Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes.
BACKGROUND
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.
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
Ben M
BM
Minogue
Stephen M
SM
Richardson
Leo Ah
LA
Zeef
Anthony J
AJ
Freemont
Judith A
JA
Hoyland
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology* / Q000378:metabolism* / Q000473:pathology; D055959:Intervertebral Disc Degeneration / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D025341:Principal Component Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic