Transcriptional profiling of equine chondrocyte de-differentiation in culture
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ABSTRACT:
ORGANISM(S): Equus caballus
PROVIDER: GSE14255 | GEO | 2012/06/01
SECONDARY ACCESSION(S): PRJNA111209
REPOSITORIES: GEO
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Project description:Monolayer cultures of primary chondrocytes undergo morphological changes that have been broadly characterized as de-differentiation. Transcriptional profiling was used to evaluate changes in gene expression during this process. Articular cartilage was harvested from a single Standardbred horse; the chondrocytes were isolated and then maintained in monolayer culture for up to 14 days. Chondrocytes were collected on Day 1 and Day 14 of culture and snap frozen in liquid nitrogen. Total RNA was isolated, subjected to one round of linear RNA amplification, and then applied to an equine-specific cDNA microarray composed of 9322 elements. Three biological replicates were analyzed at each time point using a dye-swap experimental design. One hundred six transcripts were present at levels greater than or equal to five-fold higher in Day 1 chondrocytes relative to the cells collected at Day 14. Conversely, 68 transcripts were present at levels greater than or equal to five-fold higher in Day 14 chondrocytes. Northern blot hybridization validated the microarray data by confirming the down-regulation of steady state mRNA levels for type II procollagen and aggrecan core protein and the up-regulation of type I procollagen. Gene-by-gene and cluster analyses will be used to compare the process of “de-differentiation” in primary culture to the developmental stages of normal chondrocyte differentiation and to the fibrocartilage repair tissue that forms in osteoarthritic lesions. Keywords: articular cartilage, de-differentiation, horse, cDNA microarray This is a direct comparison between one and fourteen day cultured chondrocyte transcriptomes. Three sets of technical replicates were compared. For each comparison and its dye-swap, one Day 1 technical replicate was randomly compared to one Day 14 technical replicate.
2012-05-31 | E-GEOD-14255 | biostudies-arrayexpress
Project description:Chondrocyte gene expression was analyzed to study mechanisms involved in the structural and functional adaptation of articular cartilage during postnatal maturation. Transcriptional profiling was used to compare articular chondrocytes between four neonatal and four adult horses. Expressional differences featured matrix proteins and matrix-modifying enzymes reflecting the transition from cartilage growth to cartilage homeostasis. Keywords: articular cartilage, maturation, horse, cDNA microarray
2008-11-10 | GSE10303 | GEO
Project description:Previous studies have demonstrated relative deficiencies of repair tissue within articular lesions when compared to articular cartilage. While cells occupying the lesions might be of mesenchymal origin, they do not recapitulate differentiation to the chondrogenic phenotype of normal articular chondrocytes. Nevertheless, attributes of repair tissue appear similar to chondrocytes and their precursors at various other different states. We hypothesized that analyses of gene expression profiles for these other cell phenotypes would elucidate the identity of repair tissue cells. Total RNA was isolated from repair tissue samples, neonatal articular cartilage, primary articular chondrocytes maintained in monolayer culture and passaged weekly over 28 days, and bone marrow stromal cells expanded to 80% confluence in monolayer culture. Total RNA was linearly amplified and applied to a 9413-probe set equine-specific cDNA microarray. Four repair tissue samples were compared with a dye-swap experimental design to four samples from each of the three other cell populations for a total of twelve comparisons, or twenty-four slides. Differential expression of genes of interest was validated by real-time quantitative polymerase chain reaction. Statistical analysis revealed that a total of 934 (9.9%), 1839 (19.5%), and 940 (10.0%) probe sets were differentially expressed for the bone marrow stromal cells versus repair tissue, de-differentiated chondrocytes versus repair tissue, and neonatal articular chondrocytes versus repair tissue comparisons, respectively. Transcriptional and translational machinery gene ontological categories were over-represented in transcripts demonstrating stable expression amongst the three comparisons. Biomarkers typically associated with normal articular cartilage and fibrocartilage repair tissue comprised much of the genes with the greatest levels of differential expression amongst the three comparisons. Overall, the profiles indicated that repair cells were more chondrogenic than bone marrow stromal cells and de-differentiated chondrocytes. However, transcript levels of key biomarkers and growth factors for repair tissue cells four months post-operatively fell far short of those of neonatal articular chondrocytes destined to undergo normal cartilage maturation.
2010-03-30 | GSE14252 | GEO
Project description:To identify genes that maintain the homeostasis of the articular cartilage, we compared gene expression profiles of adult articular cartilage chondrocytes with that of growth plate cartilage chondrocytes in adult (10-week-old) Sprague Dawley (SD) rats. Furthermore, to identify genes that have a potency to regenerate the articular cartilage, we compared gene expression profiles of superficial layer chondrocytes of infant epiphyseal cartilage which form articular cartilage with that of the deep layer chondrocytes which form growth plate cartilage in infant (6-day-old) SD rats.
2012-07-17 | E-MTAB-1218 | biostudies-arrayexpress
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).
2009-09-13 | E-GEOD-16464 | biostudies-arrayexpress
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.
2012-11-14 | E-GEOD-42235 | biostudies-arrayexpress
Project description:Full thickness articular cartilage lesions with penetration into the subchondral bone fill with fibrocartilage-like repair tissue. However, the repair tissue has compromised structural and biomechanical properties relative to normal articular cartilage. The objective of this study was to evaluate transcriptome differences between normal articular cartilage and repair tissue. Bilateral one-cm2 full-thickness lesions were made in the articular surface of the distal femurs of four adult horses followed by subchondral microfracture. Four months postoperatively, repair tissue from the lesion site and grossly normal articular cartilage from each stifle were collected. Total RNA was isolated from tissue samples, linearly amplified, and applied to a 9367-probeset equine-specific cDNA microarray. Eight paired comparisons matched by limb and horse were made with a dye-swap experimental design. Comparisons were validated by histological analysis and quantitative real-time polymerase chain reaction (qPCR). Statistical analysis revealed 3,327 (35.2%) differentially expressed probesets. Biomarkers typically associated with normal articular cartilage and fibrocartilage repair tissue corroborate earlier studies. Other changes in gene expression previously unassociated with cartilage repair were also revealed and validated by qPCR. The magnitude of divergence in transcriptional profiles between normal chondrocytes and the cells that populate repair tissue reveal substantial functional differences between these two cell populations. At the four-month postoperative time point, the relative deficiency within repair tissue of transcripts from genes which typically define articular cartilage indicate that while cells occupying the lesion might be of mesenchymal origin, they have not recapitulated differentiation to the chondrogenic phenotype of normal articular chondrocytes.
2009-09-10 | GSE11760 | GEO
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.
2011-07-28 | E-GEOD-23492 | biostudies-arrayexpress
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.
2012-11-14 | GSE42235 | GEO
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
2009-09-02 | GSE16464 | GEO