Abstract
The nucleus pulposus (NP) of the intervertebral disc (IVD) demonstrates substantial changes in cell and matrix composition with both ageing and degeneration. While recent transcriptomic profiling studies have helped define human NP cell phenotype, it remains unclear how expression of these markers is influenced by ageing or degeneration. Furthermore, cells of the NP are thought to derive from the notochord, although adult NP lacks identifiable notochordal (NC) cells. This study aimed to confirm expression of previously identified NP and NC marker genes in adult human NP cells from a range of ages and degenerate states. Importantly, using gene expression analysis (N = 60) and immunohistochemistry (N = 56) the study demonstrates expression of NP markers FoxF1, Pax-1, keratin-8/18, carbonic anhydrase-12, and NC markers brachyury, galectin-3 and CD24 in cells of the NP irrespective of age or degeneration. Our immunohistochemical data, combined with flow cytometry (N = 5) which identified a small number of CA12+Gal3+T+CD24+ cells, suggests the possible presence of a sub-population of cells with an NC-like phenotype in adult NP tissue. These findings suggest that the NP contains a heterogeneous population of cells, which may possess varied phenotypic and functional profiles and thus warrant further investigation to improve our understanding of IVD homeostasis and repair.
Highlights
70% of individuals in developed societies suffer from low back pain (LBP) and neck pain at some point[1, 2]
When comparing expression between nucleus pulposus (NP) cells from cervical and lumbar discs, only KRT8 and T demonstrated a significant difference, with KRT8 being higher in cervical NP and T being higher in lumbar NP (Fig. 1a and b)
Gene expression levels of notochordal cell markers T, FOXA2, chordin (CHRD), noggin (NOG) and CD24 did not differ across the various age categories, whilst LGALS3 expression was significantly lower in mature adult as compared to young adult NP samples
Summary
70% of individuals in developed societies suffer from low back pain (LBP) and neck pain at some point[1, 2]. This highlights the importance of accurate profiling of the NP cell phenotype and a number of microarray studies have been conducted in recent years in different species with a view to identifying a panel of marker genes distinguishing NP cells from other cell types, predominantly AC cells[8,9,10,11,12] Our studies using both human and bovine NP and AC cells have identified a number of differentially expressed genes[9,10,11], including forkhead box F1 (FOXF1), paired box 1 (PAX1), carbonic anhydrase 12 (CA12) and the keratins (KRT) 8, 18 and 19. Isolation of separate bovine NP and NC cell populations by size filtration with subsequent analysis of cellular gene expression identified similarities between the two cell types[10] This is important as it is suggestive of a common ontogeny between NP and NC cells, or may be indicative of a subset of NP cells within the adult human NP that are phenotypically NC cell-like. The aims of this investigation were: firstly to validate our previously described novel NP marker genes[9, 10] in a large cohort of adult human specimens, and correlate levels of gene expression with age and severity of tissue degeneration; and secondly, to analyse expression of novel NP and NC cell marker proteins in cells of the adult human NP in order to ascertain whether expression is noted in all cells or a subset of NP cells, correlating this expression to age and degenerative score
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