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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 normal human AC, AF and NP cells. Relative gene expression for (a) nucleus pulposus (NP)-specific marker genes (synaptosomal-associated protein, 25 kDa (SNAP25), cytokeratin (KRT) 8, KRT18, KRT19, N-cadherin (CDH2) and integrin-binding sialoprotein (IBSP)) and the notochord (NC) marker gene (T), and (b) IVD-specific cell marker genes ((tenomodulin (TNMD), brain abundant, membrane attached signal protein 1 (BASP1), forkhead box F1 (FOXF1), and fibulin 1 (FBLN1)) and the chondrogenic marker genes (aggrecan (ACAN) and type II collagen (COL2A1)), was normalised to the housekeeping gene and plotted on a log scale. * statistical significance between NP and annulus fibrosus (AF) cells and NP and articular cartilage (AC) cells (P < 0.05). † statistical significance between AF cells and AC cells (P < 0.05).
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Figure 5: Quantitative real-time PCR for (a) NP-specific and (b) IVD-specific cell marker genes in normal human AC, AF and NP cells. Relative gene expression for (a) nucleus pulposus (NP)-specific marker genes (synaptosomal-associated protein, 25 kDa (SNAP25), cytokeratin (KRT) 8, KRT18, KRT19, N-cadherin (CDH2) and integrin-binding sialoprotein (IBSP)) and the notochord (NC) marker gene (T), and (b) IVD-specific cell marker genes ((tenomodulin (TNMD), brain abundant, membrane attached signal protein 1 (BASP1), forkhead box F1 (FOXF1), and fibulin 1 (FBLN1)) and the chondrogenic marker genes (aggrecan (ACAN) and type II collagen (COL2A1)), was normalised to the housekeeping gene and plotted on a log scale. * statistical significance between NP and annulus fibrosus (AF) cells and NP and articular cartilage (AC) cells (P < 0.05). † statistical significance between AF cells and AC cells (P < 0.05).

Mentions: Analysis of expression of traditional marker genes in human normal IVD samples (Figure 5) showed that when compared with AC cells, ACAN and COL2A1 gene expression was significantly lower in normal AF cells (P = 0.0003, and P < 0.0001, respectively) and normal NP cells (P = 0.0018, and P < 0.0001, respectively). However, there was no significant difference for either ACAN or COL2A1 between normal AF and NP cells (P = 0.39, and P = 0.1, respectively).


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 normal human AC, AF and NP cells. Relative gene expression for (a) nucleus pulposus (NP)-specific marker genes (synaptosomal-associated protein, 25 kDa (SNAP25), cytokeratin (KRT) 8, KRT18, KRT19, N-cadherin (CDH2) and integrin-binding sialoprotein (IBSP)) and the notochord (NC) marker gene (T), and (b) IVD-specific cell marker genes ((tenomodulin (TNMD), brain abundant, membrane attached signal protein 1 (BASP1), forkhead box F1 (FOXF1), and fibulin 1 (FBLN1)) and the chondrogenic marker genes (aggrecan (ACAN) and type II collagen (COL2A1)), was normalised to the housekeeping gene and plotted on a log scale. * statistical significance between NP and annulus fibrosus (AF) cells and NP and articular cartilage (AC) cells (P < 0.05). † statistical significance between AF cells and AC cells (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Quantitative real-time PCR for (a) NP-specific and (b) IVD-specific cell marker genes in normal human AC, AF and NP cells. Relative gene expression for (a) nucleus pulposus (NP)-specific marker genes (synaptosomal-associated protein, 25 kDa (SNAP25), cytokeratin (KRT) 8, KRT18, KRT19, N-cadherin (CDH2) and integrin-binding sialoprotein (IBSP)) and the notochord (NC) marker gene (T), and (b) IVD-specific cell marker genes ((tenomodulin (TNMD), brain abundant, membrane attached signal protein 1 (BASP1), forkhead box F1 (FOXF1), and fibulin 1 (FBLN1)) and the chondrogenic marker genes (aggrecan (ACAN) and type II collagen (COL2A1)), was normalised to the housekeeping gene and plotted on a log scale. * statistical significance between NP and annulus fibrosus (AF) cells and NP and articular cartilage (AC) cells (P < 0.05). † statistical significance between AF cells and AC cells (P < 0.05).
Mentions: Analysis of expression of traditional marker genes in human normal IVD samples (Figure 5) showed that when compared with AC cells, ACAN and COL2A1 gene expression was significantly lower in normal AF cells (P = 0.0003, and P < 0.0001, respectively) and normal NP cells (P = 0.0018, and P < 0.0001, respectively). However, there was no significant difference for either ACAN or COL2A1 between normal AF and NP cells (P = 0.39, and P = 0.1, respectively).

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