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, 13 (9B), 3195-208

Novel Stem/Progenitor Cells With Neuronal Differentiation Potential Reside in the Leptomeningeal Niche

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Novel Stem/Progenitor Cells With Neuronal Differentiation Potential Reside in the Leptomeningeal Niche

Francesco Bifari et al. J Cell Mol Med.

Abstract

Stem cells capable of generating neural differentiated cells are recognized by the expression of nestin and reside in specific regions of the brain, namely, hippocampus, subventricular zone and olfactory bulb. For other brain structures, such as leptomeninges, which contribute to the correct cortex development and functions, there is no evidence so far that they may contain stem/precursor cells. In this work, we show for the first time that nestin-positive cells are present in rat leptomeninges during development up to adulthood. The newly identified nestin-positive cells can be extracted and expanded in vitro both as neurospheres, displaying high similarity with subventricular zone-derived neural stem cells, and as homogeneous cell population with stem cell features. In vitro expanded stem cell population can differentiate with high efficiency into excitable cells with neuronal phenotype and morphology. Once injected into the adult brain, these cells survive and differentiate into neurons, thus showing that their neuronal differentiation potential is operational also in vivo. In conclusion, our data provide evidence that a specific population of immature cells endowed of neuronal differentiation potential is resident in the leptomeninges throughout the life. As leptomeninges cover the entire central nervous system, these findings could have relevant implications for studies on cortical development and for regenerative medicine applied to neurological disorders.

Figures

Figure 1
Figure 1
Distribution of nestin-positive cells in rat cortex at different ages. Confocal images of coronal sections of parietal cerebral cortex: rats at embryonic day 20 (E20) (A), postnatal day +1 (P1) (B), +8 (P8) (C) and +15 (P15) (D) and adults (E). Immunolabelling with the anti-nestin (red) antibody. (F) Subventricular zone (SVZ) of P15 rats as positive control and choroid plexus. Scale bar 50 μm.
Figure 2
Figure 2
Localization of nestin-positive cells in leptomeningeal tissue. (A) Coronal section of the parietal cortex of P15 rat, stained with DAPI (blue), anti-laminin (green) and anti-nestin (red) antibodies. Scale bar 50 μm. (B, C, D) Z-stack reconstruction assembled from 10 serial 2 μm confocal sections. Sections were stained in red with anti-nestin antibodies and in green with either anti-laminin (B) or -GFAP (C) or -NG2 (D) antibodies; high magnification. None of these markers co-localizes with nestin. Scale bars 50 μm.
Figure 3
Figure 3
Tissue sampling. (A) Hematoxylin/eosin staining of a coronal section of P15 rat brain to show (arrows) the site and the extent of the biopsy. Scale bar 250 μm. (B) FACS analysis of tissue extracts from P15 rats. Viable cells, stained with Syto16, were gated and, among them, nestin-positive cells (red 40.3%) or negative (black) are shown.
Figure 4
Figure 4
In vitro expansion of the tissue extract in neural stem cell growth conditions. Floating neurospheres were obtained, with morphology and phenotype comparable to those of SVZ-NSC-derived neurospheres. (A) Transmitted light of leptomeningeal-derived neurosphere. Confocal image of neurospheres stained with MAP2 (green), nestin (red) and DAPI (blue) (B), or MAP2 (red) and GFAP (green) (C), scale bar 100 μm. (D) Relative gene expression analysis. For each sample, expression levels of different genes were normalized to levels of beta-actin mRNA. The bars show fold change ± S.D. in transcription of normalized mRNA expression levels measured for leptomeninges-derived neurosphere compared to SVZ-NSC-derived neurosphere.
Figure 5
Figure 5
In vitro expansion of the tissue extract in adherent cells culture conditions. (A) Time course experiment. Confocal images of nestin+/NG2- cells in adherent culture at 5 hrs, 1, 4, 8 and 12 days after plating. (B) CFU derived from leptomeningeal cells at different passages. Bars represent the CFU number/million of plated cells ± S.D. (B’) May–Grunwald Giemsa staining of a single colony of leptomeningeal adherent cells. (C) Transmitted light and (D) immunofluorescence, nestin (red), of adherent leptomeningeal cells. (E) FACS analysis, carried out on expanded cells obtained from adult rats, shows high expression of nestin and CD90. In vitro expanded cell population does not express neuro-glial (MAP2, GFAP and O4), leukocyte (CD45), endothelial (CD31, CD106) and haematopoietic stem cell markers (CD34). (F) Relative gene expression analysis. Leptomeningeal-derived nestin+ cells and BM-MSCs fold changes in transcription, normalized to actin mRNA, have been shown compared to SVZ-NSCs.
Figure 6
Figure 6
In vitro neural differentiation of leptomeningeal cells after adherent culture expansion (A) Real-time gene expression analysis of Nestin and Mtap2 (MAP2) in leptomeninges-derived cells in basal condition and after differentiation. The bars show folds change ± S.D. of normalized mRNA expression levels measured before and after differentiation (P < 0.01). (B, C, D) Differentiated cells, stained with antibodies against MAP2 (red). Arrows in (C) indicate dendritic spines. (D) BrdU staining (green) indicates that the MAP2-positive cells (red) derived from replicating cells. MAP2-positive cells also expressed components of the synaptic apparatus, including the presynaptic marker synaptophysin (E), the glutamate ionotropic receptor sub-unit GluR2 (G) and glutamate decarboxylase (GAD67) (F). Scale bars 50 μm.
Figure 7
Figure 7
Calcium imaging analysis of in vitro neural differentiated leptomeningeal cells. (A) Changes in intracellular free calcium are indicated by variation of 340 nm/380 nm fluorescence ratio in Fura-2-loaded cells. Depolarization induced by 55 mM KCl led to increase of 340/380 ratio in most of the cells present in the field. (B) Average responses (mean ± S.E.M.) expressed as peak and baseline values in differentiated cells and in primary neuronal cultures; ***P < 0.001.
Figure 8
Figure 8
Confocal images of injection site of transplanted leptomeningeal cells. (A) GFP-positive cells (green) into the injection site in CA2 hippocampal region stained with nuclear marker DAPI (blue), scale bar 200 μm; (B) GFP-positive cells in the injection site surrounded by glia (GFAP red signal), scale bar 100 μm; (C) Colocalization of GFP-positive cells with nestin in the injection site (yellow signal), scale bar 100 μm; (D) Colocalization of GFP-positive cells with GFAP (yellow signal) near the injection site, scale bar 50 μm; (E) Colocalization of GFP-positive cells with NG2 (yellow signal), scale bar 50 μm.
Figure 9
Figure 9
Confocal images of in vivo leptomeningeal cells neural differentiation. (A) Transplanted EGFP+ cells (green) are localized in CA1 region, scale bar 100 μm. (B) EGFP+ cells (green) showed complex phenotypes mimicking the pyramidal neurons of the hippocampus (CA1 pyramidal cell layer), scale bar 100 μm; (B’) high magnification, scale bar 20 μm. (C) Z-stack reconstruction assembled from 10 serial 1.63-μm confocal sections. EGFP (green)/MAP2 (red)/DAPI (blue)-positive cells in CA1 pyramidal layer, scale bar 50 μm. (C’, C’’, C’’’) confocal images pointing single cell (arrowhead) co-localizations (yellow) of EGFP (green) and MAP2 (red), scale bars 20 μm. EGFP+ cells co-localizing with MAP2 (yellow) (D) or not (E), scale bars 20 μm. Transplanted EGFP+ cells (green) co-localizing with MAP2 (yellow) in sub-granular zone (SGZ) and hilus of the dentatus gyrus, scale bar 200 μm (F). (F’) High magnification, scale bar 50 μm.

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