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Alpha-Ketoglutarate Influences the Self-Renewal and Differentiation of Pluripotent Stem Cells


Human pluripotent stem cells (hPSCs) hold great potential for regenerative medicine due to their ability to self-renew indefinitely in in vitro culture and to differentiate into all three germ layers. However, the use of hPSCs in the clinic has been limited by the lack of differentiation protocols that produce fully mature and functional cell types. Development of more efficient differentiation strategies therefore will be key to fully realizing the therapeutic potential of hPSCs. Differentiation occurs through epigenetic changes that turn off genes important for self-renewal and activate genes required for cellular maturation and specialization. In addition, cellular metabolism shifts to address varying energetic and biosynthetic demands. Many metabolites, whose levels are influenced by the overall metabolic network, act as cofactors for enzymes that control the epigenetic state of the cell. α-Ketoglutarate (αKG), a TCA cycle metabolite, acts as a cofactor for αKG-dependent dioxygenases which included the JmjC-domain containing family of histone demethylases (JHDMs) and the Ten-eleven translocation (TET) methylcytosine oxidases. Succinate, another TCA cycle metabolite, acts as an inhibitor of the same αKG-dependent dioxygenases. Therefore, changes in the αKG-to-succinate ratio caused by changes in cellular metabolism impact the activity of αKG-dependent dioxygenases and therefore gene expression. Both JHDMs and TETs have known roles in pluripotent stem cell self-renewal and differentiation. Recently, αKG has been reported to support self-renewal in mouse embryonic stem cells (mESCs). However, mESCs differ from traditionally maintained hPSCs in numerous ways including their stages of pluripotency. Both mESCs and human embryonic stem cells are derived from the inner cell mass, but mESCs are traditionally maintained in an earlier developmental state, called the na�ve state, corresponding to the preimplantation embryo. hESCs are traditionally maintained in a more differentiated state, called the primed pluripotent state, corresponding to the post-implantation epiblast. Therefore, because the role of αKG in hPSCs has not previously been investigated, we examined the role of αKG in primed hPSC differentiation. We discovered that αKG can accelerate the differentiation of primed hPSCs likely through its action on αKG-dependent dioxygenases. Because αKG promotes self-renewal in mESCs, we investigated whether αKG promotes differentiation of primed mouse pluripotent stem cells derived from the post-implantation embryo called Epiblast stem cells (EpiSCs). αKG also promoted differentiation in mouse EpiSCs which suggests that the role of αKG is dependent on the stage of pluripotency and is not species dependent. To further confirm the role of αKG in hPSC differentiation, we decreased the αKG-to-succinate ratio by inhibition of αKG producing enzymes or succinate consuming enzymes during hPSC differentiation. Both manipulations led to a delay of hPSC differentiation. Finally, manipulation of the αKG-to-succinate ratio led to changes in DNA hydroxymethylation and histone methylation levels suggesting an epigenetic mechanism. Taken together, my data suggests that αKG plays a context specific, differentiation promoting role in primed pluripotent stem cells.

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