Significant effort has focused on using synthetic small molecules to control stem cell development 15
, but endogenous (i.e., naturally occurring) molecules have not been extensively examined. Our untargeted metabolomics analysis has revealed a unique metabolic signature in ESCs characterized by the presence of highly unsaturated, endogenous molecules. The high degree of unsaturation makes these metabolites reactive and susceptible to oxygenation and/or hydrogenation reactions, conferring them with what may be interpreted as ‘chemical plasticity’. Examples of such metabolites include polyunsaturated fatty acids such as AA, EPA, and DHA which are rapidly released by cell membranes in response to stress or altered homeostasis, making them available for oxidative metabolism by COX, LOX, and P450 enzymes.
Our findings suggest that the redox status of ESCs is regulated during the process of differentiation, as revealed by measuring the GSH/GSSG ratio and ascorbic acid levels. The inverse relationship between the GSH/GSSG ratio and the ASA level may indicate that ASA compensates for the accumulation of GSSG to maintain homeostasis during ESC differentiation. This is consistent with the closely linked antioxidant actions of glutathione and ASA previously observed in the liver of adult mice 16
, where induction of glutathione deficiency was accompanied by a rapid increase in ASA level (contrary to other adult tissues or liver in newborn rats). Previous studies revealed the importance of ascorbic acid in promoting differentiation when present during the early stages of stem cells differentiation 17–18
We show that inhibition of 5Δ and 6Δ desaturases by curcumin and sesamin delays differentiation, which are key enzymes involved in the synthesis of ‘plastic’ ω-3 and ω-6 polyunsaturated fatty acids. Interestingly, curcumin is also known to act as an antioxidant 19
. Therefore, we cannot exclude the possibility that the increased levels of Nanog and Oct4 caused by curcumin are partially due to its regulation of the redox status.
Our experimental results also show that supplementation of ESC media with essential, naturally occurring metabolites associated with oxidative metabolism in the mitochondria 20
, such as saturated fatty acids and acyl-carnitines in β-oxidation, leads to a significant increase in neuronal and cardiac differentiation. This is consistent with previous observations that increased production of mitochondrial proteins was associated with neurogenesis 21–23
. Activation of oxidative enzymes such as NADPH oxidase, and subsequent reactive oxygen species (ROS) generation, is also a pre-requisite for cardiovascular differentiation of ESCs 24–25
Overall, our results suggest that the activation of oxidation is a metabolic signature of stem-cell differentiation. Indeed, several independent lines of evidence demonstrated that stem cells contain lower levels of ROS than their more mature progeny 23,26–27
and that ROS accumulation and signaling was required for differentiation 23
. This is consistent with previous observations that the intracellular oxidation state regulates the balance between self-renewal and differentiation 28
. Specifically, signaling molecules that promote pluripotency make stem cells more reduced while those that promote differentiation make cells more oxidized. In addition, it is well known that hypoxia maintains the pluripotent and undifferentiated phenotype of stem/precursor cells both in vitro
and in vivo
. We speculate that redox regulation, together with hypoxic conditions, makes stem cells particularly attuned to differentiate in vivo
in response to oxidative processes such as inflammation.
Finally, our results raise an intriguing possibility that specific endogenous inflammatory mediators might regulate the regenerative properties of stem cells. We found that inhibition of PLA2
, COX and LOX promotes the pluripotent, undifferentiated state of ESCs. In support of our results, a high-throughput screening assay showed that anti-inflammatory drugs promote self-renewal in human ESCs 31
. For the first time, we show that neuroprotectin D1 32
, a DHA-derived lipid mediator that activates inflammation-resolution 33
, accelerated neuronal differentiation. Pro-inflammatory lipid mediators such as leukotriene B4 and C4, in contrast, had no significant effect on mESC differentiation. Previous studies also revealed important roles of specific eicosanoids on stem cell fate, such as prostaglandin E2 34
or lipoxin A4 35
. These data suggest that specific molecular responses to injury and inflammation may regulate the proliferation and differentiation of stem cells, and this may lead to the exploration of new avenues in understanding properties associated with regeneration. Further investigation will determine the mechanisms by which pro-inflammatory and pro-resolving endogenous metabolites activate proliferation and differentiation of quiescent stem/progenitor cells in response to tissue repair or wound healing.