The mammalian brain, even at late stages of development, maintains some degree of neural plasticity that is typically credited to adult neural stem cells (aNSCs). Current literature suggests that these cells exclusively reside in specialized areas/niches of the brain including the subventricular zone (SVZ), rostral migratory stream (RMS) and the dentate gyrus (DG) of the hippocampus. However, the true phenotypic identity of these cells in situ remains unknown. In order to positively identify aNSCs, we developed a new fluorescence microscopy method that utilizes up to ten biomarkers, which have been previously used to phenotype neural cells associated with NSC niches during embryonic brain development. These include markers targeting nestin, an intermediate filament protein expressed by NSCs and immature neural progenitors, Sox2 and Pax6, two transcription factors associated with NSC self-renewal and multipotent differentiation, integrin beta1 (CD29) and HNK-1 antigen (CD57), two surface markers expressed by neuroepithelial cells and their immediate differentiating progeny, glutamate transporter (GLAST), a marker of radial glia, glial fibrillary acidic protein (GFAP), a marker of astrocytes, doublecortin (DCX), a marker of newly born neurons, isolectin IB4 (ILIB4), a marker of endothelial cells lining the brain microvasculature associated with NSC niches, and proliferating cell nuclear antigen (PCNA), a marker of actively proliferating cells. We applied these ten biomarkers to sagittal sections of postnatal day 7 (P7) and young adult (P28) rat brain. The marker expression in SVZ, RMS and DG was imaged using Volocity acquisition software and the images were then processed using CellProfiler to identify the phenotype of all proliferating (PCNA+) neural cells in the SVZ, RMS and DG in search for the elusive aNSCs. The results revealed that markers currently associated with NSCs, like nestin and Sox2, were also found on proliferating GLAST+ radial glial progenitors, GFAP+ astroglial progenitors and DCX+ neuronal progenitors, whereas Nestin+Sox2+Pax6-CD29+CD24-GLASTGFAP- DCX-ILIB4-PCNA+ phenotype uniquely identifying embryonic NSCs could not be detected in any of the above regions of interest neither in P7 nor P28 rat brains. These findings imply that neurogenic and gliogenic potentials in the early neonatal and adult rat brain are derived from a separate seminal pool of neural progenitor cells rather than embryonic-like aNSCs.
Last updated March 20, 2013