br Results br Discussion In the

Results
Discussion In the present study we show that LPA1 is expressed by stem and progenitor protease inhibitor cocktail (predominantly type 1 and type 2a) within the neurogenic niche of the dentate gyrus of adult mice, and that it outperforms Nestin, the current gold standard, as a marker for prospective isolation of cells that exhibit precursor cell properties ex vivo. Sorting LPA1-GFP+ cells in combination with prominin-1 and EGFR allows the separation of the non-proliferative from the proliferative precursor cells, the latter of which was revealed by RNA sequencing to have an unexpected immune-cell-like transcriptional profile. The intermediate filament Nestin is the most commonly used marker of neural stem cells. Although all four commonly accessible transgenic Nestin-GFP lines show expression in the adult neurogenic regions, they differ in the extent of GFP expression (Beech et al., 2004; Kawaguchi et al., 2001; Mignone et al., 2004; Yamaguchi et al., 2000). It was recently demonstrated that Nestin expression is absent from the quiescent stem cell population of the adult SVZ (Codega et al., 2014), being upregulated only after the stem cells are activated. In the present study, we demonstrate that LPA1-GFP is a more sensitive marker than Nestin-GFP and can be used to effectively isolate the proliferative hippocampal precursor population, with >99% of the neurospheres generated from the LPA1-GFP+ cells. Importantly, LPA1-GFP appears to also mark the quiescent stem cell population in the dentate gyrus. In contrast to the Nestin-GFP− population, no quiescent stem cells could be activated from the LPA1-GFP− population following in vitro depolarization, a treatment which we have previously shown to mimic neural activity (Walker et al., 2008). Combining LPA1-GFP expression with two other markers, EGFR and prominin-1, we were able to further separate the proliferative (neurosphere-forming) from the non-proliferative cells. Our flow cytometric isolation strategy has allowed the molecular characterization of proliferative, as distinct from the non-proliferative precursor cells. This distinction could previously not be made using broader markers such as Nestin and SOX2. Indeed, comparison of our list of 145 proliferative precursor-cell-enriched genes with the SOX2-enriched genes generated by Bracko et al. (2012) revealed only three common genes (Igf1, Dab2, and Txnip), and when our threshold was decreased to 2-fold enrichment only two additional commonly expressed genes were detected (Ucp2 and Hmgb2). The fact that SOX2 also marks post-mitotic astrocytes and the presence of known choroid plexus markers in their stem cell gene list highlights the limitation of sorting using a single marker. In contrast, our division of proliferative versus non-proliferative corresponds well with subpopulations identified by transcript functional profiling in another recent study (Shin et al., 2015). Our present study extends on this work by enabling prospective isolation of the proliferative and non-proliferative precursor cell populations. This will allow downstream manipulation in vitro for further characterization of factors capable of activating the quiescent stem cells, as well as for potential applications such as transplantation. Transcriptomic analysis of our isolated proliferative precursor population revealed a profile with immune-like characteristics. The presence of cytokine receptors on neural precursor cells, as well as the production of cytokines and other inflammatory molecules, supports the existence of bidirectional crosstalk between the neural stem cells and the immune system (Zhang et al., 2015). Indeed, there is emerging evidence for a direct and synergistic interaction between neural stem cells and peripheral T cells to maintain baseline neurogenesis levels as well as to promote recovery following insult (Niebling et al., 2014; Wolf et al., 2009; Zhang et al., 2015). Our analysis revealed a number of immune molecules expressed by the proliferative precursor cells, most of which have no known role in the regulation of adult neural stem cells. We had, however, previously identified one of these molecules, Oncostatin M, as a regulator of neural precursor activity (Beatus et al., 2011). In addition, as a proof of concept, we confirmed a regulatory role of one of the identified immune-related proteins in adult hippocampal precursor proliferation. More detailed studies, however, are required to further investigate the specific role that CXCL1 and the other stem-cell-specific cytokines play in this process.