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 Ϯ 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. Key words: CNS precursors; CNS stem cells; dopaminergic neurons; erythropoietin; oxygen; Parkinson's diseaseCultured CNS stem cells have proved useful in defining the pathways that lead to generation of neurons and glia (McKay, 1997). These cells self-renew, and after mitogen withdrawal, differentiate into neurons, astrocytes and oligodendrocytes in predictable proportions (Johe et al., 1996;McKay, 1997). Single extrinsic factors can shift the fate of CNS stem cells toward specific cell lineages (Johe et al., 1996;Panchision et al., 1998). The potential therapeutic application of CNS stem cells in common degenerative and ischemic diseases has become a major focus of research. The generation of dopaminergic neurons from CNS precursors is of special interest given the promising results of fetal cell transplantation in patients with Parkinson's disease (Olanow et al., 1996; Piccini at al., 1999;Freeman et al., 2000).In clinical settings, gases are appreciated as primary variables in organ survival, with O 2 as the critical gas parameter. However, traditional CNS stem cell culture (as well as virtually all other ex vivo cell culture) is performed in nonphysiologically high O 2 . Standard tissue culture incubator conditions are 5% CO 2 and 95% air, which exposes cells to a 20% O 2 environment. In mammalian brain, interstitial tissue O 2 levels range from ϳ1 to 5% (Table 1). We tested the effects of culturing CNS progenitor cells in physiological "lowered" (3 Ϯ 2%) O 2 , comparing the cultures with those grown in the usual 20% O 2 . Our results indicate that oxygen lowere...

Major problems in stem cell biology revolve around defining the developmental potential of cell populations and understanding how their potential is maintained or progressively restricted. Oxygen (O(2)) is an obvious environmental factor which has received little attention in culturing skeletal muscle progenitor cells. In this work, we examine the effects of O(2) levels on the developmental potential, proliferative capacity, and phenotype of the adult skeletal muscle fiber progenitor population (satellite cells), and cell lines that model multipotential embryonic paraxial mesoderm from which skeletal muscle develops. Both satellite cell proliferation and survival of mature fibers increased in physiologic (6%) O(2) vs. non-physiologic 20% O(2) used in virtually all traditional cell culture. Six percent O(2) conditions also accelerated the up-regulation of multiple MyoD family myogenic regulatory factors (MRFs). An unexpected finding was that fiber-adherent satellite cells could assume a non-myogenic phenotype. By the criteria of molecular markers and gross lipid accumulation, satellite cells were found to assume an adipocyte phenotype, and did so more prominently in 20% O(2) than in physiologic O(2). Selection of the adipogenic fate and execution of adipogenesis by multipotential mesenchymal cell lines was also dramatically higher in traditional 20 vs. 6% O(2), and decreased adipogenesis in physiologic O(2) was associated with significantly less expression of the adipogenic regulator, PPAR gamma. These results suggest that regulatory pathways affected by O(2) are important for satellite cell proliferation, execution of cell fate, and parent muscle survival in culture, and so may play a role in vivo under normal or pathologic conditions.

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