Abstract

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.