Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors

Abstract

The efficient generation of specific brain cells in vitro may serve as a source of cells for brain repair in several devastating neurological diseases. Production of dopaminergic neurons from precursor cells for transplantation in Parkinson's disease has become a major research goal. We found that murine mesencephalic neurospheres were viable and proliferated, preserved telomerase activity, pluripotency and dopaminergic commitment for many weeks when cultured in 3% O 2 , whereas exposing these cells to 21% oxygen prohibited long-term expansion. Microarray data suggest that a variety of genes related to the cell cycle, cell maturation and apoptosis are differentially regulated in midbrain-derived precursors cultured in 3 versus 21% oxygen after 1-2 months. Taken together, we hypothesize that sustained high oxygen has deleterious effects on the self-renewal capacity of mesencephalic neural precursors, possibly accelerating maturation and senescence resulting in overall cell loss. Gene regulation governed by low oxygen tension may be relevant to the normal development and survival of midbrain neurons. Keywords: microarray, neural precursors, neurospheres, oxygen, proliferation, telomerase. Stem cells are undifferentiated cells that retain the ability to divide and proliferate throughout postnatal life to provide precursor cells that can differentiate into specialized cells. Neural stem cells can give rise to all the cells of the CNS including glial and neuronal cells (Gage 2003). As a result of their ability to differentiate following transplantation, these cells represent a potentially valuable therapeutic tool for the treatment of genetic, traumatic, inflammatory or degenerative neurological disorders (Studer et al. 1998;Storch and Schwarz 2002;Conti et al. 2003). To enable long-term expansion, all precursor cells must maintain the capacity to self-renew and a positive balance of proliferation versus differentiation or cell death. Therefore, it is crucial to understand the mechanisms governing cell division, differentiation and senescence (Sharpless and DePinho 2004).Mammals have evolved complex circulatory systems to ensure that every cell receives sufficient oxygen for normal metabolic processes. Lack of oxygen is defined as a reduction in oxygen supply to the tissues to below physiological levels. Hypoxia can develop as a result of ischaemia, representing a pathological component of stroke and heart attack. In addition to being a consequence of the growth of a malignant tumour and so a potential marker, hypoxia also acts to promote tumour development (Brizel et al. 1996;Brat et al. 2004). Address correspondence and reprint requests to Javorina Milosevic, Department of Neurology, Max-Bürger-Forschungszentrum, Johannisallee 30, 04103 Leipzig, Germany. E-mail: javorina.Milosevic@medizin.uni-leipzig.deAbbreviations used: ADA, adenosine deaminase; bFGF, basic fibroblast growth factor; BrdU, 5-bromo-2-deoxyuridine; DA, dopamine; DAPI, 6¢-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EGF...

Oxygen Tension Within the Neurogenic Niche Regulates Dopaminergic Neurogenesis in the Developing Midbrain

“…Our in vivo data support the conclusion that the low oxygen atmospheric culture (3%) represents rather the physiological environment of the nervous tissue (average 4.6% O 2 ) than the so called hypoxic condition, because the observation that hyperoxia stimulates dopaminergic neurogenesis fits to previous in vitro studies, showing that proliferation and dopaminergic differentiation capacity of isolated midbrain dopaminergic NPCs depend on optimized oxygen levels: oxygen conditions of 3%-5% enhance long-term expansion and reduce spontaneous differentiation and cell senescence of dopaminergic progenitors in vitro [18][19][20]48]. However, severe hypoxia (<1% O 2 ) as well as normoxia (21% O 2 ) represses their proliferation and promotes spontaneous differentiation [17,34].…”

“…Low atmospheric oxygen levels of 3% confer long-term proliferation and stem cell maintenance of midbrain dopaminergic NPCs in vitro, including the conservation of their dopaminergic differentiation potential, and avoid senescence in various species, including mouse [18][19][20][21][22][23][24]. Further in vitro studies have shown that the oxygen-dependent transcription factor hypoxiainducible factor 1a (HIF-1a) is involved in these processes [15,25,26] and is responsible for the initial adaptive response of cells to oxygen variations [27,28].…”

Neuroblastoma aggressiveness in relation to sympathetic neuronal differentiation stage

“…In addition, near-physiological oxygen tensions (5-6%) improve the efficiency of induced pluripotent stem cell generation (79). Several studies on in vitro differentiation of neural stem cells have shown that low oxygen levels promote proliferation, survival and multipotency as compared to normoxic oxygen levels (80)(81)(82)(83)(84)(85)(86). Hematopoietic stem cells (HSCs) in the bone marrow have been proposed to exist in a low oxygen milieu (87,88).…”

Lack of Hypoxia-Inducible Factor-1α Impairs Midbrain Neural Precursor Cells Involving Vascular Endothelial Growth Factor Signaling

“…Tissue samples were incubated in 0.1 mg/ml papain (Roche, Mannheim, Germany)/DNase solution (100 g/ml; Roche) for 30 min at 37°C, rinsed in PBS, incubated in antipain (50 g/ml; Roche) for 30 min at 37°C, and finally homogenized by gentle triturating using a firepolished Pasteur pipette. The cells were expanded as a monolayer culture by plating onto polyornithine-fibronectin precoated dishes in a density of 20,000 cells/cm 2 (Milosevic et al, 2005). The cells were maintained in serum-free DMEM (high glucose)/F-12 mixture (1:1) medium and supplemented with 20 ng/ml human recombinant epidermal growth factor and 20 ng/ml basic fibroblast growth factor (both from PromoCell, Heidelberg, Germany).…”

“…It is necessary to understand the mechanisms governing cell proliferation, dopaminergic differentiation, and senescence of NPCs to facilitate clinical therapies (Sharpless and DePinho, 2004). Reduced oxygen tension is now recognized as a common requirement for successful expansion and dopaminergic differentiation of NPCs (Ivanovic et al, 2000;Morrison et al, 2000;Studer et al, 2000;Storch et al, 2001;Milosevic et al, 2005). Low, physiologic oxygen conditions help maintain the undifferentiated state in mouse NPCs, but the molecular mechanisms underlying oxygen effects on NPCs are essentially unknown (Milosevic et al, 2005).…”