An optimized experimental strategy for efficient conversion of embryonic stem (ES)-derived mouse neural stem (NS) cells into a nearly homogeneous mature neuronal population

Spiliotopoulos, Dimitrios; Goffredo, Donato; Conti, Luciano; Febo, F. Di; Biella, Gerardo; Toselli, Mauro; Cattaneo, Elena

doi:10.1016/j.nbd.2009.02.007

Cited by 61 publications

(89 citation statements)

References 30 publications

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“…To address this question, we treated NS cells with three different growth factor regimes to induce cell cycle exit and stimulate differentiation or quiescence, and we used microarrays to detect significantly up-and down-regulated genes. We used BMP4 to induce astrocytic differentiation (Sun et al 2011a), treatment with B27-and FGF2-containing medium to induce an early neuronal progenitor fate (Spiliotopoulos et al 2009), and BMP4 + FGF2 to induce NS cell quiescence (Sun et al 2011a;Martynoga et al 2013). In all three experiments, 1340-2054 genes were significantly down-regulated and 1442-2054 genes were up-regulated, showing the extent of transcriptional rewiring.…”

Section: Different Cre Classes Associate With Genes That Are Regulatementioning

confidence: 99%

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

Mateo

Berg²,

Haeussler

et al. 2014

Genome Res.

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The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterized. Knowledge of the central transcription factors (TFs), the noncoding gene regulatory regions that they bind to, and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq, we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX, and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence.

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Section: Different Cre Classes Associate With Genes That Are Regulatementioning

confidence: 99%

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

Mateo

Berg²,

Haeussler

et al. 2014

Genome Res.

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show abstract

“…Upon exposure to a pan-neuronal differentiation protocol, NS cells can be converted into generic MAP2 + neurons. 17,19 Here, we first differentiated wt and HD NS cell lines towards neurons, and after 7 days, the cells were fixed and immunostained with an anti-MAP2 antibody to visualize the neurites. Not surprisingly, neurite outgrowth was reduced in Q140/7 neurons compared with Q7/7 neurons, as shown by MAP2 staining (Figure 1h) and relative quantification using an automated neurite tracing (NeuriteTracer 20 ) (Figure 1i).…”

Section: Resultsmentioning

confidence: 99%

Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease

et al. 2014

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In the adult brain, neurons require local cholesterol production, which is supplied by astrocytes through apoE-containing lipoproteins. In Huntington's disease (HD), such cholesterol biosynthesis in the brain is severely reduced. Here we show that this defect, occurring in astrocytes, is detrimental for HD neurons. Astrocytes bearing the huntingtin protein containing increasing CAG repeats secreted less apoE-lipoprotein-bound cholesterol in the medium. Conditioned media from HD astrocytes and lipoprotein-depleted conditioned media from wild-type (wt) astrocytes were equally detrimental in a neurite outgrowth assay and did not support synaptic activity in HD neurons, compared with conditions of cholesterol supplementation or conditioned media from wt astrocytes. Molecular perturbation of cholesterol biosynthesis and efflux in astrocytes caused similarly altered astrocyteneuron cross talk, whereas enhancement of glial SREBP2 and ABCA1 function reversed the aspects of neuronal dysfunction in HD. These findings indicate that astrocyte-mediated cholesterol homeostasis could be a potential therapeutic target to ameliorate neuronal dysfunction in HD. Huntington's disease (HD) is an adult-onset neurodegenerative disorder characterized by cell loss mainly in the striatum and cortex. Its pathophysiology is linked to an expanded CAG repeat in the IT-15 gene, which leads to an elongated polyQ tract in huntingtin (HTT) protein. No disease-modifying treatment is available for HD and novel pathophysiological insights and therapeutic strategies are needed. 1 Lipids are vital to brain health and function. Accordingly, the brain has a local source of cholesterol, 2 and a breakdown of cholesterol synthesis causes brain malformations and impaired cognitive function. 3,4 Cholesterol metabolism is disrupted in HD 5,6 as revealed by transcriptional, biochemical, and mass spectrometry analyses in HD rodent models. 7,8 This dysregulation is linked to a specific action of mutant HTT on sterol-regulatory-element-binding proteins (SREBPs) and on its target genes, whose reduced transcription leads to lower brain cholesterol levels. 7 In HD humans, brain cholesterol homeostasis is affected since pre-symptomatic stages, as determined by measurement of the brain-specific cholesterol catabolite 24-S-hydroxy-cholesterol (24OHC). 9,10 However, it remains unclear how reduced brain cholesterol would become pathological for HD neurons.In adulthood, astrocytes produce cholesterol, which is secreted as a complex with apolipoprotein (apo) E lipoproteins and delivered to neurons. 11,12 Mutant HTT is expressed in glial cells, 13,14 and transgenic mice overexpressing mutant HTT in astrocytes show age-dependent neurological symptoms. 15,16 Additionally, primary astrocytes overexpressing full-length human mutant HTT show reduced mRNA levels of cholesterol biosynthetic genes, along with impaired cellular production and secretion of apoE. 8 Here we employed molecular and cellular tools to test the impact of cholesterol perturbation between astrocytes and neur...

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“…21 Neurons derived in this manner stop proliferating, express markers specific for TD-neurons (such as the cytoskeletal filament protein Tuj1, also known as b-tubulin-III) and can therefore be regarded as TD (Supplementary Figure S9A). 13 We irradiated these neuron cultures with 10 Gy in parallel with parental NSC and analyzed them under the same conditions.…”

Section: Resultsmentioning

confidence: 99%

“…Astrocyte differentiation and culture medium was DMEM/F12 with 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 10% FCS. Neuron differentiation was performed as described in Spiliotopoulos et al 21 NU7441 18 and KU55933 19 (Tocris, Bristol, UK) were dissolved in DMSO and used at 1 mM final concentration overnight before irradiation. X-ray irradiation of cells was performed in a Faxitron RX-650 (Faxitron Bioptics, Lincolnshire, IL, USA) device at 2 Gy/min for 5 min (total of 10 Gy) or, where indicated, for 30 sec and 25 min (total of 1 and 50 Gy, respectively).…”

Section: Methodsmentioning

confidence: 99%

Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

2011

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The impact and consequences of damage generation into genomic DNA, especially in the form of DNA double-strand breaks, and of the DNA-damage response (DDR) pathways that are promptly activated, have been elucidated in great detail. Most of this research, however, has been performed on proliferating, often cancerous, cell lines. In a mammalian body, the majority of cells are terminally differentiated (TD), and derives from a small pool of self-renewing somatic stem cells. Here, we comparatively studied DDR signaling and radiosensitivity in neural stem cells (NSC) and their TD-descendants, astrocytes -the predominant cells in the mammalian brain. Astrocytes have important roles in brain physiology, development and plasticity. We discovered that NSC activate canonical DDR upon exposure to ionizing radiation. Strikingly, astrocytes proved radioresistant, lacked functional DDR signaling, with key DDR genes such as ATM being repressed at the transcriptional level. Nevertheless, astrocytes retain the expression of non-homologous end-joining (NHEJ) genes and indeed they are DNA repair proficient. Unlike in NSC, in astrocytes DNA-PK seems to be the PI3K-like protein kinase responsible for cH2AX signal generation upon DNA damage. We also demonstrate the lack of functional DDR signaling activation in vivo in astrocytes of irradiated adult mouse brains, although adjacent neurons activate the DDR.

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An optimized experimental strategy for efficient conversion of embryonic stem (ES)-derived mouse neural stem (NS) cells into a nearly homogeneous mature neuronal population

Cited by 61 publications

References 30 publications

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

Characterization of the neural stem cell gene regulatory network identifies OLIG2 as a multifunctional regulator of self-renewal

Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease

Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

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