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.