Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

2.2 Neural Stem Cells Introduction
Conti et al [107]. This differs vastly from the neuronal subtypes identified upon
direct differentiation of ES cell with no intermediate expansion of the neural
progenitors, which are skewed towards the generation of large amounts of glu-
tamatergic neurons 33 [55]. This inconsistency shows that in vitro expansion
of NS cells may be somehow restricting the neuronal subtype differentiation
capacity of these cells and further studies will have to address whether cell
culture conditions can be altered for long-term expanded NS cells so that re-
gional identities can be re-established [400].
Previous studies have shown the derivation of glial restricted progenitors in ad-
herent culture using FGF2 for survival and expansion of the cells [76,212,276,
367]. However, these cultures change their properties over time and should not
be considered equivalent to expanded long-term stem cell lines [403]. In the
NS cell derivation from mouse ES cells described by Conti et al, the absence
of EGF causes caspase 3-lead apoptosis and an immature neuronal phenotype.
This phenomenon was found to be avoidable by culturing NS cells on laminin
in the absence of EGF, although this addressed NS cells towards neuroblast 34
commitment with differentiation upon mitogen withdrawal [290]. The NS cells
obtained in the protocol by Conti et al exhibit phenotypic similarities to ra-
dial glia in that they show no expression of neuronal or astrocyte antigens, but
uniform expression of neural precursor markers Nestin, RC2, Vimentin, 3CB2,
Lex1, Paired box gene 6 (Pax6) and Prominin (see Fig 2.2). In addition to
this set of markers considered diagnostic for neurogenic radial glia, they show
expression of the neural precursor markers Sox2, Sox3, and Emx2, and the
transcription factors Olig2 and Mash1. The absence of Sox1 and maintenance
of Sox2 is noteworthy of NS cells since the former marks all early neuroec-
todermal precursors and its absence in stem cells might indicate that Sox2
is playing the key role. In a study by Gomez-Lopez et al [169] the roles of
Sox2 and Pax6 were investigated in mouse fetal forebrain-derived NS cells,
to find that conditional deletion of either gene reduced the clonogenicity of
these cells in a gene dosage-dependent manner. Cells heterozygous for either
gene displayed moderate proliferative defects, but in the complete absence of
Sox2, cells exited the cell cycle with concomitant down-regulation of neural
progenitor markers Nestin and Blbp, and ablation of Pax6 also caused major
33 Neurons that release the main CNS excitatory neurotransmitter glutamate.
34 Neuroblasts are the descendants of NS cells in the SVZ that migrate into damaged brain
areas after strokes or other brain injuries to generate regionally appropriate new neurons
[290]
48