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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 and degenerate human 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) intervertebral disc (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 normal annulus fibrosus (AF) or NP cells and plotted on a log scale. For each gene, expression in normal NP cells or AF cells was plotted on the baseline (value = 1 +/-standard error) and the relative expression in NP or AF degenerate cells (normalised to the relevant normal cell value) was plotted adjacently. * statistical significance between normal and degenerate NP cells (P < 0.05). † statistical significance between normal and degenerate AF cells (P < 0.05).
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Figure 6: Quantitative real-time PCR for (a) NP-specific and (b) IVD-specific cell marker genes, in normal and degenerate human 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) intervertebral disc (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 normal annulus fibrosus (AF) or NP cells and plotted on a log scale. For each gene, expression in normal NP cells or AF cells was plotted on the baseline (value = 1 +/-standard error) and the relative expression in NP or AF degenerate cells (normalised to the relevant normal cell value) was plotted adjacently. * statistical significance between normal and degenerate NP cells (P < 0.05). † statistical significance between normal and degenerate AF cells (P < 0.05).

Mentions: For analysis of qRT-PCR data in human samples, the comparative (2-ΔΔCt) method was used to demonstrate differences in gene expression between normal and degenerate cells (Figure 6). No significant differences in expression for ACAN or COL2A1 were observed in degenerate samples when compared with normal samples. Analysis of NP-specific marker genes in degenerate samples (Figure 6a) showed a significant decrease in expression for SNAP25, KRT8, KRT18 and CDH2 in degenerate NP cells when compared with normal NP cells (P = 0.0002, P = 0.0003, P < 0.0001 and P = 0.0001, respectively). Only KRT18 was significantly decreased in degenerate AF cells (P < 0.0001), while KRT19 expression did not differ significantly in degenerate NP or AF cells. For the IVD-specific marker genes TNMD and BASP1 (Figure 6b) there were significant increases in degenerate AF cells when compared with normal AF cells (P = 0.03, and P < 0.0001, respectively). Interestingly, the negative IVD marker gene (potentially an AC marker) FBLN1 showed significant increases in expression in both degenerate AF cells (approximately 30 fold, P < 0.0001) and NP cells (approximately 70 fold, P < 0.0001) when compared with normal cells.


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 and degenerate human 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) intervertebral disc (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 normal annulus fibrosus (AF) or NP cells and plotted on a log scale. For each gene, expression in normal NP cells or AF cells was plotted on the baseline (value = 1 +/-standard error) and the relative expression in NP or AF degenerate cells (normalised to the relevant normal cell value) was plotted adjacently. * statistical significance between normal and degenerate NP cells (P < 0.05). † statistical significance between normal and degenerate AF cells (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Quantitative real-time PCR for (a) NP-specific and (b) IVD-specific cell marker genes, in normal and degenerate human 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) intervertebral disc (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 normal annulus fibrosus (AF) or NP cells and plotted on a log scale. For each gene, expression in normal NP cells or AF cells was plotted on the baseline (value = 1 +/-standard error) and the relative expression in NP or AF degenerate cells (normalised to the relevant normal cell value) was plotted adjacently. * statistical significance between normal and degenerate NP cells (P < 0.05). † statistical significance between normal and degenerate AF cells (P < 0.05).
Mentions: For analysis of qRT-PCR data in human samples, the comparative (2-ΔΔCt) method was used to demonstrate differences in gene expression between normal and degenerate cells (Figure 6). No significant differences in expression for ACAN or COL2A1 were observed in degenerate samples when compared with normal samples. Analysis of NP-specific marker genes in degenerate samples (Figure 6a) showed a significant decrease in expression for SNAP25, KRT8, KRT18 and CDH2 in degenerate NP cells when compared with normal NP cells (P = 0.0002, P = 0.0003, P < 0.0001 and P = 0.0001, respectively). Only KRT18 was significantly decreased in degenerate AF cells (P < 0.0001), while KRT19 expression did not differ significantly in degenerate NP or AF cells. For the IVD-specific marker genes TNMD and BASP1 (Figure 6b) there were significant increases in degenerate AF cells when compared with normal AF cells (P = 0.03, and P < 0.0001, respectively). Interestingly, the negative IVD marker gene (potentially an AC marker) FBLN1 showed significant increases in expression in both degenerate AF cells (approximately 30 fold, P < 0.0001) and NP cells (approximately 70 fold, P < 0.0001) when compared with normal cells.

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