Direct reprogramming of human astrocytes into neural stem cells and neurons
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Stefania Corti
PROVIDER: E-GEOD-36145 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2013-01-05 | E-GEOD-42908 | biostudies-arrayexpress
Project description:Cancer stem cells play key roles in human malignancies. However, their cellular and molecular origins are still poorly understood. Here we report the successful generation of cancer stem cells from primary human astrocytes by use of defined genetic factors. Combined transduction of four factors âmyc, Oct-4, p53DD, and rasâ induced efficient transformation of primary human astrocytes into malignant cells with powerful tumor-initiating capabilities. Transplantation of 100 of the cells into nude mice is sufficient for tumor formation. The cells also showed unlimited self-renewal ability with robust telomerase activities. In addition, they expressed typical glioma stem cell markers such as CD133, CD15, and CD90. Moreover, transformed cells could form spheres in culture and differentiate into neuron-astrocyte-, and oligodendrocyte-like cells. They also displayed significant resistance to temozolomide. These induced cancer stem cells (iCSCs) should be useful for both tumor biology studies and therapeutics development. This record contains data from microarray analysis of normal human astrocytes, two transduced cell lines (OCT4+3G and OCT4+6G), a patient-derived glioma stem cell line (ALPS1459) and a glioblastoma cell line (U87MG).
2016-11-07 | E-MTAB-4771 | biostudies-arrayexpress
Project description:Direct cell reprogramming has enabled the direct conversion of skin fibroblasts into functional neurons and oligodendrocytes using a minimal set of cell lineage-specific transcription factors. This approach has substantial advantages since it is rapid and simple, generating the cell type of interest in a single step. However, it remains unknown whether this technology can be applied for directly reprogramming skin cells into astrocytes, the third neural lineage. Astrocytes play crucial roles in neuronal homeostasis and their dysfunctions contribute to the origin and progression of multiple human diseases. Herein, we carried out a screening using several transcription factors involved in defining the astroglial cell fate and identified NFIA, NFIB and SOX9 to be sufficient to convert with high efficiency embryonic and post-natal mouse fibroblasts into astrocytes (iAstrocytes). We proved both by gene expression profiling and functional tests that iAstrocytes are comparable to native brain astrocytes. This protocol can be then employed to generate functional iAstrocytes for a wide range of experimental applications. Induced astrocytes (iAstro) were compared to Fibroblasts (Fibro) as negative control and to primary astrocytes (astro) as positive control. Three biological replicates were analyzed for each experimental group.
2015-06-22 | E-GEOD-59018 | biostudies-arrayexpress
Project description:It remains controversial whether the routes from differentiated cells to iPSCs are related to the reverse order of normal developmental processes or independent of them. Here, we generated iPSCs from mouse astrocytes by three (Oct3/4, Klf4 and Sox2 (OKS)), two (OK), or four (OKS plus c-Myc) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently upregulated during reprogramming and Sox1-positive cells become iPSCs. The upregulation of Sox1 is essential for OK-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during MEF reprogramming, neither Sox1 upregulation nor an increase in neurogenic potential occurs. Thus, astrocytes are reprogrammed through an NSC-like state, suggesting that reprogramming partially follows the retrograde pathway of normal developmental processes. To investigate the gene expression profile of intermediate-state cells during astrocyte reprogramming, we performed genome-wide gene expression analysis in five samples; starting astrocytes, intermediate-state cells expressing Sox1-GFP, NSCs, iPSCs established from astrocytes, and iPSCs established from MEFs (iPS-MEF-Ng-20D-17) that had previously been reported (Okita, K. et al. Nature 448: 313-317 (2007)). Two (NSCs, iPSCs from astrocytes and MEFs) or three (astrocytes, intermediate-state cells) biological replicates were prepared for microarray samples. Total RNA was extracted with an RNeasy kit (Qiagen). cDNA synthesis and transcriptional amplification were performed using 50-100 ng of total RNA with the GeneChip WT PLUS Reagent Kit (Affymetrix). Fragmented and biotin-labeled cDNA targets were hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix) according to the manufacturerâ??s protocol. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner.
2015-11-06 | E-GEOD-69237 | biostudies-arrayexpress
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). 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