Effects of oxygen on during cell propagation.

<p>Human midbrain and forebrain neural stem cells (NSCs) were propagated (EGF and bFGF) at high (20%) or low oxygen tension (3%). At day 4, cells were either trypsinized and the total cell number counted by use of a NucleoCounter (A) or cells were fixed, immunostained for Human Nuclei (HN) and the number of HN-immunoreactive (-ir) cells counted (B). For both midbrain and forebrain NSCs, the total cell numbers were significantly increased when cultures were propagated at low as compared to high oxygen tension. Data are expressed as mean±SEM with midbrain cultures at high oxygen set to 100% (*P<0.05, **P<0.01, ***P<0.001). At day 4, double immunofluorescence staining of forebrain NSCs (high and low oxygen) revealed some (<5%) glial fibrillary acidic protein (GFAP)-ir cells (green) of which a subpopulation co-expressed the neural progenitor cell marker nestin (arrows, upper panel, red) and a subpopulation co-expressed the proliferation marker Ki67 (arrows, lower panel, red). Some GFAP-ir cells did not co-express neither nestin nor Ki67 (arrowheads). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (C). Very few GFAP-ir cells were found in midbrain NSCs cultured at both high and low oxygen tension (not shown). Scale bar = 50 µm.