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Regularity of Chondrocyte Phenotypic Changes Identified by Single-cell Sequencing in vitro

ABSTRACT: Regularity of Chondrocyte Phenotypic Changes Identified by Single-cell Sequencing in vitro

PROVIDER: PRJNA1127617 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29528876_1.fastq.gz	Fastqsanger.gz
	SRR29528876_2.fastq.gz	Fastqsanger.gz
	SRR29528877_1.fastq.gz	Fastqsanger.gz
	SRR29528877_2.fastq.gz	Fastqsanger.gz
	SRR29528878_1.fastq.gz	Fastqsanger.gz

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Regularity of Chondrocyte Phenotypic Changes Identified by Single-cell Sequencing in vitro

Project description:The specific mechanism of phenotypic change of chondrocytes in vitro remains unclear. We performed single-cell sequencing of P1,P3, and P6 cells in vitro to explore the mechanisms involved.

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Changes in DNA methylation accompany changes in gene expression during chondrocyte hypertrophic differentiation in vitro.

Project description:BACKGROUND: During osteoarthritis (OA), articular chondrocytes undergo phenotypic changes that resemble developmental patterns characteristic of growth plate chondrocytes. These phenotypic alterations lead to a hypertrophy-like phenotype characterized by altered production of extracellular matrix constituents and increased collagenase activity, which in turn results in cartilage destruction in OA disease. PURPOSE: To identify transcriptomic and epigenomic changes in murine primary articular chondrocytes undergoing hypertrophy-like differentiation in vitro. METHODS: We paired an in vitro 3D/pellet culture system that mimics chondrocyte hypertrophy with RNA sequencing (RNA-Seq) and enhanced reduced representation of bisulfite sequencing (ERRBS). RESULTS: We identified hypertrophy-associated changes in DNA methylation patterns in vitro. Integration of RNA-Seq and ERRBS datasets identified associations between changes in methylation and gene expression. CONCLUSION: Our integrative analyses showed that hypertrophic differentiation of articular chondrocytes is accompanied by transcriptomic and epigenomic changes in vitro.

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Changes in DNA methylation accompany changes in gene expression during chondrocyte hypertrophic differentiation in vitro.

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Project description:Cartilage is characterized by mature chondrocyte metabolic heterogeneity in a zone-dependent manner. In this study, we used single cell mRNA sequencing (scRNA-seq) to evaluate the corresponding functional and metabolic phenotypic heterogeneity of in vitro cultured primary juvenile murine chondrocytes

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Project description:Autologous chondrocyte implantation (ACI) is an effective method to treat chronic articular cartilage injury in recent years, which requires a large number of human hyaline chondrocytes. Unfortunately, human hyaline chondrocytes often undergo dedifferentiation in vitro. Thus, it is important to elucidate the mechanism of dedifferentiation for the application of ACI technology. Long noncoding RNAs (lncRNA) play a regulatory role in gene expression in many pathological and physiological processes. However, the role of lncRNAs in human hyaline chondrocyte dedifferentiation remains unclear. The aim of this study was to investigate the expression profiles of lncRNAs in human hyaline chondrocyte dedifferentiation during in vitro culture. First, we cultured human hyaline chondrocytes in vitro and detected the expression of COL1A1, COL2A1, and SOX-9 in passage 1 (P1) and 5 (P5) chondrocytes using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting. Then, we analyzed the expression profiles of lncRNAs and mRNAs in P1 and P5 chondrocytes by microarray analysis.

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AP-1 family members act at DNA targets in conjunction with Sox9 to promote chondrocyte hypertrophy

Project description:A comprehensive analysis of Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif (Ohba et al. 2015). Here, we have explored the functional interplay between Sox9 and AP-1 in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun- and Sox9-bound regions throughout the chondrocyte genome, a reflection of direct binding of each factor to target motifs in shared enhancers, and physical interactions of AP-1 with Sox9. In vitro expression analysis indicates that direct co-binding of Sox9 and AP-1 at target motifs enhanced target gene expression, while protein-protein interactions suppressed AP-1- and Sox9-driven transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 demonstrated Sox9 was essential for hypertrophic chondrocyte development, while in vitro and ex vivo analyses showed AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model where AP-1-family members promote Sox9-action in the transition of chondrocytes to a terminal hypertrophic program. Intersection of ChIP-seq data from Sox9 and AP-1 factor Jun, RNA-seq data from developing rib chondrocytes and Col10a1mCherry positive hypertrophic chondrocytes in neonatal mice to uncover regulation of Sox9 by AP-1 factors during chondrocyte hypertrophy.

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