Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen

Abstract

Standard cell culture systems impose environmental oxygen (O(2)) levels of 20%, whereas actual tissue O(2) levels in both developing and adult brain are an order of magnitude lower. To address whether proliferation and differentiation of CNS precursors in vitro are influenced by the O(2) environment, we analyzed embryonic day 12 rat mesencephalic precursor cells in traditional cultures with 20% O(2) and in lowered O(2) (3 +/- 2%). Proliferation was promoted and apoptosis was reduced when cells were grown in lowered O(2), yielding greater numbers of precursors. The differentiation of precursor cells into neurons with specific neurotransmitter phenotypes was also significantly altered. The percentage of neurons of dopaminergic phenotype increased to 56% in lowered O(2) compared with 18% in 20% O(2). Together, the increases in total cell number and percentage of dopaminergic neurons resulted in a ninefold net increase in dopamine neuron yield. Differential gene expression analysis revealed more abundant messages for FGF8, engrailed-1, and erythropoietin in lowered O(2). Erythropoietin supplementation of 20% O(2) cultures partially mimicked increased dopaminergic differentiation characteristic of CNS precursors cultured in lowered O(2). These data demonstrate increased proliferation, reduced cell death, and enhanced dopamine neuron generation in lowered O(2), making this method an important advance in the ex vivo generation of specific neurons for brain repair.

Fig. 1.

Lowered O₂ mediates increased yield of CNS precursors. A, Precursor yield across plating densities. CNS precursors derived from the ventral mesencephalon were expanded with bFGF in lowered or 20% O₂, and total cell numbers were assessed after 5 d of proliferation, when >90% of cells are nestin+ precursors. Significantly increased cell numbers were detected at all plating densities in lowered O₂ compared with 20% O₂.B, Precursor proliferation. CNS precursors were pulsed with 10 μm BrdU for 60 min immediately before fixation and then stained for BrdU uptake. More BrdU+ cells were seen in lowered O₂ cultures during both proliferation and differentiation. Data are presented as mean ± SEM (n = 40). Differences between lowered and 20% O₂ were statistically significant at all time points and for all parameters (n = 8, p < 0.05), except percentage of BrdU+ cells at day 4 of expansion (n= 8; p = 0.10). Scale bar, 20 μm.C, Clonal growth. The yield of clones derived from single precursors was threefold higher in lowered O₂compared with 20% O₂ cultures (left). The majority of clones derived from precursors in lowered O₂cultures contained 50–500 cells, whereas clone size in 20% O₂ cultures was generally 5–50 cells (right). D, Cell death. Apoptosis was assayed by TUNEL labeling of mesencephalic precursors cultured in either lowered or 20% O₂. Representative TUNEL stains during expansion (2 and 6 d of culture) and differentiation (4 d after bFGF withdrawal) are shown. Scale bar, 20 μm. A significant decrease in the percentage of apoptotic cells in lowered O₂ compared with traditional cultures was detected (n = 8; p < 0.05).

Fig. 2.

Lowered O₂ culturing improves the yield of functional precursor-derived dopaminergic neurons.A, Precursors from E12 mesencephalon were expanded in the presence of bFGF for 5 d, followed by 5 d of differentiation, and then stained for the neuronal marker Tuj1 and for TH. A large increase in both the total number and percentage of TH+ neurons was detected in lowered O₂ compared with 20% O₂ cultures (p < 0.001). Scale bar, 20 μm. B, Western blot analysis revealed significantly more TH protein in samples from lowered (vs 20%) O₂ cultures. Each lane was loaded with 2.5 μg of total protein. C, Reverse-phase HPLC with electrochemical detection was used to quantify dopamine levels in conditioned medium (24 hr) and in buffer with 56 mm KCl after 15 min (evoked release). Significantly more dopamine was detected in cultures maintained in lowered O₂ compared with those grown in 20% O₂ (p < 0.01 in conditioned medium; p < 0.05 for evoked release).Inset shows typical chromatogram for dopamine detection in lowered and 20% O₂ culture media.

Fig. 3.

Neuronal subtype differentiation and molecular characterization of mesencephalic precursors in lowered versus 20% O₂. A, Double immunocytochemical labeling of neurons (Tuj1+, red) revealed that lowered O₂ culturing markedly increased the fractional yield of dopaminergic and serotonergic neuronal subtypes but decreased the fractional yield of GABA+ and glutamate+ neurons (all subtype labels,green). The 20% O₂ colony is an example of high GABA expression under these conditions. TH and GABA were not coexpressed as in some developing neurons in vivo. The percentage of neurons expressing the midbrain transcription factor En1 was increased in lowered O₂. Scale bars, 20 μm.B, C, Semiquantitative PCR demonstrates differential gene expression in CNS precursors cultured in lowered or 20% O₂. B, Expression of genes involved in the physiological response to changes in oxygen levels. The expression of HIF1α, VHL, Epo, and VEGF was assessed after 2 or 6 d of expansion and after 4 d of differentiation in lowered or 20% O₂. Data are normalized to GAPDH expression. A significant increase in Epo expression was detected in lowered versus 20% O₂ mostly during differentiation, whereas VEGF was upregulated during both expansion and differentiation. No O₂-dependent regulation of HIF1α or VHL messages was observed. C, Candidate genes involved in midbrain dopaminergic neuron development were also tested for O₂-dependent differential expression. Increased expression of TH and Ptx3 during differentiation confirmed the larger number of functional dopaminergic neurons in lowered O₂ cultures (compare with Fig. 2). Significant increases in expression levels of FGF8 and En1 were also detected in lowered O₂.

Fig. 4.

Epo mimics the lowered O₂ effect on dopaminergic differentiation. A, Representative images of TH+ cells grown in the presence or absence of Epo and anti-Epo antibody and in lowered or 20% O₂ conditions. All reagents were added to E12 mesencephalic precursor cultures throughout cell expansion and differentiation (10 d total) in lowered or 20% O₂. Epo supplementation significantly increased TH+ cell numbers in 20% O₂ cultures (n = 6;p < 0.05). Epo neutralizing antibody decreased TH+ cell numbers in both lowered O₂ (n = 6;p < 0.01) and 20% O₂ cultures (n = 6; p < 0.05). Scale bar, 20 μm. B, Effects of Epo and anti-Epo blocking antibody on dopamine neuron yield. C, Epo positively influences dopaminergic neuron yield in 20% O₂ in a dose-dependent manner.