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Neurospheres form at clonal density and express stem cell markers.

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posted on 20.02.2013 by Andrea J. Mothe, Tasneem Zahir, Carlo Santaguida, Douglas Cook, Charles H. Tator

NPSC cultured as an adherent monolayer for 39div were seeded at clonal density (<10 cells/µl) in EGF/FGF2 medium in suspension culture. A, Image taken on the day of seeding (d0) show single cells (arrows). B, 7d later neurospheres have formed (arrows). C, NSPC seeded at higher density (1000 cells/µl) at the time of plating form more neurospheres which are larger (arrows) (D, at 7d). Inset in D shows a high magnification image of a single neurosphere with a phase bright profile and ciliary projections (arrows). Neurospheres were plated onto matrigel and immunostained with stem cell markers. E-F, A single neurosphere showing proliferating Ki67+ NSPC (arrows) (E, green) stained with the nuclear dye Hoechst (F, merged). G-H, Three adjacent neurospheres expressing high levels of nestin (G, green) with nestin+ processes radially emanating from the neurospheres (arrows) (H, merged). I-J, Most NSPC comprising the neurosphere express Sox2 (I, green; J, merged with Hoechst nuclear counterstain).

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