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Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome [run92]

ABSTRACT: The isolation of chondrocytes from human articular cartilage for single-cell RNA sequencing requires extensive and prolonged tissue digestion at 37 °C. Modulations of the transcriptional activity likely take place during this period such that the transcriptomes of isolated human chondrocytes no longer match their original status in vivo. Here, we optimized the human chondrocyte isolation procedure to maximally preserve the in vivo transcriptome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE217870 | GEO | 2022/11/15

REPOSITORIES: GEO

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Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome [run54]

Project description:The isolation of chondrocytes from human articular cartilage for single-cell RNA sequencing requires extensive and prolonged tissue digestion at 37 °C. Modulations of the transcriptional activity likely take place during this period such that the transcriptomes of isolated human chondrocytes no longer match their original status in vivo. Here, we optimized the human chondrocyte isolation procedure to maximally preserve the in vivo transcriptome.

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Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome

Project description:Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome

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Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome [run54]

Project description:Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome [run54]

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Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome [run92]

Project description:Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome [run92]

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DNA methyltransferase 3b regulates articular cartilage homeostasis by altering chondrocyte metabolism

Project description:Osteoarthritis (OA) is the most common form of arthritis worldwide. It is a complex disease affecting the whole joint but is generally characterized by progressive degradation of articular cartilage. Recent genome-wide association screens have implicated distinct DNA methylation signatures in OA patients. We show that the de novo DNA methyltransferase (Dnmt) 3b, but not Dnmt3a, is present in healthy murine and human articular chondrocytes and expression decreases in OA mouse models and in chondrocytes from human OA patients. Targeted deletion of Dnmt3b in murine articular chondrocytes results in an early onset and progressive post-natal OA-like pathology. RNA-seq and MethylC-seq analyses of Dnmt3b loss-of-function chondrocytes shows that cellular metabolic processes are affected. Specifically, TCA metabolites and mitochondrial respiration are elevated. Importantly, a chondroprotective effect was found following Dnmt3b gain-of-function in murine articular chondrocytes in vitro and in vivo. This study shows that Dnmt3b plays a significant role in regulating post-natal articular cartilage homeostasis. Cellular pathways regulated by Dnmt3b in chondrocytes may provide novel targets for therapeutic approaches to treat OA.

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Comparison between articular chondrocytes and mesenchymal stromal cells for the production of articular chondrocyte implants

Project description:Focal lesions of articular cartilage give rise to pain and reduced joint function and may, if left untreated, lead to osteoarthritis. Implantation of in vitro generated, scaffold-free autologous cartilage discs may represent the best treatment option. Here we compare articular chondrocytes (ACs) and bone marrow-derived mesenchymal stromal cells (MSCs) for their ability to make scaffold-free cartilage discs.

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Involvement of ERK, Wnt and BMP2 signaling in human articular chondrocyte dedifferentiation in monolayer culture

Project description:Objective: When primary chondrocytes are cultured in monolayer, they undergo dedifferentiation during which they lose their phenotype and their capacity to form cartilage. Dedifferentiation is an obstacle for cell therapy for cartilage degeneration. In this study, we aimed to systemically evaluate the changes in gene expression during dedifferentiation of human articular chondrocytes to identify underlying mechanisms. Methods: RNA was isolated from monolayer-cultured primary human articular chondrocytes at serial passages. Gene expression was analyzed by microarray. Based on the microarray analysis, relevant genes and pathways were identified. Their functions in chondrocyte dedifferentiation were further investigated in detail. Results: In vitro expanded human chondrocytes showed progressive changes in gene expression during dedifferentiation. Strikingly, an overall decrease in total gene expression was detected. Genes in the Wnt and BMP pathways exhibited significant changes in expression. The non-canonical rather than the canonical Wnt pathway was found to be involved in the loss of collagen II synthesis. BMP2 was able to decelerate the dedifferentiation and reinforce the maintenance of chondrocyte phenotype in monolayer culture. DNA methylation was in part responsible for the expression downregulation of a set of genes. Conclusion: Our study revealed the roles of ERK, Wnt and BMP pathways as well as DNA methylation in chondrocyte dedifferentiation in monolayer culture. RNA human chondrocytes were allowed to dedifferentiate for 8 passages. RNA was isolated at week 0, 2 ,4, 6 and 8, which was subjected to whole genome gene expression analysis.

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Identification of chondrocyte subsets during human development

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Optimization of chondrocyte isolation from human articular cartilage to preserve the chondrocyte transcriptome

Project description:This SuperSeries is composed of the SubSeries listed below.

2022-11-15 | GSE217871 | GEO

DNA methylation profiling in chondrocyte dedifferentiation in vitro

Project description:Genome wide DNA methylation profiling of different passages of human articular chondrocytes treated with 5-azacytidine. The Illumina Infinium 450k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,578 CpGs.Samples included 6 different passages of human articular chondrocytes with 5-azacytidine treatment, 6 different passages without 5-azacytidine treatment, Bisulphite converted DNA from the 12 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip v1.2

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