Fig. 1

Neurosphere formation is enhanced under hypoxic condition compared to normoxic condition. A Experimental procedure of (B)-(C). B Microphotographs of neurospheres formed from E14.5 cortical cells under the normoxic or the hypoxic condition. Scale bar = 200 μm. C Quantification of neurosphere formation in E14.5 cortical cells under the normoxic or the hypoxic condition. The data is shown relative value of neurosphere numbers of hypoxic condition based on control value 100. The neurosphere number under the hypoxic condition was increased five times compared to under the normoxic condition (mean ± SEM, n = 5, *** p = 0.0000642). D Experimental procedure of (E). Cortical cells isolated from E14.5 brain were pre-incubated for 2 days under the normoxic or the hypoxic condition before the neurosphere formation period of the normal conventional condition. E Quantification of neurospheres after pretreatment of hypoxia in the first 2 div (mean ± SEM, n = 3, * p = 0.044). F Immunofluorescence staining of E14.5 mouse cortical cells (upper panels) and enriched NSPCs (lower panels). The cortical cells were cultured for 2 days in the presence of FGF2 under the normoxic or the hypoxic condition after the cells were isolated from E14.5 cortex (see the CM preparation in Fig. 2A). The enriched NSPCs were obtained by 2-day culture in the presence of FGF2 under the normoxic and the hypoxic conditions after 4-day culture of cortical cells under the conventional normal condition and replating (see the CM preparation in Fig. 2C). Not only nestin-positive NSPCs but also Tuj1-positive neurons were observed in the cortical cell culture, while most of the cells were NSPCs after the enrichment. Scale bar = 100 μm. G Experimental procedure of (H). H Neurospheres were formed from enriched E11.5 brain derived and E14.5 brain derived NSPCs. Scale bar = 200 μm. I, J Quantification of E11.5 NSPCs derived (I, mean ± SEM, n = 3, * p = 0.0120) and E14.5 NSPCs derived neurospheres (J, mean ± SEM, n = 4, * p = 0.0432). K Experimental procedure of (L). E14.5 enriched NSPCs were cultured for 7 days to form primary neurospheres under the normoxic or the hypoxic condition. Then, the primary neurospheres were scattered and re-plated to form secondary neurospheres under the normoxic condition. L Quantification of the secondary neurospheres (mean ± SEM, n = 3)