The subventricular zone (SVZ) is the largest neurogenic niche in the adult mammalian brain. Here we show that the brain-enriched microRNA miR-124 is an important regulator of the temporal progression of adult neurogenesis in mice. Knockdown of endogenous miR-124 maintains purified SVZ stem cells as dividing precursors, whereas ectopic expression leads to precocious and increased neuron formation. Furthermore, blocking miR-124 function during regeneration leads to hyperplasias followed by a delayed burst of neurogenesis. We identify the SRY-box transcription factor Sox9 to be a physiological target of miR-124 at the transition from transit amplifying cell to neuroblast stage. Sox9 over-expression abolishes neuronal differentiation whereas Sox9 knockdown leads to increased neuron formation. Thus, miR-124 mediated repression of Sox9 is important for progression along the SVZ stem cell lineage to neurons.

SUMMARY Adult neurogenic niches harbor quiescent neural stem cells, however their in vivo identity has been elusive. Here, we prospectively isolate GFAP+CD133+ (quiescent neural stem cells, qNSCs) and GFAP+CD133+EGFR+ (activated neural stem cells, aNSCs) from the adult ventricular-subventricular zone. aNSCs are rapidly cycling, highly neurogenic in vivo and enriched in colony-forming cells in vitro. In contrast, qNSCs are largely dormant in vivo, generate olfactory bulb interneurons with slower kinetics, and only rarely form colonies in vitro. Moreover, qNSCs are Nestin-negative, a marker widely used for neural stem cells. Upon activation, qNSCs upregulate Nestin and EGFR, and become highly proliferative. Notably, qNSCs and aNSCs can interconvert in vitro. Transcriptome analysis reveals that qNSCs share features with quiescent stem cells from other organs. Finally, small molecule screening identified the GPCR ligands, S1P and PGD2, as factors that actively maintain the quiescent state of qNSCs.

Sphere-forming assays have been widely used to retrospectively identify stem cells based on their reported capacity to evaluate self-renewal and differentiation at the single cell level in vitro. The discovery of markers that allow the prospective isolation of stem cells and their progeny from their in vivo niche allows the functional properties of purified populations to be defined. We provide an historical perspective of the evolution of the neurosphere assay, and highlight limitations in the use of sphere-forming assays, in the context of neurospheres. We discuss theoretical and technical considerations of experimental design and interpretation that surround the use of this assay with any tissue.

The ability to prospectively isolate adult neural stem cells and their progeny is crucial to study their biology and therapeutic potential. Stem cells in adult mammalian neurogenic niches are a subset of astrocytes. A major limitation in the field has been the inability to distinguish stem cell astrocytes from niche astrocytes. Here, we show that epidermal growth factor receptor (EGFR)-positive subventricular-zone (SVZ) astrocytes are activated stem cells that are eliminated by antimitotic treatment. We developed a simple strategy to simultaneously purify cells at different stages of the adult SVZ stem cell lineage by using FACS. This method combines the use of fluorescent EGF ligand, CD24, and GFP expression in GFAP::GFP transgenic mice and allows the simultaneous purification of activated stem cell astrocytes (GFP ؉ EGFR ؉ CD24 ؊ ), niche astrocytes (GFP ؉ EGFR ؊ CD24 ؊ ), transit amplifying cells (GFP ؊ EGFR ؉ CD24 ؊ ), and neuroblasts (GFP ؊ EGFR ؊ CD24 low ). One in three EGFR ؉ astrocytes gives rise to neurospheres in vitro, a 20-fold enrichment over unsorted cells. Importantly, these cells constitute the neurosphereforming population among SVZ astrocytes. This approach will be of great utility for future functional and molecular studies of the SVZ stem cell lineage.adult neurogenesis ͉ FACS ͉ astrocyte ͉ lineage ͉ differentiation

No abstract