Nature 10.1038/nature13981

The culturing of mouse embryonic stem (ES) cells requires the right mix of conditioned media, serum and cytokines to ensure self-renewal and pluripotency maintenance. ES cells have traditionally been grown in serum-containing media, but these conditions lead to heterogeneity both in the morphology and expression of pluripotency factors. Culturing ES cells in a serum-free medium (termed '2i'), which includes kinase inhibitors for MEK and GSK-3b that block differentiation signals, has been shown to reduce this heterogeneity and is currently thought to best represent the naive ground state. High-resolution genome-wide transcriptome analysis comparing ES cells cultured in serum or 2i conditions further revealed that cells grown in 2i conditions contained elevated transcripts for genes encoding metabolic functions as well as reduced H3K27me3 epigenetic marks on promoters. Carey et al. predicted that these metabolic and epigenetic outcomes might be caused by inhibition of MEK and GSK-3. To understand whether these kinases are involved in this pluripotency ground state, the authors compared the metabolism of ES cells grown in 2i or serum conditions. 2i-cultured cells contained elevated levels of the TCA cycle metabolite α-ketoglutarate (α-KG) with diminished amounts of downstream TCA metabolites, such as succinate and malate. Using metabolic tracing experiments, they found that the metabolic flux was altered with α-KG being rerouted to produce glutamate instead of progressing through the TCA cycle. The resultant high α-KG/succinate ratio observed in 2i-cultured ES cells could also explain the epigenetic observations, as this is known to promote histone demethylase activity. Indeed, inhibition of the H3K27me3 demethylase using GSK-J4 produced a greater enhancement of H3K27me3 in 2i-cultured cells versus serum-cultured cells. Finally, the authors found that the addition of α-KG to ES cells induced germline-associated gene expression and promoted the increased self-renewal of serum-treated ES cells. Although these findings implicate α-KG in the epigenetic regulation of stem cells, further experiments are needed to determine how the inhibition of GSK-3B and MEK directly alter TCA cycle flux to increase α-KG levels.