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, 29 (12), 2062-76

Nestin- And Doublecortin-Positive Cells Reside in Adult Spinal Cord Meninges and Participate in Injury-Induced Parenchymal Reaction

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Nestin- And Doublecortin-Positive Cells Reside in Adult Spinal Cord Meninges and Participate in Injury-Induced Parenchymal Reaction

Ilaria Decimo et al. Stem Cells.

Abstract

Adult spinal cord has little regenerative potential, thus limiting patient recovery following injury. In this study, we describe a new population of cells resident in the adult rat spinal cord meninges that express the neural stem/precursor markers nestin and doublecortin. Furthermore, from dissociated meningeal tissue a neural stem cell population was cultured in vitro and subsequently shown to differentiate into functional neurons or mature oligodendrocytes. Proliferation rate and number of nestin- and doublecortin-positive cells increased in vivo in meninges following spinal cord injury. By using a lentivirus-labeling approach, we show that meningeal cells, including nestin- and doublecortin-positive cells, migrate in the spinal cord parenchyma and contribute to the glial scar formation. Our data emphasize the multiple roles of meninges in the reaction of the parenchyma to trauma and indicate for the first time that spinal cord meninges are potential niches harboring stem/precursor cells that can be activated by injury. Meninges may be considered as a new source of adult stem/precursor cells to be further tested for use in regenerative medicine applied to neurological disorders, including repair from spinal cord injury.

Figures

Figure 1
Figure 1
Adult spinal cord meninges-derived cells grown in vitro as neurospheres. (A): Diagram representing the cumulative cell number as a function of time. At each time point, neurospheres were mechanically and enzymatically dissociated and cells were counted (n = 4 independent experiments). (B): FACS analysis of the vital dye CSFE dilution after 5 days in culture. Peaks represent the number of cells corresponding to each of the subsequent daughter generations (second generation 16.3%; third 49.2%; fourth 22.9%; and fifth 10.2%). The resulting proliferation index is 4.1. (C): Phase contrast image of meninges-derived neurospheres and confocal image of neurospheres stained with MAP2 (green), nestin (red), and TO-PRO3 (blue). Scale bar = 50 μm. (D): FACS analysis of the in vitro cultured meninges-derived neurospheres. Based on physical properties, such as size (FSC-H) and complexity (SSC-H), three different populations of cells (gates R1–R3) of dissociated neurospheres were drawn. For each gate, the percentage of cells positive for GFAP (blue), MAP2 (green), and nestin (orange) are shown. (E): Analysis of relative gene expression of meninges- and parenchyma-derived neurospheres (n = 3 independent experiments). The bars show relative gene expression ± SD; *, p < .05; **, p < .01; and ***, p < .001. For each sample, expression levels of different genes were normalized to the levels of β-actin mRNA. Abbreviations: CSFE, carboxyfluorescein succinimidyl ester; FACS, fluorescence-activated cell sorting; FL1, fluorescence detector 530/30 nm; MAP2, microtubule-associated protein 2.
Figure 2
Figure 2
Adult spinal cord meninges-derived cells can be differentiated into functional neuronal cells and mature oligodendrocytes. (A–H): Neuronal differentiation obtained from dissociated neurospheres cultured in neurogenic conditions (Materials and Methods section; n = 33 independent experiments). (A): Changes in morphology and Nestin (green) and MAP2 (red) expression occurring after 0, 7, and 21 days of culture in neurogenic conditions. (B): MAP2-positive cells (green) also expressed GAD67, (C) cholinergic neuron marker ChAT, and components of the synaptic apparatus, including (D) the presynaptic marker synaptophysin and (E) postsynaptic marker PSD95. (F, G): Cells differentiated in neurogenic condition for >3 weeks; the cells that express the neuronal-specific markers Tuj1 and MAP2 (red) do not express the glial marker NG2; the seldom cells that express the glial markers GFAP (green) are negative for the neuronal marker MAP2. (H): BrdU staining (green) indicates that the MAP2-positive cells (red) are derived from in vitro replicating cells. Scale bars = 50 μm (A–C, F–H) and 20 μm (D, E,). (I): In the upper panels, representative records showing single action potential elicited by a depolarizing current step from resting membrane potential (−60 mV) and voltage-dependent currents obtained in the same cell following step depolarization under whole-cell voltage clamp. An early transient inward current suggests the presence of voltage-gated Na+ channels, while the outward sustained components are consistent with the presence of voltage-gated K+ channels. The voltage step protocol with a prepulse step from −60 to −90 mV followed by a family of steps to −10, 0, and +10 mV is shown. At the bottom, a representative trace obtained probably by a more immature cell, showing a single abortive action potential elicited by a depolarizing current step from resting membrane potential (−60 mV). (J–P): Oligodendrocyte differentiation was obtained from dissociated meninges-derived neurospheres cultured in oligodendrogenic conditions (Materials and Methods section; n = 24 independent experiments). Different markers of oligodendrocyte differentiation stage progression. After 1 week, cells express the oligodendrocyte progenitor markers NG2 (red) (J), A2B5 (green) (K), and PDGFR-α (red) but were negative for the astrocyte marker GFAP (green) (L). After 2/3 weeks of culture in the differentiating medium, many cells express the early-stage oligodendrocyte marker O4 (M), the mature oligodendrocyte marker GalC (N), and the myelin basic protein MBP (O). Scale bar = 50 μm. (P): scanning electron microscope image showing cells with distinctive oligodendrocyte morphology. Scale bar = 10 μm. Abbreviations: BrdU, 5-bromo-2′-deoxyuridine; MAP2, microtubule-associated protein 2; MBP, myelin basic protein; NG2, chondroitin sulfate proteoglycan 4; PDGFR, platelet-derived growth factor receptor; PSD95, postsynaptic density protein 95.
Figure 3
Figure 3
Nestin- and DCX-positive cells are present in adult rat spinal cord (SC) meninges. (A): Meninges of adult rat SC are shown above the basal lamina of the pia mater (drawn green line) on a transverse section of SC stained with H & E or with nestin (red) antibody. Arrows indicate flattened nestin-positive cells located below the thick layer of nestin-positive cylindrical cells organized in palisade. Scale bar = 50 μm. (B): Three-dimensional image of nestin-positive cell distribution in meninges obtained from Z-stack reconstruction assembled from 20 serial 1-μm confocal sections. Tissue sections were stained with anti-nestin (red), anti-laminin (green) antibody, and with the nuclear marker TO-PRO3 (blue). Proliferation and self-renewal properties of nestin-positive cells (red) in meninges were evaluated by BrdU incorporation and observation (green nuclei) 1 (C) or 20 (D) days after administration. In (C), a Z-stack (20 μm) of a cluster of BrdU-positive cells is shown. Scale bar = 10 μm. (E): DCX-positive cells (shown by arrows) are located above the basal lamina (anti-laminin antibody, green) in the first layer of meninges. (F): In the meninges of the SC anterior medial fissure, DCX-positive cells (red) show a distinctive perivascular location. High magnification of DCX and perivascular laminin (green) is shown. Scale bar = 20 μm (F, E). (G): Gene expression of nestin and DCX in meninges (M) and SVZ (positive control) were assessed by RT-PCR (n = 3 independent experiments, n = 3 animals each). (H): DCX (red)-nestin (green) double-positive cells were also found in meninges. Scale bar = 10 μm. At least six fields (×40 objective) from six separate spinal sections for each animal were analyzed (n = 12 animals). Abbreviations: BrdU, 5-bromo-2′-deoxyuridine; RT-PCR, reverse-transcriptase polymerase chain reaction; SVZ, subventricular zone.
Figure 4
Figure 4
Meningeal reaction induced by SCI caused a significant increase in nestin-positive cells. (A): H & E-stained SC sections showing dramatic modifications of the histological organization of the SC meninges caused by SCI; arrows indicate the increase in meningeal thickness. (B): After SCI, nestin (red)/GFAP (green) double-positive cells appeared in the parenchyma but not in meninges. (C, D): Meningeal increase in thickness is coupled with an increase in proliferating Ki67-positive cells (shown at day 7 in C). Nestin and nestin/Ki67-positive cells increased significantly in number after SCI. The bars in the graphs represent the total cell number per unit of length (mm) of meninges in control conditions and at different dpi. Data are expressed as number of cells ± SEM; statistical differences between CTRL versus SCI are indicated by asterisk (*, p < .5; **, p < .01; and ***, p < .001). (E): Changes in spatio-temporal distribution of proliferating nestin-positive cells are schematically depicted. At least six fields (×40 objective) in dorsal SC region from six separate sections for each animal were analyzed (n = 3 animals for each group). Abbreviations: CTRL, CRL, control noninjured animals; dpi, days postinjury; GFAP, glial fibrillary acidic protein; SCI, spinal cord injury.
Figure 5
Figure 5
DCX-positive cells increase in number in adult SC meninges after SCI. (A, B): Following SCI, the number of meningeal DCX-positive cells increased significantly. At day 7 postinjury, transverse sections of SC were stained with anti-DCX (red) and anti-nestin (green) antibodies. The (A) panels are enlargements of the framed area in (B). DCX+/nestin− (arrows), DCX+/nestin+ (asterisks), and DCX−/nestin+ (triangles) cells are present. (C): A dramatic increase of DCX+/nestin− (arrows) cells was also observed in the layer immediately adjacent to the basal lamina. Scale bar = 20 μm. (D): The bars in the graph represent the number of DCX+ and DCX+/nestin− cells per millimeter of SC-meningeal length in control conditions and 7 days after SCI; ***, p < .001. At least six fields (×40 objective) in dorsal SC region from six separate sections for each animal analyzed (n = 3 animals for each group). (E): The increase of DCX expression at 7 dpi was confirmed by Western blot using anti-DCX antibody on protein extract from CRL-m, SCI-m, and SVZ as positive control (n = 3 independent experiments). Abbreviations: DCX, doublecortin; CRL-m, control meninges; SCI-m, meninges of SCI animals; SVZ, subventricular zone.
Figure 6
Figure 6
SCI-induced activation of stemness-related genes in meningeal cells. (A): Intensity plot representing the gene expression profile of meningeal and parenchymal cells before and 7 days after SCI. Numbers in color bar at the base of the plot indicate the relative expression levels. Raw data are in Supporting Information Table 2. (B): Relative gene expression analysis of control versus SCI meningeal cells. For each sample, expression levels of different genes were normalized to levels of β-actin mRNA. The bars show fold change ± SD of gene expression level; *, p < .05 and **, p < .01. (C): Some of the cells expressing CXCR4 in SC-meninges were also double-positive for nestin. (D): Extracellular matrix components such as agrin, fibronectin, and CSPG are shown in control and following SCI. White dotted line indicate the border between meninges and parenchyma. TO-PRO3 (blue) has been used as nuclear marker. Scale bar = 50 μm. Abbreviations: CSPG, chondroitin sulfate proteoglycan; SCI, spinal cord injury.
Figure 7
Figure 7
Meningeal nestin- and DCX-positive cells contribute to the glia scar formation. (A–C): GFP lentiviral transduction of spinal cord meninges in control animals (n = 3 animals). (A): Longitudinal spinal cord section showing GFP-expressing cells (green) restricted in meninges, above the basal lamina (red). (A, B): No GFP-positive cells were found in the parenchyma, including subpial, subependymal, and ependymal regions. (C): GFP-positive cells (green) in meninges expressing nestin (red). TO-PRO3 (blue) has been used as nuclear marker. Scale bar = 50 μm. (D–J): 7 dpi meningeal GFP-positive cells (green) migrated into the spinal cord parenchyma of injured animals (n = 3 animals). (D): Spinal cord cross-section showing GFP-positive cells cluster (green) into the glia scar coexpressing nestin (red). (E): High magnification of the cluster circled in (D) showing GFP (green)/nestin (red) double-positive cells (yellow), indicated by arrows. Scale bar = 20 μm. (F): Some of the meningeal-derived GFP-positive cells (green) coexpressed DCX (red). Scale bar = 20 μm. (G): High magnification showing a DCX-positive cell (red, lower left corner) and a DCX/GFP double-positive cell (upper right corner). Scale bar = 10 μm. (H, J): Meninges-derived GFP-positive cells (green) in the glial scar did not express either NG2 (red in panel H) or GFAP (red in panels I (Z-stack reconstruction and J). Scale bars = 20 μm (H, I) and 10 μm (J). (K, L): Meningeal GFP-positive cells (green) distribution into cross spinal cord section 30 dpi (n = 3 animals). (K): Meningeal GFP-positive cells (green) distribution into: the fibrotic scar (indicated by dashed line); the glial scar, among the GFAP (red)-positive cells surrounding the scar; and the perilesioned parenchymal region (GFP-positive cells indicated by arrows). The drawn square in the schematic representation of the cross spinal cord section describes the location of the area represented in (K). (L): Meningeal-derived GFP-positive cells migrated to the dorsal horn. The circle in the schematic representation of the cross spinal cord section represents the location of the panel (L). Scale bar = 50 μm. TO-PRO3 (blue) has been used as nuclear marker. At least six fields (×40 objective) in dorsal spinal cord region from six separate sections for each animal were analyzed (n = 3 animals for each group). Abbreviations: DCX, doublecortin; dpi, days postinjury; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; NG2, chondroitin sulfate proteoglycan 4.

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