies on the relative levels of two physiological metabolites, namely ascorbic acid (vitamin C, VitC) and l-proline (l-Pro), which affect global DNA methylation, transcriptional profile, and energy metabolism. Specifically, while a high VitC/l-Pro ratio drives ESCs toward a naive state, the opposite condition (l-Pro excess) captures a fully reversible early primed pluripotent state, which depends on autocrine fibroblast growth factor and transforming growth factor β signaling pathways. Our findings highlight the pivotal role of metabolites availability in controlling the pluripotency continuum from naive to primed states.