Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes

Abstract

IntroductionNucleus 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.MethodsMicroarrays were conducted on bovine AC, AF and NP cells, using Affymetrix Genechip® 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.ResultsMicroarray comparisons between NP/AC&AF and NP/AC identified 34 NP-specific and 49 IVD-specific genes respectively that were differentially expressed ≥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.ConclusionsThis 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.

Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc

“…These genes exhibited the highest differential expression in our study. In comparison to a bovine micro-array transcriptional profiling study using three donors [6], we also found differential expression of bKrt19 between NP and AC tissue, but not for bFoxf1 between NP and AF tissue and bCD24 between NP and AC tissue. The lack of detectable CA12 expression in the bovine tail IVD is supported by literature [6].…”

“…The IVD is a unique tissue that differs from Articular Cartilage (AC) in embryonic development, nutrient supply, oxygen tension and biochemical composition [3]. Moreover, whole transcriptome analyses of IVD and AC cells revealed distinct gene expression profiles for the NP, AF and AC in rat, canine, bovine and human IVDs [4][5][6][7]. These studies began to unravel the NP cell phenotype at the transcriptome level leading to a first consensus paper regarding definition of healthy young NP cells [8].…”

“…Therefore, model organisms, such as the cow, are used for in vitro cellular and ex vivo biomechanical studies of the IVD [14,15]. The bovine tail IVD represents a good model for the non-degenerate human IVD: the bovine NP retains few notochordal cells and whole transcriptome analyses identified similar phenotypic marker genes as in humans [6,16,17]. This study aimed to establish whether IVD phenotypic marker genes, which we previously used to distinguish non-degenerate human NP from AF cells, can be used to describe the cell phenotype in the outer AF, inner AF and NP in situ in the bovine tail IVD and this was compared to passage 0 monolayer cells.…”

The evolutionary importance of cell ratio between notochordal and nucleus pulposus cells: an experimental 3-D co-culture study

“…There have been several morphological papers published, which describe the morphological differences between these cells [19,20]. Transcriptomics, on the other hand, comparing the two cell populations revealed that there are only about two dozen genes really distinct between these two cell types [27,30]. The search for specific markers to distinguish these two cell populations has just started [13].…”

“…It has been speculated that these cells have precursor character and might belong to the exact same cell lineage as the disc cells since there were not too many differences reported between these two lineages [27,32]. Other research groups are convinced that these cells are originating from another cell layer than the mesoderm, but are rather ectodermal origin.…”