real time polymerase chain reaction (qrt-pcr) analyses was conducted to assess mrna expression of Pax-1, FoxF1, Ovo-2, Ker-19, Lam-5 and IBSP using

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1 Is There A True Nucleus Pulpous Marker Which Can Be Used To Determine Stem Cell Differentiation Towards A Nucleus Pulposus Phenotype Rather Than Chondrocytes? Abbey A. Thorpe, BSc 1, Laura Creemers 2, Ashley A. Cole 3, Lee M. Breakwell 3, Antony L R Michael 3, Neil Chiverton 3, Chris Sammon, BSc PhD 4, Christine L. Le Maitre, BSc PhD 1. 1 Biomedical Research Institute, Sheffield Hallam University, Sheffield, United Kingdom, 2 Orthopaedics Department, UMC Utrecht, Utrecht, Netherlands, 3 Sheffield Teaching Hospitals, Sheffield, United Kingdom, 4 Materials and Engineering Research Institute, Sheffield Hallam University, Sheffield, United Kingdom. Disclosures: A.A. Thorpe: None. L. Creemers: None. A.A. Cole: None. L.M. Breakwell: None. A.L. Michael: None. N. Chiverton: None. C. Sammon: None. C.L. Le Maitre: None. Introduction: Despite recent progress in the development of tissue engineering stem cell therapies with the potential to promote the repair and regeneration of the nucleus pulposus (NP), the ability of such biomaterial scaffolds to induce NP cell differentiation cannot be characterised due to incomplete understanding of the molecular markers that define NP cells of the healthy IVD. The vast majority of studies characterise NP cell differentiation using traditional chondrogenic genes such as aggrecan, collagen type II (Col II) and sex determining region box 9 (Sox-9). Despite this, the cells and tissues of articular cartilage (AC) and the NP demonstrate significant differences in terms of ontogeny, extracellular matrix (ECM) disposition and biomechanical behaviour (Mwale et al 20104). Consequently vast amounts of current research has focused on identifying novel NP differential markers from AC, with a catalogue of potential markers being proposed; those with most significant potential include cytokeratin-19 (Ker-19), paired box protein (Pax-1), ovostatin-2 (Ovo-2) and forkhead box-1 (Foxf1), with integrin binding sialoprotein (IBSP) being identified as a potential negative NP marker (Minogue et al 2010, Rutges et al 2010). Here we evaluated the gene and protein expression of proposed NP markers, aiming to elucidate an NP phenotypic profile that could be used to determine adult stem cell differentiation. We additionally investigated laminin-5 (lam-5) as a potential ECM marker for NP cells, as this has been previously shown to be highly expressed in NP tissue in comparison to annulus fibrosis (AF) tissue (Gilchrist et al 2007, Bridgen et al 2013). Methods: AC samples were obtained from patients undergoing total knee replacements and intervertebral disc (IVD) samples were collected at post mortem or from patients undergoing microdisectomy for nerve root compression, cauda equine syndrome or sciatica. Quantitative

2 real time polymerase chain reaction (qrt-pcr) analyses was conducted to assess mrna expression of Pax-1, FoxF1, Ovo-2, Ker-19, Lam-5 and IBSP using isolated AC cells directly extracted from 12 patients, all of which were classified as macroscopically and histologically non-degenerate, and isolated NP cells directly extracted from 56 patients across a spectrum of histological grades of degeneration: non-degenerate (<3); moderately degenerate (3-7); severely degenerate (>7) and those containing infiltrated cells (Inf). All IVD samples were independently histologically graded by two researchers according to previously published criteria (Le Maitre et al 2005). Results from qrt-pcr were used to select potential NP marker candidates for protein expression analysis. Immunohistochemistry for PAX-1, Ker-19, Lam-5 and FoxF1 were performed on tissue sections from the same 12 AC and 56 NP samples as well as 25 additional PM samples, Thompson grades 1-5 n=5. Results: qrt-pcr data demonstrated significantly higher expression of Foxf1 and Pax-1 in NP cells than AC cells, with Pax-1 undetected in all AC samples, with no difference in expression observed across the different grades of degeneration (Fig 1). Lam-5 was found to be highly expressed in one AC and non-degenerate NP sample, with an increasing percentage of samples expressing Lam-5 observed with increasing grade of IVD degeneration (Fig 1). NP and AC cells displayed similar expression levels for IBSP, ovo-2 and Ker-19 although an increase in percentage of positive samples for ovo-2 and Ker-19 was seen with increasing IVD degeneration (Fig 1). Pax-1, Foxf1 and Lam-5 were subsequently assessed at protein level with Ker-19 also included due to its use as a proposed NP marker in a number of studies. Immunohistochemistry detection of Pax-1 demonstrated punctate staining within the cytoplasm of some NP cells with a higher percentage of immunopositive cells present in the NP than annulus fibrosus (AF) and cartilage endplate cells. Pax-1 immunopositive cells were localised to pockets within the NP, with greater staining intensity seen in cell clusters. Low levels of Pax-1 immunopositivity was observed in Thompson grade 1 and 2, with an increased percentage of immunopositive cells in Thompson grade 3, which was increased further in grade 4 discs (fig 2). Lam-5 immunopositivity was also limited to cells in the NP, with low immunopositivity observed in Thompson grades 1 and 2, but increased staining accompanied by matrix staining was observed in grades 3 and 4 (fig 2). Few immunopositive cells for Pax-1 and Lam-5 were observed in Thompson grade 5 samples, although significant loss of NP tissue was identified in these severely degenerate tissue sections. Ker-19 expression was also restricted to NP cells, however less than 5% of NP cells were positive in Thompson grades 1, 2 and 3 discs, with the exception of one grade 1 sample where the majority of NP

3 cells were immunopositive. Interestingly immunopositive cells appeared to be larger than neighbouring NP cells present within the same sample. No immunopositive cells for Ker-19 were identified in Thompson grade 4 and 5 samples. Discussion: This study investigated a number of proposed NP markers in a large cohort of NP and AC samples to validate their use as potential NP markers. Foxf1, Pax-1 and Lam-5 mrna were differentially expressed between NP and AC cells, with Foxf1 and Pax-1 showing particular promise as differential markers since their gene expression did not alter with stage of degeneration. Despite this we have demonstrated that expression of these genes did not correspond to protein expression in all NP cells within a sample, with very few immunopostive cells identified in non-degenerate samples with increasing immunopositivity observed with increasing grade of degeneration. Interestingly Ker-19, which has been reported to be an NP positive marker was only detected in 1 non-degenerate NP sample at mrna and with less than 5% immunopositvity observed at protein level this agrees with the study by Rugtes et al., who showed limited Ker-19 expression except in young individuals. Yet groups have begun accepting Ker-19 as an NP marker for assessing NP differentiation of stem cells. It is clear from these results that despite considerable research efforts to identify novel molecular characteristics of NP cells, a definitive differential marker still remains elusive. This study also highlights the need to conduct NP marker studies on large sample cohorts with varying degrees of degeneration, since the markers presented here were shown to be differentially expressed at gene and protein level with increasing grade of degeneration. Current investigations are focused on expanding these studies on a greater population of surgical samples to give a greater insight into the phenotypic profile of NP cells. Significance: In order to develop regenerative strategies for intervertebral disc repair appropriate markers for the NP phenotype are required, these to date remain elusive and may not exist, raising the question for what is the ideal characteristics to develop in differentiating stem cells for NP repair.

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5 ORS 2015 Annual Meeting Poster No: 1597