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.

Objective. Development of stem cell therapies for regenerating the nucleus pulposus (NP) are hindered by the lack of specific markers by which to distinguish NP cells from articular chondrocytes (ACs). The purpose of this study was to define the phenotype profile of human NP cells using gene expression profiling and to assess whether the identified markers could distinguish mesenchymal stem cell (MSC) differentiation to a correct NP cell phenotype.Methods. Affymetrix MicroArray analyses were conducted on human NP cells and ACs, and differential expression levels for several positive (NP) and negative (AC) marker genes were validated by real-time quantitative polymerase chain reaction (PCR) analysis. Novel marker gene and protein expression was also assessed in human bone marrow-derived MSCs (BM-MSCs) and adipose tissue-derived MSCs (AD-MSCs) following differentiation in type I collagen gels.Results. Analysis identified 12 NP-positive and 36-negative (AC) marker genes that were differentially expressed >20-fold, and for a subset of them (NPpositive genes PAX1, FOXF1, HBB, CA12, and OVOS2; AC-positive genes GDF10, CYTL1, IBSP, and FBLN1), Conclusion. This study is the first to use gene expression profiling to identify the human NP cell phenotype. Importantly, these markers can be used to determine the in vitro differentiation of MSCs to an NP-like, rather than an AC-like, phenotype. Interestingly, these results suggest that AD-MSCs may be a more appropriate cell type than BM-MSCs for use in engineering intervertebral disc tissue.Low back pain is a major public health problem in the Western World, with over 80% of the population estimated to report low back pain during their lifetime (1). This has a serious impact on economies, with total costs related to this condition exceeding $100 billion per year in the US alone (2). One of the principal causes of low back pain is degeneration of the intervertebral disc (IVD), whereby adverse changes in the matrix results in dehydration of the nucleus pulposus (NP), loss of disc height, and loss of mechanical function, leading to traumatic damage and associated pain (3-5).Current treatments are aimed at relieving symptoms of low back pain, rather than halting progression of the disease by inhibiting the underlying disease mechanisms or by restoring disc structure and function. Thus, there is an urgent need to provide more-effective treatments for IVD degeneration. Since the NP is the region of the IVD that is most affected during degeneration,

IntroductionMatrix metalloproteinases (MMPs) are known to be involved in the degradation of the nucleus pulposus (NP) during intervertebral disc (IVD) degeneration. This study investigated MMP-10 (stromelysin-2) expression in the NP during IVD degeneration and correlated its expression with pro-inflammatory cytokines and molecules involved in innervation and nociception during degeneration which results in low back pain (LBP).MethodsHuman NP tissue was obtained at postmortem (PM) from patients without a history of back pain and graded as histologically normal or degenerate. Symptomatic degenerate NP samples were also obtained at surgery for LBP. Expression of MMP-10 mRNA and protein was analysed using real-time polymerase chain reaction and immunohistochemistry. Gene expression for pro-inflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-α), nerve growth factor (NGF) and the pain-associated neuropeptide substance P were also analysed. Correlations between MMP-10 and IL-1, TNF-α and NGF were assessed along with NGF with substance P.ResultsMMP-10 mRNA was significantly increased in surgical degenerate NP when compared to PM normal and PM degenerate samples. MMP-10 protein was also significantly higher in degenerate surgical NP samples compared to PM normal. IL-1 and MMP-10 mRNA demonstrated a significant correlation in surgical degenerate samples, while TNF-α was not correlated with MMP-10 mRNA. NGF was significantly correlated with both MMP-10 and substance P mRNA in surgical degenerate NP samples.ConclusionsMMP-10 expression is increased in the symptomatic degenerate IVD, where it may contribute to matrix degradation and initiation of nociception. Importantly, this study suggests differences in the pathways involved in matrix degradation between painful and pain-free IVD degeneration.

We have modified the surface topography of poly e-caprolactone (PCL) and polylactic acid (PLA) blended films to improve cell proliferation and to guide the regeneration of peripheral nerves. Films with differing shaped grooves were made using patterned silicon templates, sloped walls (SL), V-shaped (V), and square-shaped (SQ), and compared with nongrooved surfaces with micropits. The solvent cast films were tested in vitro using adult adipose-derived stem cells differentiated to Schwann cell-like cells. Cell attachment, proliferation, and cell orientation were all improved on the grooved surfaces, with SL grooves giving the best results. We present in vivo data on Sprague-Dawley rat sciatic nerve injury with a 10-mm gap, evaluating nerve regeneration at 3 weeks across a polymer nerve conduit modified with intraluminal grooves (SL, V, and SQ) and differing wall thicknesses (70, 100, 120, and 210 mm). The SL-grooved nerve conduit showed a significant improvement over the other topographical-shaped grooves, while increasing the conduit wall thickness saw no positive effect on the biological response of the regenerating nerve. Furthermore, the preferred SL-grooved conduit (C) with 70 mm wall thickness was compared with the current clinical gold standard of autologous nerve graft (Ag) in the rat 10-mm sciatic nerve gap model. At 3 weeks postsurgery, all nerve gaps across both groups were bridged with regenerated nerve fibers. At 16 weeks, features of regenerated axons were comparable between the autograft (Ag) and conduit (C) groups. End organ assessments of muscle weight, electromyography, and skin reinnervation were also similar between the groups. The comparable experimental outcome between conduit and autograft, suggests that the PCL/PLA conduit with inner lumen microstructured grooves could be used as a potential alternative treatment for peripheral nerve repair.

Background Little is understood of the molecular mechanisms involved in the earliest cell fate decision in human development, leading to the establishment of the trophectoderm (TE) and inner cell mass (ICM) stem cell population. Notably, there is a lack of understanding of how transcriptional networks arise during reorganisation of the embryonic genome post-fertilisation. Results We identified a hierarchical structure of preimplantation gene network modules around the time of embryonic genome activation (EGA). Using network models along with eukaryotic initiation factor (EIF) and epigenetic-associated gene expression we defined two sets of blastomeres that exhibited diverging tendencies towards ICM or TE. Analysis of the developmental networks demonstrated stage specific EIF expression and revealed that histone modifications may be an important epigenetic regulatory mechanism in preimplantation human embryos. Comparison to published RNAseq data confirmed that during EGA the individual 8-cell blastomeres are transcriptionally primed for the first lineage decision in development towards ICM or TE. Conclusions Using multiple systems biology approaches to compare developmental stages in the early human embryo with single cell transcript data from blastomeres, we have shown that blastomeres considered to be totipotent are not transcriptionally equivalent. Furthermore we have linked the developmental interactome to individual blastomeres and to later cell lineage. This has clinical implications for understanding the impact of fertility treatments and developmental programming of long term health. Electronic supplementary material The online version of this article (10.1186/s12864-019-5558-8) contains supplementary material, which is available to authorized users.

Modular Hierarchy -Biological networks form regions of higher connectivity than would be expected by chance, known as modules. Modules represent functionally related elements of a network and their relative influence in a system can be estimated by their centrality.Metanode -The most central ten connected genes within a module.Connectivity -The number of links existing between a given node and its neighbours. An increased connectivity is indicative of a gene which is involved in numerous processes.Community Centrality -A measure of the relative 'importance' of a node, characterised by high connectivity or connections between areas of high connectivity.Bridgeness -A property of a node in a network which sits between two areas of high connectivity, such that if removed, it would cause the separation of a single module into two. These nodes act as 'bridges' between modules and an increased bridgeness identifies a node which connects multiple modules.Party hub -A node with multiple connections which, in a biological system, is thought to represent a gene with many active simultaneous interactions, such as protein complexes. It is characterised by a node which has a reduced bridgeness at a given centrality when compared to a date-hub. Date hub -A node with multiple connections which, in a biological system, has non-concurrent interactionswith other nodes. These are thought to represent transcription factors. It is characterised by a node which has an increased bridgeness at a given centrality when compared to a party-hub. Similarity Network Fusion -A network approach which uses nearest neighbour relationships to combinedatasets and identify regions of similarity within and between them. In the context of this manuscript, coherency between datasets represents genes whose expression patterns are conserved between cells derived from embryonic tissue and human embryonic stem cell lines. An interactome network model of gene expression unique to ICM can be used as a framework to 100 assess similarity with human embryonic stem cells. 101An interactome network model can be used to consider the proteins derived from the differentially 102 expressed genes and the proteins that they interact with. Using this approach allowed us to consider 103 the wider context of biological influence generated by the gene expression unique to either the ICM 104 or TE and to implement these models as a framework to assess similarity with the hESC lines. We first 105 used the genes with unique expression in either ICM or TE to generate interactome network models 106 by inference to known protein-protein interactions (Figure 4A & 4B). As interactome networks 107 account for inferred interactions these may be shared between models. Comparing the TE and ICM 108 interactome network models an overlap of 5659 inferred genes was present that represented 109 potentially shared protein:protein interactions, accounting for 72% of the ICM interactome and 66% 110 of the TE interactome. 111 Both networks were enriched for genes associated with pluripotency, f...