Final Activity Report Summary - ENDONSC (Regulation of stem cell proliferation and differentiation by endothelial cells)
Stem cells reside in localised areas in the tissue, called niches. Neural stem cell (NSC) niches in the adult mammalian brain include the subventricular zone (SVZ), the dentate gyrus and the olfactory bulb. NSCs have been shown to localise around the blood vessels and endothelial cells (EC) can regulate the proliferation, self-renewal and the differentiation of these NSC into neurons in vitro, although the mechanism is unknown. We have analysed the structure of the mouse NSC niche by using different markers for NSC (including Sox2, Mash1, CD133, GFAP and Nestin), neural progenitors and astroglial cells and established their relationship with the surrounding endothelium in the SVZ in both healthy brains and after ischemic injury, which mimics a stroke scenario. In the adult and newborn SVZ most Sox2+ cells are close to the blood vessels with some of them directly contacting the vessel wall.
We found that the Sox2+ NSC population is more complex than anticipated. Whereas Sox2+/Mash1+ neural progenitors are preferentially found surrounding the blood vessels, Sox2+/CD133+ cells are localised in the subependymal layer. Ki67+ staining, a marker of cell proliferation, showed that most Sox2+ cells are in a proliferating status. We identified a percentage of the Sox2+ cells which localise close to the vessels and are able to retain the proliferation marker BrdU for up to three months, suggesting that they are true stem cells. In co-culture experiments in which NSC and EC are allowed to exchange soluble factors, we identified a number of unexpected EC-secreted factors that influence the proliferation status of NSC. We observed that these proteins increased the number of neurospheres (NSC-rich structures) in vitro and we are currently performing in vivo assays to study their effect in vivo. In addition, we performed microarrays analysis to study the transcription profile of the NSC that had been co-culture with the endothelial cells and found an up-regulation of genes involved in cell proliferation and cell migration and a down-regulation of genes involved in stem cell differentiation, suggesting that endothelial cells recruit NSC and prevent their differentiation.
In animals in which a stroke-like injury had been induced by occluding the middle cerebral artery, we see an increase in the expression of Sox2, Sox9, and Mash-1 in the SVZ and rostral migratory stream (RMS). The NSC sub-populations change in the SVZ of these animals and we are currently performing BrdU-staining studies to identify what genes are expressed by the BrdU retaining cells.
We found that the Sox2+ NSC population is more complex than anticipated. Whereas Sox2+/Mash1+ neural progenitors are preferentially found surrounding the blood vessels, Sox2+/CD133+ cells are localised in the subependymal layer. Ki67+ staining, a marker of cell proliferation, showed that most Sox2+ cells are in a proliferating status. We identified a percentage of the Sox2+ cells which localise close to the vessels and are able to retain the proliferation marker BrdU for up to three months, suggesting that they are true stem cells. In co-culture experiments in which NSC and EC are allowed to exchange soluble factors, we identified a number of unexpected EC-secreted factors that influence the proliferation status of NSC. We observed that these proteins increased the number of neurospheres (NSC-rich structures) in vitro and we are currently performing in vivo assays to study their effect in vivo. In addition, we performed microarrays analysis to study the transcription profile of the NSC that had been co-culture with the endothelial cells and found an up-regulation of genes involved in cell proliferation and cell migration and a down-regulation of genes involved in stem cell differentiation, suggesting that endothelial cells recruit NSC and prevent their differentiation.
In animals in which a stroke-like injury had been induced by occluding the middle cerebral artery, we see an increase in the expression of Sox2, Sox9, and Mash-1 in the SVZ and rostral migratory stream (RMS). The NSC sub-populations change in the SVZ of these animals and we are currently performing BrdU-staining studies to identify what genes are expressed by the BrdU retaining cells.