Movie S4

The effect of calcium-free environment on neural rosette maintenance.
Neural rosettes were derived from human embryonic stem cells stably transfected with the pEF1a-AcGFP1-N1 vector. Time lapse imaging of a neural rosette responding to calcium-free HBSS buffer was filmed before HBSS treatment for 10 min in N2B27 medium, then changed to HBSS buffer for 3 hr, and in N2B27 medium after replacing HBSS buffer overnight. The movie was extracted from a full-length film, which was recorded at 1 picture/5 min using the confocal microscope system (LSM 700, Carl Zeiss GmbH, Germany). The movie depicts the structure of the neural rosette is undergoing dismantling in HBSS buffer, but restructuring and forming smaller neural rosettes in N2B27 medium overnight after lifting HBSS buffer, suggesting the effect of the absence of calcium in neural rosette maintenance is temporary if rescued in time (i.e. reinstalling the environment in N2B27 medium).