Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics (original) (raw)
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Scientific reports, 2016
Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals, they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However, little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ), we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months, the proliferative status of activated NSCs was already altered by 6 months, with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signa...
Stem Cells, 2008
It is now generally accepted that continuous neurogenesis occurs in the adult mammalian brain, including that of humans. Modulation of adult neurogenesis can provide therapeutic benefits for various brain disorders, including stroke and Parkinson's disease. The subventricular zone-olfactory bulb pathway is one of the preferred model systems by which to study neural stem cell proliferation, migration, and differentiation in adult rodent brain. Research on adult neurogenesis would greatly benefit from reliable methods for long-term noninvasive in vivo monitoring. We have used lentiviral vectors encoding firefly luciferase to stably mark endogenous neural stem cells in the mouse subventricular zone. We show that bioluminescence imaging (BLI) allows quantitative follow-up of the migration of adult neural stem cells into the olfactory bulb in time. Moreover, we propose a model to fit the kinetic data that allows estimation of migration and survival times of the neural stem cells using in vivo BLI. Long-term expression of brain-derived neurotrophic factor in the subventricular zone attenuated neurogenesis, as detected by histology and BLI. In vivo monitoring of the impact of drugs or genes on adult neurogenesis is now within reach.
TGFβ lengthens the G1 phase of stem cells in aged mouse brain
Stem cells (Dayton, Ohio), 2014
Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique, we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition, we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells, but not in transit-amplifying cells, and directly impacts on neurogenesis. Finally, we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of ...
The Journal of Physiological Sciences
Neural stem cells (NSCs) are maintained in the adult mammalian brain throughout the animal’s lifespan. NSCs in the subependymal zone infrequently divide and generate transit amplifying cells, which are destined to become olfactory bulb neurons. When transit amplifying cells are depleted, they are replenished by the quiescent NSC pool. However, the cellular basis for this recovery process remains largely unknown. In this study, we traced NSCs and their progeny after transit amplifying cells were eliminated by intraventricular infusion of cytosine β-D-arabinofuranoside. We found that although the number of neurosphere-forming NSCs decreased shortly after the treatment, they were restored to normal levels 3 weeks after the cessation of treatment. More importantly, the depletion of transit amplifying cells did not induce a significant expansion of the NSC pool by symmetric divisions. Our data suggest that the size of the NSC pool is hardly affected by brain damage due to antimitotic dru...
Cell Stem Cell
Until now, limitations in the ability to enrich adult NSCs (aNSCs) have hampered meaningful analysis of these cells at the transcriptome level. Here we show via a split-Cre technology that coincident activity of the hGFAP and prominin1 promoters is a hallmark of aNSCs in vivo. Sorting of cells from the adult mouse subependymal zone (SEZ) based on their expression of GFAP and prominin1 isolates all self-renewing, multipotent stem cells at high purity. Comparison of the transcriptome of these purified aNSCs to parenchymal nonneurogenic astrocytes and other SEZ cells reveals aNSC hallmarks, including neuronal lineage priming and the importance of cilia- and Ca-dependent signaling pathways. Inducible deletion of the ciliary protein IFT88 in aNSCs validates the role of ciliary function in aNSCs. Our work reveals candidate molecular regulators for unique features of aNSCs and facilitates future selective analysis of aNSCs in other functional contexts, such as aging and injury.► Split-Cre fate mapping identifies GFAP/prominin1-expressing cells as aNSCs in vivo ► FACS sorting of GFAP/prominin1-expressing cells isolates aNSCs at high purity ► This approach distinguishes aNSCs from niche astrocytes and ependymal cells ► Transcriptome analysis revealed molecular characteristics of aNSCs
Neuron, 2014
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 PGD 2 , as factors that actively maintain the quiescent state of qNSCs.
Journal of Neuroscience, 2007
We determined the embryonic origins of adult forebrain subventricular zone (SVZ) stem cells by Cre-lox fate mapping in transgenic mice. We found that all parts of the telencephalic neuroepithelium, including the medial ganglionic eminence and lateral ganglionic eminence (LGE) and the cerebral cortex, contribute multipotent, self-renewing stem cells to the adult SVZ. Descendants of the embryonic LGE and cortex settle in ventral and dorsal aspects of the dorsolateral SVZ, respectively. Both populations contribute new (5-bromo-2'-deoxyuridine-labeled) tyrosine hydroxylase- and calretinin-positive interneurons to the adult olfactory bulb. However, calbindin-positive interneurons in the olfactory glomeruli were generated exclusively by LGE-derived stem cells. Thus, different SVZ stem cells have different embryonic origins, colonize different parts of the SVZ, and generate different neuronal progeny, suggesting that some aspects of embryonic patterning are preserved in the adult SVZ. This could have important implications for the design of endogenous stem cell-based therapies in the future.
Visualizing Cell Cycle Phase Organization and Control During Neural Lineage Elaboration
Cells, 2020
In neural precursors, cell cycle regulators simultaneously control both progression through the cell cycle and the probability of a cell fate switch. Precursors act in lineages, where they transition through a series of cell types, each of which has a unique molecular identity and cellular behavior. Thus, investigating links between cell cycle and cell fate control requires simultaneous identification of precursor type and cell cycle phase, as well as an ability to read out additional regulatory factor expression or activity. We use a combined FUCCI-EdU labelling protocol to do this, and then apply it to the embryonic olfactory neural lineage, in which the spatial position of a cell correlates with its precursor identity. Using this integrated model, we find the CDKi p27KIP1 has different regulation relative to cell cycle phase in neural stem cells versus intermediate precursors. In addition, Hes1, which is the principle transcriptional driver of neural stem cell self-renewal, surpr...
Journal of Neuroscience, 2009
in SVZ and the functional characteristics of their neuronal progeny. Here, we have compared the properties of NSCs isolated from embryonic lateral ganglionic eminence and adult and aged SVZ in mice using in vivo and in vitro systems, analyzed their gene expression profile, and studied their electrophysiological characteristics before and after differentiation into neurons. We show a loss of NSCs in SVZ from aged mice accompanied by reduced expression of genes for NSC markers, developmentally important transcription factors, and neurogenic factors. However, when isolated in vitro, the NSCs from SVZ of aged animals have capacity for proliferation and multilineage differentiation, including production of functional neurons, similar to that of NSCs in adult mice, albeit with lower efficacy. These properties are of major importance when considering therapeutic applications of neuronal replacement from endogenous NSCs in the injured, aged brain.