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

Standard cell culture systems impose environmental oxygen (O-2) levels of 2 0%, whereas actual tissue O-2 levels in both developing and adult brain are an order of magnitude lower. To address whether proliferation and differen tiation of CNS precursors in vitro are influenced by the O-2 environment, w e analyzed embryonic day 12 rat mesencephalic precursor cells in traditiona l cultures with 20% O-2 and in lowered O-2 (3 +/- 2%). Proliferation was pr omoted and apoptosis was reduced when cells were grown in lowered O-2, yiel ding greater numbers of precursors. The differentiation of precursor cells into neurons with specific neurotransmitter phenotypes was also significant ly altered. The percentage of neurons of dopaminergic phenotype increased t o 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 n inefold net increase in dopamine neuron yield. Differential gene expression analysis revealed more abundant messages for FGF8, engrailed-1, and erythr opoietin in lowered O-2. Erythropoietin supplementation of 20% O-2 cultures partially mimicked increased dopaminergic differentiation characteristic o f 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 gene ration of specific neurons for brain repair.