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.
Highlights
Stem cells are capable of generating identical progeny through unlimited numbers of cell divisions whilst retaining the ability to respond to physiological demands by producing daughters committed to differentiate
Mouse embryonic stem (ES) cells differentiate efficiently into neural precursor cells upon withdrawal of serum in adherent monolayer culture [8] or via treatment of embryoid bodies with retinoic acid [30,31]. These precursors have previously been expanded in fibroblast growth factor 2 (FGF-2) with transient retention of neuronal differentiation potential [31,32], but continuous propagation is accompanied by restriction to glial fates ([33] and unpublished data)
After 7 d, cells were re-plated in basal medium (NS-A plus N2) in the presence of either FGF-2 alone or FGF-2 plus epidermal growth factor (EGF)
Summary
Stem cells are capable of generating identical progeny through unlimited numbers of cell divisions whilst retaining the ability to respond to physiological demands by producing daughters committed to differentiate. In tissues that utilise stem cells to sustain cell turnover, the stem cell compartment must be renewed in balance with the production of transit-amplifying progenitors [5] This requires either equivalence between symmetrical self-renewal and commitment divisions, or an asymmetric mode of stem cell division. Epidermal stem cells [6] and neural stem cells [7] can be expanded in vitro, accompanied by differentiation It is unclear whether this reflects a dependence of tissue stem cells on a cellular niche, an intrinsic bias of tissue stem cells towards asymmetric division, or a failure to develop appropriate culture conditions to suppress commitment and sustain symmetrical self-renewal, as has been achieved for ES cells [8]
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