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 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...

Effect of inhibitors of NADPH oxidase complex and mitochondrial ATP‐sensitive potassium channels on generation of dopaminergic neurons from neurospheres of mesencephalic precursors

“…The present results suggest that Apocynin and 5-HD act by stimulating differentiation of mesencephalic precursors toward the DA phenotype. The results are consistent with those of previous studies, which have suggested that the redox state modulates differentiation of mesencephalic precursors (Studer et al, 2000;Rodriguez-Pallares et al, 2001Tsatmali et al, 2005), and with those of studies that have suggested that ROS levels affect the phenotype of cells in culture (Li et al, 2006;Xiao et al, 2009), including neurons (Tsatmali et al, 2006).…”

“…It is known that the number of proliferating DAergic precursors in culture is maximal during the first week and decreases sharply thereafter (Bouvier and Mytilineou, 1995). The number of proliferating cells further decreases after induction of the differentiation, as expected after withdrawal of the mitogens from the cell culture (Studer et al, 2000;Ebert et al, 2008). Several previous studies have suggested that intracellular levels of ROS modulate neuronal differentiation, and it has been observed that cell fractions exhibiting low levels of ROS contain proliferative progenitor cells, while newly generated neurons express high levels of ROS (Katoh et al, 1997;Goldsmit et al, 2001;Wang et al, 2007;Tsatmali et al, 2005); this was also confirmed in the present study by DHE labeling.…”

“…Previous studies in our laboratory have shown that treatment of ventral mesencephalic progenitors with antioxidants such as N-acetylcysteine and dipyridamole induces an increase in the number of DAergic neurons generated from neurospheres of mesencephalic precursors (Rodriguez-Pallares et al, 2001, and several findings suggest that the redox state of the cell and ROS levels may be responsible for some of these effects (Studer et al, 2000;Riaz and Bradford, 2005). However, the source of ROS and the mechanisms involved in the effects have not yet been elucidated.…”

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“…For example, hematopoietic stem cells are maintained in hypoxic niches in bone marrow (Parmar et al, 2007). Interestingly, hypoxia also prevents the differentiation of neural stem cells and promotes the maintenance of self-renewal potential of embryonic stem (ES) cells (Studer et al, 2000;Marrison et al, 2000;Ezashi et al, 2005;Santilli et al, 2010). In addition, restricted oxygen concentrations have been shown to enhance the production of induced pluripotent stem cells (iPSC) (Yoshida et al, 2009).…”

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