A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells

Ferrón, Sacri R.; Andreu-Agulló, Celia; Mira, Helena; Sánchez, Pilar Fernández; Marqués-Torrejón, María Ángeles; Fariñas, Isabel

doi:10.1038/nprot.2007.104

Cited by 89 publications

(114 citation statements)

References 37 publications

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“…Multipotent self-renewing NSCs can be grown in vitro as clonal aggregates named "neurospheres" in the presence of EGF and FGF2 and differentiated to the three major neural lineages when seeded without EGF onto an adhesive substrate (Reynolds and Weiss, 1992;Ferró n et al, 2007). Neurosphere cultures isolated from the SEZ of 12-month-old mice produced numbers of neurons equivalent to those of young mice (percentage of ␤III-tubulinϩ cells, 16.7 Ϯ 0.4 vs 15.4 Ϯ 1.1, respectively; n ϭ 3 independent cultures per age) when they were seeded in Matrigel and differentiated for 7 div, in agreement with previous reports (Ahlenius et al, 2009).…”

Section: Resultsmentioning

confidence: 99%

“…Methods for adult NSC culture and self-renewal assessment have been previously described in detail (Ferró n et al, 2007). Neurosphere growth medium contained 20 ng/ml epidermal growth factor (EGF) and 10 ng/ml FGF2.…”

Section: Animalsmentioning

confidence: 99%

“…The following primary antibodies were used: mouse anti-BrdU (Dako; 1:250) and ␤III-tubulin (clone Tuj1; 1:300; Covance); rabbit anti-GFAP (1:200; Dako), S100␤ (1:100; Dako), and CR (1:2000; Swant); and goat anti-Sox2 (1:50; R&D Systems). The immunocytochemical procedures were done according to published protocols (Ferró n et al, 2004(Ferró n et al, , 2007. Control samples without the addition of the primary antibodies yielded no signal.…”

Section: Animalsmentioning

confidence: 99%

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Telomere Shortening in Neural Stem Cells Disrupts Neuronal Differentiation and Neuritogenesis

Ferrón¹,

Marqués-Torrejón²,

Mira³

et al. 2009

J. Neurosci.

157

129

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Proliferation in the subependymal zone (SEZ) and neurogenesis in the olfactory bulb decline in the forebrain of telomerase-deficient mice. The present work reveals additional effects of telomere shortening on neuronal differentiation, as adult multipotent progenitors with critically short telomeres yield reduced numbers of neurons that, furthermore, exhibit underdeveloped neuritic arbors. Genetic data indicate that the tumor suppressor protein p53 not only mediates the adverse effects of telomere attrition on proliferation and selfrenewal but it is also involved in preventing normal neuronal differentiation of adult progenitors with dysfunctional telomeres. Interestingly, progenitor cells with short telomeres obtained from fetal brains do not exhibit any replicative defects but also fail to acquire a fully mature neuritic arbor, demonstrating cell cycle-independent effects of telomeres on neuronal differentiation. The negative effect of p53 on neuritogenesis is mechanistically linked to its cooperation with the Notch pathway in the upregulation of small GTPase RhoA kinases, Rock1 and Rock2, suggesting a potential link between DNA damage and the Notch signaling pathway in the control of neuritogenesis. We also show that telomerase expression is downregulated in the SEZ of aging mice leading to telomere length reductions in neurosphere-forming cells and deficient neurogenesis and neuritogenesis. Our results suggest that age-related deficits could be caused partly by dysfunctional telomeres and demonstrate that p53 is a central modulator of adult neurogenesis, regulating both the production and differentiation of postnatally generated olfactory neurons.

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Section: Resultsmentioning

confidence: 99%

Section: Animalsmentioning

confidence: 99%

Section: Animalsmentioning

confidence: 99%

See 1 more Smart Citation

Telomere Shortening in Neural Stem Cells Disrupts Neuronal Differentiation and Neuritogenesis

Ferrón¹,

Marqués-Torrejón²,

Mira³

et al. 2009

J. Neurosci.

157

129

View full text Add to dashboard Cite

show abstract

“…Transfections were performed by electroporation (1500 V, 20 msec, 1 pulse), and 0.3 µg luciferase construct was cotransfected with 0.1 µg pSV40PK Renilla (Promega) into 2 × 10 5 neurospheres derived from E16.5 brains as described (Ferrón et al 2007). Neurospheres were cultured in RHB basal media (Takara) supplemented with N2, Gentamycin, BSA, EGF, and FGF.…”

Section: Line-1 Transcription Studiesmentioning

confidence: 99%

Targeted deletion of a 170-kb cluster of LINE-1 repeats and implications for regional control

et al. 2018

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Approximately half the mammalian genome is composed of repetitive sequences, and accumulating evidence suggests that some may have an impact on genome function. Here, we characterized a large array class of repeats of long-interspersed elements (LINE-1). Although widely distributed in mammals, locations of such arrays are species specific. Using targeted deletion, we asked whether a 170-kb LINE-1 array located at a mouse imprinted domain might function as a modulator of local transcriptional control. The LINE-1 array is lamina associated in differentiated ES cells consistent with its AT-richness, and although imprinting occurs both proximally and distally to the array, active LINE-1 transcripts within the tract are biallelically expressed. Upon deletion of the array, no perturbation of imprinting was observed, and abnormal phenotypes were not detected in maternal or paternal heterozygous or homozygous mutant mice. The array does not shield nonimprinted genes in the vicinity from local imprinting control. Reduced neural expression of protein-coding genes observed upon paternal transmission of the deletion is likely due to the removal of a brain-specific enhancer embedded within the LINE array. Our findings suggest that presence of a 170-kb LINE-1 array reflects the tolerance of the site for repeat insertion rather than an important genomic function in normal development.

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“…In culture, NSCs form free-floating aggregates called "neurospheres" (Figure 1C). Self-renewal and multipotency characteristics of NSCs are assessed in vitro by clonal analysis in which single cells give rise to neurospheres [10,11] ( Figure 1C). …”

Section: Neurogenic Nichesmentioning

confidence: 99%

Epigenetic regulation of stemness maintenance in the neurogenic niches

Montalbán-Loro

Domingo-Muelas

Bizy

et al. 2015

WJSC

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In the adult mouse brain, the subventricular zone lining the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus are two zones that contain neural stem cells (NSCs) with the capacity to give rise to neurons and glia during the entire life of the animal. Spatial and temporal regulation of gene expression in the NSCs population is established and maintained by the coordinated interaction between transcription factors and epigenetic regulators which control stem cell fate. Epigenetic mechanisms are heritable alterations in genome function that do not involve changes in DNA sequence itself but that modulate gene expression, acting as mediators between the environment and the genome. At the molecular level, those epigenetic mechanisms comprise chemical modifications of DNA such as methylation, hydroxymethylation and histone modifications needed for the maintenance of NSC identity. Genomic imprinting is another normal epigenetic process leading to parentalspecific expression of a gene, known to be implicated in the control of gene dosage in the neurogenic niches. The generation of induced pluripotent stem cells from NSCs by expression of defined transcription factors, provide key insights into fundamental principles of stem cell biology. Epigenetic modifications can also occur during reprogramming of NSCs to pluripotency and a better understanding of this process will help to elucidate the mechanisms required for stem cell maintenance. This review takes advantage of recent studies from the epigenetic field to report knowledge regarding the mechanisms of stemness maintenance of neural stem cells in the neurogenic niches.

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A combined ex/in vivo assay to detect effects of exogenously added factors in neural stem cells

Cited by 89 publications

References 37 publications

Telomere Shortening in Neural Stem Cells Disrupts Neuronal Differentiation and Neuritogenesis

Telomere Shortening in Neural Stem Cells Disrupts Neuronal Differentiation and Neuritogenesis

Targeted deletion of a 170-kb cluster of LINE-1 repeats and implications for regional control

Epigenetic regulation of stemness maintenance in the neurogenic niches

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