RNaseq of primary human OA chondrocytes alones or in transwell cocultures with primary human Adipose-derived Mesenchymal Stromal Cells, in presence of 10nM rapamycin or DMSO as vehicle control
Ontology highlight
ABSTRACT: 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 joint condition associated with articular cartilage loss, low-grade synovitis and alterations in subchondral bone and periarticular tissues. In OA, the interest for mesenchymal stem cell (MSC)-EV therapeutic applications has increased. We have assessed the immunomodulary properties of adipose-derived MSCs (AD-MSCs) microvesicles (MV) and exosomes (EX) in interleukin (IL)-1β stimulated OA chondrocytes and cartilage explants and characterized them by mass spectrometry in order to uncover novel mediators in (AD-MSC)-EV immunomodulation.
Project description:Examination of the genome-wide distribution of 5hmC in osteoarthritic chondrocytes compared to normal chondrocytes in order to elucidate the effect on OA-specific gene expression. 5hmC-sequencing was performed and data was compared with microarray gene expression data to identify genes with differential expression between normal and OA chondrocytes that are potentially under epigenetic regulation. High-throughput sequencing of 5hmC in 4 normal and 4 OA chondrocyte samples.
Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, Ahlbäck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Experiment Overall Design: Gene expression profiles of monolayer cultures (ML; passage 2) and Hyaff-11 scaffold cultures (3D; 14 days in vitro) of chondrocytes from 3 normal donors (ND; underwent ACT treatment) and 3 donors suffering from Osteoarthritis (OA; underwent knee replacement surgery) were determined. Comparative analyses between 3D and ML cultures (3D vs. ML) were performed to assess differentiation capacity of ND and OA chondrocytes. Furthermore, OA-related differences were determined comparing OA and ND monolayers as well as scaffold cultures (each OA vs. ND).
Project description:Primary human OA chondrocytes placed in micropellets for 7 days in order to in vitro reconstituate cartilage tissue. Then cells are treated or not with IL1b for 5 days.
Project description:Examination of the genome-wide distribution of 5hmC in osteoarthritic chondrocytes compared to normal chondrocytes in order to elucidate the effect on OA-specific gene expression. 5hmC-sequencing was performed and data was compared with microarray gene expression data to identify genes with differential expression between normal and OA chondrocytes that are potentially under epigenetic regulation. Gene expression patterns were examined by comparing the 5 normal samples to the 2 OA samples to assess the changing expression profiles between normal and OA chondrocytes. We analyzed the changes in gene expression in OA; genes with a fold-change ≥ or ≤1.5 or 1.2, with a difference in intensity of >100 and within the lower 90% confidence bound, were selected.
Project description:We used chondrocytes exposed to osteoarthritic synovial fluid as a chronic disease model to study the effects of a chronic disease microenvironment on 2’-O-me rRNA heterogeneity. Human non-OA human articular chondrocytes (5 individual donors) were cultured either in the presence of OA-SF (20% (v/v), pool of 14 donors) or in a normal culture medium and were refreshed every other day. After 14 days of culture, RNA was isolated for 2’-O-methylation profiling of ribosomal RNAs.
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:Long non-coding RNAs (lncRNAs) play pivotal roles in diseases such as osteoarthritis (OA). However, knowledge of the biological roles of lncRNAs is limited in OA. We aimed to explore the biological function and molecular mechanism of HOTTIP in chondrogenesis and cartilage degradation. We used the human mesenchymal stem cell (MSC) model of chondrogenesis, in parallel with, tissue biopsies from normal and OA cartilage to detect HOTTIP, CCL3, and miR-455-3p expression in vitro. Biological interactions between HOTTIP and miR-455-3p were determined by RNA silencing and overexpression in vitro. We evaluated the effect of HOTTIP on chondrogenesis and degeneration, and its regulation of miR-455-3p via competing endogenous RNA (ceRNA). Our in vitro ceRNA findings were further confirmed within animal models in vivo. Mechanisms of ceRNAs were determined by bioinformatic analysis, a luciferase reporter system, RNA pull-down, and RNA immunoprecipitation (RIP) assays. We found reduced miR-455-3p expression and significantly upregulated lncRNA HOTTIP and CCL3 expression in OA cartilage tissues and chondrocytes. The expression of HOTTIP and CCL3 was increased in chondrocytes treated with interleukin-1β (IL-1β) in vitro. Knockdown of HOTTIP promoted cartilage-specific gene expression and suppressed CCL3. Conversely, HOTTIP overexpression reduced cartilage-specific genes and increased CCL3. Notably, HOTTIP negatively regulated miR-455-3p and increased CCL3 levels in human primary chondrocytes. Mechanistic investigations indicated that HOTTIP functioned as ceRNA for miR-455-3p enhanced CCL3 expression. Taken together, the ceRNA regulatory network of HOTTIP/miR-455-3p/CCL3 plays a critical role in OA pathogenesis and suggests HOTTIP is a potential target in OA therapy.
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.
Project description:In order to study the impact of rapamycin on AD-MSC under inflammatory or resting conditions, primary human Adipose-derived Mesenchymal Stromal Cells (AD-MSC) at passage 1 were plated at 1.5x10^4 in MEM-alpha, 5% Human Platelet Lysate, 1% Penicillin-Streptomycin and allowed to stand for 24h. The next day, AD-MSC were stimulated for 2 days (or not) with 20ng/mL IFN-gamma in presence of 10nM rapamycin (from 10.000X stock in DMSO) or DMSO (at 1:10000 dilution) as vehicle control. After 2 days, media were removed and RNA were extracted using RNeasy kit from QIAGEN with an on-column DNAse I digestion as manufacturer's instructions. RNA intergrity number were estimated on Agilent Bioanalyzer and were all above 9 before sequencing. Libraries and RNA sequencing were performed by the Beijing Genomics Institute (BGI)