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Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers.

Gilson A, Dreger M, Urban JP - Arthritis Res. Ther. (2010)

Bottom Line: Notochordal nucleus pulposus cells from pig, phenotypically similar to human infant nucleus pulposus cells, were all CK8-positive.The mesenchymal intermediate filament protein vimentin was present in all bovine and porcine nucleus pulposus cells.The notochordal cell population is reported to disappear from the nucleus pulposus of bovine discs before birth and from human discs in childhood.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK. audrey.gilson@dpag.ox.ac.uk

ABSTRACT

Introduction: Development of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes.

Methods: We searched for markers using the differential in-gel electrophoresis proteomic technology to compare proteins of bovine nucleus pulposus cells, phenotypically similar to mature human nucleus cells, with those of bovine articular chondrocytes. In the cohort of the differentially expressed proteins identified by mass spectrometry, cytokeratin 8 (CK8) was further validated by immunostaining of freshly isolated cells and frozen tissue sections using monoclonal antibodies.

Results: We identified a set of 14 differentially expressed proteins. Immunohistochemistry showed that only a subset of cells (approximately 10%) was positive for one of these proteins, CK8, an intermediate filament protein present in epithelial but not mesenchymal cells. In tissue sections, CK8-positive cells were seen in all discs examined and appeared as small isolated clusters surrounded by gelatinous matrix. Notochordal nucleus pulposus cells from pig, phenotypically similar to human infant nucleus pulposus cells, were all CK8-positive. The mesenchymal intermediate filament protein vimentin was present in all bovine and porcine nucleus pulposus cells.

Conclusions: The notochordal cell population is reported to disappear from the nucleus pulposus of bovine discs before birth and from human discs in childhood. However our finding of the co-expression of vimentin and CK8 in small isolated clusters of the bovine nucleus pulposus cells indicates that a subpopulation of notochordal-like cells remains in the mature bovine disc. This finding agrees with reports in the literature on co-expression of cytokeratins and vimentin in adult human discs. As notochordal cells produce factors that promote matrix production, the CK8-positive subpopulation could have important implications for activity and survival of the nucleus pulposus, and should be considered in development of cell therapies for disc repair. In addition, the finding of differential expression of proteins in the cell population of nucleus pulposus has implications with regard to the search for specific markers.

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Proteome map of freshly isolated bovine nucleus pulposus (NP) cells and identification of cytokeratin 8 (CK8) by mass spectrometry. (a) Representative two-dimensional CyDye3-stained gel of total NP cell proteins separated according to their isoelectric point and molecular weight and (b) analysis of the NP and articular chondrocyte (AC) spot patterns by DeCyder software. (c) Mass spectrum of the spot indicated by an arrowhead in (a) and (b) identified by peptide mass fingerprinting as CK8.
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Figure 1: Proteome map of freshly isolated bovine nucleus pulposus (NP) cells and identification of cytokeratin 8 (CK8) by mass spectrometry. (a) Representative two-dimensional CyDye3-stained gel of total NP cell proteins separated according to their isoelectric point and molecular weight and (b) analysis of the NP and articular chondrocyte (AC) spot patterns by DeCyder software. (c) Mass spectrum of the spot indicated by an arrowhead in (a) and (b) identified by peptide mass fingerprinting as CK8.

Mentions: To identify specific markers of freshly isolated bovine NP cells, the Cy3- and Cy5-labelled water-soluble protein fractions of NP cells and ACs were resolved by 2D gel electrophoresis. In total, six analytical 2D gels corresponding to independent cell isolations were analyzed using the DeCyder software, which was initially set up to detect an estimated 2,500 protein spots on a single gel (Figure 1a, representative 2D spot pattern of NP Cy3-labelled protein sample). A corresponding enlarged view of the gel area boxed in Figure 1a has been depicted for the two different cell types (NP and AC) on three different gels to emphasize the high level of protein resolution and to demonstrate the reproducibility of the 2D profiles where the spot indicated by an arrowhead is consistently present in the NP fraction and absent in the AC fraction (Figure 1b). Individual gel spot intensity was converted into a volumetric map for a three-dimensional visualization of the spot pattern (Figure 1b). The relatively abundant spot indicated by an arrowhead in Figure 1a and 1b was excised manually from a colloidal Coomassie Blue-stained preparative gel and digested with trypsin. The resulting peptide mixture was subjected to peptide mass fingerprinting analysis to generate a mass spectrum (Figure 1c) with experimentally measured peptide masses. The protein was identified as CK8 based on 22 peptide matches, 42% sequence coverage, a Mascot score of 107, and molecular mass and pI values of 55 kDa and 5.7, respectively. Similarly, 13 other proteins found either only in nucleus or only in cartilage cells were identified. These differences arose mainly from post-translational modifications of metabolic enzymes (the study of which will be the subject of a separate publication, which is in preparation), and this is possibly why they were not identified on mRNA microarrays [14,15].


Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers.

Gilson A, Dreger M, Urban JP - Arthritis Res. Ther. (2010)

Proteome map of freshly isolated bovine nucleus pulposus (NP) cells and identification of cytokeratin 8 (CK8) by mass spectrometry. (a) Representative two-dimensional CyDye3-stained gel of total NP cell proteins separated according to their isoelectric point and molecular weight and (b) analysis of the NP and articular chondrocyte (AC) spot patterns by DeCyder software. (c) Mass spectrum of the spot indicated by an arrowhead in (a) and (b) identified by peptide mass fingerprinting as CK8.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875658&req=5

Figure 1: Proteome map of freshly isolated bovine nucleus pulposus (NP) cells and identification of cytokeratin 8 (CK8) by mass spectrometry. (a) Representative two-dimensional CyDye3-stained gel of total NP cell proteins separated according to their isoelectric point and molecular weight and (b) analysis of the NP and articular chondrocyte (AC) spot patterns by DeCyder software. (c) Mass spectrum of the spot indicated by an arrowhead in (a) and (b) identified by peptide mass fingerprinting as CK8.
Mentions: To identify specific markers of freshly isolated bovine NP cells, the Cy3- and Cy5-labelled water-soluble protein fractions of NP cells and ACs were resolved by 2D gel electrophoresis. In total, six analytical 2D gels corresponding to independent cell isolations were analyzed using the DeCyder software, which was initially set up to detect an estimated 2,500 protein spots on a single gel (Figure 1a, representative 2D spot pattern of NP Cy3-labelled protein sample). A corresponding enlarged view of the gel area boxed in Figure 1a has been depicted for the two different cell types (NP and AC) on three different gels to emphasize the high level of protein resolution and to demonstrate the reproducibility of the 2D profiles where the spot indicated by an arrowhead is consistently present in the NP fraction and absent in the AC fraction (Figure 1b). Individual gel spot intensity was converted into a volumetric map for a three-dimensional visualization of the spot pattern (Figure 1b). The relatively abundant spot indicated by an arrowhead in Figure 1a and 1b was excised manually from a colloidal Coomassie Blue-stained preparative gel and digested with trypsin. The resulting peptide mixture was subjected to peptide mass fingerprinting analysis to generate a mass spectrum (Figure 1c) with experimentally measured peptide masses. The protein was identified as CK8 based on 22 peptide matches, 42% sequence coverage, a Mascot score of 107, and molecular mass and pI values of 55 kDa and 5.7, respectively. Similarly, 13 other proteins found either only in nucleus or only in cartilage cells were identified. These differences arose mainly from post-translational modifications of metabolic enzymes (the study of which will be the subject of a separate publication, which is in preparation), and this is possibly why they were not identified on mRNA microarrays [14,15].

Bottom Line: Notochordal nucleus pulposus cells from pig, phenotypically similar to human infant nucleus pulposus cells, were all CK8-positive.The mesenchymal intermediate filament protein vimentin was present in all bovine and porcine nucleus pulposus cells.The notochordal cell population is reported to disappear from the nucleus pulposus of bovine discs before birth and from human discs in childhood.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK. audrey.gilson@dpag.ox.ac.uk

ABSTRACT

Introduction: Development of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes.

Methods: We searched for markers using the differential in-gel electrophoresis proteomic technology to compare proteins of bovine nucleus pulposus cells, phenotypically similar to mature human nucleus cells, with those of bovine articular chondrocytes. In the cohort of the differentially expressed proteins identified by mass spectrometry, cytokeratin 8 (CK8) was further validated by immunostaining of freshly isolated cells and frozen tissue sections using monoclonal antibodies.

Results: We identified a set of 14 differentially expressed proteins. Immunohistochemistry showed that only a subset of cells (approximately 10%) was positive for one of these proteins, CK8, an intermediate filament protein present in epithelial but not mesenchymal cells. In tissue sections, CK8-positive cells were seen in all discs examined and appeared as small isolated clusters surrounded by gelatinous matrix. Notochordal nucleus pulposus cells from pig, phenotypically similar to human infant nucleus pulposus cells, were all CK8-positive. The mesenchymal intermediate filament protein vimentin was present in all bovine and porcine nucleus pulposus cells.

Conclusions: The notochordal cell population is reported to disappear from the nucleus pulposus of bovine discs before birth and from human discs in childhood. However our finding of the co-expression of vimentin and CK8 in small isolated clusters of the bovine nucleus pulposus cells indicates that a subpopulation of notochordal-like cells remains in the mature bovine disc. This finding agrees with reports in the literature on co-expression of cytokeratins and vimentin in adult human discs. As notochordal cells produce factors that promote matrix production, the CK8-positive subpopulation could have important implications for activity and survival of the nucleus pulposus, and should be considered in development of cell therapies for disc repair. In addition, the finding of differential expression of proteins in the cell population of nucleus pulposus has implications with regard to the search for specific markers.

Show MeSH
Related in: MedlinePlus