Characterization of neurosphere cell phenotypes by flow cytometry.
BACKGROUND
Neural stem cell research regularly utilizes neurosphere cultures as a continuous source of primitive neural cells. Results from current progenitor cell assays show that these cultures contain a low number of neural 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 based on their relative fluorescence intensity in the blue and red regions of the spectrum. Individual sorted subpopulations were reanalyzed after 7 days in culture.


RESULTS
Neurosphere cultures contain a relatively high number of cells that stain weakly with Hoechst 33342. This subpopulation is present when cultured as an entire batch in the presence of epidermal growth factor (EGF). When cultured separately, this subpopulation gives rise to a neurosphere population with essentially the same characteristics as freshly isolated embryonic mouse brain cells but contains substantially fewer weakly Hoechst-stained cells.


CONCLUSIONS
Similar to hemopoietic systems, neurosphere cultures contain a subpopulation that can be characterized by a low emission of Hoechst fluorescence. When cultured separately, this subpopulation gives rise to a phenotype similar to freshly isolated, uncultured neural cells.
Hulspas
R
R
Cancer Center and Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. Ruud.Hulspas@umassmed.edu
Quesenberry
P J
PJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology; D001562:Benzimidazoles; D015415:Biomarkers / Q000032:analysis; D001854:Bone Marrow Cells / Q000276:immunology; D001921:Brain / Q000166:cytology* / Q000196:embryology; D002469:Cell Separation; D002478:Cells, Cultured; D002999:Clone Cells; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006410:Hematopoiesis; D051379:Mice; D008807:Mice, Inbred BALB C; D009474:Neurons / Q000145:classification / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D010641:Phenotype; D013194:Staining and Labeling; D013234:Stem Cells / Q000145:classification / Q000166:cytology* / Q000187:drug effects / Q000276:immunology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D001562:Benzimidazoles; D015415:Biomarkers; D005456:Fluorescent Dyes; D004815:Epidermal Growth Factor; C017807:bisbenzimide ethoxide trihydrochloride