Project description:Cartilage tissue is comprised of extracellular matrix and chondrocytes, a cell type with very low cellular turnover in adults, providing limited capacity for regeneration. However, in development a significant number of chondrocytes actively proliferate and remodel the surrounding matrix. Uncoupling the microenvironmental influences that determine the balance between clonogenic potential and terminal differentiation of these cells is essential for the development of novel approaches for cartilage regeneration. Unfortunately, most of the existing methods are not applicable for the analysis of functional properties of chondrocytes at a single cell resolution. Here we demonstrate that a novel 3D culture method provides a long-term and permissive in vitro niche that selects for highly clonogenic, colony-forming chondrocytes which maintain cartilage-specific matrix production, thus recapitulating the in vivo niche. As a proof of concept, clonogenicity of Sox9IRES-EGFP mouse chondrocytes is almost exclusively found in the highest GFP+ fraction known to be enriched for chondrocyte progenitor cells. Although clonogenic chondrocytes are very rare in adult cartilage, we have optimized this system to support large, single cell-derived chondrogenic organoids with complex zonal architecture and robust chondrogenic phenotype from adult pig and human articular chondrocytes. Moreover, we have demonstrated that growth trajectory and matrix biosynthesis in these organoids respond to a pro-inflammatory environment. This culture method offers a robust, defined and controllable system that can be further used to interrogate the effects of various microenvironmental signals on chondrocytes, providing a high throughput platform to assess genetic and environmental factors in development and disease.
Project description:Cartilage tissue is comprised of extracellular matrix and chondrocytes, a cell type with very low cellular turnover in adults, providing limited capacity for regeneration. However, in development a significant number of chondrocytes actively proliferate and remodel the surrounding matrix. Uncoupling the microenvironmental influences that determine the balance between clonogenic potential and terminal differentiation of these cells is essential for the development of novel approaches for cartilage regeneration. Unfortunately, most of the existing methods are not applicable for the analysis of functional properties of chondrocytes at a single cell resolution. Here we demonstrate that a novel 3D culture method provides a long-term and permissive in vitro niche that selects for highly clonogenic, colony-forming chondrocytes which maintain cartilage-specific matrix production, thus recapitulating the in vivo niche. As a proof of concept, clonogenicity of Sox9IRES–EGFP mouse chondrocytes is almost exclusively found in the highest GFP+ fraction known to be enriched for chondrocyte progenitor cells. Although clonogenic chondrocytes are very rare in adult cartilage, we have optimized this system to support large, single cell-derived chondrogenic organoids with complex zonal architecture and robust chondrogenic phenotype from adult pig and human articular chondrocytes. Moreover, we have demonstrated that growth trajectory and matrix biosynthesis in these organoids respond to a pro-inflammatory environment. This culture method offers a robust, defined and controllable system that can be further used to interrogate the effects of various microenvironmental signals on chondrocytes, providing a high throughput platform to assess genetic and environmental factors in development and disease.
Project description:Atelocollagen gel is often used for three-dimensional culture of articular cartilage-derived cells, but further knowledge is needed about the effect of atelocollagen gel on cells. We performed a microarray analysis using human articular cartilage-derived cells cultured in three different methods (2D culture, 3D culture with atelocollagen gel, and 3D culture without atelocollagen gel).
Project description:An organoid culture system can better recapitulate the cellular structure, function, and interaction between cells and the extracellular matrix (ECM) than the traditional two-dimensional (2D) culture system. We here constructed a condylar cartilage organoid and utilized it to explore the regulatory role of primary cilia at the organoid level. RNA sequencing unveiled the differences of transcriptomics between the condylar cartilage organoid and 2D culture chondrocytes.
Project description:To investigate the paracrine effects of stromal elements on cancer cells, we developed a “stromal” culture system, which incorporates structural and diffusible stroma-derived elements into homotypic cultures amenable to functional genomics and metabolomics. We show that microenvironmental cues co-regulate cancer metabolism and gene expression. Stromal inputs broadly influence histone acetylation in the cancer epigenome, coinciding with induction of genes implicated in anabolic metabolism and inflammation. The gene expression and metabolic changes induced by stromal factors overlap with those previously identified following oncogenic Kras, suggesting functional complementarity between cell-autonomous and microenvironmental pathways. We implicate the BET family of epigenetic readers as key transducers of stromal inputs to drive alterations in gene expression and suggest paracrine epigenome regulation as a conduit through which stromal signals drive metabolic and immune adaptation to a challenging tumor microenvironment.
Project description:RATIONALE: Shark cartilage extract may help shrink or slow the growth of colorectal cancer or breast cancer cells.
PURPOSE: Randomized phase III trial to determine the effectiveness of shark cartilage in treating patients who have advanced colorectal cancer or advanced breast cancer.
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.