The Journal of Neuroscience, April 12, 2006, ():
The Unique 473HD-Chondroitinsulfate Epitope Is Expressed by Radial Glia and Involved in Neural Precursor Cell Proliferation
J. Neurosci. von Holst et al.
26: 4082
Supplemental data
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Supplemental tables
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Fig.1: The 473HD-positive cell population gives rise to significantly more neurospheres than the non-selected cell population
E13 mouse cortical (Cortex) and ganglionic eminence (GE) cells were maintained under neurosphere-forming conditions either directly after acute dissociation (non-selected) or used for immunopanning with mAb 473HD (selected) and subsequently kept under analogous conditions, that is in the presence of EGF and bFGF. Within 7div, 473HD-positive immunoselected cortical (B) and GE (D) cell populations displayed an increased capacity to generate neurospheres when compared to the non-selected cortical (A) and GE (C) cells. For quantitative assessment, only neurospheres with a diameter larger than 50 µm were taken into account. Numbers in the micrographs (A-D) indicate examples of neurospheres included for quantification; Scale bar: 100 µm.
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Fig.2: The 473HD-positive cell-derived neurospheres are multipotent
Photomicrographs of immunolabeled cells are shown. Neurospheres derived from immunopanned 473HD-positive cells were allowed to differentiate for 5 div and the adherent cultures were studied by immunocytochemistry with established markers, as indicated. Note the emergence of neurons (B, red colour), oligodendrocytes (C, green colour) and astrocytes (D, blue colour), which emphasizes the multipotential character of neurospheres generated from 473HD-sorted cells, as visible in the merged image (A). Scale bar: 25 µm.
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Fig.3: Dose-responses analysis of chondroitinase ABC as added to neurosphere cultures
Cell suspensions from E13 mouse forebrain were cultured in growth factor-containing media in the presence or absence of the enzyme ChABC for 5 days. Chondroitinase ABC was added to neurosphere cultures once at the beginning of the experiment at concentrations of 10, 20, 50, 100 or 200 mU/ml. Neural stem cells began to divide and neurospheres formed after 2 div. The bar diagrammes illustrate the numbers of neurospheres generated after 2 and 5 div. The numbers of neurospheres per visual field observed in control cultures as compared to neurospheres growing in the presence of different concentrations of ChABC are given. The addition of ChABC decreased the number of neurospheres, with a saturation of the effect observed at a concentration of 50 mU/ml. The column graphs represent the absolute number of neurospheres pro visual field (mean ± SD of the neurospheres counted in ten different visual fields and two independent experiments).
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Fig. 4: Chondroitinase ABC treatment of 473HD-immunopanned cells reduces the number of generated neurospheres.
473HD epitope expressing cells from dorsal (Cor) and ventral (GE) regions of E13 forebrain were enriched by immunopanning and cultivated under neurosphere-forming conditions either in the absence (A, C) or in the presence of 50 mU/ml chondroitinase ABC (ChABC, B, D) for 5 div. The removal of CS GAGs by ChABC-treatment led to a reduction in the number of cortical- (B) and striatal-derived neurospheres (D), in comparison to the control situation (A and C). Scale bar: 100 µm.
