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Fig. 1. Cleavage of CS-GAGs reduces the proliferation rate of mouse E13
telencephalic neural stem/progenitor cells and interferes with the generation
of neurospheres. The efficacy of the CS-GAG removal after ChABC treatment
of Nsph cultures was controlled by double immunofluorescence and western blot
analyses. (A) Cryosections obtained from Nsphs grown for 7 days in
vitro (div) in the absence (w/o, upper panels) or presence (lower panels) of
50 mU ChABC were immunostained with 473HD and pk-anti-phosphacan antibodies
(KAF13) that detect the unique DSD-1 epitope and the proteins encoded by the
Rptpß gene, respectively. The 473HD epitope was strongly
expressed in the outer cell layers of the Nsph under control conditions and
was absent after ChABC treatment, which resulted in enhanced reactivity of the
polyclonal antibodies with the RPTP-ß protein (KAF13). (B) Western
blot analysis confirmed the efficient removal of the 473HD epitope by ChABC,
which was not achieved by keratanase (Ker) treatment. (C-E)
Phase-contrast micrographs documenting the formation of Nsphs grown in
serum-free medium in the presence of EGF and bFGF (C) and upon addition of
ChABC (D) or keratanase (E) after 5 div. ChABC treatment caused an almost
complete settling of Nsphs on the culture substrate and subsequent cellular
outgrowth. (F) Quantification of the numbers of cortical (Cor) and
striatal (GE) Nsphs present per visual field in bulk cultures. Note the
significant decrease in the number of primary Nsphs grown in the presence of
ChABC in comparison with control (w/o) or keratanase-treated cultures.
(G) The total number of secondary Nsphs generated from cortical and
striatal control or ChABC-treated primary Nsphs was determined in clonal
density assays. In comparison with control cultures (w/o), the number of
secondary Nsphs obtained from primary ChABC-treated Nsphs appeared
significantly reduced. (H) Photomicrographs of dissociated,
BrdU-labeled primary control (w/o, upper panels) and ChABC-treated (lower
panels) Nsphs that had received a 15-hour BrdU pulse. (I) The fraction
of BrdU-incorporating cycling cells in H was determined relative to the total
number of cell nuclei (DAPI), expressed as percentage fraction. Note that
approximately twice as many actively cycling cells were detected in the
control population (w/o), as compared with the cells that had been exposed to
ChABC. (A,H) Cell nuclei were counterstained with bisbenzimide and are shown
in blue (DAPI). **, P<0.01; ***,
P<0.001. Scale bars: 50 µm in A; 150 µm in C-E; 50 µm in
H.
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