Single Cell RNA Sequencing of Adult Mouse Intervertebral Disc Cells

Intervertebral disc degeneration is a leading cause of chronic low back pain. Cell-based strategies that seek to treat disc degeneration by regenerating the central nucleus pulposus hold significant promise, but key challenges remain. One of these is the inability of therapeutic cells to effectively mimic the performance of native nucleus pulposus cells, which are unique amongst skeletal cell types in that they arise from the embryonic notochord. In this study we use single cell RNA sequencing to demonstrate emergent heterogeneity amongst notochord-derived nucleus pulposus cells in the postnatal mouse disc. Specifically, we established the existence of early and late stage nucleus pulposus cells, corresponding to notochordal progenitor and mature cells, respectively. Late stage cells exhibited significantly higher expression levels of extracellular matrix genes including aggrecan, and collagens II and VI, along with elevated TGF-β and PI3K-Akt signaling. Additionally, we identified Cd9 as a novel surface marker of late stage nucleus pulposus cells, and demonstrated that these cells were localized to the nucleus pulposus periphery, increased in numbers with increasing postnatal age, and co-localized with emerging glycosaminoglycan-rich extracellular matrix. Cells were isolated from intervertebral discs of P30 shh-cre:R26R-tdTomato mice. TdTomato+ cells were enriched using fluorescence activated cell sorting and analyzed using single cell RNA-sequencing. Three replicate sequencing batches were performed, each comprising pooled cells from litter matched animals. Libraries were generated using the 10x Genomics Chromium Single Cell 3’ Reagent kit v3 per manufacturer’s instructions. Libraries were uniquely indexed using the Chromium dual Index Kit, and paired-end sequencing was performed at a targeted depth for each library of 20,000 mean reads per cell (NovaSeq 6000; Illumina, San Diego, USA). Nucleus pulposus cells were identified within the total sequenced cell population by expression of established markers Krt8, Krt18, Krt19 and T.