Characterization of neurosphere cell phenotypes by flow cytometry

Background: Neural stem cell research regularly utilizes neurosphere cultur es as a continuous source of primitive neural cells. Results from current p rogenitor cell assays show that these cultures contain a low number of neur al progenitors. Our goal is to characterize neurosphere cultures and define subpopulations in order to purify neural progenitor cells. Methods: Cells from embryonic mouse neurosphere cultures were stained with Hoechst 33342 and analyzed by flow cytometry. Subpopulations were sorted ba sed on their relative fluorescence intensity in the blue and red regions of the spectrum. Individual sorted subpopulations were reanalyzed after 7 day s in culture. Results: Neurosphere cultures contain a relatively high number of cells tha t stain weakly with Hoechst 33342. This subpopulation is present when cultu red as an entire batch in the presence of epidermal growth factor (EGF). Wh en cultured separately, this subpopulation gives rise to a neurosphere popu lation with essentially the same characteristics as freshly isolated embryo nic mouse brain cells but contains substantially fewer weakly Hoechst-stain ed cells. Conclusions: Similar to hemopoietic systems, neurosphere cultures contain a subpopulation that can be characterized by a low emission of Hoechst fluor escence. When cultured separately, this subpopulation gives rise to a pheno type similar to freshly isolated, uncultured neural cells. (C) 2000 Wiley L iss, Inc.