Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

2.2 Neural Stem Cells Introduction
A proof-of-principle experiment performed by Conti et al [107] demonstrated
the presence of NS cells within neurospheres, and consisted in allowing passage
40 mouse neurospheres derived from fetal forebrain to attach to gelatin-coated
plastic in the presence of EGF and FGF2. Since under these conditions Conti et
al had already proven the generation of NS cells in adherent monoculture (see
Section 2.2), the appearance of bipolar cells that were indistinguishable from
NS cells and could be serially propagated as uniform RC2 + /GFAP - populations
and then induced to differentiate into astrocytes or neurons, concluded that
radial glia-like cells present in neurospheres give rise to NS cells in adherent
culture in the presence of FGF2 and EGF. Conversely, they observed that
NS cells of either ES cell or fetal brain origin readily formed neurospheres if
detached from the substratum mechanically or due to overgrowth, confirming
that NS cells and thus radial glia are likely the neurosphere forming stem cells,
although in neurospheres they constitute only a fraction of the cell population.
Analogously to the embryoid body (EB) differentiation observed in ES cell
aggregates, the differentiation observed within neurospheres is presumably due
to aggregation [125].
An important limitation of the neurosphere culture system is that, when used
to screen compounds that affect NS cell expansion, human NS cells expand
more slowly in suspension culture in vitro than do their mouse counterparts,
which makes quantification of cell proliferation harder due to variable cell
death. A second important limitation of neurosphere assays is that it is dif-
ficult to identify the precise cellular target due to the presence of restricted
progenitors and differentiated cell types, and real-time monitoring of cellu-
lar responses is not possible in aggregates. Finally, fusion of neurospheres is
a common occurrence in suspension, which confounds analyses based solely
on sphere numbers or size [404]. To summarise, the neurosphere paradigm
is invaluable in that it has demonstrated the existence of progenitors within
cultured tissues, but it is accompanied by several important shortcomings:
· the cellular complexity created by the mixed environment is a barrier for
dissecting the mechanisms responsible for the self-renewal and commit-
ment processes;
· the heterogeneity of the cellular population pollutes global expression
profiling experiments and makes it hard to identify a precise cellular tar-
get;
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