Human Astrocytes Exhibit Tumor Microenvironment, Age- and Sex-Related Transcriptomic Signatures
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE168375 | GEO | 2022/01/05
REPOSITORIES: GEO
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Project description:Astrocytes were purified from mouse cortices and human cortex tissue by immunopanning with HepaCAM. All treatments were proceeded on 3DIV in serum-free medium. Astrocytes were treated by TNFα for 48hr, by Poly I:C for 72hr, or by hypoxia for 72hr. Acutely purified atrocytes RNA was harvest immediately after HepaCAM immunopanning. Serum selected astrocytes RNA was collected after 4-6 days culturing in serum medium. Serum-free cultured astrocytes RNA was collected on 5-6 DIV. Serum-selection purified human astrocytes were transplanted into P2-11 Rag2 immunodeficient mouse brains, and after about 8 months, transplanted human and host mouse astocytes RNA was collected acutely on HepaCAM immunopanning dish.
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Project description:Single-cell RNA-seq: We used single-cell RNAseq to investigate the maturation of astrocytes within human cortical spheroids Bulk RNA-seq: Bulk sequencing from astrocytes and neurons purified (via immunopanning) from iPSC-derived coritical spheroids at varying in vitro differentiation states
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Project description:Astrocytes were purified from fetal and adult human brain tissue using an immunopanning method with the HepaCAM antibody. Samples were taken from otherwise 'healthy' pieces of tissue, unless otherwise specified. 6 fetal astrocyte samples, 12 adult astrocyte samples, 8 GBM or sclerotic hippocampal samples, 4 whole human cortex samples, 4 adult mouse astrocyte samples, and 11 human samples of other purified CNS cell types
2015-12-11 | E-GEOD-73721 | biostudies-arrayexpress
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2011-09-06 | GSE26066 | GEO
Project description:Comparison of expression data of rat forebrain astrocytes from P1, P7 acutely isolated by immunopanning or cultured with astrocytes prepared by McCarthy and de Vellis' (1980) method. Elucidating the genes induced by serum in immunopannedrat astrocytes. Three biological replicates for each sample were done. MD-astrocytes were prepared as described in McCarthy and de Vellis 1980 and harvested for mRNA after 7DIV. IP-astrocytes were isolated from P1 or P7 Sprague Dawley rats and processed for RNA immediately (IP-astrocytes P1/P7), or cultured for 7 days in HBEGF before harvesting (Cult. IP-astrocytes P1/P7). For the serum studies, we plated IP-astrocytes P7 in MD-astrocyte media containing 10% fetal calf serum immediately after isolation and cultured them for 7 days. After 7 days, the cultures were either processed for total RNA or washed 3x with dPBS and astrocyte base media with HBEGF was added. The cells were cultured for an additional 7 days and then processed for RNA. We isolated total RNA with the QIAshredder and Qiagen RNeasy Mini Kit. We used the 3’IVT Express kit for preparation of the RNA and the Rat Genome 230 2.0 Array chip (Affymetrix, Santa Clara). RT-PCR was used to elucidate the level of contamination in each cell sample.
2011-09-05 | E-GEOD-26066 | biostudies-arrayexpress
Project description:Astrocytes were purified from postnatal day 2 mouse cortices by immunopanning with HepaCAM. Inhibitors and DMSO were added on in-vitro day 2. HBEGF was depleted on the cell prep day and till in-vitro day 7. In-vitro day 2, 7 and 14 samples were collected on designed timepointed.
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Project description:Human Astrocytes Exhibit Tumor Microenvironment, Age- and Sex-Related Transcriptomic Signatures
| PRJNA707056 | ENA
Project description:Research regarding the role of astrocytes as M-NM-2-amyloid (AM-NM-2) degrading cells has broadened our view about the mechanisms how these common glia cells function in AlzheimerM-bM-^@M-^Ys disease (AD). Based on previous studies adult mouse astrocytes are able to degrade AM-NM-2 deposits from AD mouse model and human brain tissue sections ex vivo, contrary to neonatal astrocytes. In this study, we studied the possible altered gene expression profiles of adult and neonatal astrocytes cultured for 22 h on top of the AM-NM-2 burdened tg APdE9 or wild-type mouse brain sections using whole genome microarrays. Quantitative RT-PCR analysis confirmed the significant up-regulation of HtrA serine peptidase 1 (Htra1), metallopeptidase 9 (Mmp9), phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex) and scavenger receptor class A, member 5 (Scara5) in adult astrocytes, whereas neonatal astrocytes up-regulated Mmp9 and down-regulated genes related to cholesterol transport and synthesis: apolipoprotein E (Apoe), 24-dehydrocholesterol reductase (Dhcr24) and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcs1). These findings brought out novel genes which expression is altered during AM-NM-2 clearance in adult and neonatal astrocytes ex vivo. Astrocyte cultures: For the adult astrocyte cultures, hippocampi and cortices were isolated from 6-8-week-old C57Bl/6j wild-type and tg eGFP mice and the tissue was suspended in DMEM/F12 (3:1, Gibco BRL, NY, USA) containing 10 % heat-inactivated fetal bovine serum (Gibco BRL) and 100 U / ml penicillin-streptomycin (Gibco BRL). The cells were treated with Percoll (Sigma-Aldrich, St.Louis, USA) and plated onto poly-L-lysine coated flasks in DMEM/F12 (3:1) containing 10 % heat-inactivated fetal bovine serum, 100 U / ml penicillin-streptomycin and G5 supplement (Gibco BRL). Before the experiments the glial cell cultures were shaken to remove microglia at 200 rpm for 2 h. For the neonatal astrocyte cultures, hippocampi and cortices were isolated from 2-day-old wild-type and eGFP mice according to the protocol described above, except that the culture medium was DMEM (+4500mg/l glucose, + L-glutamine, -pyruvate, Gibco BRL) without G5 supplement and the isolation method included no Percoll treatment. Standard anti-GFAP (1:500; Dako Cytomation, Glostrup, Denmark) immunocytochemistry was used to determine the purity of astrocyte cultures. The cells were incubated with biotinylated goat anti-rabbit IgG (1:200 dilution; Vector Laboratories, CA) secondary antibody and Vectastain ABC Elite kit was used (Vector Laboratories) according to manufacturerM-bM-^@M-^Ys protocol. Hydroxen peroxide (H2O2) and nickel enhanced diaminobenzidine (Ni-DAB) was added to detect the immunoreactivity. Three fields per well and at least three wells per cell type were analyzed for the percentage of Ni-DAB stained cells of all cellular profiles in the field. Adult and neonatal astrocyte cultures both contained on average 99.6 % M-BM-1 0.6 % anti-GFAP immunoreactive astrocytes. Anti-Iba (2 M-BM-5g/ml; Wako, Germany) and anti-CD11b (1:500 dilution; Serotec, UK) antibodies were used to detect possible microglial cells. The cultures contained less than 0.4 % of microglial cells in all experiments. Ex vivo treatments: tg APdE9 / wild-type mouse brain section incubated with neonatal (age 3 d) or adult (age 6-8 wks) mouse eGFP expressing astrocytes (4 x 105 cells / well), n=6. The mouse brain sections used were prepared from tg APdE9 mouse line (created by co-injection of chimeric mouse/human APPSwe and PS1-dE9 vectors, both controlled by their own mouse prion protein promoter element [APdE9 mice (Jankowsky et al. 2004) provided by Prof. Heikki Tanila] and their wild type C57Bl/6j littermates. Before the mRNA isolation process, the samples were pooled (n=3). The purity of the samples was checked with FACS (FACSCalibur, Becton Dickinson, USA) using the analyzing program CellQuestTM version 3.3 (Becton Dickinson, USA). After 22 h incubation the culture medium was removed from the wells and the brain sections with cultured eGFP astrocytes were incubated with 1 x trypsin-EDTA (500 M-BM-5l /well) for up to 15 min at 37M-BM-0 C. Partial detachment (at this point, the astrocytes should not lose the grip completely) of the astrocytes from the underlying brain section was monitored with a standard light microscope. After the incubation the brain sections with cultured astrocytes were carefully removed into small petri dishes filled with FBS-containing culture medium to inactivate the trypsin. Subsequently, the brain sections were immediately examined with fluorescence microscope (Olympus IX-71, Japan). The eGFP astrocytes were separated from the underlying brain section using a special suction hose, which was a modified version from suction hoses typically used for the microinjection techniques. In the other end of the tube there is a pipet tip as a mouthpiece, in the middle of the tube a 0.22 M-BM-5m filter and in the other end a stretched glass capillary (prepared with a Bunsen burner). The green fluorescent eGFP astrocytes were picked up gently from the underlying brain section (without breaking the brain tissue) using wavelength 468 nm of the fluorescence microscope and simultaneous light microscopy. Data normalization and analysis: The raw data was preprocessed with the robust multichip algorithm (Irizarry et al. 2003), normalized per chip to the median. 12 samples were grouped into 4 sample types: adult_wt, adult_tg, neonatal_wt, neonatal_tg. The change in the astrocyte gene expression was identified according to the sections the astrocytes were incubated (WT or TG). The differentially expressed probe sets were further processed using statistical analysis with Welch unpaired t-test assuming unequal variances (p<0.05; fold change > 2.0 or < 0.5). Benjamini-Hochberg false-discovery rate (FDR) correction for multiple testing was used to control the number of false-positives results.
2012-07-13 | E-GEOD-29317 | biostudies-arrayexpress
Project description:Astrocytes are critical for the development and function of synapses. There are notable species differences between human astrocytes and commonly used animal models. Yet, it is unclear whether astrocytic genes involved in synaptic function are stable or exhibit dynamic changes associated with disease states and age in humans, which is a barrier in understanding human astrocyte biology and its potential involvement in neurologic diseases. To better understand the properties of human astrocytes, we acutely purified astrocytes from the cerebral cortices of over 40 humans across various ages, sexes, and disease states. We performed RNA sequencing to generate transcriptomic profiles of these astrocytes and identified genes associated with these biological variables. We found that human astrocytes in tumor-surrounding regions downregulate genes involved in synaptic function and sensing of signals in the microenvironment, suggesting involvement of peritumor astrocytes in tumor-associated neural circuit dysfunction. In aging, we also found downregulation of synaptic regulators and upregulation of markers of cytokine signaling, while in maturation we identified changes in ionic transport with implications for calcium signaling. In addition, we identified subtle sexual dimorphism in human cortical astrocytes, which has implications for observed sex differences across many neurologic disorders. Overall, genes involved in synaptic function exhibit dynamic changes in the peritumor microenvironment and aging. These data provide powerful new insights into human astrocyte biology in several biologically relevant states that will aid in generating novel testable hypotheses about homeostatic and reactive astrocytes in humans.SIGNIFICANCE STATEMENT Astrocytes are an abundant class of cells playing integral roles at synapses. Astrocyte dysfunction is implicated in a variety of human neurologic diseases. Yet our knowledge of astrocytes is largely based on mouse studies. Direct knowledge of human astrocyte biology remains limited. Here, we present transcriptomic profiles of human cortical astrocytes, and we identified molecular differences associated with age, sex, and disease state. We found that peritumor and aging astrocytes downregulate genes involved in astrocyte-synapse interactions. These data provide necessary insight into human astrocyte biology that will improve our understanding of human disease.
| S-EPMC8883850 | biostudies-literature