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. 2015 Nov;93(11):1703-12.
doi: 10.1002/jnr.23615. Epub 2015 Jul 3.

Physiologically normal 5% O2 supports neuronal differentiation and resistance to inflammatory injury in neural stem cell cultures

Xiaoyun Sun  1 , Ludmila A Voloboueva  1 , Creed M Stary  1 , Rona G Giffard  1
Affiliations

Physiologically normal 5% O2 supports neuronal differentiation and resistance to inflammatory injury in neural stem cell cultures

Xiaoyun Sun et al. J Neurosci Res. 2015 Nov.

Abstract

Recent studies have demonstrated that neural stem cell (NSC) culture at physiologically normoxic conditions (2-5% O2) is advantageous in terms of neuronal differentiation and survival. Neuronal differentiation is accompanied by a remarkable shift to mitochondrial oxidative metabolism compared with preferentially glycolytic metabolism of proliferating cells. However, metabolic changes induced by growth in a normoxic (5%) O2 culture environment in NSCs have been minimally explored. This study demonstrates that culturing under 5% O2 conditions results in higher levels of mitochondrial oxidative metabolism, decreased glycolysis, and reduced levels of reactive oxygen species in NSC cultures. Inflammation is one of the major environmental factors limiting postinjury NSC neuronal differentiation and survival. Our results show that NSCs differentiated under 5% O2 conditions possess better resistance to in vitro inflammatory injury compared with those exposed to 20% O2. The present work demonstrates that lower, more physiologically normal O2 levels support metabolic changes induced during NSC neuronal differentiation and provide increased resistance to inflammatory injury, thus highlighting O2 tension as an important determinant of cell fate and survival in various stem cell therapies.

Keywords: metabolism; mitochondria; neurogenesis; oxygen; stem cell.

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Conflict of interest statement

Conflict of interest statement: The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
5% O2 increases neuronal differentiation. (A) NSC cultures differentiated under 5% O2 demonstrated higher levels of neuronal differentiation as indicated by neuronal markers Dcx (young neurons) and MAP2 (mature neurons), without significant changes in GFAP glial staining (nuclei are labeled with DAPI (blue), Dcx+, MAP2+, GFAP+ and NG2+ cells are green). Quantification of and the percentage of neuronal Dcx+ and MAP2+ and glial cells (B) the total cell density (per 300×400 μm field of view) (C). The data show three independent experiments, with at least 500 total analyzed cells in each experiment (**p=0.062 for Dcx+, *p=0.014 for MAP2+, *p=0.04 for NG2+, ***p<0.001 for total cell number compared to the corresponding cell type or total cell number at 20% O2, calculated by unpaired t-test).
Figure 2
Figure 2
Mitochondrial changes induced by low oxygen. 5% O2 conditions did not promote significant changes in mitotracker staining (green), but induced significant increase in mitochondrial potential sensitive dye TMRE (Fig. 2A) indicating higher mitochondrial energetics. Quantification of mitotracker intensity (B), TMRE intensity (C) and TMRE/mitotracker ratio (D). The data show three independent experiments, with at least 10 fields of view in each experiment (**p<0.0014, ***p=0.0006 compared to 20% O2, calculated by unpaired t-test).
Figure 3
Figure 3
Changes in glycolysis and oxidative stress induced by 5% O2. Culturing under low oxygen promoted decrease in glycolytic rates as evidenced by decreased rates of lactate production (A), and decrease in ROS levels as indicated by Cytox staining. Representative images of Cytox staining (B). Quantification of Cytox staining (C) The data show three independent experiments, with 2-3 independent samples per experiment (***p<0.001 compared to 20% O2, calculated by unpaired t-test).
Figure 4
Figure 4
The effect of 5% O2 on Dcx+ cell survival during CM in vitro inflammatory injury. Representative changes of normal control (NC) and CM treated cultures. Nuclei are labeled with DAPI (blue), Dcx+, cells are green (A). Quantification of Dcx+ cells (B) and total cell numbers (C). The data show three independent experiments with at least 500 total analyzed cells in each experiment (*p<0.05 ANOVA compared to 20% O2, #p<0.05 ANOVA compared to the corresponding control condition).
Figure 5
Figure 5
The effect of 5% O2 on Dcx+ cell survival during TNF-α inflammatory injury. Representative changes of normal control (NC) and TNF-α treated cultures. Nuclei are labeled with DAPI (blue), Dcx+, cells are red (A). Quantification of Dcx+ cells (B) and total cell numbers (C). The data show three independent experiments with at least 500 total analyzed cells in each experiment (*p<0.05 ANOVA compared to 20% O2, #p<0.05 ANOVA compared to the corresponding control condition).
Figure 6
Figure 6
5% O2 increases Dcx+ survival, suppresses apoptotic rates and protects mitochondrial function during antimycin A treatment. Representative images Dcx+ staining in normal control (NC) and Antimycin A (AA) treated cultures (nuclei are labeled with blue DAPI, Dcx+ cells are green) (A). Quantification of Dcx+ cells (B) and total cell numbers (C). Representative images of TUNEL staining in control and Antimycin A (AA) treated cultures (TUNEL+ cells are green) (D). Quantification of apoptotic staining (E). Time course of mitochondrial TMRE intensity in control, TNF-α and antimycin A (AA) treated cultures (F). Quantification of survival of Dcx+ cells (G) and total cell numbers (H) of NSC cultures grown at 20% O2 or 5% O2, and shifted to 5% O2 for the antimycin A treatment. The data show three independent experiments with at least 500 total analyzed cells in each experiment (*p<0.05 ANOVA compared to 20% O2, #p<0.05 ANOVA compared to the corresponding control condition).
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