Fig 1. Experimental design and preparation of neural stem cells by the neural stem sphere (NSS) method.

(A) Preparation of mouse neural stem cells (NSCs) by NSS method and the times of exposure to heat shock (HS). Large numbers of NSCs were produced by the NSS method directly from mouse ES cells. These cells were cultured on dishes as monolayers and expanded in medium supplemented with FGF2. NSCs in the proliferation phase were exposed to HS. (B) Experimental schedule. NSCs were exposed to HS by immersing the culture dishes in thermostat baths at 37°C, 42°C, 43°C or 44°C after culture at 37°C under an atmosphere of 5% CO₂ for 24 h (day 0) and 96 h (day 3). 3 h after HS, gene expression and caspase activities were assessed; after 24 h, the numbers of adherent cells, cell viability were measured and immunofluorescence staining was performed.