Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells.
Embryonic stem (ES) cells proliferate and maintain their pluripotency for over 1 year in vitro and may therefore provide a sufficient source for cell therapies. However, most of the previously reported methods for obtaining a source for cell therapies have not been simple. We describe here a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells cocultured with the PA6 stromal cell line for at least 3 days were capable of differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP-expressing D3-ES cells. The spheres contained nestin-positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self-renewal ability. The cells differentiated into microtubule-associated protein-2 (MAP2)-positive neuronal cells and glial fibrillary acidic protein (GFAP)-positive glial cells. gamma-Aminobutyric acid-positive cells and tyrosine hydroxylase-positive cells were also observed in the spheres. The percentages of the MAP2- or GFAP-positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue.
Kitajima
Hideomi
H
Department of Neurosurgery, Division of Neuroscience, Graduate School of Medicine, Gifu University, Gifu, Japan.
Yoshimura
Shinichi
S
Kokuzawa
Jouji
J
Kato
Masayasu
M
Iwama
Toru
T
Motohashi
Tsutomu
T
Kunisada
Takahiro
T
Sakai
Noboru
N
D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D001973:Bromodeoxyuridine / Q000378:metabolism; D002452:Cell Count / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D064231:Nestin; D009457:Neuroglia / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D050814:Octamer Transcription Factor-3; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D055748:SOXB1 Transcription Factors; D013234:Stem Cells / Q000502:physiology*; D017154:Stromal Cells / Q000502:physiology*; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D004268:DNA-Binding Proteins; D005904:Glial Fibrillary Acidic Protein; D006609:High Mobility Group Proteins; D007381:Intermediate Filament Proteins; D008869:Microtubule-Associated Proteins; C079240:Mtap2 protein, mouse; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D012333:RNA, Messenger; D055748:SOXB1 Transcription Factors; C098693:Sox1 protein, mouse; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; D049452:Green Fluorescent Proteins; D005680:gamma-Aminobutyric Acid; D004815:Epidermal Growth Factor; D014446:Tyrosine 3-Monooxygenase; D001973:Bromodeoxyuridine