Hypoxia Induces a Metabolic Shift and Enhances the Stemness and Expansion of Cochlear Spiral Ganglion Stem/Progenitor Cells

Previously, we demonstrated that hypoxia (1% O-2) enhances stemness markers and expands the cell numbers of cochlear stem/progenitor cells (SPCs). In this study, we further investigated the long-termeffect of hypoxia on stemness and the bioenergetic status of cochlear spiral ganglion SPCs cultured at low oxygen tensions. Spiral ganglion SPCs were obtained from postnatal day 1 CBA/CaJ mouse pups. The measurement of oxygen consumption rate, extracellular acidification rate (ECAR), and intracellular adenosine triphosphate levels corresponding to 20% and 5% oxygen concentrations was determined using a Seahorse XF extracellular flux analyzer. After low oxygen tension cultivation for 21 days, the mean size of the hypoxia-expanded neurospheres was significantly increased at 5% O-2; this correlated with high-level expression of hypoxia-inducible factor-1 alpha (Hif-1 alpha), proliferating cell nuclear antigen (PCNA), cyclin D1, Abcg2, nestin, and Nanog proteins but downregulated expression of p27 compared to that in a normoxic condition. Low oxygen tension cultivation tended to increase the side population fraction, with a significant difference found at 5% O-2 compared to that at 20% O-2. In addition, hypoxia induced a metabolic energy shift of SPCs toward higher basal ECARs and higher maximum mitochondrial respiratory capacity but lower proton leak than under normoxia, where the SPC metabolism was switched toward glycolysis in long-term hypoxic cultivation.