title: Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.
abstract: Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.
authors.affiliation: Institute for Stem Cell Research, University of Edinburgh, Edinburgh, United Kingdom.
authors.forename: Luciano
authors.initials: L
authors.lastname: Conti
authors.forename: Steven M
authors.initials: SM
authors.lastname: Pollard
authors.forename: Thorsten
authors.initials: T
authors.lastname: Gorba
authors.forename: Erika
authors.initials: E
authors.lastname: Reitano
authors.forename: Mauro
authors.initials: M
authors.lastname: Toselli
authors.forename: Gerardo
authors.initials: G
authors.lastname: Biella
authors.forename: Yirui
authors.initials: Y
authors.lastname: Sun
authors.forename: Sveva
authors.initials: S
authors.lastname: Sanzone
authors.forename: Qi-Long
authors.initials: QL
authors.lastname: Ying
authors.forename: Elena
authors.initials: E
authors.lastname: Cattaneo
authors.forename: Austin
authors.initials: A
authors.lastname: Smith
mesh_terms: D000818:Animals; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D033581:Stem Cell Transplantation
chemical_list: D016222:Fibroblast Growth Factor 2; D004815:Epidermal Growth Factor