Isolation and characterization of mouse neural precursor cells in primary culture.
Primary cultures of mouse neural precursor cells were established by enzymatic dissociation of embryonic Day 10 fetal heads followed by negative selection of non-neural contaminating cells. The latter were allowed to attach and spread on a plastic substrate under conditions that permitted neural precursor cells to remain suspended in the culture medium. The resulting neuroepithelial cell enriched suspension then was plated on dishes coated with poly-D-lysine. Growth of fibroblastic cells was inhibited in a selective medium. Cell proliferation was measured by immunoperoxidase staining of nuclei after bromodeoxyuridine labeling. The proportion of labeled cells declined from 50% on Day 1 until Day 5 when it approached zero, and after 7 days in culture a fourfold increase in cell number was achieved in medium containing 1% fetal bovine serum, transferrin, insulin, cholera toxin, and sodium selenite. Differentiation of neural precursor cells was studied by indirect immunofluorescence microscopy for the appearance of neuron- and astrocyte-specific cytoskeletal proteins at successive intervals in culture. Cells bearing neuritic processes and expressing neurofilaments as well as microtubule-associated protein 2 were present in low numbers on Day 1, increasing through Day 14. Stellate cells with morphologic features of astrocytes and immunoreactive for glial fibrillary acidic protein were not detected until Day 5 and did not become abundant until Day 11. No differences in morphology or immunocytochemical staining characteristics were found between neural precursor cells processed by enzymatic dissociation of whole fetal heads and those recovered by manual dissection of fetal neuroepithelia. The large number of neural precursor cells obtained by this rapid, simple method makes possible the production of mass cultures for molecular analysis of the regulatory factors that control proliferation and differentiation during early development of the mouse central nervous system.
Laboratory of Cell Biology, Institute of Physical and Chemical Research RIKEN, Tsukuba Life Science Center, Ibaraki, Japan.
H
H
Kitani
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R
Shiurba
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T
Sakakura
Y
Y
Tomooka
D000818:Animals; D001921:Brain / Q000166:cytology / Q000196:embryology*; D002455:Cell Division; D002469:Cell Separation; D002478:Cells, Cultured; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods; D005333:Fetus; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D007700:Kinetics; D051379:Mice; D008813:Mice, Inbred ICR; D008869:Microtubule-Associated Proteins / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis*; D009474:Neurons / Q000166:cytology*
D003470:Culture Media; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D016650:Fluorescein-5-isothiocyanate