The naive to lineage primed transition

The naive to lineage primed transition was proposed as a gradual continuum of pluripotent states with distinct functional and transcriptional signatures and biases (Tesar, 2016). Several stem cell types with different degrees of pluripotency can be established under artificial culture conditions (Wu and Izpisua Belmonte, 2015). Specifically, naive and primed (EpiSCs) cells rely on chemical inhibitors (2i) or growth factors (F/A), respectively, and are largely used as in vitro models of pluripotency. Here we provide evidence that the relative levels of two physiological metabolites, namely VitC and l-Pro, push FBS/LIF ESCs toward naive (low l-Pro/high VitC) or early primed (high l-Pro/low VitC) states, between naive/2i and F/A primed states of the pluripotency continuum. In particular, similarly to the F/A primed state (EpiSCs), the high l-Pro/low VitC condition is associated with a metabolic reprogramming toward glycolysis and is strictly dependent on the activation and maintenance of autocrine bFGF and TGF-β signaling pathways. Interestingly, while F/A EpiSCs show molecular features of late pre-gastrulation epiblast and are difficult to revert to naive ESCs (Guo et al., 2009), PiCs display molecular, metabolic, and functional features (Table S5) of a transitional/reversible primed state in the pluripotency continuum, which has been hypothesized to define the boundary beyond which the transition becomes irreversible (Hackett and Surani, 2014; Martello and Smith, 2014). Of note, our findings indicate that VitC and l-Pro are both limiting in ESCs even in complete FBS/LIF culture conditions. This is in line with our recent findings that an autoregulatory loop limits l-Pro biosynthesis in ESCs (D\'Aniello et al., 2015); nevertheless, whether and how intracellular levels of VitC are regulated in ESCs is still unknown and deserves further investigation. VitC enhances epigenetic modifications, including DNA and histone demethylation, and promotes cell reprogramming (Esteban et al., 2010; Stadtfeld et al., 2012; Wang et al., 2011). Here we show that VitC and l-Pro supplementation oppositely modify DNA methylation at genomic regions that normally gain methylation during the rotenone to epiblast transition (Blaschke et al., 2013). The DNA demethylation effect of VitC is well explained by its ability to positively regulate Fe(II)/oxoglutarate-dependent dioxygenase enzymes of the TET family (5mC to 5hmC conversion) (Blaschke et al., 2013; von Meyenn et al., 2016). Conversely, no data have been reported so far linking l-Pro and DNA methylation. Although the molecular mechanism by which a sudden increase of l-Pro induces DNA methylation in ESCs is still far from being fully elucidated, we suggest that l-Pro may influence VitC homeostasis, reducing its availability/activity for the TET demethylases. First, the vast majority of the genomic regions that are hypomethylated in VitC-treated ESCs are in turn hypermethylated upon l-Pro supplementation. Moreover, at appropriate stoichiometric ratios, VitC fully counteracts l-Pro-induced DNA methylation. How may l-Pro affect VitC availability? l-Pro could alter the redox balance and eventually induce VitC oxidation, i.e., its conversion to dehydroascorbic acid, by generating reactive oxygen species, which are by-product of the activity of mitochondrial l-Pro oxidase enzyme (PRODH/POX) (Phang et al., 2010). However, previous findings argue against a primary role of oxidative stress/redox signaling in this process (Comes et al., 2013; D\'Aniello et al., 2015), and further evidence comes from metabolomics analysis, which reveals no sign of global oxidation and even higher levels of reduced glutathione (GSH) in l-Pro-treated cells (Figure 3G and Table S4), suggesting that l-Pro supplementation does not exert an oxidizing effect in these culture conditions. Thus, we propose that the mechanism by which VitC and l-Pro antagonistic effects capture alternative states in the pluripotency continuum does not rely, at least primarily, on altered redox balance, which would favor a reducing (VitC) versus an oxidant (l-Pro) state. Of note, this is consistent with the idea that pluripotency is linked to a reduced state, while activation of oxidation is a metabolic signature of ESC differentiation (Yanes et al., 2010). Indeed, the GSH/glutathione disulfide (GSSG) ratio and VitC levels are inversely correlated in ESC cardiac and neural differentiation, suggesting that VitC compensates for GSSG accumulation to maintain homeostasis (Yanes et al., 2010). Since l-Pro activity depends on protein synthesis (D\'Aniello et al., 2015), we hypothesize that a sudden increase of l-Pro could influence VitC homeostasis/availability, at least in part, by inducing the synthesis of l-Pro-rich proteins such as collagens. Indeed, nascent collagens are modified by VitC-dependent Fe(II)/oxoglutarate-dependent prolyl hydroxylase enzymes (Gorres and Raines, 2010). Thus, an abrupt increment of collagen biosynthesis/hydroxylation may reduce VitC availability for TET demethylases as well as for other VitC-dependent enzymes, such as the prolyl hydroxylases that regulate the hypoxia-inducible factor (HIF) steady-state level (Fong and Takeda, 2008). Interestingly, HIF is a key regulator of the metabolic reprogramming and is stabilized in primed but not in naive human ESCs (Sperber et al., 2015; Zhou et al., 2012). Of note, some of the HIF targets are induced in l-Pro-treated cells, such as Ldha, Pfkfb3, Plin2, Gbe1, and Pygl (Table S2). Although future studies are needed to clarify the molecular mechanisms, our findings have important implications for stem cell biology as they provide insights into how the availability of natural metabolites contribute to global epigenetic changes regulating alternative pluripotent states (Carey et al., 2015; Ryall et al., 2015; Wang et al., 2009). Furthermore, they attest to the definition of the optimal culture conditions for the capture in vitro of an early primed state of pluripotency.