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Standard cell culture systems impose environmental oxygen (O2) levels of 20%, whereas actual tissue O2 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 O2 environment, we analyzed embryonic day 12 rat mesencephalic precursor cells in traditional cultures with 20% O2 and in lowered O2 (3 ± 2%). Proliferation was promoted and apoptosis was reduced when cells were grown in lowered O2, 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 O2 compared with 18% in 20% O2. 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 O2. Erythropoietin supplementation of 20% O2cultures partially mimicked increased dopaminergic differentiation characteristic of CNS precursors cultured in lowered O2. These data demonstrate increased proliferation, reduced cell death, and enhanced dopamine neuron generation in lowered O2, making this method an important advance in the ex vivogeneration of specific neurons for brain repair.

Enhanced Proliferation, Survival, and Dopaminergic Differentiation of CNS Precursors in Lowered Oxygen