Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptomic analysis of osteoblastic cells derived from human mesenchymal stem cells, incubated with secreted factors release by adipocytes against non-incubated controls

ABSTRACT: Our preliminary findings lead us to propose bone marrow adipocyte secretions as new actors of bone loss. Indeed, using a coculture model based on human bone marrow stromal cells, we previously showed that soluble factors secreted by adipocytes induced the conversion of osteoblasts towards an adipocyte-like phenotype. To further decipher the molecular changes triggered by the coculture, we performed transcriptional analyses using Agilent 60-mer Sure Print G3 Human Gene Expression Microarray Kit containing a 50, 599 probes. Transcriptional changes were monitored in 7 biological replicates each consisting of osteoblasts grown alone (OB) and cocultured osteoblasts (OB-CC) upon 9h or 48h and adipocytes (AD) at day 14 of differentiation placed in serum-free medium for 9h or 48h.

ORGANISM(S): Homo sapiens

SUBMITTER: ALINE CLABAUT

PROVIDER: E-MTAB-8849 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Transcriptional profiling of MSC, Osteoblasts and U2OS cells. The aim was to quantitate relative gene expression in MSC, osteoblasts and U2OS. MSC and osteoblasts were used as normal cells in this study because osteosarcoma most likely originates from MSC or osteoblasts. Cell culture and treatment: U2OS cells were cultured in McCoys 5A media (Invitrogen) containing 10% FBS. MSC and osteoblasts were cultured as described by Jaiswal et al. (J Cell Biochem 64, 295-312; 1997). Cells were treated with 5 uM 5-Azadeoxycytidine (5-Aza-CdR) or 300 nM Trischostatin-A (TSA) for 96 hours or 18 hours, respectively. Microarray analysis: Total RNA was extracted using the Qiagen kit according to the manufacturer's instructions, including a DNase step. RNA was quantified using the NanoDrop ND-100, followed by quality assessment with the 2100 Bioanalyzer (Agilent Technologies). RNA concentrations for individual samples were greater than 200ng/ul, with 28s/18s ratios greater than 2.2 and RNA integrity numbers of 10 (highest). Sample amplification and labeling procedures were carried out using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) according to the manufacturer's instructions. The labeled cRNA was purified using the RNeasy mini kit (Qiagen) and quantified. RNA spike-in controls (Agilent Technologies) were added to the RNA samples before amplification. 0.75 microgram of samples labeled with Cy3 or Cy5 were mixed with control targets (Agilent Technologies), assembled on Oligo Microarray, hybridized, and processed according to the Agilent microarray protocol. Scanning was performed with the Agilent Scanner G2505B under the default settings recommended by Agilent Technologies. Data analysis: All arrays were subject to the quality checks recommended by the manufacturer. Images were visually inspected for artifacts, and distributions of signal and background intensity of both red and green channels were examined to identify anomalous arrays. No irregularities were observed, and all arrays were retained and used. All calculations were performed using the R statistical computing platform and packages from the Bioconductor bioinformatics software project. To determine the expression levels of genes in MSC, Osteoblasts and U2OS, the data were LOWESS-normalized, the probe intensities of the mock channel (no drug treatment) were extracted and set to the same scale by quantile normalization. Mean of replicate samples were calculated for each probe.

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Transcriptomic analysis of osteoblastic cells derived from human mesenchymal stem cells, incubated with secreted factors release by adipocytes against non-incubated controls

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