Effects of oxygen concentration on the proliferation and differentiation of mouse neural stem cells in vitro

Abstract

Background and purpose: Cerebral ischemia is known to elicit the activation of neural stem cells (NSCs); however its mechanism is not fully determined. Although oxygen concentration is known to mediate many ischemic actions, there has been little attention given to the role of pathological oxygen changes under cerebral ischemia on the activation of NSCs. We investigated the effects of various oxygen concentrations on mouse neural stem cells in vitro.

Methods: NSCs were cultured from the ganglionic eminence of fetal ICR mice on embryonic day 15.5 using a neurosphere method. The effects of oxygen concentrations on proliferation, differentiation, and cell death of NSCs were evaluated by bromodeoxyuridine (BrdU) incorporation, immunocytochemistry, and TUNEL assay, respectively.

Results: The highest proliferation and the neuronal differentiation of the NSCs were observed in 2% oxygen, which yielded significantly higher proportions of both BrdU-labeled cells and Tuj1-positive cells when compared with 20% and 4% oxygen. On the other hand, the differentiation to the astrocytes was not affected by oxygen concentrations, except in the case of anoxia (0% oxygen). The cell death of the NSCs increased in lower oxygen conditions and peaked at anoxia. Furthermore, the switching of the neuronal subtype differentiation from GABA-positive to glutamate-positive neurons was observed in lower oxygen conditions.

Conclusions: These findings raise the possibility that reduced oxygen levels occurring with cerebral ischemia enhance NSC proliferation and neural differentiation, and that mild hypoxia (2% oxygen), which is known to occur in the ischemic penumbra, is suitable for abundant neuronal differentiation.

Fig. 1

The effects of oxygen concentrations on the proliferation of cultured NSCs. NSCs were treated with BrdU (10 μM) for 6 h under various oxygen concentrations and were processed for BrdU immunocytochemistry. The representative immunofluorescence images (a) and the proportions of BrdU-labeled cells (b) are shown. Arrows in (a) indicated the BrdU-labeled cells. When compared to 20% oxygen, 10%, 4%, 3%, 2%, and 1%, but not 0% oxygen, significantly increased EGF-induced proliferation. The highest proliferation was observed at 2% oxygen. The asterisks indicate significant difference compared to 20% (P < 0.05; one-way ANOVA followed by Fisher’s PLSD test), n = 8

Fig. 2

The effects of oxygen concentrations on cell death of the cultured NSCs. After incubation under various oxygen concentrations, NSCs were processed with the TUNEL assay and the representative immunofluorescence images (a) and the proportions of dead cells (b) are shown. Arrows in (a) indicated the TUNEL-positive cells. The low oxygen culture significantly increased the proportion of dead cells. The asterisks indicate significant difference compared to 20% (P < 0.05; one-way ANOVA followed by Fisher’s PLSD test), n = 8

Fig. 3

The effects of oxygen concentrations on the differentiation of cultured NSCs. After the NSCs were differentiated under various oxygen concentrations, they were processed for immunocytochemistry against a neuronal marker, Tuj1, and an astrocyte marker, GFAP, and the representative immunofluorescence images (a) and the proportion of Tuj1—(b) and GFAP—(c) positive cells are shown. When compared to 20% oxygen, 3%, 2%, and 1% significantly increased the proportion of Tuj1-positive cells. The highest proportion of Tuj1-positive cells was observed at 2% oxygen. In contrast, the proportion of GFAP-positive cells significantly decreased in 0% oxygen when compared to 3% and 2% oxygen concentrations. The asterisks indicate significant difference compared to 20% (P < 0.05; one-way ANOVA followed by Fisher’s PLSD test), n = 6

Fig. 4

The effects of oxygen concentrations on neuronal subtype differentiation of the cultured NSCs. After the NSCs were differentiated under various oxygen concentrations, they were processed for immunocytochemistry against a neuronal marker, Tuj1 and GABA (a and b) or glutamate (c and d). The representative immunofluorescence images of Tuj1 and GABA (a) and of Tuj1 and glutamate (c) are shown. The proportion of GABA-positive cells (b) and glutamate positive cells (d) against the number of Tuj1-positive cells is also shown. Arrows in (a) and (c) indicate the double-positive cells. The proportion of GABA-positive neurons was significantly decreased in lower oxygen concentrations, whereas the proportion of glutamate-positive neurons was significantly increased in lower oxygen concentrations. The asterisks indicate significant difference compared to 20% (P < 0.05; one-way ANOVA followed by Fisher’s PLSD test), n = 8