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

Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA - Arthritis Res. Ther. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tissue Injury and Repair, School of Biomedicine, Faculty of Medical and Human Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK. Ben.Minogue@manchester.ac.uk

ABSTRACT

Introduction: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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Quantitative real-time PCR for (a) NP-specific and (b) IVD-specific cell marker genes in separated bovine NP and NC cells. Relative gene expression for (a) bovine nucleus pulposus (NP)-specific marker genes (synaptosomal-associated protein, 25 kDa (SNAP25), cytokeratin (KRT) 8, KRT18, KRT19, N-cadherin (CDH2) and sclerostin domain containing 1 (SOSTDC1)) and the notochord (NC) marker gene (T), and (b) intervertebral disc (IVD)-specific cell marker genes (tenomodulin (TNMD), brain abundant, membrane attached signal protein 1 (BASP1), tumor necrosis factor, alpha-induced protein 6 (TNFAIP6), forkhead box F1 (FOXF1), forkhead box F2 (FOXF2) and aquaporin (AQP1)), was normalised to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and plotted on a log scale. * statistical significance between NP and NC cells (P < 0.05).
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Figure 4: Quantitative real-time PCR for (a) NP-specific and (b) IVD-specific cell marker genes in separated bovine NP and NC cells. Relative gene expression for (a) bovine nucleus pulposus (NP)-specific marker genes (synaptosomal-associated protein, 25 kDa (SNAP25), cytokeratin (KRT) 8, KRT18, KRT19, N-cadherin (CDH2) and sclerostin domain containing 1 (SOSTDC1)) and the notochord (NC) marker gene (T), and (b) intervertebral disc (IVD)-specific cell marker genes (tenomodulin (TNMD), brain abundant, membrane attached signal protein 1 (BASP1), tumor necrosis factor, alpha-induced protein 6 (TNFAIP6), forkhead box F1 (FOXF1), forkhead box F2 (FOXF2) and aquaporin (AQP1)), was normalised to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and plotted on a log scale. * statistical significance between NP and NC cells (P < 0.05).

Mentions: Although the NP of mature bovine caudal disc is considered to be populated by chondrocyte-like cells, histological assessment of the tissues used here (18 to 36 months old) revealed a small population of resident notochordal cells. To determine whether the identified NP or IVD markers were expressed in either chondrocyte-like NP cells or these larger notochordal cells, the two cell types were isolated and the marker genes detected by qRT-PCR along with the molecular NC marker, brachyury homolog (T) [36,37]. Comparisons of NP (Figure 4a) and IVD (Figure 4b) specific genes in NP cells and NC cells clearly demonstrated expression of each gene by both cell types. Interestingly, NP-specific genes (with the exception of SNAP25) demonstrated significantly higher expression by NC cells than NP cells (all genes P < 0.0001), while IVD-specific genes demonstrated significantly higher expression by NP cells than NC cells (all genes P < 0.0001). The proposed NC marker gene T, demonstrated expression in both NP and NC cells, but expression was significantly higher in NC cells (P < 0.0001).


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

Minogue BM, Richardson SM, Zeef LA, Freemont AJ, Hoyland JA - Arthritis Res. Ther. (2010)

Quantitative real-time PCR for (a) NP-specific and (b) IVD-specific cell marker genes in separated bovine NP and NC cells. Relative gene expression for (a) bovine nucleus pulposus (NP)-specific marker genes (synaptosomal-associated protein, 25 kDa (SNAP25), cytokeratin (KRT) 8, KRT18, KRT19, N-cadherin (CDH2) and sclerostin domain containing 1 (SOSTDC1)) and the notochord (NC) marker gene (T), and (b) intervertebral disc (IVD)-specific cell marker genes (tenomodulin (TNMD), brain abundant, membrane attached signal protein 1 (BASP1), tumor necrosis factor, alpha-induced protein 6 (TNFAIP6), forkhead box F1 (FOXF1), forkhead box F2 (FOXF2) and aquaporin (AQP1)), was normalised to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and plotted on a log scale. * statistical significance between NP and NC cells (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875656&req=5

Figure 4: Quantitative real-time PCR for (a) NP-specific and (b) IVD-specific cell marker genes in separated bovine NP and NC cells. Relative gene expression for (a) bovine nucleus pulposus (NP)-specific marker genes (synaptosomal-associated protein, 25 kDa (SNAP25), cytokeratin (KRT) 8, KRT18, KRT19, N-cadherin (CDH2) and sclerostin domain containing 1 (SOSTDC1)) and the notochord (NC) marker gene (T), and (b) intervertebral disc (IVD)-specific cell marker genes (tenomodulin (TNMD), brain abundant, membrane attached signal protein 1 (BASP1), tumor necrosis factor, alpha-induced protein 6 (TNFAIP6), forkhead box F1 (FOXF1), forkhead box F2 (FOXF2) and aquaporin (AQP1)), was normalised to the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and plotted on a log scale. * statistical significance between NP and NC cells (P < 0.05).
Mentions: Although the NP of mature bovine caudal disc is considered to be populated by chondrocyte-like cells, histological assessment of the tissues used here (18 to 36 months old) revealed a small population of resident notochordal cells. To determine whether the identified NP or IVD markers were expressed in either chondrocyte-like NP cells or these larger notochordal cells, the two cell types were isolated and the marker genes detected by qRT-PCR along with the molecular NC marker, brachyury homolog (T) [36,37]. Comparisons of NP (Figure 4a) and IVD (Figure 4b) specific genes in NP cells and NC cells clearly demonstrated expression of each gene by both cell types. Interestingly, NP-specific genes (with the exception of SNAP25) demonstrated significantly higher expression by NC cells than NP cells (all genes P < 0.0001), while IVD-specific genes demonstrated significantly higher expression by NP cells than NC cells (all genes P < 0.0001). The proposed NC marker gene T, demonstrated expression in both NP and NC cells, but expression was significantly higher in NC cells (P < 0.0001).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Tissue Injury and Repair, School of Biomedicine, Faculty of Medical and Human Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK. Ben.Minogue@manchester.ac.uk

ABSTRACT

Introduction: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus