Comparison of primary vs immortalized HUVEC
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PROVIDER: PRJNA432724 | ENA |
REPOSITORIES: ENA
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Project description:Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors and species. Cell type specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research and tailored drug development. In these experiments primary HUVEC were compared to immortalized HUVEC with respect to their global gene expression pattern.
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Project description:Fundamental research and drug development for personalized medicine necessitates cell cultures from defined genetic backgrounds. However, providing sufficient numbers of authentic cells from individuals poses a challenge. Here, we present a new strategy for rapid cell expansion that overcomes current limitations. Using a small gene library, we expanded primary cells from different tissues, donors and species. Cell type specific regimens that allow the reproducible creation of cell lines were identified. In depth characterization of a series of endothelial and hepatocytic cell lines confirmed phenotypic stability and functionality. Applying this technology enables rapid, efficient and reliable production of unlimited numbers of personalized cells. As such, these cell systems support mechanistic studies, epidemiological research and tailored drug development. In these experiments primary murine hepatocytes were compared to immortalized murine hepatocytes with respect to their global gene expression pattern.
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Project description:Rationale: Immortalized cells may exhibit important differences relative to their primary cell counterparts. Microarrays were used compare primary human umbilical vein endothelial cells (HUVECs) and the immortalized HUVEC cell line EA.hy926, in their response to inhibition of the mevalonate pathway by a HMG-CoA reductase inhibitor (atorvastatin). The effects of atorvastatin were reversed by the addition of mevalonate, to subtract non-specific changes in gene expression. Methods: Confluent cell cultures of HUVECs and EA.hy926 cells (two independent experiments in each cell type) were incubated with 1) statin-free media; 2) 10 microM atorvastatin; 3) 500 microM mevalonate; or 4) a combination of 10 microM atorvastatin and 500 microM mevalonate. All cells were harvested at 24 h. Total RNA was isolated using Trizol reagent (Invitrogen). cDNA was prepared from 10 microgram total RNA using a double-stranded cDNA synthesis kit (Life Technologies) with a T7-dT24 primer for first-strand synthesis. cRNA was synthesized from cDNA and biotinylated using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics). Twenty microgram of cRNA was fragmented by heating at 94°C for 35 min in fragmentation buffer, containing 40 mM Tris-acetate, pH 8.1, 125 mM KOAc, 30 mM MgOAc. Fifteen micrgram of fragmented cRNA, together with control cRNAs and grid alignment oligonucleotides, were hybridized overnight to GeneChip Human Genome U133A 2.0 arrays (Affymetrix) at 45°C under constant rotation. Arrays were washed and incubated with an anti-streptavidin antibody. Fluorescent signals were measured using an Agilent Gene Array Laser Scanner and analyzed with MicroArray Suite 5.0 (Affymetrix). Microarrays were scaled using default settings.
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Project description:Comparison of mRNA expression in human EPC vs. HUVEC vs. human monocytes. Cell-type specific gene expression under basal cell culture conditions (no stimulation). The hybridization was performed with three samples of EPC vs. three samples of HUVEC vs. 3 samples of CD14+ monocytes. • The origin of the biological sample: Human endothelial progenitor cells (EPC): EPC were ex vivo cultivated from human peripheral blood-derived mononuclear cells (PBMC). PBMC were isolated by density gradient centrifugation from healthy human volunteers as previously described (Dimmeler et al., 2001). Pooled human umbilical vein endothelial cells (HUVEC) were purchased from Cambrex (Verviers, Belgium). CD14+ monocytes were purified from PBMC by positive selection with anti-CD14-microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Purity assessed by FACS analysis was greater than 95%. • Manipulation of biological samples and protocols used: for example, growth conditions, treatments, separation techniques: EPC: 8000000 PBMC/ml were plated on human fibronectin (Sigma, Taufkirchen, Germany) and maintained in endothelial basal medium (Cambrex) with EGM SingleQuots and 20% fetal calf serum (FCS). After 3 days, nonadherent cells were removed and adherent cells were incubated in fresh medium for 24 h before starting experiments. HUVEC: HUVEC were cultured in endothelial basal medium (Cambrex) supplemented with hydrocortisone, bovine brain extract, gentamicin, amphotericin B, epidermal growth factor, and 10% FCS until the third passage according to the manufacturer’s recommendations. CD14+ monocytes: No culture. • Protocol for preparing the hybridization extract: for example, the RNA or DNA extraction and purification protocol. Total RNA was extracted from EPC, HUVEC, and CD14+ monocytes using the RNeasy cleanup system (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Quantity and quality of total RNA was analyzed by the 2100 Bioanalyzer system (Agilent Technologies, Waldbronn, Germany) and agarose gel electrophoresis. • Labeling protocol(s): The detailed protocol for the sample preparation and microarray processing is available from Affymetrix (Santa Clara, CA). Briefly, 10 µg of purified total RNA was reverse transcribed by Superscript II reverse transcriptase (Life Technologies, Grand Island, NY) using T7-(dT)24 primer containing a T7 RNA polymerase promoter. After synthesis of the second complementary DNA (cDNA) strand, this product was used in an in vitro transcription reaction to generate biotinylated complementary RNA (cRNA). • The protocol and conditions used during hybridization, blocking and washing: Fifteen micrograms of fragmented, biotinylated cRNA were hybridized to a HG-U95Av2 microarray (Affymetrix Inc.) for 16 hours at 45° C with constant rotation at 60 rpm. This high-density oligonucleotide array targets 9,670 human genes as selected from the National Center for Biotechnology Information (NCBI) Gene Bank database with a total of 12,000 oligonucleotide sets. Each microarray was used to assay a single sample. After hybridization, the microarray was washed and stained on an Affymetrix fluidics station and scanned with an argon-ion confocal laser, with a 488 nm emission and detection at 570 nm. • GeneChip image analysis was performed using the Microarray Analysis Suite 5.0 (Affymetrix, Inc.). Expression data were analyzed utilizing the GeneSpring™ software version 4.2 (Silicon Genetics Inc., San Carlos, CA). Keywords: parallel sample
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Project description:We used microarrays to detail the global gene expression of primary RPE and immortalized RPE. We investigated highly expression genes in immortalized RPE.
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