Control of embryonic stem cell metastability by l-proline catabolism

The molecular mechanisms controlling mouse embryonic stem cell (ESC) metastability, i.e. their capacity to fluctuate between different states of pluripotency, are not fully resolved. We developed and used a novel automation platform, the Cell(maker), to screen a library of metabolites on two ESC-based phenotypic assays (i.e. proliferation and colony phenotype) and identified two metabolically related amino acids, namely l-proline (l-Pro) and l-ornithine (l-Orn), as key regulators of ESC metastability. Both compounds, but mainly l-Pro, force ESCs toward a novel epiblast stem cell (EpiSC)-like state, in a dose- and time-dependent manner. Unlike EpiSCs, l-Pro-induced cells (PiCs) contribute to chimeric embryos and rely on leukemia inhibitor factor (LIF) to self-renew. Furthermore, PiCs revert to ESCs or differentiate randomly upon removal of either l-Pro or LIF, respectively. Remarkably, PiC generation depends on both l-Pro metabolism (uptake and oxidation) and Fgf5 induction, and is strongly counteracted by antioxidants, mainly l-ascorbic acid (vitamin C, Vc). ESCs ↔ PiCs phenotypic transition thus represents a previously undefined dynamic equilibrium between pluripotent states, which can be unbalanced either toward an EpiSC-like or an ESC phenotype by l-Pro/l-Orn or Vc treatments, respectively. All together, our data provide evidence that ESC metastability can be regulated at a metabolic level.