De novo and salvage pathways of DNA synthesis in primary cultured neurall stem cells.
We studied the de novo and salvage pathways of DNA synthesis in sphere-forming neural stem cells obtained from mouse embryos by a neurosphere method. The former pathway needs folic acid (FA) for nucleotide biosynthesis, while the latter requires deoxyribonucleosides (dNS). We examined the proliferative activity of sphere-forming cells in E14.5 embryos by counting the number of spheres formed in media that lacked FA and/or dNS. Proliferation failure and apoptosis occurred in a deficient medium lacking of both FA and dNS. Spheres formed in the deficient medium supplemented with dNS, without FA, did not produce neuron, but rather only seem to generate astrocytes and oligodendrocytes when plated under differentiation condition in culture. On the other hand, a subpopulation of cultured cells formed spheres in the deficient medium supplemented with FA alone in an appropriate concentration, and did possess the self-renewing and multipotential characteristics of neural stem cells. Spheres formed in the media containing low dose Azathioprine and methotrexate, inhibitors of de novo DNA synthesis, were selectively prevented from producing neurons even in the presence of FA. These results suggested that activating de novo DNA synthesis was needed for neural stem cells to proliferate with multipotentiality.
Sato
Kenichi
K
Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai 980-8574, Japan.
Kanno
Junko
J
Tominaga
Teiji
T
Matsubara
Yoichi
Y
Kure
Shigeo
S
D000367:Age Factors; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001379:Azathioprine / Q000494:pharmacology; D001921:Brain / Q000166:cytology; D002452:Cell Count / Q000379:methods; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay / Q000379:methods; D004247:DNA / Q000096:biosynthesis*; D004261:DNA Replication / Q000187:drug effects; D003853:Deoxyribonucleosides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique / Q000379:methods; D005492:Folic Acid / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D020287:In Situ Nick-End Labeling / Q000379:methods; D007381:Intermediate Filament Proteins / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D019081:O Antigens / Q000378:metabolism; D013234:Stem Cells / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism; D014803:Vitamin B Complex / Q000494:pharmacology
D000963:Antimetabolites; D003853:Deoxyribonucleosides; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D008869:Microtubule-Associated Proteins; C079240:Mtap2 protein, mouse; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D019081:O Antigens; D014404:Tubulin; C462941:beta3 tubulin, mouse; D014803:Vitamin B Complex; D004247:DNA; D005492:Folic Acid; D001379:Azathioprine; D008727:Methotrexate