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Fetal cartilage selective genes identified in a genome-scale analysis

ABSTRACT: Cartilage plays a fundamental role in the development of the human skeleton. Early in embryogenesis, mesenchymal cells condense and differentiate to chondrocytes to shape the early skeleton. Subsequently, the cartilage anlagen differentiate to form the growth plates, which are responsible for linear bone growth, and the articular chondrocytes, which facilitate joint function. However, despite the multiplicity of roles of cartilage during human fetal life, surprisingly little is known about its transcriptome. To address this, a whole genome microarray expression profile was generated using RNA isolated from 18-22 week human distal femur fetal cartilage and compared with a database of control normal human tissues aggregated at UCLA, termed CELSIUS. From the wealth of data, 161 cartilage-selective genes were identified, defined as genes significantly expressed in cartilage with low expression and little variation across a panel of 34 non-cartilage tissues. Among these 161 genes were cartilage-specific genes such as collagen genes and 25 genes which have been associated with skeletal phenotypes in humans and/or mice. Many of the other cartilage-selective genes do not have established roles in cartilage or are novel, unannotated genes. Quantitative RT-PCR confirmed the unique pattern of gene expression observed by microarray analysis. Defining the gene expression pattern for cartilage has identified new genes that may contribute to human skeletogenesis as well as provided further candidate genes for skeletal dysplasias. The data suggest that fetal cartilage is a complex and transcriptionally active tissue and demonstrate that the set of genes selectively expressed in the tissue has been greatly underestimated. Keywords: Normal Tissue expression profile, tissue-selective Genes, tissue-specific genes

ORGANISM(S): Homo sapiens

PROVIDER: GSE6565 | GEO | 2007/07/06

SECONDARY ACCESSION(S): PRJNA98737

REPOSITORIES: GEO

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Project description:Cartilage plays a fundamental role in the development of the human skeleton. Early in embryogenesis, mesenchymal cells condense and differentiate to chondrocytes to shape the early skeleton. Subsequently, the cartilage anlagen differentiate to form the growth plates, which are responsible for linear bone growth, and the articular chondrocytes, which facilitate joint function. However, despite the multiplicity of roles of cartilage during human fetal life, surprisingly little is known about its transcriptome. To address this, a whole genome microarray expression profile was generated using RNA isolated from 18-22 week human distal femur fetal cartilage and compared with a database of control normal human tissues aggregated at UCLA, termed CELSIUS. From the wealth of data, 161 cartilage-selective genes were identified, defined as genes significantly expressed in cartilage with low expression and little variation across a panel of 34 non-cartilage tissues. Among these 161 genes were cartilage-specific genes such as collagen genes and 25 genes which have been associated with skeletal phenotypes in humans and/or mice. Many of the other cartilage-selective genes do not have established roles in cartilage or are novel, unannotated genes. Quantitative RT-PCR confirmed the unique pattern of gene expression observed by microarray analysis. Defining the gene expression pattern for cartilage has identified new genes that may contribute to human skeletogenesis as well as provided further candidate genes for skeletal dysplasias. The data suggest that fetal cartilage is a complex and transcriptionally active tissue and demonstrate that the set of genes selectively expressed in the tissue has been greatly underestimated. Experiment Overall Design: RNA from 7 normal fetal cartilage samples was isolated. 5 samples were run in duplicate on U133 2.0 arrays, while 2 were run on U133A and U133B subsets. In brief, cartilage selective genes were identified by comparing 5 normal cartilage samples (GSM149638,GSM149639 ,GSM149640,GSM149641 ,GSM149642) to normal tissue expression profiles found on CELCIUS at UCLA. Genes which were found expressed at significantly higher levels were validated for their elevated and specific expression using the three platforms. The 9 validation arrays are: 5 U133 2.0 (GSM149643 ,GSM149644 ,GSM149645 ,GSM149646,GSM149647), 2 U133A (GSM149651, GSM149652), 2 U133B (GSM149653, GSM149654)

Project description:We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFB3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development. RNA fhMSCs of chondrogenically differentiating fhMSCs was isolated at week, 0, 1, 2, 3, 4 and 5. Moreover, RNA of 4 human growth plates from was isolated.

Project description:IL-1B is an important cytokine that is often found to be up-regulated during osteoarthritic and rheumatoid joint diseases. It is viewed as a catabolic factor, inducing enzymes that allow for the degradation of the cartilage extracellular matrix and also has essential roles as an autocrine and paracrine factor in fibronectin fragment-mediated degradation. It can also reduce the synthesis of the major cartilage components, type II collagen and aggrecan. On the other hand, IL-1B also has the ability to induce the growth and morphogenic factor BMP-2. During joint diseases, IL-1B is synthesized by both synovial cells and chondrocytes. Addition of IL-1 biological antagonists such as IL-1 receptor antagonists can suppress cartilage degradation in vitro. Thus, the production of IL-1B could act as the first step in mediating a cascade of other mediators in cartilage which could be relevant to the fate of the cartilage. In order to obtain a global picture of the effect of IL-1B production on human adult articular chondrocytes, we analyzed changes in gene expression induced by IL-1B by microarray analysis. We found that IL-1B has a diverse effect on gene expression profile in chondrocytes. One of the predominant responses that we observed in adult human articular chondrocytes on exposure to IL-1B is a dramatic increase in a large set of chemokines and other genes related to the inflammatory cascade. Keywords: Gene response to IL-1B (10 ng/ml) Cartilage was obtained from adult human tissue donors with above the knee amputations due to chondrosarcoma or traumatic injury or from autopsy. Chondrocytes were isolated following established protocols, maintained in high density, and treated with IL-1B (10 ng/ml). Chondrocytes treated with buffer only served as the untreated control. The experiment was carried out in duplicate. Total RNA was extracted from these chondrocytes, labeled with fluorophores (Cy3 or Cy5) and analyzed for expression changes using the Human Operon/Qiagen v3.0 oligonucleotide array. The analysis was repeated with the fluorophore dyes exchanged between the untreated and experimental RNAs.

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Fetal cartilage selective genes identified in a genome-scale analysis

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