Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics - PubMed (original) (raw)

Comparative Study

Adult mammalian forebrain ependymal and subependymal cells demonstrate proliferative potential, but only subependymal cells have neural stem cell characteristics

B J Chiasson et al. J Neurosci. 1999.

Abstract

The adult derivatives of the embryonic forebrain germinal zones consist of two morphologically distinct cell layers surrounding the lateral ventricles: the ependyma and the subependyma. Cell cycle analyses have revealed that at least two proliferating populations exist in this region, one that is constitutively proliferating and one that is relatively quiescent and thought to include the endogenous adult neural stem cells. Earlier studies demonstrated that specific dissection of the region surrounding the lateral ventricles was necessary for the in vitro isolation of multipotent, self-renewing neural stem cells. However, in these studies, the ependymal layer was not physically separated from the subependymal layer to identify the specific adult laminar localization of the neural stem cells around the lateral ventricles. To determine which cellular compartment in the adult forebrain contained the neural stem cells, we isolated and cultured the ependyma separately from the subependyma and tested for the presence of neural stem cells using the in vitro neurosphere assay. We demonstrate that the ependymal cells can proliferate in vitro to form sphere-like structures. However, the ependymal cells generating spheres do not have the ability to self-renew (proliferate to form secondary spheres after dissociation) nor to produce neurons, but rather only seem to generate glial fibrillary acidic protein-positive ependymal cells when plated under differentiation conditions in culture. On the other hand, a subpopulation of subependymal cells do possess the self-renewing and multipotential characteristics of neural stem cells. Therefore, the adult forebrain neural stem cell resides within the subependymal compartment.

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Figures

Fig. 1.

Fig. 1.

Schematic representation of a coronal section though the forebrain of the adult mouse showing the ependyma (thick black line) lining of the lateral ventricles (LV) surrounded by the subependyma (cross-hatched area). Dissections of the ependyma and/or subependyma were performed on the medial wall closest to the septum (Sep). CC, Corpus callosum;Ctx, cortex; Str, striatum.

Fig. 2.

Fig. 2.

The ependyma can be dissected free of surrounding tissue. A, A 14 μm coronal section through the lateral ventricle demonstrates that S-100β (red) antibodies selectively stain all of the ependymal cells adjacent to the lateral ventricle and that BrdU (green) antibodies only stain a subpopulation of the cells in the subependyma. A few cells appear to be double-labeled cells in 14-μm-thick sections (yellow, indicated by arrow in_A_), but on closer inspection, it is clear that some of the BrdU-labeled nuclei (green) can still be seen beyond the edge of (and thus are distinct from) the S-100β-positive ependyma cytoplasmic staining (red) as shown in_A′_ (higher magnification of cells indicated in_A_). Thus, these apparent double-labeled cells are actually separate cells sitting on top of one another within the 14 μm sections. Further support for this conclusion comes from the complete lack of any (even suggestive) double labeling seen in thinner 6 μm sections. The same nonoverlapping populations of BrdU+ subependymal and S-100β+ependymal cells were observed after 7 d of BrdU labeling and then a 31 d survival. B, From a partially dissected coronal section, S-100β+ ependymal cells (red) and some BrdU+ subependymal cells (green; a few are marked with_arrowheads_) can be seen on the striatal (str) wall of the lateral ventricle (undissected), whereas only BrdU+ cells in the subependyma (arrowheads) remain after the ependyma was removed (arrow) from the septal (sep) wall of the lateral ventricle. C, Dissected ependymal sheets (from the septal wall of the lateral ventricle) that were stained for S-100β (red) and BrdU (green) demonstrate only S-100β+ cells. D, Hoechst 33258 labeling of cell nuclei confirmed that all of the cells that were present from the dissected ependyma stained intensely for S-100β (compare C, D), strongly suggesting that none of the proliferating cells in the subependyma have been transferred in the dissection and that all of the cells present were of ependymal origin. Note that, during cell culture experiments, only the areas of subependyma that had the adjacent ependymal lining removed was sampled for the subependymal treatment group. Scale bars, 100 μm.

Fig. 3.

Fig. 3.

Ependymal spheres form in no exogenous growth factor, and subependymal spheres arise only in EGF or FGF2. The generation of spheres in FGF2 from tissue containing both the ependyma and subependyma exceeds the number of spheres generated by ependymal and subependymal cultures separately (ependyma alone plus subependyma alone < ependyma with subependyma; p < 0.01). A similar, but nonsignificant, trend to increased spheres for the ependymal and subependymal cocultures is seen in EGF, as well. The increased numbers of spheres in cocultures can all be attributed to the generation of additional subependymal (larger, nonciliated) spheres.

Fig. 4.

Fig. 4.

Subependymal (A) and ependymal (B) spheres demonstrate distinct morphologies after 8 d in vitro. Ependymal spheres were small and measured <100 μm in diameter compared with the larger (500–1000 μm in diameter) spheres generated from subependyma alone. One very obvious difference between the ependymal spheres and the subependymal spheres was the fact that ependymal spheres were composed of ciliated cells, which caused the spheres to rotate in the cell culture media. C, A small number of nonsphere-forming ependymal cells derived from a primary dissection adhered to the culture plate and displayed an elongated, bipolar morphology in the presence of FGF2. Scale bars, 100 μm.

Fig. 5.

Fig. 5.

Ependymal spheres produce differentiated ependymal cells as revealed by expression of GFAP. A, GFAP+ immunostaining of cells from a differentiated ependymal sphere originally grown in EGF and subsequently plated in 1% FBS for 1 week. B, The same field stained with MAP2 antibodies revealed that no neurons were derived from the differentiated ependymal sphere. Bright spots are noncellular, nonspecific fluorescence. Scale bars, 50 μm.

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