UC Irvine

Embryonic stem cells (ESC) are distinguished by their capacity for self-renewal and differentiation, abilities that are foundational to emerging research on cell-based therapies and regenerative medicine. The mechanisms underlying stem cell pluripotency and differentiation however, remain poorly understood. The discovery of induced pluripotent stem cells (iPSCs) advanced our understanding of pluripotency and broadened the applications of stem cell; it also bypassed the ethical issue of using embryonic stem cells. Although the technique has been broadly applied and modified in the last decade, the mechanistic details remain elusive. It has been demonstrated that activation of phosphatidylinositol-3 kinase/Akt signaling enhanced stem cell pluripotency and iPSC reprogramming. Activation of Akt1 has been reported translocating from cytoplasm to mitochondria. However, the roles of mitochondrial Akt1 plays in the regulation of stem cell pluripotency and iPSC reprogramming have not yet been determined. Using Yamanaka factor reprogramming, we demonstrated that activation of mitochondrial Akt1 signaling enhanced reprogramming efficiency. In addition, we also observed that the DNA methylation status at the OCT4 and NANOG promoters of the resulting Yamanaka factors- mitochondrial Akt1-reprogrammed cell displayed a more profound de-methylation profile. We also demonstrated that the resulting Yamanaka-mitochondrial Akt1-reprogrammed cells displayed a respiration profile more similar to that in ESCs. Increasing evidence has implicated the importance of mitochondria and cellular metabolism in iPSC reprogramming. Interestingly, we observed that activation of mitochondrial Akt1 signaling reduced cellular oxygen consumption, NADH levels and maintained ATP levels, leading to lower reactive oxygen species production and lower apoptosis levels under stress conditions. We further identified the interaction of Akt1 with pyruvate dehydrogenase (PDH) complex (PDC) in mitochondria, that the dihydrolipoamide dehydrogenase (E3) subunit of the PDC preferentially bound to Akt1. The activation of mitochondrial Akt1 signaling enhanced PDH activity. Using computational method, we identified a protein-protein interaction site on Akt1 and further screened and obtained two potential small molecule compounds, NSC 34766 and NSC 628725, that may bind to Akt1 via this site. Follow up studies indicated that the compounds disrupted the Akt1-E3 interaction and reduced the effects of mitochondrial Akt1 and insulin on PDH activity.