Nitric Oxide And Hypoxia Response In Pluripotent Stem Cells

Abstract

The expansion of pluripotent cells (ESCs and iPSCs) under conditions that maintain their pluripotency is necessary to implement a cell therapy program. Previously, we have described that low nitric oxide (NO) donor diethylenetriamine/nitric oxide adduct (DETA-NO) added to the culture medium, promote the expansion of these cell types. The molecular mechanisms are not yet known. We present evidences that ESC and iPSCs in normoxia in presence of low NO triggers a similar response to hypoxia, thus maintaining the pluripotency. We have studied the stability of HIF-1α (Hypoxia Inducible Factor) in presence of low NO. Because of the close relationship between hypoxia, metabolism, mitochondrial function and pluripotency we have analyzed by q RT-PCR the expression of genes involved in the glucose metabolism such as: HK2, LDHA and PDK1; besides other HIF-1α target gene. We further analyzed the expression of genes involved in mitochondrial biogenesis such as PGC1α, TFAM and NRF1 and we have observed that low NO maintains the same pattern of expression that in hypoxia. The study of the mitochondrial membrane potential using Mito-Tracker dye showed that NO decrease the mitochondrial function. We will analyze other metabolic parameters, to determinate if low NO regulates mitochondrial function and mimics Hypoxia Response. The knowledge of the role of NO in the Hypoxia Response and the mechanism that helps to maintain self-renewal in pluripotent cells in normoxia, can help to the design of culture media where NO could be optimal for stem cell expansion in the performance of future cell therapies.