Project description:To investigate the regulatory role of MLO-Y4 mitochondria on OA chondrocytes, we isolated mitochondria from MLO-Y4 and transplanted to chondrocytes treated with IL-1β. We then performed gene expression profiling analysis using data obtained from RNA-seq of chondrocytes from vehicle group and mitochondria transplanted group.
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. Keywords: time course, cell type comparison, tissue engineered cartilage; osteoarthritis; Hyaff-11 scaffold; human chondrocytes; gene expression profiling; regenerative medicine; differentiation potential
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:To identify DOT1L targets, associated signaling pathways and networks in chondrocytes, we used genome-wide gene expression microarray analysis in human articular chondrocytes of 5 different donors (without known or documented joint disease) treated with EPZ-5676 or vehicle for 4 days. It is known that DOT1L inhibitors require longer time of treatment in order to show effect and influence the expression of MLL target genes in leukemia cells, but we opted for this relatively short inhibition time to be able to identify early changes induced by DOT1L inhibition. Human articular chondrocytes were obtained from 5 non-OA hip fracture patients. The cells were treated with 3 μM EPZ-5676 or vehicle (DMSO) for 4 days.
Project description:Genome-wide association studies (GWAS) have identified over 100 loci associated with OA risk, but the majority of OA risk variants are non-coding, making it difficult to identify the impacted genes for further study and therapeutic development. To address this need, we used a multi-omic approach and genome editing to identify and functionally characterize putative OA risk genes. Computational analysis of GWAS and ChIP-seq data revealed that chondrocyte regulatory loci are enriched for OA risk variants. Mapping active enhancers in primary human chondrocytes and intersecting them with OA GWAS variants produced a refined list of putative causal variants. Identifying DNA loops in the chondrocyte cell line C28/I2 using in situ Hi-C allowed us to connect those putative causal variants to target genes and revealed SOCS2 as a putative mediator of OA risk. CRISPR-Cas9-mediated deletion of SOCS2 in primary human chondrocytes from three independent donors led to heightened expression of inflammatory markers in response to a cartilage matrix breakdown product of fibronectin. In total, we identified 56 putative OA risk genes—including 20 that are connected to OA risk SNPs via DNA looping—for further research and potential therapeutic development.
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: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:Objective: Assuming that mesenchymal stem cells adapt to the osteoarthritic joint environment to exert a chondroprotective effect, we aimed at investigating the molecular response set up by MSCs after priming by OA chondrocytes in cocultures. Design: We used primary human OA chondrocytes and adipose stem cells (ASCs) in mono- and cocultures and performed a high throughput secretome analysis. Among secreted proteins differentially induced in cocultures, we identified thrombospondin-1 (THBS1) as a potential candidate that could be involved in the chondroprotective effect of ASCs. Results: Secretome analysis revealed significant induction of THBS1in ASCs/chondrocytes cocultures at the mRNA and protein levels. Interestingly, we showed that THBS1 was up-regulated at late stages of MSC chondrogenic differentiation while recombinant THBS1 exerted a prochondrogenic effect on MSC. However, down-regulation of THBS1 in ASCs did not revert OA chondrocyte phenotype by decreasing hypertrophic and inflammatory markers. Nevertheless, down-regulation of THBS1 in ASCs reduced their immunosuppressive activity while recombinant THBS1 exerted an anti-inflammatory role on T lymphocytes. THBS1 function was evaluated in vivo in the collagenase-induced OA (CIOA) model by comparing ASCs expressing siTHBS1 and control ASCs. The OA protective effect of ASCs was reversed when THBS1 was down-regulated in ASCs indicating that THBS1 plays a role in the therapeutic effect of ASCs Conclusions: Our data gather some evidence that THBS1 exerts a pro-chondrogenic and anti-inflammatory function in vitro, which could partially explain a chondroprotective effect of ASCs in OA.
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.
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.