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First published online June 7, 2007
Stem Cells Vol. 25 No. 9 September 2007, pp. 2291 -2301
doi:10.1634/stemcells.2006-0609; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Oxygen Tension Regulates Survival and Fate of Mouse Central Nervous System Precursors at Multiple Levels

Hui-Ling Chena, Francesca Pistollatoa,b, Daniel J. Hoeppnerc, Hsiao-Tzu Nid, Ronald D.G. McKayc, David M. Panchisiona

aCenter for Neuroscience Research, Children's National Medical Center, Washington, DC, USA;
bHemato-Oncology Laboratory, Department of Pediatrics, University of Padua, Padua, Italy;
cLaboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA;
dR&D Systems, Inc., Minneapolis, Minnesota, USA

Key Words. Neural stem cells • Oxygen • Hypoxia • Multipotent • Expansion • Oligodendrocyte

Correspondence: David M. Panchision, Ph.D., 5th Floor, Suite 5340, 111 Michigan Avenue NW, Washington, DC 20010, USA. Telephone: 202-884-2269; Fax: 202-884-4988; e-mail: dpanchision{at}cnmcresearch.org

Received on September 28, 2006; accepted for publication on May 30, 2007.

First published online in STEM CELLS EXPRESS  June 7, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Despite evidence that oxygen regulates neural precursor fate, the effects of changing oxygen tensions on distinct stages in precursor differentiation are poorly understood. We found that 5% oxygen permitted clonal and long-term expansion of mouse fetal cortical precursors. In contrast, 20% oxygen caused a rapid decrease in hypoxia-inducible factor 1{alpha} and nucleophosmin, followed by the induction of p53 and apoptosis of cells. This led to a decrease in overall cell number and particularly a loss of astrocytes and oligodendrocytes. Clonal analysis revealed that apoptosis in 20% oxygen was due to a complete loss of CD133loCD24lo multipotent precursors, a substantial loss of CD133hiCD24lo multipotent precursors, and a failure of remaining CD133hiCD24lo cells to generate glia. In contrast, committed neuronal progenitors were not significantly affected. Switching clones from 5% to 20% oxygen only after mitogen withdrawal led to a decrease in total clone numbers but an even greater decrease in oligodendrocyte-containing clones. During this late exposure to 20% oxygen, bipotent glial (A2B5+) and early (platelet-derived growth factor receptor {alpha}) oligodendrocyte progenitors appeared and disappeared more quickly, relative to 5% oxygen, and late stage O4+ oligodendrocyte progenitors never appeared. These results indicate that multipotent cells and oligodendrocyte progenitors are more susceptible to apoptosis at 20% oxygen than committed neuronal progenitors. This has important implications for optimizing ex vivo production methods for cell replacement therapies.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Oxygen is an important energy source for cell metabolism [1], and oxygen utilization is tightly regulated in the central nervous system (CNS) [2]. PO2 values in the CNS are similar among all adult mammalian species tested and range from 0.55% (4.1 mmHg) in the midbrain to 8.0% (60 mmHg) in the pia; the measured PO2 of cortex is 2.53%–5.33% (19–40 mmHg) [3]. Deviations from these levels occur in stroke, which reduces oxygen availability to tissues [4], and can occur in hyperbaric therapies, which are intended to increase oxygen availability in stroke patients and neonates but may cause oxygen toxicity and neurological complications [5, 6].

Although cellular responses in the adult brain to changes in oxygen availability are well-described [4, 7, 8], oxygen changes appear to have complex but poorly understood effects on precursor cell fate. Most in vitro studies of fetal CNS precursors are performed in a nonphysiological, near-room (20%) oxygen tension. By comparison, lowered oxygen in the physiological range (2%–5%) increases the expansion of rat or human ventral midbrain precursors and promotes the generation of tyrosine hydroxylase (TH)+ dopaminergic neurons, a cell type that is lost in Parkinson disease [9, 10]. Culture of rat multipotent neural crest precursors in lowered oxygen promotes their survival and the generation of TH+ sympathoadrenal cells [11]. Lowered oxygen decreases apoptosis and promotes long-term expansion of mouse midbrain neurospheres and the subsequent generation of neurons [12]. Conversely, lowered oxygen also prevents neuronal differentiation of rat precursors as measured by ßIII-tubulin expression [13]. The pleiotropic nature of these responses suggests a lineage-dependent component to how oxygen regulates precursor fate. This has been difficult to ascertain since the effect of changing oxygen tension has not been tested at defined steps in CNS expansion and differentiation or on distinct CNS precursor subtypes within the same tissue.

To address this, we combined optimized flow cytometric methods [14] with a customized system to control gas composition during incubation, microscopy-aided recording, and experimental manipulation. Using clonal analysis, we found that 5% oxygen, which is within the physiologically measured range, specifically promoted the survival and clonal expansion of mouse multipotent CNS stem cells. In contrast, 20% oxygen caused stem cell apoptosis and limited fates of surviving multipotent cells but did not affect neuronal progenitors. We also found multiple effects of oxygen on the oligodendrocyte lineage. These novel findings show that oxygen tension regulates distinct precursor subtypes at different stages during proliferation and differentiation.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Gas-Controlled Incubation
For 20% oxygen culturing, cells were incubated in a Forma Series II CO2 incubator; CO2 was added to maintain a 20% O2, 5% CO2, 75% N2 balance. For 5% or 2% oxygen culturing, cells were incubated in a customized, computer-controlled system (Biospherix, Ltd., Syracuse, NY, http://www.biospherix.com). This permitted gas regulation to within 0.1% O2 increments during all phases of culturing, image acquisition and experimentation (5% O2, 5% CO2, 90% N2 balance or 2% O2, 5% CO2, 93% N2 balance), allowed pre-equilibration of medium, and eliminated artifacts introduced by periodic room air reperfusion. The system consists of (a) modular remote chambers for maintaining cultures; (b) a gas-controlled glove box for cell manipulation; and (c) an attached chamber containing a microscope (Axiovert 10; Carl Zeiss, Jena, Germany, http://www.zeiss.com), with a mounted digital camera (QColor3; Olympus, Tokyo, http://www.olympus-global.com), for cell visualization and recording.

Isolation, Expansion, and Differentiation of Mouse Cortical Precursors
Animal tissue was acquired with an approved protocol in accordance with animal care and use committee guidelines. Embryonic day 13.5 (E13.5) mouse cortex was freshly dissociated and monolayer-cultured as described [15]. Cells were expanded by daily addition of 20 ng/ml basic fibroblast growth factor (bFGF; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) for 5 days prior to first passage. For subsequent expansion, cells were replated at 100 cells per mm2 and passaged in 3-day iterations. Clonal analysis was performed by plating at 0.3 cells per mm2.

In some experiments, cultures were supplemented with erythropoietin (10 ng/ml; R&D Systems), N-acetylcysteine (1 mM; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), ascorbic acid (200 µM; Sigma-Aldrich), or B27 with or without retinoic acid (2% vol/vol; Invitrogen, Carlsbad, CA, http://www.invitrogen.com). For terminal fate and oxygen switching experiments, cells were expanded for 3 days, and then precursor cell differentiation was induced by culturing cells in Dulbecco's modified Eagle's medium/Ham's F-12 medium/N2 supplements without mitogen. After 2 days post-mitogen withdrawal, cells were switched to Neurobasal medium supplemented with 2% B27 (Invitrogen) and 10 ng/ml neurotrophin-3 (R&D Systems) to promote maturation of postmitotic cells for an additional 4 days (6 days of differentiation, total). Where proliferation was measured, 5-bromo-2'-deoxyuridine (BrdU; 10 µM; Roche, Indianapolis, IN, http://www.roche-applied-science.com) was added to cultures 1 hour prior to fixation.

Immunocytochemistry
Cells were fixed in cold 4% paraformaldehyde for 15 minutes and rinsed three times with phosphate-buffered saline. Cells were incubated with primary antibodies against nestin (mouse IgG1, 1:100; Chemicon, Temecula, CA, http://www.chemicon.com), BrdU (mouse IgG1, 1:100; Roche), activated caspase-3 (rabbit IgG, 1:4,000; Wyeth Pharmaceuticals, http://www.wyeth.com), microtubule-associated protein 2 subunits a and b (MAP2a+b; mouse IgG1, 1:200; Sigma-Aldrich), ßIII-tubulin (TuJ1; mouse IgG1, 1:500; Covance, Princeton, NJ, http://www.covance.com), polysialated neural cell adhesion molecule (polysialated/embryonic neural cell adhesion molecule (PSA-NCAM/E-NCAM); mouse, 1:400; Chemicon), glial fibrillary acidic protein (GFAP; rabbit IgG, 1:800; Dako, Fort Collins, CO), A2B5 (mouse IgM, 1:10,000; Chemicon), platelet-derived growth factor receptor {alpha} (PDGFR{alpha}; rabbit, 1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), or O4 (mouse IgM, 1:100; Sigma-Aldrich), followed by fluorescent secondary antibodies (Alexa dyes, 1:1,500; Invitrogen). Cells were nuclear-counterstained with 4',6-diamidino-2-phyenylindole (DAPI; 100 ng/ml) to measure total cell number. Staining of up to four simultaneous colors was visualized by epifluorescence using appropriate filters (BX60 upright microscope; Olympus); digital images were compiled for figures using Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, http://www.adobe.com).

Protein Analysis
Cells were harvested in denaturing lysis buffer (2% SDS, 10% glycerol, 50 mM Tris [pH 6.8], 50 mM dithiothreitol, protease, and phosphatase inhibitors); isolation procedures for 5% oxygen samples were done in the gas-controlled glovebox. Lysates were aliquoted and frozen at –80°C until use. Protein was measured by an enzyme-linked immunosorbent assay (ELISA) as directed (R&D Systems). Microtiter dishes (Nunc, Rochester, NY, http://www.nuncbrand.com) were coated with lysate in 0.1 M sodium bicarbonate at 4°C overnight before incubating with antibodies against hypoxia-inducible factor 1{alpha} (anti-HIF1{alpha}; rabbit; Novus Biologicals, Littleton, CO, http://www.novusbio.com), nucleophosmin (NPM; rabbit; as directed; Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), p53 (rabbit; as directed; Cell Signaling Technology), or phospho-serine-specific p53 (Ser6, Ser9, Ser15, Ser20, Ser37, Ser45, and Ser392; rabbit; as directed; Cell Signaling Technology). Intensity of staining was measured using a microplate reader set to a 450 nm wavelength. The color intensity was normalized to protein concentration, measured by Micro BCA protein kit (Pierce, Rockford, IL, http://www.piercenet.com).

Flow Cytometry
Cells were assayed as described [14]. Briefly, 1 x 106 cells per milliliter in flow cytometry buffer were stained for CD133 (biotinylated rat IgG2b, 10 µg/ml; R&D Systems) and CD24 (phycoerythrin-conjugated rat IgG2b, 0.1 µg/ml; BD Biosciences, San Diego, http://www.bdbiosciences.com). Between washes, fluorochrome-streptavidin-conjugated secondary antibody (Alexa dyes; Invitrogen) was incubated against CD133. For viability gating, 7-amino-actinomycin-D (final concentration, 50 ng/ml; BD Biosciences) and/or Annexin V-allophycocyanin (1:200, as directed; BD Biosciences) was added prior to analysis. Cells were analyzed on a FACSCalibur cytometer (BD Biosciences) with appropriate negative and compensation controls. Fluorescent intensities for cells were point-plotted on two-axis graphs or histograms using CellQuest software (BD Biosciences).

Prospective Cell Isolation
Cells were freshly isolated from cortex using Liberase-1 (Roche), labeled for CD133/CD24, and run on a fluorescence-activated cell sorter, either a FACSAria (BD Biosciences) or an Influx (Cytopeia, Seattle, http://www.cytopeia.com) sorter, as previously described [14]. Single viable cells were gated based on Annexin V exclusion and pulse width and then physically sorted into collection tubes for low density plating and clonal analysis. Postsort purity analysis was performed on aliquots from each sort group. FACSDiva or FlowJo software was used for analysis.

Cell Cycle Analysis
After culture at 5% or 20% oxygen, passaged cells were fixed by adding 4 volumes of ice-cold 100% ethanol. Cells were pelleted at 500g for 5 minutes, washed, spun again, and then resuspended in 1x Hanks' balanced salt solution to a final density of 1 x 106 cells per milliliter. Cells were incubated with 20 µg/ml RNase A (Sigma-Aldrich) for 30 minutes at 37°C to digest RNA and then with propidium iodide (80 µg/ml; Invitrogen). Flow cytometric analysis was performed on a FACSCalibur (BD Biosciences). DNA content in each event (cell) was measured to determine the proportion of cells in G0/G1 versus S/G2/M phases of the cell cycle.

Statistical Analysis
Data were quantified as total cell number or marker-expressing cells as a percentage of total (nuclear DAPI-stained) cells. Graphs and statistics were generated using Prism 3.0 software (GraphPad, Inc., San Diego, http://www.graphpad.com). Statistical significance was measured by simple t tests or one-way analysis of variance with post hoc test. Asterisks in figures denote p value: *, p < .05; **, p < .01; ***, p < .001. For all graphs, an asterisk directly above a column or data point indicates a significant difference from its 5% oxygen counterpart; an asterisk over a bracket indicates a significant difference from another variable, as indicated.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mouse CNS Precursor Expansion Increases at Lowered Oxygen Tension
Fetal CNS precursors proliferate extensively in the presence of mitogens such as bFGF [16]. With freshly isolated mouse cortical precursors, we observed a significant increase in cell numbers at 5% compared with 20% oxygen at all plating densities tested (2.6–180 cells per mm2; Fig. 1A). These differences were amplified at the lowered densities typically used for clonal analysis. In 5% oxygen, precursors survived and divided from near-clonal plating densities (2.6 cells per mm2), whereas in 20% oxygen, cell viability dropped to near-zero at densities below 20 cells per mm2. At the highest density tested, enhancement of colony size and number at 5% oxygen was first noticeable during the second day of expansion (not shown). Since oxygen tensions in cortex range from 2.53% to 5.33% (19–40 mmHg) and are even lower in other regions [3], we tested the possibility that 2% oxygen would promote even greater expansion. We found no significant difference in cell numbers (Fig. 1A), and the frequency of nestin expression was 98.9% ± 0.3% in 2% oxygen and 98.3% ± 0.8% in 5% oxygen. This indicates that precursor expansion is similar within the 2%–5% physiological range of oxygen tensions. The capacity for long-term expansion was also affected by oxygen. In 5% oxygen, precursors expanded over extended periods of time with no reduction in numbers, whereas 20% oxygen caused a decrease in precursor number (Fig. 1B, 1C).

We also tested the possibility that initial culture in 5% oxygen for two passages would increase the tolerance of cells to subsequent exposure to 20% oxygen. At the third passage, cells were split and grown in either 5% or 20% oxygen (supplemental online Fig. 1A). Exposure to 20% oxygen after this initial low-oxygen culture period still led to reduced precursor numbers (supplemental online Fig. 1B). We also tested a number of commonly used additives for CNS precursor culture at previously published concentrations (supplemental online Fig. 1C, 1D). We cultured cells in 5% or 20% with standard medium alone or with (a) erythropoietin (Epo), which is induced under low oxygen conditions [9] and binds to a receptor that is highly expressed in the neuroepithelium [17] and required for neural precursor survival and expansion in vivo [18]; (b) the antioxidants N-acetylcysteine [19] and ascorbic acid [20]; or (c) B27 supplements, which contain a number of antioxidants and survival factors; we used variants with and without retinol, which promotes neuronal differentiation [21]. We found that Epo had no added effect on cortical precursors at 10 ng/ml (supplemental online Fig. 2C) or 30 ng/ml (not shown), unlike its effect on ventral midbrain progenitor cells [9], nor did 1 mg/ml N-acetylcysteine. Ascorbic acid decreased cell numbers at both 5% and 20% oxygen compared with 5% oxygen control, possibly because of repression of hypoxia-inducible factor 1{alpha} [22]. We did, however, find a surprising strain-dependent difference in the response of precursors to B27. Although precursors from C57Bl/6J and C57Bl/6N mice expanded similarly in all other conditions, B27 without retinol significantly reduced C57Bl/6J cell numbers in 5% oxygen while not increasing cell numbers in 20% oxygen (compared with 5% control; supplemental online Fig. 1C). In contrast, B27 (with or without retinol) had no effect on C57Bl/6N precursors at 5% oxygen but increased cell numbers at 20% oxygen numbers to those of the 5% oxygen control (supplemental online Fig. 1D). Thus, many commonly used additives are suboptimal in duplicating the growth-promotion effects of 5% oxygen, but B27 may increase or reduce CNS precursor numbers depending on the mouse strain.


Figure 1
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  		Figure 1. Lowered oxygen promotes expansion of mouse cortical precursors at all densities and over extended times. (A): Expansion of acutely dissociated embryonic day 13.5 mouse cortical cells, plated at varying plating densities and expanded for 5 days in 2%, 5% or 20% oxygen; mean ± SEM; n = 3. (B): Highest density acute cultures were passaged and expanded sequentially in 3-day intervals. (C): Cell recovery after successive passaging of 5% or 20% oxygen cultures; mean ± SEM; n = 4–6 for all data points except n = 2 at day 17. No cells were recovered from 20% oxygen culture by day 14. All graphs shown in logarithmic scale with cubic spline curve. Scale bar = 100 µm.

 


Figure 2
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  		Figure 2. Acute response of Hif1{alpha}, NPM, and p53 to increased oxygen. Early passage cells were initially expanded in 5% oxygen and then exposed to 20% oxygen for 10 minutes to 24 hours prior to protein lysis. (A–C): Enzyme-linked immunosorbent assay (ELISA) using antibodies against Hif1{alpha} (A), NPM (B), and p53 (C). (D, E): ELISA using antibodies against phosphorylated forms of p53 at Ser6 (D), Ser15 (E), and Ser20 (F). Asterisks indicate a significant difference from 0 minutes (control). Abbreviations: Hif1{alpha}, hypoxia-inducible factor 1{alpha}; NPM, nucleophosmin; P-Ser, phosphorylated serine.

 
Acute Changes in HIF1{alpha}, Nucleophosmin, and p53 After Increasing Oxygen
A well-characterized rapid cellular response to oxygen is the induction of HIF1{alpha} during hypoxia and its post-translational degradation at high oxygen tensions [1]. HIF1{alpha} promotes the transcription of glycolytic enzyme and prosurvival genes and can repress CNS precursor differentiation [1, 13, 23]. Since HIF1{alpha} is highly unstable, we initially cultured early passage precursors in 5% oxygen using the low oxygen glovebox to prevent even transient exposure to higher oxygen levels. One day after the last medium change, we exposed replicate dishes to 20% oxygen for 10 minutes to 24 hours prior to harvest for ELISA. We found that HIF1{alpha} protein expression was significantly decreased within 10 minutes of acute exposure to 20% oxygen (Fig. 2A), consistent with its reported half-life of 5–8 minutes at room oxygen [24]. NPM is a nucleolar cell-cycle and apoptosis-control protein [25] that is induced by HIF1{alpha} and that inhibits p53-induced apoptosis [26]. NPM was more slowly downregulated to 50% of control levels within 4.5 hours of acute 20% oxygen exposure (Fig. 2B). In contrast, p53 was upregulated within 24 hours of acute exposure to 20% oxygen (Fig. 2C). Several serine residues on p53 can be phosphorylated in response to stressors, leading to p53 activation [27]. We found that of seven serine sites tested, three (Ser6, Ser15, and Ser20) showed significantly increased phosphorylation by 24 hours of acute 20% oxygen exposure (Fig. 2D–2F). Of these, p53 (Ser15) is reported to be phosphorylated during both anoxia and hyperoxia in tumor cell lines [28, 29], suggesting that it is activated by extremes in oxygen tension.

Apoptosis Is Increased in 20% Oxygen Compared with 5% Oxygen
Since p53 activation can lead to apoptosis or mitotic arrest, we analyzed these cells by (a) BrdU incorporation into newly synthesized DNA, marking proliferative S-phase entry; (b) nestin expression, identifying all neural precursor cells; (c) activated, proteolytically cleaved caspase 3 to identify apoptosis; and (d) pyknotic nuclei, which identify dead cells and can be easily distinguished by their condensed, intensely bright DAPI+ appearance. We observed equivalent percentages of BrdU+ cells in both 5% and 20% oxygen (Fig. 3A, 3B), even though overall colony size and number was much larger in 5% oxygen. Living cells were nearly all nestin+ in 5% oxygen, and this percentage was only modestly reduced in 20% oxygen (Fig. 3A, 3B), indicating that precursors had not differentiated. However, the number of cleaved caspase-3+ and/or pyknotic cells was greater in 20% than 5% oxygen (Fig. 3A, 3C). Since BrdU analysis does not give a complete picture of the distribution of cells in the cell cycle, we also performed flow cytometric analysis of cells using the DNA binding dye propidium iodide. We found no difference between oxygen tensions in the proportion of cells in each cell cycle phase (Fig. 3D, 3E). Although we did not measure senescence [30], the cell cycle analysis and the high frequency of cell death indicated that this was not a prominent response to 20% oxygen. These results indicate that apoptosis, rather than mitotic arrest, is the principal mechanism causing different expansion rates between 5% and 20% oxygen.


Figure 3
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  		Figure 3. Apoptosis is increased in 20% oxygen compared with 5% oxygen. (A): Acutely dissociated cells were initially expanded in 5% oxygen, passaged, and expanded for 3 days in either 5% or 20% oxygen. Staining shows BrdU incorporation (1 hour, red), act. Casp3 (green, upper panel), or the precursor marker nestin (green, lower panel). (B, C): BrdU and nestin staining measured as percentage of live 4',6-diamidino-2-phyenylindole-positive (DAPI+) cells (B) or act. Casp3+ and pyknotic cells measured as percentage of all DAPI+ nuclei (live plus pyknotic) (C); mean ± SEM. (D): Cell cycle analysis of passaged cells by flow cytometry after 3 days of expansion in 5% (purple) or 20% (green overlay) oxygen. (E): Cell cycle analysis, percentage of cells in each cell cycle phase; mean ± SEM. Scale bar = 200 µm (main images), 74 µm (insets). Abbreviations: act. Casp3, activated caspase-3; BrdU, 5-bromo-2'-deoxyuridine; Ox, oxygen; P.I., propidium iodide.

 
Low Oxygen Promotes Multilineage Competence
We next tested the role of oxygen in the differentiation of mouse cortical precursors. Freshly isolated and expanded cells were differentiated by mitogen withdrawal for 6 days. Expansion and differentiation in 5% oxygen yielded large colonies that generated neurons (ßIII-tubulin+, MAP2a+b), astrocytes (GFAP+), and oligodendrocytes (O4+; Fig. 4A, 4C). Although expansion did not differ between 2% and 5% oxygen (Fig. 1A), we found that the percentages of astrocytes and oligodendrocytes generated were greater in 2% than 5% oxygen (Fig. 4C). In contrast, culture in 20% oxygen resulted in small colonies containing almost exclusively neurons (Fig. 4B, 4C). Even in the rare larger colonies generated in 20% oxygen, few astrocytes were found. This is notable since high cell density promotes glial differentiation via contact-dependent signals [31, 32]. Since oligodendrocytes were generated in dense and sparse areas alike in 5% oxygen, it is unlikely that differences in clone density could account for the absence of oligodendrocytes in 20% oxygen.


Figure 4
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  		Figure 4. Lowered oxygen promotes multilineage competence. Acutely dissociated cells from embryonic day 13.5 mouse cortex were cultured at low density in 2%, 5%, or 20% oxygen for 5 days of expansion and 6 days of differentiation. Colonies of equivalent size are shown for direct comparison, although colonies in 20% oxygen were nearly all smaller. (A): Examples of colonies stained for neurons (MAP2a+b or ßIII-Tub), astrocytes (GFAP), and O4 after culture in 5% oxygen. (B): Examples of similar sized colonies cultured in 20% oxygen (nearly all colonies were smaller). (C): Quantitation after four-way staining for MAP2a+b, GFAP, O4, and 4',6-diamidino-2-phyenylindole; mean ± SEM; n = 3. Asterisks over brackets indicate a significant difference between connected columns. Abbreviations: ßIII-Tub, ßIII-tubulin; GFAP, glial fibrillary acidic protein; MAP2a+b, microtubule-associated protein 2 subunits a and b; O4, oligodendrocytes.

 
Since cells from the C57Bl/6N strain showed improved expansion in B27, we also tested the differentiation of these cells. Cells from both mouse strains appeared more differentiated in the presence of B27 even during expansion (supplemental online Fig. 1E), so we examined the expression of cell type markers both in expansion and after differentiation. None of the markers tested differed significantly between control and B27-treated among cells in expansion (supplemental online Fig. 1F). Analysis of differentiated cells showed that B27 (with or without retinol) significantly decreased the percentage of MAP2ab+ neurons and increased the number of O4+ oligodendrocytes compared with the control (supplemental online Fig. 1G). However, the percentage of oligodendrocytes in these conditions still was less than that seen after expansion in either 5% or 2% oxygen (Fig. 4C).

These results indicate that lowered oxygen promotes the generation of multiple differentiated neural lineages. To account for these results, we hypothesized two mechanisms: (a) 20% oxygen is detrimental to the expansion of multipotent stem cells, but not neuronal-restricted progenitors, which results in the generation of neurons only, or (b) 20% oxygen tension inhibits the expansion of all precursors equally, but also selectively inhibits the maturation of glia.

Two Multipotent Precursor Populations Show Differing Responses to 20% Oxygen
A substantial proportion of expanded fetal CNS precursors are multipotent stem cells [33], but culture can also support committed progenitors that give rise to either neurons or glia [19, 34]. The cell surface marker CD133 is expressed in multipotent mouse [35] and human [36] postnatal CNS precursors, whereas CD24 has low expression in multipotent stem cells [37] but higher expression in neuronal progenitors and neurons [38]. We previously showed that acutely isolated CD133hiCD24hi mouse cortical cells are predominantly a novel type of neuronal progenitor, whereas multipotent cells are enriched in the CD133hiCD24lo and CD133loCD24lo population [14]. This provided a way of testing the first hypothesis that stem cells are selectively more vulnerable to apoptosis than neuronal progenitors in 20% oxygen.

We dissociated cortical tissue (Fig. 5A, 5B), expanded cells initially in 5% oxygen, and then passaged and expanded for 3 days in either 5% or 20% oxygen. The cells were then lifted, labeled, and analyzed by flow cytometry (Fig. 5C–5E). We found that cultured cells all expressed CD133 but not at the highest levels seen in freshly isolated cells (Fig. 5B); this is due to the limited expansion of CD133hiCD24hi cells compared with CD24lo cells [14]. Cultures contained none of the CD133–CD24hi neurons found in freshly isolated cells (Fig. 5C, 5D, arrowheads), since these neurons do not survive passaging [15]. Instead, cultures expanded in 5% oxygen contained substantial numbers of CD133loCD24lo cells, which we demarcated as region 1 (R1). This analysis showed that the total number of recovered cells was reduced threefold in 20% compared with 5% oxygen (Fig. 5E); of these, cells within R1 were reduced eightfold. The remaining population (R2) was reduced twofold in absolute numbers but was increased 46% as a percentage of cells remaining in 20% culture. This reduction in total cell number was not due to terminal differentiation, since most surviving cells were nestin+ and the cell cycle profile was unchanged between oxygen tensions (Fig. 3). In addition, the maximal intensity of CD24 expression in the total population was unchanged. This suggests that CD133loCD24lo cells most frequently undergo cell death in 20% oxygen.


Figure 5
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  		Figure 5. Differential response of two types of multipotent cell to 5% versus 20% oxygen. (A): Flow cytometric profile of freshly isolated embryonic day 13.5 mouse cortical cells; polygon (main population, 92% of all events) demarcates intact cells, 95% of which were viable by 7-amino-actinomycin-D exclusion. (B): Coexpression of CD133 and CD24 in these cells; vertical line is for comparison with expanded cells in the next panels. (C, D): Freshly dissociated cells were expanded in 5% or 20% oxygen for 5 days and then passaged, antibody-labeled, and analyzed by flow cytometry. Arrowheads indicate the absence of CD133–CD24hi neurons in both conditions. Long arrow indicates the presence of an expanded population of cells expressing very low levels of CD24 in 5% oxygen, which was lost in 20% oxygen. Vertical line demarcates border between R1 and R2. (E): Cell number after expansion, in R1 versus R2; mean ± SEM; n = 3. (F): Fluorescence-activated cell sorter gates overlaid on CD133/CD24 expression profile for prospective enrichment of multipotent cells (CD133loCD24lo and CD133hiCD24lo), neuronal progenitors (CD133hiCD24hi), and neurons (CD133loCD24hi). (G, H): Clonal analysis of sorted cells cultured solely in 5% oxygen (G) or 20% oxygen (H) for 3 days of expansion and 6 days of differentiation. Characterization and counting of clone types 1–7 was performed exactly as described in Panchision et al. [14]. Asterisks over 20% oxygen column (H) indicate a significant difference from the corresponding clone type at 5% oxygen (G). Abbreviations: AO, astrocyte-oligodendrocyte clone; N, neuron-only clone; NAO, neuron-astrocyte-oligodendrocyte clone; NO, neuron-oligodendrocyte clone; R, region.

 
To further test this hypothesis, we sorted acutely dissociated E13.5 cortical cells using the same gating methods (Fig. 5F) and clonal analysis (Table 1; supplemental online Fig. 2; Fig. 5G, 5H) as previously described [14]. We found that the numbers of CD133loCD24hi neurons and clone-forming CD133hiCD24hi neuronal progenitors were not significantly changed by culture in 5% oxygen (Fig. 5G) versus 20% oxygen (Fig. 5H). In contrast, multipotent CD133loCD24lo cells predominantly generated type 4 (simple three-fate) clones in 5% oxygen [14] but died during expansion in 20% oxygen. CD133hiCD24lo cells were also multipotent and generated type 4, 5, and 7 clones in 5% oxygen [14]. Fewer of these clones were generated in 20% oxygen, and the surviving clones, surprisingly, included only neurons with short bipolar morphologies, similar to type 3 clones from CD133hiCD24hi cells [14]. These results indicate that two multipotent precursor populations show differing fates in 20% oxygen. Glia-only clones were not generated in any condition, consistent with limited gliogenesis at this early stage of development [39], but oligodendrocyte-rich three-fate clones were completely eliminated in 20% oxygen. Based on these results, we conclude that oligodendrocytes in E13.5 cortical cultures are derived primarily from multipotent cells but are missing due to stem cell apoptosis or to loss during oligodendrocyte differentiation in 20% oxygen.


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  		Table 1. Clone types after embryonic day 13.5 (E13.5) mouse cortical cell sorting

 
Distinct Stages of Oxygen Sensitivity During Expansion Versus Differentiation
Since glial-restricted progenitors were not present in acutely isolated cortical cells, we tested our second hypothesis using expanded cultures. We plated first passage mouse cells at clonal density, expanded them for 4 days, and differentiated for 6 days. In some replicate wells, we switched oxygen exposure after 2 days post-mitogen withdrawal, at a time when precursor cells are undergoing differentiation (Fig. 6A, 6B). As with freshly isolated cultures, passaged cells grown entirely in 5% oxygen (Fig. 6, 5->5%) during both phases generated clones with multiple cell types. In contrast, clones grown entirely in 20% oxygen (Fig. 6, 20->20%) generated only neurons. Switching from 5% to 20% (Fig. 6, 5->20%) oxygen after 2 days postwithdrawal caused a 30% decrease in total clone number and a two-fold decrease in oligodendrocyte-containing clones. Switching from 20% to 5% (Fig. 6, 20->5%) oxygen after 2 days postwithdrawal still did not yield clones containing GFAP+ or O4+ cells. These results indicate that later steps of oligodendrocyte maturation or survival are also inhibited by exposure to 20% oxygen.


Figure 6
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  		Figure 6. Effects of changing oxygen during expansion versus differentiation of the O4 lineage. (A, B): Passaged cells were plated at clonal density, expanded for 3 days at 5% or 20% oxygen, and then differentiated by mitogen withdrawal for another 6 days. Some clones were exposed to the same oxygen tension throughout (5->5%, 20->20%), whereas others were switched to a different oxygen tension at 2 days postwithdrawal (5->20%, 20->5%). Clones were then stained for lineage markers and quantified. Mean ± SEM, n = 3 for all data points. Asterisks indicate significant difference from corresponding 5->5% oxygen culture. (C–E): Time course of O4 lineage marker expression in mass culture after mitogen withdrawal (i.e., days of differentiation), including A2B5 (C), PDGFR{alpha} (D), and O4 (E). All cells were expanded in 5% oxygen prior to withdrawal. Asterisks over 20% oxygen column indicate a significant difference from corresponding time points at 5% oxygen. Abbreviations: GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; O4, oligodendrocyte; PDGFR{alpha}, platelet-derived growth factor receptor {alpha}.

 
To determine the stage of oligodendrocyte differentiation that was affected by 20% oxygen, we examined the expression of several oligodendrocyte lineage markers. These included A2B5, a marker for bipotent astrocyte-oligodendrocyte progenitors [40]; PDGFR{alpha}, a marker for immature oligodendrocyte progenitors [41, 42]; and O4, a marker of late oligodendrocyte progenitors [43–45]. Since expression of each of these markers was low in actively expanding cultures, we also measured expression at 1–4 days after mitogen withdrawal (Fig. 6C–6E). In cell cultured only in 5% oxygen, A2B5 expression increased quickly by 1 day after withdrawal and was lost by 3 days, consistent with a transient generation and differentiation of bipotent glial progenitors. In cultures switched to 20% oxygen upon bFGF withdrawal, these cells were still generated by day 1 but were no longer present by day 2 (Fig. 6C). Maximum numbers of PDGFR{alpha}+ cells appeared at 3 days postwithdrawal in 5% oxygen; surprisingly, these cells appeared and disappeared more rapidly in 20% oxygen (Fig. 6D). Finally, O4+ cells did not begin to increase until 2 days postwithdrawal and continued increasing through day 4 in 5% oxygen. This increase was completely abolished in 20% oxygen (Fig. 6E). These results indicate that acute exposure to 20% oxygen accelerates the progressive differentiation of the oligodendrocyte lineage but leads to loss of these cells by the O4+ stage.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Oxygen tension is an important regulator of cell metabolism, survival, and fate. Although extreme fluctuations in oxygen availability are a major cause of cell death [4, 46], physiological (2%–5%) oxygen tensions promote long-term CNS neurosphere expansion [12], neural crest stem cell survival [11], and the generation of specific CNS and peripheral neuron types [9–11] and inhibit neuronal differentiation [13]. These results suggest that oxygen effects differ depending on the commitment or differentiation stage of precursors. In this study, we examined the role of oxygen tension in the fate of several precursor subtypes in E13.5 mouse cortical tissue during expansion and differentiation. Although 2% and 5% oxygen similarly promoted the expansion of precursors, 20% oxygen caused a rapid decrease in HIF1{alpha} and NPM, two proteins implicated in low oxygen response and cell survival. Within 24 hours, p53 was induced, and cell death occurred within 40 hours to 5 days, predominantly among CD133loCD24lo multipotent cells. Another multipotent population marked by CD133hiCD24lo was less strongly reduced, but surviving cells no longer generated three-lineage clones but instead only neurons. Neuron-committed progenitors survived equivalently in either 5% or 20% oxygen. Bipotent glial and oligodendrocyte progenitors were not generated in large numbers until bFGF is withdrawn; increasing oxygen tension from 5% to 20% accelerated the differentiation of these early progenitors and then caused the loss of late oligodendrocyte progenitors.

These results not only indicate differing cell responsiveness to high oxygen levels but also suggest an active role of changing oxygen tensions in driving lineage progression. This is seen in hematopoietic development, where hypoxia accelerates the generation of mesoderm and hemangioblasts from embryonic stem cells [47]. Oxygen response via HIF1{alpha}, ARNT, and vascular endothelial growth factor is required for proper hematopoietic development [47, 48]. The HIF1{alpha}-related molecule HIF2{alpha} is required to maintain germ cell numbers and does so by inducing Oct4, a positive regulator of pluripotency [49].

Analogous intracellular mechanisms may work in the CNS. Midbrain precursors from nestin-Cre HIF1{alpha} conditionally null mice show defects in survival, proliferation, and dopaminergic differentiation [50]. However, cortical neurosphere expansion was not limited because of this defect or because of culture in 20% oxygen [12, 50], unlike the results we report in monolayer culture. Cortical defects from nestin-Cre HIF1{alpha} conditionally null mice are not obvious until E19 [51], when most precursors have already differentiated. In contrast, lowered oxygen represses neuronal differentiation of rat cortical precursors by the combined activity of HIF1{alpha} and Notch; dominant-negative block of HIF1{alpha} function prevents Notch activation [13]. In addition, we find that HIF1{alpha} is rapidly reduced in response to acute increases from 5% to 20% oxygen (Fig. 2A). One interpretation of these conflicting results is that HIF1{alpha} is nonredundant in midbrain but acts in concert with the related protein HIF2{alpha} or HIF3{alpha} in the cortex. The weak and pial-localized expression of HIF2{alpha} in the brain [52] makes it an unlikely candidate to duplicate HIF1{alpha} functions in neural precursors. HIF3{alpha} [53] may play such a role, but it has not yet been characterized in the CNS. We also find that decreased NPM and activation of p53 are part of the precursor response leading to apoptosis in 20% oxygen. These changes are consistent with studies in tumor cell lines, showing that NPM is induced by HIF1{alpha} at low oxygen levels [26] and that NPM acts to repress p53-mediated apoptosis [26]. Our future studies will determine whether similar mechanisms regulate oxygen responses in neural precursors.

The present results extend our prior findings showing heterogeneous behavior within the CD24lo population. Previously [14], we found that CD133loCD24lo cells generated three-fate clones with simpler (bipolar) morphologies and weaker neuronal/glial marker staining than CD133hiCD24lo cells. We surmised that the former may be a more primitive multipotent cell population. Consistent with this idea, the majority of neurospheres from postnatal SVZ comes from CD24lo/– transit-amplifying type C cells rather than type B stem cells; expansion in epidermal growth factor promotes multipotency in these type C cells [38]. Likewise, fetal cortical CD133hiCD24lo cells may retain potency that they do not exhibit in vivo, whereas the CD133loCD24lo population may be stem cells both in vitro and in vivo. The generation of neuron-only clones in 20% oxygen may result from the premature differentiation or death of glial lineages (Fig. 5C–5E) or the reversion of these CD133hiCD24lo precursors to a potency closer to that occurring in vivo.

Our results here, our parallel study of human CNS precursors [54], and a recent study showing oligodendrocyte death in rats exposed to hyperbaric (80%) oxygen [55] show the high degree to which the oligodendrocyte lineage is sensitive to changes in oxygen levels. Addition of factors that promote the oligodendrocyte lineage, such as platelet-derived growth factor or tri-iodothyronine (T3) [56, 57], may promote survival in part by mimicking a hypoxic response via HIF1{alpha} stabilization [58]. Generation of rat [59] and human [56, 57, 60] oligodendrocytes in 20% oxygen may be further facilitated by the dense neurospheres in which these precursors are often grown. The high density of the spheres likely reduces local oxygen tension [61] or provides a paracrine activity that limits apoptosis or senescence of these cells [30, 62, 63]. The rapid cell death we report in monolayer cultures, compared with mouse midbrain neurospheres [12], may result from a lower variability in cell exposure to 20% oxygen.

Although hypoxia slows rat precursor differentiation after mitogen withdrawal [13], rat precursors can still expand (supplemental online Fig. 3) and generate multifate clones [31, 33] using the identical culture conditions that cause mouse precursor apoptosis in our study. We recently showed that human oligodendrocyte generation is limited in 20% oxygen by premature differentiation rather than death of stem cells and oligodendrocyte progenitors, but this occurred even in the presence of mitogens [54]. Among mouse precursors, we found evidence of accelerated differentiation of mouse oligodendrocyte precursors prior to the loss of this lineage in 20% oxygen (Fig. 6D). These results suggest that intracellular mechanisms of oxygen-dependent differentiation are conserved among rat, mouse, and human but yield different outcomes based on mitogen responsiveness and susceptibility to apoptosis.

Our results reinforce the idea that lowered oxygen tensions are a more physiologically relevant approach for in vitro analysis and manipulation of CNS precursors. The enhanced survival and multipotency of mouse precursors at lower oxygen tensions will facilitate clonal analysis using genetic mouse mutants. Our parallel studies of CNS precursors from human [54] and mouse both show that changes in oxygen tension regulate CNS stem cell expansion and the generation of oligodendrocytes. Thus, dynamic variation of oxygen tension may be a critical component in developing effective cellular therapies for demyelinating disorders. Finally, recent studies support an endogenous oxygen-sensitive regenerative capacity in the CNS [64–69], suggesting that clinical modulation of oxygenation after stroke may be important for stem cell recruitment and neurogenesis in these patients [70, 71].


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
This work was supported by funds from the NIH Mental Retardation and Developmental Disabilities Research Center (Grant P30HD40677), the Children's National Medical Center (CNMC) Research Advisory Council, and the CNMC Board of Visitors and by the intramural program of National Institute of Neurological Disorders and Stroke, NIH. H.-L.C. is supported by a Haseltine postdoctoral fellowship. F.P. is supported by the Frank and Nancy Parsons Fund, the Georgia Derrico and Rod Porter Fund for the CNMC/University of Padova Sister Program, an Associazione Italiana Ricerca sul Cancro (Italy) regional grant, and a Fondo per gli Investimenti della Ricerca di Base (Italy) grant. We thank Ann Kennedy for technical assistance; Teresa Hawley, Daniela Papini, and Bhargavi Rajan for flow cytometry support; and Drs. Vittorio Gallo, Michael Bell, and Ramesh Chittajallu for constructive advice on the manuscript. H.-L.C. is currently affiliated with Institute of Behavioral Medicine, College of Medicine and Hospital, National Cheng-Kung University, Tainan, Taiwan, R.O.C.


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 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS