Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

2.2 Neural Stem Cells Introduction
and RC2, an antibody recognising the presence of Nestin [310]. Until the late
1990s, the only cell line that could consistently generate human neuronal cells
in vitro was NTERA-2, a teratocarcinoma 28 derived cell line that required the
performance of complex manipulations to induce differentiation [22,397]. In
1997, Sah et al. established the first immortalized adherent human fetal neu-
ral precursor cell line using retrovirally expressed avian v-myc [441] that led
to subsequent independent reports using similar strategies [114,145,513], until
the possibility of expanding human fetal neural precursors in suspension cul-
tures was explored [83,428,483]). The floating aggregates of cells were termed
"neurospheres" [427] and only recently adherent monoculture protocols have
been developed as an alternative.
Neurospheres With the exception of ES cells, it has always proven difficult
to obtain homogeneous propagation of stem cell cultures ex vivo since they tend
to be accompanied by differentiation. In 1992 Weiss and Reynolds discovered
that cells from fetal mouse CNS could be propagated in suspension culture
with EGF, as a cluster of floating cells that they termed "neurospheres" (Fig
2.4). A neurosphere represents a clonal single cell-derived floating cluster of
Figure 2.4: (a,b) Contrast microscopy images of early phase neurosphere formation,
in which individual cells form small clusters. (c,d) Immunofluorescence microscopy
images in which (c) EGFR (green) and the Nestin protein (red) are detected on an
intact neurosphere, and (d) nuclei (DAPI staining, blue) and cell mitosis (5-bromo-
2’-deoxyuridine (BrdU) incorporation, green) are detected on a frozen neurosphere
section. Image adapted from [375].
proliferating cells [427] that contains thousands of cells and is a mixture of
stem and progenitor cells, with only up to 5% stem cells [122]. The number of
stem cells in a neurosphere is evaluated in a clonogenic assay by determining
28 A germ cell tumor.
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