Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

2.2 Neural Stem Cells Introduction
tors FGF2 and EGF as described by several previous studies [107,165,403,481].
In their study, Sun et al [479] have shown that NS cells and their progenitors
grew on adherent substrate significantly faster in the first four passages than
the NS cells in neurospheres, but slowing down and plateauing their growth
rate after the sixth or seventh passage, while in neurospheres their growth kept
increasing slowly but steadily for more than 10 passages.
Immunocytochemical analysis using multiple lineage-specific antibodies in early
primary cultures for both neurosphere and adherent methods showed about
60% MAP2 + neurons and about 40% Nestin + progenitors, indicating that
both cultures were initially composed of neural progenitor and mature neu-
ronal populations. However, proliferating neural progenitors became dominat-
ing in adherent culture over the next few days to finish off on the ninth day
with a Nestin + progenitor percentage of 93.76%, clearly identifying the type
of cells that eventually compose the culture [479]. In order to test for the
ability of both culture systems to differentiate into neuronal and glial cells,
growth factor withdrawal was induced for seven days and differentiation was
assessed through the use of antibodies against Tuj1, GFAP and Olig-4 for the
identification of neurons, astrocytes and oligodendrocytes, respectively. The
data gathered from passages 1, 3 and 5 revealed no significant differences in
the differentiation potential of cells cultured with either method [479].
Self-renewal capacity was assessed in the two culture systems through a clono-
genic assay that evaluated the capacity of primary neurospheres to form sec-
ondary neurospheres, as well as the capacity of plating from a single cell from
the original adherent culture. The clone forming rates at passage 1 were found
to be 63% for neurospheres and 44% for adherent culture, a number signifi-
cantly higher for neurospheres than the 20% reported by Reynolds et al [525].
Interestingly, as discussed in the study by Marten et al [319], in the fetal
mouse forebrain geminal zone there seem to be two differently responding self-
renewing cells that grow neurospheres with greater diameters in the presence
of EGF alone in their culture in vitro than FGF. Since in the culture systems
developed by Sun et al both EGF and FGF were added, the two differently
responsive stem cells might have been produced together, increasing the clone
forming rates observed [479].
Finally, neurosphere cultures were found to have a greater passage potential
than adherent cultures, possibly due to the three-dimensional reproduction of
the niche environment experienced by the cells in vivo. In fact, the regulatory
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