The findings reported here establish that the growth factors EGF and FGF-2, plus insulin provided in N2, are sufficient to sustain robust expansion of neural stem cells in defined monoculture, liberated from any requirement for a specific cellular niche. Previous propagation of mammalian CNS precursor cells [64
] has predominantly relied on short-term progenitor cell cultures [22
], genetic immortalization of progenitors [23
], or maintenance of stem cells within neurospheres [7
]. Progenitor cells from the adult rat hippocampus have been propagated in adherent culture with FGF-2 whilst retaining ability to form neurons [65
], albeit at low efficiency without inductive astrocytes [66
]. These hippocampal-derived cells are heterogeneous by immunohistochemical staining and “undergo asymmetric cell divisions to continually replenish the supply of multipotent progenitors” [65
]. In contrast, the uniformity and stability of marker expression in NS cells, combined with the long-term retention of neuronal differentiation efficiency, indicate that differentiation is completely suppressed by FGF-2 plus EGF in adherent culture. Consequently, the NS cells undergo continuous symmetrical self-renewal divisions.
The critical difference between the adherent culture system we describe and those used by others appears to be the use of EGF in addition to FGF-2. Although often applied to neurosphere cultures, EGF is typically omitted for attached cells. We find that continuous provision of EGF is essential for the derivation and propagation of NS cells, whether sourced from mouse ES cells or foetal brain. This may be related to the role of ErbB signalling in establishment of radial glia in vivo [67
] and crosstalk with the Notch pathway [68
]. Also, EGF functions to suppress apoptosis in NS cells (B and D).
The evidence suggests that NS cells are closely related to a radial glia lineage. Whether any radial glia actually function as self-renewing stem cells in vivo is uncertain, since they exist only transiently during foetal development. However, recent fate mapping analysis [69
] indicates that a subset of radial glia give rise to the sub-ventricular zone astrocytes that function as adult neural stem cells [70
]. A possible relationship has also been proposed between hippocampal progenitors and radial glia [72
]. The derivation of NS cells may reflect this intrinsic potential of radial glia to convert into stem cells. EGF has been reported to down-regulate expression of Dlx2 in transit-amplifying cells of the sub-ventricular zone and promote their conversion into neurosphere forming cells [55
]. Consistent with this, we find that Dlx2 is not expressed in NS cells. Intriguingly, evidence has recently been presented that the EGF receptor undergoes asymmetric segregation in a proportion of RC2-staining neural progenitor cell divisions in vivo [28
]. The retention or loss of the EGF receptor is suggested to facilitate alternative fate choices. Retention by both daughters of full responsiveness to EGF may be a central aspect of NS cell propagation. This could represent a crucial divergence from the circumstances of radial progenitor cells in vivo that underpins self-renewal in vitro.
NS cells do not express pluripotent cell-specific transcription factors Oct-4 and Nanog, but show appropriate expression of neural genes and absence of mesoderm and endoderm-specific genes. Their close relationship to a defined endogenous neural precursor cell, radial glia, adds further interest. NS cells can be cryopreserved, and may be transiently or stably transfected by electroporation or lipofection (unpublished data), or transduced with lentiviral vectors. They may be derived from previously engineered ES cells or transgenic mice or be genetically modified after derivation, opening new windows for genetic intervention into self-renewal and lineage commitment decisions in the nervous system and for investigation of neurodegenerative processes and oncogenic transformation. The potential of NS cells to generate different neuronal sub-types has yet to be determined, but their engraftment after transplantation into the adult brain suggests the potential for delivery of cell replacement and gene therapies. Whilst long-term stability and functional integration in vivo will have to be established in future studies, the preliminary data of human analogues to mouse NS cells provides encouragement for this approach.
In the context of fundamental stem cell biology, homogenous expansion of any stem cell in defined conditions has hitherto been the exclusive preserve of the ES cell. Ex vivo propagation of tissue stem cells has invariably been accompanied by differentiation, often interpreted as reflecting an intrinsic bias towards asymmetric division. The findings reported here show that this is definitively not the case for at least one class of neural stem cell. NS cells undergo sustained symmetrical self-renewal divisions with complete suppression of differentiation in response to FGF-2 and EGF. They thus provide a directly accessible system for molecular characterization and experimental manipulation of the stem cell state. Therefore, NS cells offer the first known tissue stem cell resource for direct comparison with ES cells in order to delineate common and distinct mechanistic features of lineage-restricted and pluripotent stem cells.