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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.

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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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Cytokeratin 8 (CK8) and vimentin expression in bovine and porcine disc cells. Dual immunofluorescence staining of CK8 and vimentin (VIM) performed on freshly isolated bovine nucleus pulposus cells (a-c), bovine annulus fibrosus cells (d-f), and porcine notochordal cells (g-i). Nuclei were visualized with DAPI (4'-6-diamidino-2-phenylindole) (scale bar = 60 μm). All cells examined were positive for VIM (b,e,h). No annulus cells were positive for CK8 (d). Some bovine nucleus cells -- forming clusters, as indicated by an arrowhead in (a) -- and all porcine cells (g) were positive for CK8; these cells co-expressed VIM (c,i).
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Figure 4: Cytokeratin 8 (CK8) and vimentin expression in bovine and porcine disc cells. Dual immunofluorescence staining of CK8 and vimentin (VIM) performed on freshly isolated bovine nucleus pulposus cells (a-c), bovine annulus fibrosus cells (d-f), and porcine notochordal cells (g-i). Nuclei were visualized with DAPI (4'-6-diamidino-2-phenylindole) (scale bar = 60 μm). All cells examined were positive for VIM (b,e,h). No annulus cells were positive for CK8 (d). Some bovine nucleus cells -- forming clusters, as indicated by an arrowhead in (a) -- and all porcine cells (g) were positive for CK8; these cells co-expressed VIM (c,i).

Mentions: It has been suggested that mature NP cells could originate and migrate from the surrounding cells of the annulus or the cartilaginous end-plates [32] which are mesenchymal in origin. The type III intermediate filament vimentin is used as a marker of cells of mesodermal origin in contrast to the type II to which the epithelial CK8 belongs [33,34]. We therefore examined whether any cells were specific for this type III intermediate filament. Dual-immunofluorescence labelling of freshly isolated cells for CK8 and vimentin was performed in NP and AF from bovine and porcine tissues. The bovine NP cell population, as reported elsewhere [35], all appeared to express vimentin; it was found in both CK8-positive and -negative cells (Figure 4a-c), while the cells of the AF were positive for vimentin only (Figure 4d-f). Co-expression of vimentin and CK8 was consistently restricted to a fraction of the cell population in bovine cells, whereas the porcine notochordal cells stained ubiquitously for both CK8 and vimentin (Figure 4g-i). At higher magnification, the spatial distribution of CK8 and vimentin differed slightly between the two cell types. In bovine NP cells, both intermediate filaments formed extensive networks throughout the cytoplasm (Figure 5c,e,f) while they were mainly compactly organized at the periphery of porcine notochordal cells (Figure 5d,g,h).


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)

Cytokeratin 8 (CK8) and vimentin expression in bovine and porcine disc cells. Dual immunofluorescence staining of CK8 and vimentin (VIM) performed on freshly isolated bovine nucleus pulposus cells (a-c), bovine annulus fibrosus cells (d-f), and porcine notochordal cells (g-i). Nuclei were visualized with DAPI (4'-6-diamidino-2-phenylindole) (scale bar = 60 μm). All cells examined were positive for VIM (b,e,h). No annulus cells were positive for CK8 (d). Some bovine nucleus cells -- forming clusters, as indicated by an arrowhead in (a) -- and all porcine cells (g) were positive for CK8; these cells co-expressed VIM (c,i).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Cytokeratin 8 (CK8) and vimentin expression in bovine and porcine disc cells. Dual immunofluorescence staining of CK8 and vimentin (VIM) performed on freshly isolated bovine nucleus pulposus cells (a-c), bovine annulus fibrosus cells (d-f), and porcine notochordal cells (g-i). Nuclei were visualized with DAPI (4'-6-diamidino-2-phenylindole) (scale bar = 60 μm). All cells examined were positive for VIM (b,e,h). No annulus cells were positive for CK8 (d). Some bovine nucleus cells -- forming clusters, as indicated by an arrowhead in (a) -- and all porcine cells (g) were positive for CK8; these cells co-expressed VIM (c,i).
Mentions: It has been suggested that mature NP cells could originate and migrate from the surrounding cells of the annulus or the cartilaginous end-plates [32] which are mesenchymal in origin. The type III intermediate filament vimentin is used as a marker of cells of mesodermal origin in contrast to the type II to which the epithelial CK8 belongs [33,34]. We therefore examined whether any cells were specific for this type III intermediate filament. Dual-immunofluorescence labelling of freshly isolated cells for CK8 and vimentin was performed in NP and AF from bovine and porcine tissues. The bovine NP cell population, as reported elsewhere [35], all appeared to express vimentin; it was found in both CK8-positive and -negative cells (Figure 4a-c), while the cells of the AF were positive for vimentin only (Figure 4d-f). Co-expression of vimentin and CK8 was consistently restricted to a fraction of the cell population in bovine cells, whereas the porcine notochordal cells stained ubiquitously for both CK8 and vimentin (Figure 4g-i). At higher magnification, the spatial distribution of CK8 and vimentin differed slightly between the two cell types. In bovine NP cells, both intermediate filaments formed extensive networks throughout the cytoplasm (Figure 5c,e,f) while they were mainly compactly organized at the periphery of porcine notochordal cells (Figure 5d,g,h).

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