Title: Adult Neural Stem Cells and Their Perivascular Niche
Description: Stem cells reside in specialized niches that support their selfrenewal and differentiation. A balance between intrinsic and extrinsic signals mediates stem cell quiescence, activation and proliferation. In the mammalian subventricular zone (SVZ), the stem cells are a subset of GFAP+ astrocytes. A quiescent pool of GFAP+ stem cell astrocytes generates activated (actively dividing) GFAP+EGFR+ stem cell astrocytes. These in turn generate EGFR+ transit amplifying cells, which give rise to neuroblasts that migrate to the olfactory bulb. In the SVZ niche, dividing cells localize next to blood vessels. SVZ stem cells and transit amplifying cells also directly contact blood vessels at sites that lack glial end feet and pericyte coverage, a feature unique to SVZ vasculature. Diffusible signals from transformed endothelial cell lines have been shown to increase survival, proliferation and neurogenic differentiation of SVZ neural stem cells and their progeny in vitro. However, the effect of primary endothelial cells is unknown.

Furthermore, previous studies have not elucidated whether vascular signals from neurogenic and non-neurogenic regions are different and/or act on specific stages of the neural stem cell lineage. Moreover, the role of pericytes in the SVZ stem cell niche has not been defined. Here we describe a FACS methodology to isolate pure, primary endothelial cells and pericytes from neurogenic and non-neurogenic brain regions and perform studies in vitro to examine their effect on distinct stages of the SVZ neural stem cell lineage. Primary endothelial cells from both cortex and SVZ support proliferation and neuronal differentiation of activated stem cell astrocytes and transit amplifying cells in the absence of any exogenous growth factors. Notably, their signals are more potent than those secreted from the immortalized bend.3 endothelial cell line. Proliferation of activated stem cell astrocytes and transit amplifying cells with conditioned medium from primary cortical cells was shown to depend on EGFR in vitro.

Here we define for the first time the effect of pericytes on SVZ neural stem cells. Pericytes promote the proliferation of activated stem cell astrocytes and transit amplifying cells, but to a lesser extent than endothelial cells. Strikingly, activated stem cell astrocytes and transit amplifying cells generate proportionally more neurons in response to pericyte conditioned medium than other conditions, and SVZ pericyte signals are particularly potent on activated stem cell astrocytes. Little is known about the heterogeneity of pericytes in the brain. After culturing FACS-purified pericytes, we observed multiple in vitro phenotypes of pericytes from both cortex and SVZ. Over time, both cortical and SVZ pericyte cultures became dominated by a rapidly proliferating cell with a progenitor morphology, which could be serially passaged. In preliminary studies, this passaged pericyte exhibited features of mesenchymal stem cells. To probe pericyte heterogeneity in the brain, we used mesenchymal stem cell markers.

Novel pericyte subpopulations could be prospectively purified from both the cortex and SVZ using CD13, CD146, and CD105. Interestingly, CD13+CD105-CD146- pericytes were the most proliferative population from both the SVZ and cortex, but only those from SVZ could be passaged. Staining with these markers in vivo demonstrated specific morphologies and staining patterns on different sized vessels in the SVZ. Fractones, an ECM structure unique to the SVZ, arose from pericytes. As an endothelial marker, CD146 displayed different patterns of staining on different sized vessels, and stained naked vessels that lacked a basement membrane. While the SVZ vascular bed is largely quiescent, we also detected rare CD146+ tip cells. Collectively, these studies demonstrate the use of a powerful methodology to directly purify endothelial cells and pericytes from the brain in a neurogenic region, the SVZ, and a non-neurogenic region, the cortex. We uncover previously undescribed vascular cell diversity in the brain, and a novel role for brain pericytes on neural stem cells and their progeny.

In addition to elucidating novel roles of vascular cells in the SVZ niche, this protocol offers a flexible and effective platform to obtain pure and contextually precise cells for future experiments in other brain regions or stem cell niches. Defining the different components of the niche is central to understanding the regulation of stem cells under homeostatic conditions and conversely how these signals are lost or perturbed during aging and tumorigenesis.