Transcription profiling by array to investigate dose-dependent effects of human recombinant CCL25 on porcine chondrocytes isolated from 3D micromasses
Ontology highlight
ABSTRACT: Porcine chondroytes were stimulated with human recombinant CCL25 for 7 days with different concentrations (two samples). To mimic osteoarthritic like conditions, we also added TNF-alpha (two samples). Three samples were used as control samples. It is the first time, that chondrocytes were stimulated with CCL25 in order to find a save dose range for further experiments.
Project description:To study chondrogenesis, we used a chicken limb bud model: We used RNA sequencing, and examined the differences between gene expression patterns during cartilage formation in micromass cultures of embryonic limb bud-derived progenitors. We sequenced in triplicate at Day 0,1,2,3,4,6,10,15 and also from mature birds.
Project description:In mammals, temporally coordinated daily rhythms of behaviour and physiology are generated by a multi-oscillatory circadian system, entrained through cyclic environmental cues (e.g. light). Presence of niche-dependent physiological time cues have been proposed to allow local tissues flexibility of adopting a different phase relationship if circumstances require. Up till now, such tissue-unique stimuli have remained elusive. Here we show that cycles of mechanical loading and osmotic stimuli within physiological range drive rhythmic expression of clock genes and reset clock phase and amplitude in cartilage and intervertebral disc tissues.
Project description:The aim of the current study was to identify molecular markers for articular cartilage that can be used for the quality control of tissue engineered cartilage. Therefore a genom-wide expression analysis was performed using RNA isolated from articular and growth plate cartilage, both extracted from the knee joints of minipigs. Keywords: Native material or primary cells isolated from articular cartilage and growth plate cartilage Articular and growth plate cartilage were taken for RNA extraction and hybridization on Affymetrix microarrays. Furthermore chondrocytes from each type of cartilage were isolated and cell culture was started and terminated at day 10 or day 20. Total RNA from cultivated cells was extracted, and hybridization on Affymetrix microarrays was performed.
Project description:Responses of human adult articular chondrocytes under conditions of lack or excess of bone morphogenetic protein-7 also called osteogenic protein 1. Original processed data file is in E-MTAB-571.additional.zip archive.
Project description:Osteoarthritis is a degenerative joint disease that ranks among the leading causes of pain, adult disability, shortening of working life, and socioeconomic costs worldwide. The mechanisms underlying osteoarthritis pathogenesis are yet to be fully elucidated, thus limiting current disease management and treatment. Galectin-1 is an endogenous carbohydrate-binding protein central to adhesion via glycan-bridging, glycoconjugate-mediated signaling, cell proliferation, differentiation, apoptosis, cancers, and host-pathogen interactions. The chondrocyte glycophenotype, which can act as a system of counter-receptors for galectin binding, is compromised in osteoarthritis. We here investigated Galectin-1 and associated gene network's role in human osteoarthritis pathogenesis. Immunohistochemical analysis showed that Galectin-1 associates with osteoarthritic cartilage and subchondral bone histopathology and severity (p<0.0001, n=29 patients). Glycan-dependent Galectin-1 binding to osteoarthritic chondrocytes' cell surface led to marked upregulation of matrix metalloproteinases and activation of NF-κB. Biochemical, molecular and genome-wide analyses showed that Galectin-1 strongly activates a large inflammatory gene network (p<10-16). Bioinformatic analyses of gene promoters up-regulated by Galectin-1 unveiled an overwhelming NF-κB signaling signature. Inhibition of any of several components of the NF-κB pathway using dedicated inhibitors led to dose-dependent impairment of Galectin-1-mediated transcriptional activation. This study identifies for the first time Galectin-1 as an activator of clinically relevant inflammatory-response genes co-regulated by NF-κB. Since inflammation is critical to cartilage degeneration in osteoarthritis, this report is also first to put glycobiology at the center-stage of osteoarthritic cartilage degeneration. Finally, this is the first report to uncover a Galectin-1 gene signature and associated gene network in any biological setting or species. For microarray experiments, osteoarthritic chondrocytes were isolated from five male patients (47-78 years). Following starvation, cells were incubated in the presence of 50 µg/ml recombinant Galectin-1 for 24 h. For each donor population, untreated cells were included as control. In total, 10 samples were analyzed.
Project description:Polycomb repressive complex 2 (PRC2) controls maintenance and lineage determination of stem cells by suppressing genes that regulate cellular differentiation and tissue development. However, the role of PRC2 in lineage-committed somatic cells is largely unknown. In this study, we ablated Eed, an essential component of PRC2, in growth plate chondrocytes to study the role of PRC2 in skeletal development. In this study, we profiled genes assocaited with H3K27me3 histone 3 modification in primary rib chondrocytes. Mouse primary chodnrocytes were subjected to ChIP analysis using H3K27me3 antibody. ChIP'ed DNA and Input DNA were sequenced by Otogenetics (Necros, GA)
Project description:We have previously demonstrated that a mixture of curcuminoids extract, hydrolyzed collagen and green tea extract (COT) inhibited inflammatory and catabolic mediatorâ??s synthesis by osteoarthritic (OA) human chondrocytes. The objectives of this study were to identify new targets of COT using genomic approaches. We compared gene expression profiles of chondrocytes treated with COT and/or with interleukin(IL)-1β. The proteins coded by the most important COT sensitive genes were then quantified by specific immunoassays. Cartilage specimens were obtained from 12 patients (10 women and 2 men; mean age 67 years old, range 54-76 years old) with knee OA. Primary human chondrocytes were cultured in monolayer until confluence and then incubated for 24 hours in the absence or in the presence of human IL-1β (10e-11M) and with or without COT, each compound at the concentration of 4 µg/ml. Microarray gene expression profiling between control, COT, IL-1β and COT IL-1β conditions was performed.
Project description:BBF2H7 (BBF2 human homolog on chromosome 7), an ER-resident basic leucine zipper transcription factor, is activated in response to ER stress and abundantly expresses in chondrocytes. While BBF2H7 is widely expressed in many tissues and organs, the most intense signals were detected in the proliferating zone of the cartilage. We compared gene expressions in primary cultured chondrocytes prepared from rib cartilage between WT and BBF2H7-/- mice at E18.5. Primary cultured chondrocytes were prepared from E18.5 rib cartilage of WT and BBF2H7-/- mice. Chondrocytes were isolated using 0.2% collagenase D (Roche) after adherent connective tissue was removed by 0.2% trypsin (Sigma) and collagenase pretreatment. Isolated chondrocytes were maintained in α-MEM (Gibco) supplemented with 10% FCS and 50 µg/mL ascorbic acid. Adenovirus vectors expressing the mouse p60 BBF2H7 (1-377 aa, BBF-N) were constructed with the AdenoX Expression system (Clontech), according to the manufacturerâs protocol. The cells were infected with adenoviruses 30 h before analysis. We compared gene expressions in primary cultured chondrocytes prepared from rib cartilage between WT and BBF2H7-/- mice at E18.5 using a microarray and various genes associated with protein secretory pathway and ER biogenesis were significantly down-regulated in BBF2H7-/- chondrocytes. We infected primary cultured chondrocytes prepared from BBF2H7-/- mice with adenovirus expressing p60 BBF2H7. Several genes were up-regulated and we picked up them as the direct target of BBF2H7.
Project description:The objective was to study the combined effects of rapamycin and primary human Adipose-derived Mesenchymal Stromal Cells (AD-MSC) on primary human OA chondrocytes (versus their effects separetely), in an in-vitro model that reproduce an intra-articular injection. For this purpose, P1 OA chondrocytes were seeded in 6-well plates (5x10^5 cells/well) in their proliferative medium for 24h. In parallel, P1 AD-MSC were seeded in 0.4 µm inserts (7.2x10^4) in their proliferative medium for 24h. The next days, all media were removed, cells were washed twice with PBS and media were replaced by miminal chondrogenic medium. Finally, OA chondrocytes were cocultured with AD-MSC (versus alone) in presence of 10nM rapamycin or DMSO (at 1:10000 dilution) as vehicle control for 3 days. At the end, media and inserts were removed and RNA of OA chondrocytes were extracted using RNeasy kit from QIAGEN with an on-column DNase I digestion as manufacturer's instructions. Because of poor quality RNA, one sample (25_DMSO_AD10) and his control (25_RA10_AD10) have been excluded from this analysis. Otherwise, all RNA integrity numbers were above 9 before libraries construction.
Project description:Osteoarthritis (OA) is a degenerative joint disease with a substantial health economic burden. There is no current treatment; instead, disease management targets the main symptoms (pain and stiffness) and culminates in joint replacement surgery. OA is a disease of cartilage degeneration, but the molecular changes leading to the development of OA are still poorly understood. In this study we compare methylation, gene transcription and protein abundance at the genome-wide level in individually-matched samples of chondrocytes extracted from affected and relatively healthy articular cartilage across 12 OA patients undergoing total knee replacement. Integration analysis highlights genes that are consistently affected at multiple levels, including AQP1, CLEC3B and COL1A1, and also relevant biological pathways such as extracellular matrix organization, collagen catabolism and proteolysis. Collectively these results provide a first view of the comprehensive molecular landscape underpinning OA development and point to potential therapeutic avenues.