The unique 473HD-Chondroitinsulfate epitope is expressed by radial glia and involved in neural precursor cell proliferation

Abstract

Neural stem cells have been documented in both the developing and the mature adult CNSs of mammals. This cell population holds a considerable promise for therapeutical applications in a wide array of CNS diseases. Therefore, universally applicable strategies for the purification of this population to further its cell biological characterization are sought. Here, we report that the unique chondroitin sulfate epitope recognized by the monoclonal antibody 473HD is surface expressed on actively cycling, multipotent progenitor cells of the developing telencephalon with radial glia-like properties. When used for immunopanning, the antibody enriched at least threefold for neural stem/progenitor cells characterized by the ability to self-renew as neurospheres that generated all major neural lineages in differentiation assays. In contrast, the 473HD-depleted cell fraction was mostly devoid of neurosphere-forming cells. The isolation of 473HD-positive adult multipotent progenitors from the subependymal zone of the lateral ventricle wall revealed a substantial overlap with the known adult neural stem cell marker LewisX. When the chondroitin sulfates were removed from immunoselected 473HD-positive neural stem/progenitor cell surfaces by chondroitinase ABC treatment or perturbed by the monoclonal antibody 473HD that recognizes the unique DSD-1 chondroitin sulfate epitope, the generation of neurospheres was significantly reduced. Thus, the 473HD epitope could not only be used for the isolation of multipotent neural progenitors during forebrain development as well as from the adult neurogenic niche but may also constitute a functionally important entity of the neural stem cell niche.

Figure 1.

Developmental expression of the 473HD epitope in the telencephalic VZ of the mouse forebrain is closely associated with radial glia. Fluorescence micrographs of frontal sections after double immunostaining with mAb 473HD and various markers, as indicated, are shown. A, B, Inspection of the VZ of the forebrain close to the lateral ventricle revealed radially oriented surface expression of the 473HD epitope on cells that had incorporated BrdU (A, cortical VZ; A′, inset at a higher magnification; B, VZ of the GE). D–F, Double immunolabeling with antibodies against nestin (E) demonstrated that many precursor cells in the VZ were 473HD positive (D–F). H–J, Staining with 473HD (H) and the radial glia marker RC2 (I) yielded colocalization with filament-like radially oriented RC2-positive structures (J). L–N, Staining with 473HD (L) and the radial glia marker BLBP (M), which marks radial glia cells positioned closer to the ventricular surface of the VZ, also showed extensive colocalization with 473HD-expressing cells (N). C, G, K, Cell nuclei were counterstained with bisbenzimide and visualized using DAPI (4′,6-diamidino-2-phenylindole) filters. O–Q, Cryosections of E18 mouse forebrains were studied by double immunofluorescence and confocal analysis with various markers. The boxed areas in the VZ and SVZ are enlarged in the O′–Q′ and O″–Q″, respectively. The 473HD epitope was preferentially detected on radially oriented nestin-positive precursor cells (O) but was barely present on newborn βIII-tubulin-positive neurons (P). Note that there is one 473HD, βIII-tubulin-positive cell in P″. NG2 could mainly be attributed to cells in the perivascular regions (Q, arrowhead), whereas the 473HD-immunoreactive cells in the VZ appeared NG2 negative. Individual NG2-positive cells can be observed in the nascent SVZ (Q″). Scale bars: A, B, 50 μm; C–N, 30 μm; O–Q, 20 μm. LV, Lateral ventricle; Cor, cortex; βIII, βIII-tubulin.

Figure 2.

Immunoselection of 473HD-positive cells from E13 forebrain proves their radial glia identity. Acutely dissociated cells from an E13 cortex were immunostained 2 h after adhesion and compared with the immunoselected 473HD-positive cell fraction. Representative high-power micrographs are shown to better reveal the immunoreactivity of individual cells. A, B, The 473HD epitope (A) is only expressed by a fraction of the nestin-positive cells (B). E, G–I, Analysis of the 473HD-positive cell population shows that most cells are nestin positve (E) and that many of them are actively cycling because they incorporated BrdU during a 2 h pulse in utero (G–I). J, K, M, N, The majority of immunoselected 473HD-positive cells also expressed the radial glia cell markers RC2 (J), GLAST (K), and BLBP (N) as well as the adult NSC marker 487/LeX (M). The quantitative analysis of immunofluorescence studies with various markers using control cells compared with 473HD-immunopanned cells from an E13 and E18 cortex and GE is shown in the supplemental material (available at www.jneurosci.org). Note that the immunopanning procedure clearly enriches for the 473HD epitope in conjunction with several radial glia markers. C, F, I, L, O, All cell nuclei were counterstained with bisbenzimide and are shown in blue [DAPI (4′,6-diamidino-2-phenylindole)]. Scale bar: (in A) 50 μm.

Figure 3.

Selectively isolated 473HD-positive precursor cells differentiate into neurons or form neurospheres in suspension cultures. Cells were sorted from E13 mouse cortex and GE cell suspensions by immunopanning using mAb 473HD and were subsequently cultured on laminin for 2 d. A–C, Immunocytochemical staining with established markers clearly indicated that the majority of cells differentiated into immature neurons (C) under these conditions (A–C). A and B show the phase contrast and labeled nuclei images, respectively, corresponding to the βIII-tubulin staining shown in C. D, E, When cultured under neurosphere-forming conditions in the presence of EGF and bFGF, the sorted 473HD epitope-expressing cells began to divide within 24 h (D) and formed multicellular neurospheres after 7 d (E). F, These neurospheres differentiated morphologically and gave rise to neurons and glia (see supplemental Fig. 2, available at www.jneurosci.org as supplemental material) revealing their multipotency. G, The graph shows the percentage of βIII-tubulin-immunoreactive neurons (mean ± SD) obtained from 473HD-immunoselected (s, light gray bars) compared with nonselected control (ns, dark gray bars) cells from dorsal (Cor) and ventral (GE) telencephalic tissue. H, The graph shows the fraction of neurosphere-forming cells (mean ± SD) in the nonselected control (ns, dark gray bars) compared with the 473HD-enriched (s, light gray bars) cell population from both the cortex (Cor) and GE that increases threefold in the immunoselected 473HD-positive cell populations derived from cortical and striatal tissues. The cell nuclei were counterstained with bisbenzimide and are shown in blue [DAPI (4′,6-diamidino-2-phenylindole)] (B). Scale bars: A–F, 50 μm.

Figure 4.

The 473HD epitope is expressed in neurospheres on actively cycling radial glia cells that comprise the neurosphere-forming cell population. Photomicrographs of cryosections of forebrain-derived neurospheres labeled with mAb 473HD and antibodies to nestin are shown. A–D, Note the extensive codistribution of the 473HD epitope with nestin-positive cells in the outer cell layer of the neurosphere. E–H, Using confocal laser-scanning microscopy, many of the cells labeled with BrdU after a 15 h pulse were found to express the 473HD epitope (E). Similarly, a coexpression of 473HD with the radial glia cell markers RC2 (F), BLBP (G), and GLAST (H) could be demonstrated. Quantitative analyses of dissociated cell suspensions obtained from E13 neurospheres were performed and compared with those enriched by immunopanning with mAb 473HD. All cells were counterstained with bisbenzimide and are shown in blue [DAPI (4′,6-diamidino-2-phenylindole)] (A), except for the confocal images (E–H). I, J, The percentage of positive cells for the various markers is shown for the acutely dissociated nonselected (ns, dark gray bars) compared with the immunoselected (s, light gray bars) 473HD-positive cell population. This set of data revealed that >50% of the 473HD-positive cells were BrdU positive (collected from neurospheres labeled with a 15 h BrdU pulse) and expressed the marker nestin. Interestingly, many 473HD-immunoselected cells coexpressed the radial glial cell markers RC2 and GLAST, and almost all were BLBP and 487/LeX positive. The bar graph displays the results of the quantitative analysis (mean ± SD) of cells obtained from cortical (I) and striatal (J) neurospheres. K, The cell biological comparison of the immunoselected 473HD epitope-expressing cells (s, light gray bars) with the nonselected control population (ns, dark gray bars) from cortical (Cor) and striatal (GE) neurospheres showed an increase in the relative number of βIII-tubulin-positive neurons rising from 25 to 80% when grown for 2 d on laminin. L, In parallel, the emergence of neurospheres from these cell populations was monitored in clonal density assays. Note that the fraction of neurosphere-forming cells (mean ± SEM) could be significantly increased (n = 3) in the positively selected 473HD-positive cell population (s, light gray bars), whereas the negatively selected cell population (n, open bars) was almost completely depleted of neurosphere-forming cells (n = 3) in cell populations obtained from both cortical and striatal neurospheres after immunopanning with 473HD. Scale bar: (in A) A–D, 50 μm; E–H, 10 μm. βIII tub, βIII-Tubulin.

Figure 5.

The 473HD epitope is expressed in the neurogenic niches of the adult brain. A–D, The photomicrographs show double-immunofluorescence images of 473HD epitope expression in red (CY3) and GFAP expression in green (CY2) in the adult SVZ of the lateral ventricular wall (A) and of the subgranular layer (SGL) of the dendate gyrus (D) taken by conventional or confocal microscopy, respectively. The 473HD epitope is clearly present in both neurogenic adult NSC niches, in which it partially colocalizes with GFAP expression (white arrowheads). B′–B″, D′–D′″, This colocalization is better appreciated after higher magnification in the SVZ (B′–B″) and in the SGL (D′–D′″); the white arrowheads indicate the same cells as in A and D, respectively. An example of a 473HD-negative, GFAP-positive cell is shown by the yellow arrowhead in A and C. E–G, Note that the 473HD epitope also colocalizes with GLAST. All cell nuclei were counterstained with bisbenzimide and are shown in blue [DAPI (4′,6-diamidino-2-phenylindole)], except for the confocal images (D–G). Scale bar: (in A) A, 50 μm; D, 100 μm; high-power images, 10 μm. EL, Ependymal layer; g, granular layer; h, hilus; LV lateral ventricle; m, molecular layer.

Figure 6.

The 473HD epitope is expressed on actively cycling 487/LeX-positive cells of the SVZ and in adult SVZ-derived neurospheres. A–D, This set of data revealed that ∼90% of the 473HD-positive cells were BrdU positive (A, B) and expressed the adult NSC 487/LeX (C, D). Interestingly, the immunoselected 473HD-positive cell population comprised ∼50% βIII-tubulin-positive cells 2 h after plating, suggestive of type A cells (neuroblasts). E, The quantitative analysis (mean ± SD) of dissociated cell suspensions obtained from a microdissected adult SVZ was performed and compared with the one derived from cell suspensions enriched by immunopanning with mAb 473HD. The percentage of positive cells for the various markers is shown for the acutely dissociated nonselected (ns, dark gray bars) compared with the immunoselected (s, light gray bars) 473HD-positive cell population. F, G, Consequently, when grown for 2d on laminin, most of the immunoselected 473HD epitope-expressing cells became immature βIII-tubulin-positive neurons. I, J, M, N, Photomicrographs of cryosections of adult neurospheres derived from SVZ cells after 14 d in culture labeled by double immunofluorescence with mAb 473HD (I) and antibodies to nestin (J, N) and 487/LeX (M). K, O, Note the extensive codistribution of the 473HD epitope with nestin- and 487/LeX-positive cells in the outer cell layer of the neurospheres. H, L, All cell nuclei were counterstained with bisbenzimide and are shown in blue [DAPI (4′,6-diamidino-2-phenylindole)]. Scale bar: (in A) A–G, 30 μm; H–O, 50 μm. βIII tub and βIII, βIII-Tubulin.

Figure 7.

Interference with CS-GAGs including the 473HD epitope reveals a requirement for these carbohydrate structures in neurosphere formation. A–H, Photomicrographs of double-immunofluorescence stainings using the antibodies 473HD (red) and pk-anti-phosphacan (KAF13, green), which detect defined CS-GAGs and the core proteins of the RPTPβ gene products, respectively. Immunoselected 473HD-positive cells from E13 cortical tissue were cultured for 2 d in the absence (A–D) or presence (E–H) of the GAG-lyase ChABC on a laminin substrate. In control cultures, 473HD-positive cells are present (B) and colocalize with KAF13 immunoreactivity (C, D). As expected, ChABC treatment leads to a loss of the 473HD epitope (F) without affecting the core protein immunoreactivity (G, H). Rather, an enhanced KAF13 staining could be observed after CS-GAG removal (compare D, H). Note the efficient CS-GAG removal within 2 d after the singular addition of ChABC after plating. When immunoselected 473HD-positive cells from E13 cortical or striatal tissue were cultured under neurosphere-forming conditions in the continuous presence of ChABC, the number of neurospheres was clearly reduced. Cell nuclei were counterstained with bisbenzimide and are shown in blue [DAPI (4′,6-diamidino-2-phenylindole)] I, The capacity to form neurospheres was threefold reduced for 473HD-positive cells derived from the cortex and twofold for GE-derived 473HD-positive cells. J, Data from antibody pertubation experiments in clonal density assays of neurosphere formation. The cultivation in the presence of the mAb 473HD in the culture medium resulted in the specific threefold reduction in the number of neurospheres, comparable with the effect of ChABC treatment of parallel sister cultures. Note that the addition of the 487/LeX isotype control antibody did not significantly alter the number of neurospheres. All data are expressed as mean ± SD from at least three independent experiments. Scale bar: (in A) A–H, 50 μm.