Adult spinal cord has little regenerative potential that appears to be limited to the central canal where a spinal cord stem cell population has been shown to reside. This population shows capacity of multipotential neural differentiation in vitro whereas differentiation in vivo is mostly restricted to glial lineage. We have previously described stem/progenitor cells with neural differentiation potential in postnatal rat brain located in the leptomeninges (arachnoid and pia mater). Based on this finding we hypothesize that adult spinal cord meninges could host a neural stem cell-like population. Leptomeninges were isolated by microdissection from adult rat spinal cord. Dissociated leptomeningeal cells were successfully growth in vitro as neurospheres up to several month. Under differentiation conditions in vitro, expanded neurosphere cells were induced to differentiate into neuro-glia lineages. In vitro differentiated neurons displayed typical neuronal morphology, phenotype and functional properties, as confirmed by electrophysiological studies. GFAP positive astrocytes and O4/MBP/GalC-positive oligodendrocytes were also observed. We analyzed the expression of neural stem cell markers (nestin /sox2 /sox9) in meningeal tissue of adult rat spinal cord by immunofluorescence confocal microscopy. We found a layer of nestin-positive cells over the basal lamina marked by laminin; among the nestin positive cells we observed clusters expressing the self renewal marker Oct4 or the proliferating marker Ki67. These nestin-positive cells were seen to be in tight contact with immature neurons (dcx). These data suggest that meninges are niches harboring neural stem cells in vivo. Spinal cord stem cells have been shown to be activated following mechanical injury. To further confirm that leptomeninges are niches of stem cells, adult rats were exposed to standardized injury. Our data indicate that the nestin-positive cells in the meninges respond to the injury by increasing their proliferation with maximal effect at 3 days post injury. We also observed an increase of dcx-positive neuroblasts and of the ECM components, such as CSPG. In conclusion we found in adult spinal cord meninges a new niche of stem/progenitor cells with neural differentiation potential. As compared to other known CNS neural stem cell niche (central canal spinal cord, SVZ), adult spinal cord meningeal niche can be a more accessible and efficient source of neural stem/progenitors cells and should be considered an important alternative source of stem/progenitor cells in regenerative medicine for spinal cord injury therapy.
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