Pharmacological inhibition of mTORC1 activity protects against inflammation-induced apoptosis of nucleus pulposus cells

Lumbar disc herniation is a common disease characterized by the degeneration of intervertebral discs (IVDs), accompanied by imbalance of metabolic and inflammatory homeostasis. Current studies establish that IVD degeneration is induced by increased apoptosis of nucleus pulposus (NP) cells. However, the underlying mechanisms of NP cell survival/apoptosis are not well elucidated. Here, we reveal a novel mechanism by which mTORC1 signaling controls NP cell survival through regulating metabolic homeostasis. We demonstrated that hyperactivated mTORC1 activity induced by inflammatory cytokines engenders the apoptosis of NP cells, whereas pharmacological inhibition of mTORC1 activity promotes NP cell survival. Using an integrative approach spanning metabolomics and biochemical approaches, we showed that mTORC1 activation enhanced glucose metabolism and lactic acid production, and therefore caused NP cell apoptosis. Our study identified mTORC1 in NP cells as a novel target for IVD degeneration, and provided potential strategies for clinical intervention of lumbar disc herniation.     

Role of growth differentiation factor-5 and bone morphogenetic protein type II receptor in the development of lumbar intervertebral disc degeneration

The present study was designed to evaluate the role of growth differentiation factor-5 (GDF-5) and bone morphogenetic protein type II receptor (BMPR-II) in the development of lumbar intervertebral disc degeneration (IDD). A total of 24 patients with lumbar IDD (experiment group) and 6 patients with lumbar vertebral fracture (control group) were enrolled in the study. Tissue samples of IVD from the experiment group and control group were obtained during lumbar fusion operation, respectively. Fixation and decalcification of IVD tissue were performed, and then HE staining was carried out to observe the morphological changes of the lumbar IVD tissues. The expression of GDF-5 and BMPRII in human lumbar IVD was detected by immunohistochemical staining. HE staining results showed that non- and minimal degeneration was found in 11 cases (score range, 0-3), moderate degeneration in 12 cases (score range, 4-8), and severe degeneration in 7 cases (score range, 9-12). According to the immunohistochemical results, the positive expression rates of GDF-5 and BMPRII in NP were higher than those in AF of the non- and minimal degeneration group, moderate degeneration group and severe degeneration group (all P < 0.05). However, no significant difference in GDF-5 or BMPRII positive expression was observed among the normal, non- and minimal, moderate and severe degeneration groups in neither NP area nor AF area (all P > 0.05). In conclusion, our results showed that GDF-5 and BMPRII expressed both in normal and degenerated IVD tissues, and GDF-5 might have an inhibition effect on degenerated lumbar IVD, suggesting that gene therapy may be a useful approach in producing physiological effects during early- and late-phase of lumbar IDD.     

Demineralized Bone Particle Impregnated Poly(l-Lactide-co-Glycolide) Scaffold for Application in Tissue-Engineered Intervertebral Discs

Abstract

Demineralized bone particle (DBP) contains powerful bioactive molecules that facilitate new bone or cartilage growth. We developed hybrid scaffolds of poly(l-lactide-co-glycolide) (PLGA) with various concentrations of DBP (DBP/PLGA), of which phenotypes on intervertebral disc (IVD) cells were investigated. The hybrid scaffold has a cylindrical donut shape with two distinct parts; the inner is for the nucleus pulposus (NP) and the outer is for annulus fibrosus (AF). Rabbit NP and AF cells were seeded into the inner and outer regions of the DBP/PLGA scaffolds separately. Disc cell viability in DBP/PLGA scaffolds was superior to pure PLGA scaffold and increased with increasing DBP concentration. In vitro- and in vivo-formed tissues were characterized by RT-PCR, Safranin-O, Masson’s trichrome staining and immunohistochemi- cal staining for type-I and type-II collagen. DBP/PLGA hybrid scaffolds revealed more active expression of disc phenotypes, as characterized by protein and mRNA expression, than the PLGA control. This study provides valuable information for potential disc replacement using DBP and PLGA.     

Monitoring of the effect of intervertebral disc nucleus pulposus ablation by MRI

In order to investigate intervertebral disc (IVD) degeneration and repair, a quantitative non‐invasive tool is needed. Various MRI methods including qCPMG, which yields dipolar echo relaxation time (T_DE), magnetization transfer contrast (MTC), and ¹H and ²H double quantum filtered (DQF) MRI were used in the present work to monitor changes in rat IVD after ablation of the nucleus pulposus (NP), serving as a model of severe IVD degeneration. In the intact IVD, a clear distinction between the annulus fibrosus (AF) and the NP is obtained on T₂ and T_DE weighted images as well as on MTC maps, reflecting the high concentration of ordered collagen fibers in the AF. After ablation of the NP, the distinction between the compartments is lost. T₂ and T_DE relaxation times are short throughout the disc and MTC is high. ¹H and ²H DQF signal, which in intact discs is obtained only for the AF, is now observable throughout the tissue. These results indicate that after ablation, there is an ingression of collagen fibers from the AF into the area that was previously occupied by the NP, as was confirmed by histology. Copyright © 2010 John Wiley & Sons, Ltd.     

Expression of Laminin Isoforms,Receptors, and Binding Proteins Unique to Nucleus Pulposus Cells of Immature Intervertebral Disc

Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. Previously, we found that the laminin γ1 chain was more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin α6 subunit, as compared to adjacent anulus fibrosus region. This result suggests that cell-matrix interactions may be unique to the immature NP. However, the identity of laminin isoforms specific to immature or mature NP tissues, their associated receptors, and functional significance are still poorly understood. In this study, we evaluated the zonal-specific expression of the laminin chains, receptors (i.e., integrins), and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc. Our goal was to reveal features of cellular environment and cell-matrix interactions in the immature NP. Results from both immunohistochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin α5 chain, laminin receptors (integrin α3, α6, β4 subunit, and CD239), and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus and that laminins may be important contributors to region-specific IVD biology. The revealed laminin isoforms, their receptors, and related binding proteins may be used as distinguishing features of these immature NP cells in the intervertebral disc.     

Bone morphogenetic protein-2 and -7 mediate the anabolic function of nucleus pulposus cells with discrete mechanisms

Bone morphogenetic proteins (BMPs) play roles in promoting cell anabolism, especially in extracellular matrix production. The difference between BMP members in their capacity to modulate intervertebral disc cell activity is yet to be defined. BMP-7/OP-1 has been shown to retard disc degeneration. We compared the activity of BMP-7 with that of BMP-2 on nucleus pulposus (NP) cell phenotype and function, and investigated how they differentially affect the gene expression profiles of signaling cascade components in human NP cells under degenerative states. We found that while both BMP-2 and BMP-7 enhanced matrix production of bovine NP cells, BMP-7 is more potent than BMP-2 at various dosages (50–800 ng/ml). BMP-7 exerted a relatively stronger stimulation on sulfated glycosaminoglycan production and proliferation in human NP cells. Degenerated NP cells showed an overall weaker response to the BMPs than non-degenerated cells, and were more sensitive to BMP-7 than BMP-2 stimulation. Compared to BMP-2, BMP-7 not only induced the gene expression of canonical BMP components, but also evoked changes in MAPKs as well as CREB1 and EP300 gene expression in degenerated NP cells, suggesting potential activation of the cAMP dependent protein kinase related pathways. In contrast to BMP-2, BMP-7 concomitantly inhibited the expression of profibrotic genes. We propose that BMP-2 and BMP-7, and likely other BMPs, may operate multifaceted but discrete molecular machineries that give rise to their different capacity in regulating NP cell phenotype. Further investigations into such differential capacity may possibly derive alternative cues important for IVD repair or engineering.     

Screening of hyaluronic acid–poly(ethylene glycol) composite hydrogels to support intervertebral disc cell biosynthesis using artificial neural network analysis

Hyaluronic acid (HA)–poly(ethylene glycol) (PEG) composite hydrogels have been widely studied for both cell delivery and soft tissue regeneration applications. A very broad range of physical and biological properties have been engineered into HA–PEG hydrogels that may differentially affect cellular “outcomes” of survival, synthesis and metabolism. The objective of this study was to rapidly screen multiple HA–PEG composite hydrogel formulations for an effect on matrix synthesis and behaviors of nucleus pulposus (NP) and annulus fibrosus (AF) cells of the intervertebral disc (IVD). A secondary objective was to apply artificial neural network analysis to identify relationships between HA–PEG composite hydrogel formulation parameters and biological outcome measures for each cell type of the IVD. Eight different hydrogels were developed from preparations of thiolated HA (HA–SH) and PEG vinylsulfone (PEG–VS) macromers, and used as substrates for NP and AF cell culture in vitro. Hydrogel mechanical properties ranged from 70 to 489 kPa depending on HA molecular weight, and measures of matrix synthesis, metabolite consumption and production and cell morphology were obtained to study relationships to hydrogel parameters. Results showed that NP and AF cell numbers were highest upon the HA–PEG hydrogels formed from the lower-molecular-weight HA, with evidence of higher sulfated glycosaminoglycan production also upon lower-HA-molecular-weight composite gels. All cells formed more multi-cell clusters upon any HA–PEG composite hydrogel as compared to gelatin substrates. Formulations were clustered into neurons based largely on their HA molecular weight, with few effects of PEG molecular weight observed on any measured parameters.     

Engineering alginate for intervertebral disc repair

Alginate is frequently studied as a scaffold for intervertebral disc (IVD) repair, since it closely mimics mechanical and cell-adhesive properties of the nucleus pulposus (NP) of the IVD. The aim of this study was to assess the relation between alginate concentration and scaffold stiffness and find preparation conditions where the viscoelastic behaviour mimics that of the NP. In addition, we measured the effect of variations in scaffold stiffness on the expression of extracellular matrix molecules specific to the NP (proteoglycans and collagen) by native NP cells. We prepared sample discs of different concentrations of alginate (1%–6%) by two different methods, diffusion and in situ gelation. The stiffness increased with increasing alginate concentration, while the loss tangent (dissipative behaviour) remained constant. The diffusion samples were ten-fold stiffer than samples prepared by in situ gelation. Sample discs prepared from 2% alginate by diffusion closely matched the stiffness and loss tangent of the NP. The stiffness of all samples declined upon prolonged incubation in medium, especially for samples prepared by diffusion. The biosynthetic phenotype of native cells isolated from NPs was preserved in alginate matrices up to 4 weeks of culturing. Gene expression levels of extracellular matrix components were insensitive to alginate concentration and corresponding matrix stiffness, likely due to the poor adhesiveness of the cells to alginate. In conclusion, alginate can mimic the viscoelastic properties of the NP and preserve the biosynthetic phenotype of NP cells but certain limitations like long-term stability still have to be addressed.     

Enhancement of Energy Production of the Intervertebral Disc by the Implantation of Polyurethane Mass Transfer Devices

Insufficient nutrient supply has been suggested to be one of the etiologies for intervertebral disc (IVD) degeneration. We are investigating nutrient transport into the IVD as a potential treatment strategy for disc degeneration. Most cellular activities in the IVD (e.g., cell proliferation and extracellular matrix production) are mainly driven by adenosine-5′-triphosphate (ATP) which is the main energy currency. The objective of this study was to investigate the effect of increased mass transfer on ATP production in the IVD by the implantation of polyurethane (PU) mass transfer devices. In this study, the porcine functional spine units were used and divided into intact, device and surgical groups. For the device and surgical groups, two puncture holes were created bilaterally at the dorsal side of the annulus fibrosus (AF) region and the PU mass transfer devices were only implanted into the holes in the device group. Surgical groups were observed for the effects of placing the holes through the AF only. After 7 days of culture, the surgical group exhibited a significant reduction in the compressive stiffness and disc height compared to the intact and device groups, whereas no significant differences were found in compressive stiffness, disc height and cell viability between the intact and device groups. ATP, lactate and the proteoglycan contents in the device group were significantly higher than the intact group. These results indicated that the implantation of the PU mass transfer device can promote the nutrient transport and enhance energy production without compromising mechanical and cellular functions in the disc. These results also suggested that compromise to the AF has a negative impact on the IVD and must be addressed when treatment strategies are considered. The results of this study will help guide the development of potential strategies for disc degeneration.     

MicroRNA-16 inhibits the lipopolysaccharide-induced inflammatory response in nucleus pulposus cells of the intervertebral disc by targeting TAB3

IntroductionA variety of inflammatory mediators are produced by the degenerative human intervertebral disc (IVD) tissues spontaneously, suggesting their role in the development of intervertebral disc degeneration (IDD). Our present study was designed to investigate the regulatory effect of microRNA-16 (miR-16) on the lipopolysaccharide (LPS)-induced inflammatory response in nucleus pulposus (NP) cells of the IVD.Material and methodsNP cells were treated with LPS to induce inflammatory responses. The expression of miRNA and genes was determined by qRT-PCR. Western blot and an ELISA kit were used to detect the proteins and protein expression, respectively. A dual luciferase reporter assay was applied to identify the correlation between a miRNA and a gene, and to test nuclear factor-κB (NF-κB) activity.ResultsThe results suggested that miR-16 positively regulated the mRNA and protein expression of extracellular matrix (ECM) genes (including aggrecan and collagen II) in NP cells, while it was negatively correlated with ECM degrading enzymes (including MMP3, MMP13, ADAMTS4, ADAMTS5) and related genes of nitric oxide (NO) reaction. Further studies revealed that miR-16 could oppositely regulate NF-κB and MAPK pathways by directly mediating their upstream gene TAB3.ConclusionsOur study suggested that, in NP cells of the IVD, miR-16 could exert inhibitory effects on the LPS-induced inflammatory response through NF-κB and MAPK pathways by directly mediating TAB3. In this way, miR-16 would play a protective role against LPS-induced IDD and inflammation. Therefore, miR-16 may be a novel therapeutic target for the inhibit of the ECM in the IVD.     

Photocrosslinkable laminin-functionalized polyethylene glycol hydrogel for intervertebral disc regeneration

Intervertebral disc (IVD) disorders and age-related degeneration are believed to contribute to lower back pain. There is significant interest in cell-based strategies for regenerating the nucleus pulposus (NP) region of the disc; however, few scaffolds have been evaluated for their ability to promote or maintain an immature NP cell phenotype. Previous studies have shown that NP cell–laminin interactions promote cell adhesion and biosynthesis, which suggests a laminin-functionalized biomaterial may be useful for promoting or maintaining the NP cell phenotype. Here, a photocrosslinkable poly(ethylene glycol)–laminin 111 (PEG-LM111) hydrogel was developed. The mechanical properties of PEG-LM111 hydrogel could be tuned within the range of dynamic shear moduli values previously reported for human NP. When primary immature porcine NP cells were seeded onto PEG-LM111 hydrogels of varying stiffnesses, LM111-presenting hydrogels were found to promote cell clustering and increased levels of sGAG production as compared to stiffer LM111-presenting and PEG-only gels. When cells were encapsulated in 3-D gels, hydrogel formulation was found to influence NP cell metabolism and expression of proposed NP phenotypic markers, with higher expression of N-cadherin and cytokeratin 8 observed for cells cultured in softer (<1 kPa) PEG-LM111 hydrogels. Overall, these findings suggest that soft, LM111-functionalized hydrogels may promote or maintain the expression of specific markers characteristic of an immature NP cell phenotype.     

Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc

The histological and biochemical changes that occur in the extracellular matrix of the intervertebral disc (IVD) during ageing and degeneration have been investigated extensively. However, the mechanisms behind these changes are not fully understood. A number of studies have suggested the involvement of matrix metalloproteinases (MMPs) and ADAMTS in IVD degeneration, but few have localized the site of production of these enzymes to the cells of the degenerate disc. This study uses immunohistochemical techniques to localize and quantify the production of degrading enzymes (MMPs 1, 3, and 13, and ADAMTS 4) and their inhibitors (TIMPS 1, 2, and 3) within non-degenerate and degenerate discs of varying severity of degeneration. In all discs investigated, the cells that produced the enzymes and their inhibitors were the chondrocyte-like cells of the nucleus pulposus and inner annulus fibrosus (AF), with little immunopositivity in the outer AF. Non-degenerate discs showed low numbers of cells expressing the degradative enzymes MMP 1 and ADAMTS 4, suggesting a role for these enzymes in normal homeostasis. No MMP 3 or MMP 13 immunopositivity was observed in non-degenerate discs. In degenerate discs, the number of cells immunopositive for MMPs 1, 3, 13 and ADAMTS 4 increased with the severity of degeneration. This increase in degrading enzymes was also accompanied by increases in the number of cells immunopositive for TIMPs 1 and 2 but not TIMP 3. This study highlights that although the expression of a number of MMPs increases with degeneration, this is accompanied by an increase in their inhibitors. However, the increase in the number of cells immunoreactive for ADAMTS 4 with increasing degeneration was not paralleled by a rise in its inhibitor TIMP 3. This finding indicates that the aggrecanases, rather then the MMPs, are a possible therapeutic target for the inhibition of disc degeneration.     

Intervertebral disc ageing and degeneration: The antiapoptotic effect of oestrogen

As an important part of the spinal column, the intervertebral disc (IVD) plays an important role in the intervertebral juncture and spinal movement in general. IVD degeneration (IVDD), which mimics disc ageing but at an accelerated rate, is a common and chronic process that results in severe spinal symptoms, such as lower back pain. It is generally assumed that lower back pain caused by IVDD can also develop secondary conditions, including spinal canal stenosis, spinal segmental instability, osteophyte formation, disc herniation and spinal cord and nerve root compression. Over the past few years, many researchers around the world have widely studied the relevance between oestrogen and IVDD, indicating that oestrogen can effectively alleviate IVDD development by inhibiting the apoptosis of IVD cells. Oestrogen can decrease IVD cell apoptosis in multiple ways, including the inhibition of the inflammatory cytokines IL-1β and TNF-α, reducing catabolism because of inhibition of matrix metalloproteinases, upregulating integrin α₂β₁ and IVD anabolism, activating the PI3K/Akt pathway, decreasing oxidative damage and promoting autophagy. In this article, we perform an overview of the literature regarding the antiapoptotic effect of oestrogen in IVDD.     

Intervertebral disc degeneration induced by intradiscal poly(methyl methacrylate) leakage after spine augmentation in an in vivo rabbit model

Although intradiscal cement leakage is a common complication of spine augmentation, the effects of cement leakage into the intervertebral disc (IVD) have not been well investigated. This study aimed to determine the effects of cement leakage on IVD degeneration in rabbits. Poly(methyl methacrylate) (PMMA) particles were injected into lumbar discs of rabbits using 26G needles. Tissue effects were assessed using disc height, sagittal T2-weighted images, histology and immunohistochemistry. The results showed that stimulation with PMMA particles significantly reduced disc height compared with that in the sham-operation group at 3 weeks after injection. The mean signal intensity of the operated discs showed little to no changes among all groups at 3 weeks post-operation. After 6 weeks, the signal intensity of the PMMA-injected group decreased by 22% compared with that in the sham-operation group. Histological and quantitative immunohistochemical examination indicated phenotypic tissue changes from cartilaginous tissue into fibrotic tissue, with apparent degeneration in the PMMA group. Additionally, more collagen type II-containing tissues, but fewer matrix metalloproteinase-7-positive cells or apoptotic cells, were detected in the sham-operation group. The PMMA particle-induced degeneration rate was slower than that of the degeneration group, whereas the histologic data showed no difference in the progression of degeneration between the two groups. These data suggest that PMMA particles can moderately accelerate disc degeneration compared with the 18G needle puncture model. In conclusion, intradiscal injection of PMMA particles induced significant IVD degeneration in vivo. Therefore, further study of the adverse effects of PMMA leakage on IVD degeneration is required.     

Clarifying the nomenclature of intervertebral disc degeneration and displacement: from bench to bedside

As a significant determinant of low back pain, intervertebral disc degeneration (IDD) has attracted more and more attention of both investigators and physicians. Disc herniation, termed as intervertebral disc displacement, is amongst the most prevalent spinal diseases closely linked with IDD. Due to the same origins and similar pathophysiology, the ambiguity regarding the similarity and difference of IDD and intervertebral disc displacement thus remains. The aim of this study was to clarify the nomenclature of IDD and disc herniation in terms of molecular etiology, pathophysiology, nature history and clinical outcomes. Collectively, IDD is a type of multifaceted, progressive spinal disease with or without clinical symptoms as back pain, characterized by extracellular matrix and the integrity of NP and AF lost, fissures formation. Disc herniation (termed as intervertebral disc displacement) is a type of spinal disease based on IDD or not, with local pain and/or sciatica due to mechanical compression and autoimmune cascades upon the corresponding nerve roots. Clarifying the nomenclature of intervertebral disc degeneration and displacement has important implications both for investigators and for physicians.     

The role of nerve fibers and their neurotransmitters in regulating intervertebral disc degeneration

Intervertebral disc degeneration (IVDD) has been the major contributor to chronic lower back pain (LBP). Abnormal apoptosis, senescence, and pyroptosis of IVD cells, extracellular matrix (ECM) degradation, and infiltration of immune cells are the major molecular alternations during IVDD. Changes at tissue level frequently occur at advanced IVD tissue. Ectopic ingrowth of nerves within inner annulus fibrosus (AF) and nucleus pulposus (NP) tissue has been considered as the primary cause for LBP. Innervation at IVD tissue mainly included sensory and sympathetic nerves, and many markers for these two types of nerves have been detected since 1940. In fact, in osteoarthritis (OA), beyond pain transmission, the direct regulation of neuropeptides on functions of chondrocytes have attracted researchers’ great attention recently. Many physical and pathological similarities between joint and IVD have shed us the light on the neurogenic mechanism involved in IVDD. Here, an overview of the advances in the nervous system within IVD tissue will be performed, with a discussion on in the role of nerve fibers and their neurotransmitters in regulating IVDD. We hope this review can attract more research interest to address neuromodulation and IVDD itself, which will enhance our understanding of the contribution of neuromodulation to the structural changes within IVD tissue and inflammatory responses and will help identify novel therapeutic targets and enable the effective treatment of IVDD disease.     

Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold

Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells-fibrin suspension and polymerized by dropping thrombin-sodium chloride (CaCl(2)) solution. A PLGA-cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold-cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.     

Potential biomarkers of the mature intervertebral disc identified at the single cell level

Intervertebral disc (IVD) degeneration and trauma is a major socio‐economic burden and the focus of cell‐based regenerative medicine approaches. Despite numerous ongoing clinical trials attempting to replace ailing IVD cells with mesenchymal stem cells, a solid understanding of the identity and nature of cells in a healthy mature IVD is still in need of refinement. Although anatomically simple, the IVD is comprised of heterogeneous cell populations. Therefore, methods involving cell pooling for RNA profiling could be misleading. Here, by using RNA in situ hybridization and z proportion test, we have identified potential novel biomarkers through single cell assessment. We quantified the proportion of RNA transcribing cells for 50 genetic loci in the outer annulus fibrosus (AF) and nucleus pulposus (NP) in coccygeal bovine discs isolated from tails of four skeletally mature animals. Our data reconfirm existing data and suggest 10 novel markers such as Lam1 and Thy1 in the outer AF and Gli1, Gli3, Noto, Scx, Ptprc, Sox2, Zscan10 and LOC101904175 in the NP, including pluripotency markers, that indicate stemness potential of IVD cells. These markers could be added to existing biomarker panels for cell type characterization. Furthermore, our data once more demonstrate heterogeneity in cells of the AF and NP, indicating the need for single cell assessment by methods such as RNA in situ hybridization. Our work refines the molecular identity of outer AF and NP cells, which can benefit future regenerative medicine and tissue engineering strategies in humans.