ABSTRACT: To identify factors involved in OPC senescence, we compared gene expressions between OPC-CG4, OPC-FCS and OPC-Rev. We established OPC senescence model system, in which OPC become senescent in the presence of high concentration of FCS. This phenotypes were kept even when the medium was switched to their optimal serum-free medium.
Project description:To identify factors involved in OPC senescence, we compared gene expressions between OPC-CG4, OPC-FCS and OPC-Rev. Overall design: We established OPC senescence model system, in which OPC become senescent in the presence of high concentration of FCS. This phenotypes were kept even when the medium was switched to their optimal serum-free medium.
Project description:To identify factors involved in tumorigenicity of glioma-initiating cells (GICs), we compared gene expression in GIC-like cells with and without sox11 expression. We established sox11-expressing mouse glioma-initiating cell (GIC)-like cell line (NSCL61s), NSCL61s-sox11, which lost tumorigenicity when transplanted in vivo. We think that genes, which are differently expressed between NSCL61s and NSCL61s-sox11, would be new targets for glioma therapy.
Project description:To identify factors involved in glioma-initiating cells (GICs), we compared gene expression between GIC-like cells and non-GICs. Neural stem cells (NSCs) were transfected with pCMS-EGFP-HRasL61 and pBabe-neo by electroporation and cultured in 0.5 mg/ml neomycin. The GFP-positive stable NSCs (NSCL61s) were purified by flow cytometry. Total RNA was prepared using RNeasy Mini Kit (QIAGEN).
Project description:To identify factors involved in glioma-initiating cells (GICs), we compared gene expressions between GIC-like cells and non-GICs. p53-deficient Neural stem cells (NSCs), oligodendrocyte precursor cells (OPCs), and astrocytes (ASTs) were transfected with pCMS-EGFP-HRasL61 and pBabe-neo by electroporation and cultured in 0.5 mg/ml neomycin. The GFP-positive stable NSCs (NSCL61s), OPCs (OPCL61s) and ASTs (ASTL61s) were purified by flow cytometry. Total RNA was prepared using a RNeasy Mini Kit (QIAGEN) and used for the expression analysis.
Project description:To quantitative analysis of transcriptome changes caused by lnc-OPC knockdown during OPC differentiation from NSC, lentivirus-based short hairpin RNAs were used to knockdown the lnc-OPC expression in a neural stem cell culture . Subsequently, puromycin-selected NSCs were differentiated to OPC in culture for three days.RNA-Seq was performed on the polyadenylated fraction of RNA isolated from cell samples. DEseq was used for differential gene expression analysis caused by lnc-OPC knockdown. GO functional term enrichment analysis of differential gene expression caused by lnc-OPC knockdown, revealed significant enrichment of ‘oligodendrocyte development’, ‘oligodendrocyte differentiation’, ‘glia cell development’, and ‘axon ensheathment’ terms that are associated with oligodendrogenesis. mRNA profiles of differentiiated NSC samples after lnc-OPC knockdown by RNA-sequencing.
Project description:Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neuron. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. Overall design: DEG analysis of primary OPC and OL populations, 5 biological replicates per population
Project description:Like neurons, oligodendrocytes (OL) are cells with elaborate morphology that probably require asymmetrical spatial regulation of biological processes. Formation of membrane protrusions is critical for OL development and interaction with axons. We hypothesized that the enrichment of specific mRNAs in protrusions of oligodendrocyte precursor cells (OPC) is important for morphological differentiation, thus having an impact in myelination. To explore this hypothesis, we established a modified Boyden chamber system to physically separate soma from membrane protrusions of rat primary OPC cultured in vitro for 24h. We performed a whole transcriptome analysis (RNAseq) of primary rat OPC soma and membrane protrusion fractions and found a subcellular enrichment of mRNAs in these structures during initial protrusion formation. At the very initial stage of OPC protrusion extension, there is a significant subcellular enrichment of transcripts encoding proteins related to cellular component assembly and cytoskeleton organization, particularly of actin-related molecules. This suggests that the regulation of the cytoskeleton dynamics may be locally controlled in OPCs and probably relevant for their differentiation program. Overall design: Rat OPCs were plated on a modified Boyden chamber system for 24h consisting of a transwell with 1μm pore size with chemo- and haptotactic gradients to physically separate OPC soma and protrusion fractions for RNA sequencing. RNAseq was done on 2 samples for OPC soma and 2 samples of OPC protrusions. Due to very low RNA yields in OPC protrusions samples (typically 9ng per well), each sample consisted in a pool of several experiments.
Project description:In this study we analyzed the myeloma cell contact-mediated changes on the transcriptome of skeletal precursor cells. Therefore, human mesenchymal stem cells (MSC) and osteogenic precursor cells (OPC) were co-cultured with the representative myeloma cell line INA-6 for 24 h. Afterwards, MSC and OPC were separated from INA-6 cells by fluorescence activated cell sorting. Total RNA of MSC and OPC fractions was used for whole genome array analysis. Overall design: Primary MSC from the cancellous bone from the acetabulum received from donors after total hip arthroplasty were isolated as described by Noth et al., 2002 (PMID:12382974) and expanded in DMEM/Ham´s F12 medium including 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 50 µg/ml L-Ascorbic acid 2-phosphate. MSC were passaged at least once before they were used for experiments. Osteogenic precursor cells (OPC) were generated by incubating MSC with DMEM High Glucose medium for two weeks supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mmol/l beta-glycerophosphate, 100 nmol/l dexamethasone, and 50 µg/ml L-Ascorbic acid-2-phosphate. The myeloma cell medium consisted of RPMI 1640 medium, 20% FCS, 100 µg/ml gentamicin, 2 mmol/l L-glutamine, and 1 mmol/l sodium pyruvate. One day before co-culturing, confluent MSC and OPC monolayers were adapted to cell culturing conditions by incubating them with a 1:1 (v/v) mixture of MSC/myeloma cell medium and OPC/myeloma cell medium and INA-6 cells were stained with 5 µmol/l Cell Tracker® Green 5-chloromethylfluorescein diactetate. Afterwards, INA-6 cells were washed with PBS and used for co-culture. After 24 h, cells were trypsinized and separated by fluorescence activated cell sorting (BD FACS AriaTM III cell sorter). The control cells were treated accordingly. Cell pellets of MSC and OPC fractions were lysed in RA1 buffer containing 1% 2-mercaptoethanol and stored at -80°C until total RNA extraction. Total RNA was isolated with the NucleoSpin® RNA II kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) according to the manufacturer´s instructions. The whole genome array analysis was performed using the Affymetrix HG-U133 Plus 2.0 GeneChips (Affymetrix, High Wycombe, United Kingdom). Each 5 control specimens and 5 co-cultured samples of both MSC and OPC were used for hybridization.