Dataset Information

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes.

ABSTRACT: Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.Microarrays were conducted on bovine AC, AF and NP cells, using Affymetrix Genechip(R) Bovine Genome Arrays. Differential expression levels for a number of genes were confirmed by quantitative real time polymerase chain reaction (qRT-PCR) on bovine, AC, AF and NP cells, as well as separated bovine NP and notochordal (NC) cells. Expression of these novel markers were further tested on normal human AC, AF and NP cells, and degenerate AF and NP cells.Microarray comparisons between NP/AC&AF and NP/AC identified 34 NP-specific and 49 IVD-specific genes respectively that were differentially expressed > or =100 fold. A subset of these were verified by qRT-PCR and shown to be expressed in bovine NC cells. Eleven genes (SNAP25, KRT8, KRT18, KRT19, CDH2, IBSP, VCAN, TNMD, BASP1, FOXF1 & FBLN1) were also differentially expressed in normal human NP cells, although to a lesser degree. Four genes (SNAP25, KRT8, KRT18 and CDH2) were significantly decreased in degenerate human NP cells, while three genes (VCAN, TNMD and BASP1) were significantly increased in degenerate human AF cells. The IVD negative marker FBLN1 was significantly increased in both degenerate human NP and AF cells.This study has identified a number of novel genes that characterise the bovine and human NP and IVD transcriptional profiles, and allows for discrimination between AC, AF and NP cells. Furthermore, the similarity in expression profiles of the separated NP and NC cell populations suggests that these two cell types may be derived from a common lineage. Although interspecies variation, together with changes with IVD degeneration were noted, use of this gene expression signature will benefit tissue engineering studies where defining the NP phenotype is paramount.

SUBMITTER: Minogue BM

PROVIDER: S-EPMC2875656 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes.

Minogue Ben M BM Richardson Stephen M SM Zeef Leo Ah LA Freemont Anthony J AJ Hoyland Judith A JA

Arthritis research & therapy 20100211 1

<h4>Introduction</h4>Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.<h4>Methods</h4>Microarrays were ...[more]

PMID: 20149220

Similar Datasets

Project description:IntroductionThe goal of this study is to characterize transcriptome changes and gene regulation networks in an organ culture system that mimics early post-traumatic intervertebral disc (IVD) degeneration.MethodsTo mimic a traumatic insult, bovine caudal IVDs underwent one strike loading. The control group was cultured under physiological loading. At 24 hours after one strike or physiological loading, RNA was extracted from nucleus pulposus (NP) and annulus fibrosus (AF) tissue. High throughput next generation RNA sequencing was performed to identify differentially expressed genes (DEGs) between the one strike loading group and the control group. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes analyses were performed to analyze DEGs and pathways. Protein-protein interaction (PPI) network was analyzed with cytoscape software. DEGs were verified using qRT-PCR. Degenerated human IVD tissue was collected for immunofluorescence staining to verify the expression of DEGs in human disc tissue.ResultsOne strike loading resulted in significant gene expression changes compared with physiological loading. In total 253 DEGs were found in NP tissue and 208 DEGs in AF tissue. Many of the highly dysregulated genes have known functions in disc degeneration and extracellular matrix (ECM) homeostasis. ACTB, ACTG, PFN1, MYL12B in NP tissue and FGF1, SPP1 in AF tissue were verified by qRT-PCR and immunofluorescence imaging. The identified DEGs were involved in focal adhesion, ECM-receptor interaction, PI3K-AKT, and cytokine-cytokine receptor interaction pathways. Three clusters of PPI networks were identified. GO enrichment revealed that these DEGs were mainly involved in inflammatory response, the ECM and growth factor signaling and protein folding biological process.ConclusionOur study revealed different DEGs, pathways, biological process and PPI networks involved in post-traumatic IVD degeneration. These findings will advance the understanding of the pathogenesis of IVD degeneration, and help to identify novel biomarkers for the disease diagnosis.

Project description:INTRODUCTION:Intervertebral disc (IVD) degeneration is considered a major underlying factor in the pathogenesis of chronic low back pain. Although the healthy IVD is both avascular and aneural, during degeneration there is ingrowth of nociceptive nerve fibres and blood vessels into proximal regions of the IVD, which may contribute to the pain. The mechanisms underlying neural ingrowth are, however, not fully understood. Semaphorin 3A (sema3A) is an axonal guidance molecule with the ability to repel nerves seeking their synaptic target. This study aimed to identify whether members of the Class 3 semaphorins were expressed by chondrocyte-like cells of the IVD addressing the hypothesis that they may play a role in repelling axons surrounding the healthy disc, thus maintaining its aneural condition. METHODS:Human IVD samples were investigated using reverse transcription polymerase chain reaction (RT-PCR) to identify gene expression of sema3A, 3F and their receptors: neuropilins (1 and 2) and plexins (A1-4). Sema3A protein was also localised within sections of normal and degenerate human IVD and immunopositivity quantified. Serial sections were stained using PGP9.5 and CD31 to correlate semaphorin 3A expression with nerve and blood vessel ingrowth, respectively. RESULTS:Sema3A protein was expressed highly in the healthy disc, primarily localised to the outer annulus fibrosus. In degenerate samples, sema3A expression decreased significantly in this region, although cell clusters within the degenerate nucleus pulposus exhibited strong immunopositivity. mRNA for sema3A receptors was also identified in healthy and degenerate tissues. CD31 and PGP9.5 were expressed most highly in degenerate tissues correlating with low expression of sema3A. CONCLUSIONS:This study is the first to establish the expression of semaphorins and their receptors in the human IVD with a decrease seen in the degenerate painful IVD. Sema3A may therefore, amongst other roles, act as a barrier to neuronal ingrowth within the healthy disc.

Project description:Asporin, also known as periodontal ligament-associated protein 1 (PLAP1), is a member of the family of small leucine-rich proteoglycan (SLRP) family. It is present within the cartilage extracellular matrix (ECM), and is reported have a genetic association with osteoarthritis. Its D14 allele has recently been found to be associated with lumbar disc degeneration in Asian subjects. There have been no studies, however, of this gene’s normal immunohistochemical localization within the human intervertebral disc, nor of expression levels in Caucasian individuals with disc degeneration. Studies were approved by our human subjects Institutional Review Board. Methods included immunohistochemical localization of asporin in the disc of humans and the sand rat (a small rodent with spontaneous age-related disc degeneration), and Affymetrix microarray analysis of asporin gene expression in vivo and in vitro. mmunohistochemical studies of human discs revealed that some, but not all, cells of the outer annulus expressed asporin. Fewer cells in the inner annulus contained asporin, and it was rarely present in cells in the nucleus pulposus. Similar patterns were found for the presence of asporin in lumbar discs of sand rats. Substantial relative gene expression levels were seen for asporin in both disc tissue and in annulus cells grown in three-dimensional culture. More degenerate human discs (Thompson grade 4) showed higher expression levels of asporin than did less degenerate (grade 1, 2 and 3) discs, p = 0.004. In the discs of Caucasian subjects studied here, and in the sand rat, greater immunolocalization levels were found in the outer compared to inner annulus. Localization was rare in the nucleus. Gene expression studies showed greatest expression of asporin in the more degenerate human discs in vivo. Disc Tissue samples were obtained via the National Cancer Institute Cooperative Tissue Network (CHTN) as well as surgical disc procedures performed on patients with herniated discs and degenerative disc disease. Tissue was fixed and paraffin embedded. Standard laser capture microdissection (LCM) techniques were used to collect the cells from which total RNA was isolated and analyzed via microarray. Based on the Thompson scouring system, unhealthy discs (grade 4) were compared to healthy discs (grades 2,3).

Project description:Bovine caudal discs have been widely used in spine research due to their increased availability, large size, and mechanical and biochemical properties that are comparable to healthy human discs. However, despite their extensive use, the radial variations in bovine disc composition have not yet been rigorously quantified with high spatial resolution. Previous studies were limited to qualitative analyses or provided limited spatial resolution in biochemical properties. Thus, the main objective of this study was to provide quantitative measurements of biochemical composition with higher spatial resolution than previous studies that employed traditional biochemical techniques. Specifically, traditional biochemical analyses were used to measure water, sulfated glycosaminoglycan, collagen, and DNA contents. Gravimetric water content was compared to data obtained through Raman spectroscopy and differential scanning calorimetry. Additionally, spatial distribution of lipids in the disc's collagen network was visualized and quantified, for the first time, using multi-modal second harmonic generation (SHG) and Coherent anti-Stokes Raman (CARS) microscopy. Some heterogeneity was observed in the nucleus pulposus, where the water content and water-to-protein ratio of the inner nucleus were greater than the outer nucleus. In contrast, the bovine annulus fibrosus exhibited a more heterogeneous distribution of biochemical properties. Comparable results between orthohydroxyproline assay and SHG imaging highlight the potential benefit of using SHG microscopy as a less destructive method for measuring collagen content, particularly when relative changes are of interest. CARS images showed that lipid deposits were distributed equally throughout the disc and appeared either as individual droplets or as clusters of small droplets. In conclusion, this study provided a more comprehensive assessment of spatial variations in biochemical composition of the bovine caudal disc.

Dataset Information

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes.

Publications

Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets