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

<jats:title>Abstract</jats:title><jats:p>Intervertebral disc (<jats:styled-content style="fixed-case">IVD</jats:styled-content>) 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 <jats:styled-content style="fixed-case">IVD</jats:styled-content> cells with mesenchymal stem cells, a solid understanding of the identity and nature of cells in a healthy mature <jats:styled-content style="fixed-case">IVD</jats:styled-content> is still in need of refinement. Although anatomically simple, the <jats:styled-content style="fixed-case">IVD</jats:styled-content> is comprised of heterogeneous cell populations. Therefore, methods involving cell pooling for <jats:styled-content style="fixed-case">RNA</jats:styled-content> profiling could be misleading. Here, by using <jats:styled-content style="fixed-case">RNA </jats:styled-content><jats:italic>in situ</jats:italic> hybridization and z proportion test, we have identified potential novel biomarkers through single cell assessment. We quantified the proportion of <jats:styled-content style="fixed-case">RNA</jats:styled-content> transcribing cells for 50 genetic loci in the outer annulus fibrosus (<jats:styled-content style="fixed-case">AF</jats:styled-content>) and nucleus pulposus (<jats:styled-content style="fixed-case">NP</jats:styled-content>) in coccygeal bovine discs isolated from tails of four skeletally mature animals. Our data reconfirm existing data and suggest 10 novel markers such as <jats:italic>Lam1</jats:italic> and <jats:italic>Thy1</jats:italic> in the outer <jats:styled-content style="fixed-case">AF</jats:styled-content> and <jats:italic>Gli1, Gli3</jats:italic>,<jats:italic> Noto</jats:italic>,<jats:italic> Scx</jats:italic>,<jats:italic> Ptprc</jats:italic>,<jats:italic> Sox2</jats:italic>,<jats:italic> Zscan10</jats:italic> and <jats:italic><jats:styled-content style="fixed-case">LOC</jats:styled-content>101904175</jats:italic> in the <jats:styled-content style="fixed-case">NP</jats:styled-content>, including pluripotency markers, that indicate stemness potential of <jats:styled-content style="fixed-case">IVD</jats:styled-content> 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 <jats:styled-content style="fixed-case">AF</jats:styled-content> and <jats:styled-content style="fixed-case">NP</jats:styled-content>, indicating the need for single cell assessment by methods such as <jats:styled-content style="fixed-case">RNA <jats:italic>in situ</jats:italic> hybridization</jats:styled-content>. Our work refines the molecular identity of outer <jats:styled-content style="fixed-case">AF</jats:styled-content> and <jats:styled-content style="fixed-case">NP</jats:styled-content> cells, which can benefit future regenerative medicine and tissue engineering strategies in humans.</jats:p>