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. 2000 Oct 1;20(19):7370-6.
doi: 10.1523/JNEUROSCI.20-19-07370.2000.

Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells

S J Morrison  1 , M Csete, A K Groves, W Melega, B Wold, D J Anderson
Affiliations

Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells

S J Morrison et al. J Neurosci. .

Abstract

Isolated neural crest stem cells (NCSCs) differentiate to autonomic neurons in response to bone morphogenetic protein 2 (BMP2) in clonal cultures, but these neurons do not express sympathoadrenal (SA) lineage markers. Whether this reflects a developmental restriction in NCSCs or simply inappropriate culture conditions was not clear. We tested the growth and differentiation potential of NCSCs at approximately 5% O(2), which more closely approximates physiological oxygen levels. Eighty-three percent of p75(+)P(0-) cells isolated from embryonic day 14.5 sciatic nerve behaved as stem cells under these conditions, suggesting that this is a nearly pure population. Furthermore, addition of BMP2 plus forskolin in decreased oxygen cultures elicited differentiation of thousands of cells expressing tyrosine hydroxylase, dopamine-beta-hydroxylase, and the SA lineage marker SA-1 in nearly all colonies. Such cells also synthesized and released dopamine and norepinephrine. These data demonstrate that isolated mammalian NCSCs uniformly possess SA lineage capacity and further suggest that oxygen levels can influence cell fate. Parallel results indicating that reduced oxygen levels can also promote the survival, proliferation, and catecholaminergic differentiation of CNS stem cells (Studer et al., 2000) suggests that neural stem cells may exhibit a conserved response to reduced oxygen levels.

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Figures

Fig. 1.
Fig. 1.
Decreased oxygen promotes sympathoadrenal lineage differentiation. Sciatic nerve p75+P0− cells were cultured in decreased oxygen or in 20% oxygen, for a total of 12 d, either under standard conditions or with 5 μmforskolin and 1 ng/ml BMP2 added after 6 d of standard culture. Cultures were fixed and stained with antibodies against TH, DBH, and the sympathoadrenal lineage marker SA-1. The barsshow the percentage of stem cell colonies that contained any cells expressing the given markers. Error bars show SEs. Sympathetic markers were expressed at a significantly higher frequency in decreased oxygen and in the presence of forskolin and BMP2 (p < 0.05). In addition, in the presence of BMP2 plus forskolin, there were considerably more TH+ cells per colony than in the absence of these factors (see Fig.2A,C).
Fig. 2.
Fig. 2.
Induction of TH expression by culture in BMP2 plus forskolin in decreased oxygen. A, C, andE are epifluorescence illumination of anti-TH antibody staining of the phase contrast fields shown in B,D, and F, respectively. A,B, A colony grown in decreased oxygen plus 1 ng/ml BMP2 plus 5 μm forskolin. C, D, A colony grown in decreased oxygen without added factors.E, F, A colony grown in 20% oxygen plus BMP2 plus forskolin. No TH expression was seen in cultures grown in 20% oxygen without BMP2 or forskolin in this experiment (data not shown). In general, only rare TH+ cells were observed in 20% oxygen cultures. A–F, 10× objective magnification.
Fig. 3.
Fig. 3.
Coexpression of SA lineage markers. Allpanels are from cultures grown in decreased oxygen plus 1 ng/ml BMP2 plus 5 μm forskolin. Cultures were double-labeled with anti-TH (A, B) and anti-DBH (C) or anti-SA1 (D). E and F show the merged images in A, C andB, D, respectively. Thus,A, C, and E represent the same field of one colony, and B, D, andF represent the same field of a second colony.C and D were contrast-adjusted to subtract out background attributable to “bleed through” of R-phycoerythrin secondary antibody (A, B) into the FITC channel (C, D); this underestimates the number of DBH+ and SA-1+ cells. Arrowheads inE and F indicate double-labeled cells.A–F, 40× objective magnification.
Fig. 4.
Fig. 4.
Neuronal differentiation of SA lineage cells. NFM (green) and TH (red) staining of stem cell colonies cultured from p75+P0− cells in decreased oxygen in the presence of BMP2 plus forskolin, followed by additional growth in NT-3 plus NGF. A–C show the same field of a single colony photographed with a 40× objective.D–F show one field of a different colony photographed with a 20× objective. A and D show superimpositions of the NFM and TH staining; B andE show tyrosine hydroxylase staining; Cand F show NFM staining. Arrow points(A-C) indicate SIF-like cells with polygonal morphology, short or absent neurites, high levels of TH staining, and low levels of NFM staining. Arrowheads indicate cells that appear transitional between SIF-like cells and sympathetic neurons, with polygonal morphology, long neurites, high levels of TH staining, and high NFM staining. Arrows indicate cells that resemble sympathetic neurons, with rounded cell bodies, long neurites, moderate TH staining, and high NFM staining.
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References

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