title: Establishment by an original single-cell cloning method and characterization of an immortal mouse melanoblast cell line (NCCmelb4).
abstract: We devised a unique new single-cell cloning method which uses microscope cover glasses and established a melanoblast cell line derived from mouse neural crest cells. A microscope cover glass was nicked and broken into small pieces and put on a dish. Culture medium and a suspension of 20-30 cells/ml were dropped in the dish. After 1-3 d, a piece of glass to which only one cell was adhered was picked up and transferred to another dish containing culture medium. The greatest advantage of this method is that the derivation of a colony from a single cell can be directly confirmed by microscopy and there is no risk of migratory cells being contaminated by other colonies. Using this single-cell cloning method, in this study we established a cell line derived from a neural crest cell line (NCC-S4.1) and designated it as NCCmelb4. When the culture medium was supplemented with stem cell factor (SCF) alone, NCCmelb4 cells were KIT-positive and tyrosinase-negative melanocyte precursors; they remained at an immature and undifferentiated stage. When the medium was supplemented with phorbol 12-o-tetradecanoyl-13-acetate (TPA) + cholera toxin (CT), the cell morphology changed and became L-3,4-dihydroxyphenylalanine (DOPA)-positive. This observation indicates that the NCCmelb4 cells are capable of further differentiation with suitable stimulation. NCCmelb4 cells derived from the mouse neural crest has characteristics of melanocyte precursors (melanoblasts), and is a cell line which can be utilized to study differentiation-inducing factors and growth factors without the effects of feeder cells.
authors.affiliation: Department of Dermatology, St Marianna University School of Medicine, Kawasaki, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Ito
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kawa
authors.forename: Hidenori
authors.initials: H
authors.lastname: Watabe
authors.forename: Hirotake
authors.initials: H
authors.lastname: Ono
authors.forename: Shiho
authors.initials: S
authors.lastname: Ooka
authors.forename: Masayuki
authors.initials: M
authors.lastname: Nakamura
authors.forename: Yoshinao
authors.initials: Y
authors.lastname: Soma
authors.forename: Masako
authors.initials: M
authors.lastname: Mizoguchi
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000096:biosynthesis; D002453:Cell Cycle / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D019334:Endothelin-3 / Q000494:pharmacology; D007150:Immunohistochemistry; D008544:Melanocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009432:Neural Crest / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010641:Phenotype; D019089:Stem Cell Factor / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000943:Antigens, Differentiation; D019334:Endothelin-3; D019089:Stem Cell Factor; D004247:DNA; D002772:Cholera Toxin; D013755:Tetradecanoylphorbol Acetate