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

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 similar to 5% O-2, which more closely approximates p hysiological oxygen levels. Eighty-three percent of p75(+) P-o(-) cells iso lated from embryonic day 14.5 sciatic nerve behaved as stem cells under the se conditions, suggesting that this is a nearly pure population. Furthermor e, addition of BMP2 plus forskolin in decreased oxygen cultures elicited di fferentiation of thousands of cells expressing tyrosine hydroxylase, dopami ne-beta-hydroxylase, and the SA lineage marker SA-1 in nearly all colonies. Such cells also synthesized and released dopamine and norepinephrine. Thes e data demonstrate that isolated mammalian NCSCs uniformly possess SA linea ge capacity and further suggest that oxygen levels can influence cell fate. Parallel results indicating that reduced oxygen levels can also promote th e survival, proliferation, and catecholaminergic differentiation of CNS ste m cells (Studer et al., 2000) suggests that neural stem cells may exhibit a conserved response to reduced oxygen levels.