br Experimental Procedures br Author Contributions

Experimental Procedures
Author Contributions
Acknowledgments
Introduction In recent years, a number of small molecules have been identified that control stem cell differentiation and/or cell reprogramming. Surprisingly, only a few metabolites have been identified in this context, possibly because their relevance has been underappreciated. The “physiological” compounds identified so far include epigenetic modifiers or regulators of the cell energy/redox status acting on mitochondrial oxidative metabolism. For example, saturated fatty acids and the acylcarnitines were shown to improve embryonic stem cell (ESC) differentiation (Yanes et al., 2010). Butyrate, a short-chain fatty acid, increases the efficiency of cell reprogramming by inhibiting histone deacetylases (Mali et al., 2010). Finally, ascorbic Bafetinib (vitamin C), a redox controller and a cofactor for histone demethylases, is a key regulator of stem cell differentiation and reprogramming (Cao et al., 2012; Esteban et al., 2010; Wang et al., 2011; Yang et al., 2008). However, it has recently become evident that also some naturally occurring amino acids can regulate stem cell behavior. Indeed, it has been shown that (1) ESC self-renewal depends on L-threonine catabolism (Shyh-Chang et al., 2013; Wang et al., 2009) and (2) L-proline (L-Pro) forces ESCs toward an epiblast stem cell (EpiSC)-like state (Washington et al., 2010) and regulates ESC metastability (Casalino et al., 2011). L-Pro is particularly interesting because its mitochondrial oxidative catabolism is linked to cell survival/cell death in cancer cells (Liu et al., 2012; Phang et al., 2012) and to life span extension in C. elegans (Zarse et al., 2012). Here, we provide evidence that physiological concentrations of L-Pro are sufficient to convert ESCs into mesenchymal-like, highly motile, invasive pluripotent stem cells, which acquire metastatic potential in vivo. This previously unforeseen embryonic-stem-cell-to-mesenchymal-like transition (esMT) is fully reversible either after L-Pro withdrawal or by addition of vitamin C. Most remarkably, L-Pro extensively remodels both the transcriptome and the epigenome, thus acting as a potent signaling metabolite in pluripotent stem cells.
Results
Discussion How metabolites regulate stem cell biology represents a major challenge and is still poorly understood. Here, we uncover an unexpected role of L-Pro as a key regulator of mouse ESC migratory and invasive phenotype, revealing that L-Pro is a potent epigenetic modifier. L-Pro, and in particular its mitochondrial catabolism, has become an important area of study being at the crossroads of key metabolic pathways, including citric acid cycle, polyamines biosynthesis, and urea cycle (Phang et al., 2012). Despite that L-Pro is a nonessential amino acid (Phang et al., 2008), we demonstrate that the addition of L-Pro in ESC culture medium is sufficient to induce a previously unforeseen esMT, which converts compacted/clustered ESCs into scattered, freely motile pluripotent stem cells. Of note, this phenotype was not observed in previous studies (Casalino et al., 2011; Washington et al., 2010), most likely because it markedly increased when ESCs are seeded at clonal density in the presence of L-Pro. Accordingly, PiCs generated at higher density (PiCs500) showed poor migratory and invasive properties compared to PiCs generated at clonal density (PiCs50) indicating that seeding density strongly influences the EMT-like phenotype of PiCs. The L-Pro-induced esMT is accompanied by a global remodeling of the ESC transcriptome, which nicely correlates with the phenotypic variation observed. Indeed, genes involved in the control of cell cytoarchitecture, adhesion, and migration/invasiveness are largely affected by L-Pro. Notably, despite this extensive remodeling of the transcriptome, expression levels of the pluripotency-associated factors (Oct4, Nanog, and Sox2) and the developmental pluripotency-associated gene Dppa3 (Stella) are comparable in PiCs and ESCs. Furthermore, PiCs, like ESCs, are REX1 (Zfp42)-positive (≥95%) and show similar in vitro differentiation kinetics. This molecular signature and the fact that PiCs strictly depend on LIF for self-renewal (Casalino et al., 2011) suggest that they define a population of inner cell mass-like cells that have acquired a motile and invasive phenotype.