Abstract
Severe and chronic low back pain is often associated with intervertebral disc (IVD) degeneration. While imposing a considerable socio-economic burden worldwide, IVD degeneration is also severely impacting on the quality of life of affected individuals. Cell-based regenerative medicine approaches have moved into clinical trials, yet IVD cell identities in the mature disc remain to be fully elucidated and tissue heterogeneity exists, requiring a better characterization of IVD cells. The bovine coccygeal IVD is an accepted research model to study IVD mechano-biology and disc homeostasis. Recently, we identified novel IVD biomarkers in the outer annulus fibrosus (AF) and nucleus pulposus (NP) of the mature bovine coccygeal IVD through RNA in situ hybridization (AP-RISH) and z-proportion test. Here we follow up on Lam1, Thy1, Gli1, Gli3, Noto, Ptprc, Scx, Sox2 and Zscan10 with fluorescent RNA in situ hybridization (FL-RISH) and confocal microscopy. This permits sub-cellular transcript localization and the addition of quantitative single-cell derived values of mRNA expression levels to our previous analysis. Lastly, we used a Gaussian mixture modeling approach for the exploratory analysis of IVD cells. This work complements our earlier cell population proportion-based study, confirms the previously proposed biomarkers and indicates even further heterogeneity of cells in the outer AF and NP of a mature IVD.
Highlights
Cell-based tissue regeneration approaches to address degeneration of intervertebral discs (IVD)are a glimpse of hope for a large percentage of the population suffering from severe and chronic lower back pain due to age or injury related degenerative disc disease (DDD)
In need for further characterization of resident cells in the mature IVD, we recently proposed a set of novel IVD biomarkers based on the proportion of cells within the outer annulus fibrosus (AF) and nucleus pulposus (NP) tissue of bovine coccygeal IVDs being either positive or negative for the proposed biomarker transcript [3]: Laminin1 (Lam1) belongs to a group of glycoproteins of high molecular weight and is present in the extracellular matrix (ECM) of the basal lamina with the ability to bind to collagens, integrins and proteoglycans [25]
Some of our biomarkers showed a non-normal distribution in transcriptionally active outer AF cells: Glioma-associated oncogene 3 (Gli3), Noto, and Zscan10 (Figure 5A) or NP cells: Thymocyte differentiation antigen 1 (Thy1), Glioma-associated oncogene 1 (Gli1), Gli3, Scx, Sex determining region Y-box 2 (Sox2) and Zscan10 (Figure 5B) as determined through the use of the Shapiro–Wilk test; k-means clustering was performed when data was not normally distributed to fit data to a Gaussian mixture model (GMM) (Tables S2 and S3). Using this approach and assessing one gene at a time we identified distinct sub-populations with at least a two-fold difference of transcript levels as represented by the total fluorescence intensity of Gli3, Noto and Zscan10 expressing cells in the outer AF cell population (Figure 6A) and for Thy1, Gli1, Gli3, Scx, Sox2 and Zscan10 in the NP cell population (Figure 6B)
Summary
Cell-based tissue regeneration approaches to address degeneration of intervertebral discs (IVD). The IVD is a tissue of strength, resilience and capable of reducing the impact of movement on the rigid vertebral bodies that stabilize our spine [1]. In this field of regenerative medicine, strategies are pursued to refurbish ailing IVDs with autologous or allogenic cells, derived either from the disc itself or other sources, with special interest in mesenchymal stem cells (MSC) derived from bone marrow, articular cartilage or adipose tissue. On first glance the IVD might appear as an anatomically simple structure, comprised of concentric rings in the annulus fibrosus (AF) and gradually transitioning towards a central nucleus pulposus (NP) [1,7,8].
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