Nitric Oxide Mediated Signal Transduction in Networks of Human ...

Fig. 1 Cells migrate out of NT2 spheres
that were seeded on PDL and Matrigelcoated
microdishes. Representative photomicrographs
of NT2 spheres treated with
10 lM RA for 1 week (a–c) and 2 week (d–
f). The white lines in c delineate distance of
migration from the rim of the sphere to the
furthest migrated cells. Arrow heads in (f)
show representative neuronal processes. A
higher magnification of the migrated cells
shows single cells that bear no or few
neurite in 1-week-old spheres (g) when
compared with 2-week-old spheres that
elaborated long neurites (h). The use of RA
for 2 weeks significantly decreased cell
migration as compared with 1-week-old
spheres (i). Scale bars: 100 lm
(***p < 0.001).
dispersed mechanically into single cells and stained
against both markers. While 10.3% of the cells stained
positively for bIII-tubulin (Fig. 2e and h), about 84.3%
remained nestin-positive (Fig. 2g). Upon further incubation
with RA for an additional week, the spheres displayed
an increasing amount of bIII-tubulin-stained cells (Fig. 2d).
The immunoreactivity was not only expressed in the cell
bodies but also in the emerging neuronal processes (Fig. 2d
and f). We counted about 46% bIII-tubulin- and 63%
nestin-positive cells (Fig. 2g and h). An increase in the
level of bIII-tubulin, elaboration of neuronal processes
together with reduction in the level of nestin upon longer
incubation with RA indicated the progress of neuronal
differentiation.
(a) (b) (c)
(d) (e)
Ó 2009 The Authors
Journal Compilation Ó 2009 International Society for Neurochemistry, J. Neurochem. (2009) 110, 1828–1841
(g)
(i)
NO signaling regulates neuronal precursor motility | 1831
(h)
Studies on gene expression of NT2 cell during and
shortly after differentiation with RA showed down-regulations
of several stem cell markers and up-regulation of
nestin and neurofilament transcripts that support our present
observations at the protein levels (Houldsworth et al. 2002;
Przyborski et al. 2003). Because of their high cellular
motility we further characterized the type of cells that
migrated out of 1-week-old NT2 spheres. Only 3.5 ± 0.5%
of the migratory cells were bIII-tubulin-positive and almost
all of the furthest migrated cells lacked the bIII-tubulin
staining (Fig. 2c). Hence, the cells at the front of the
migration expressed mainly the precursor marker nestin.
Costaining of BrdU together with nestin indicated that most
of the proliferating cells were nestin-positive neuronal
(f)