Transcriptomic evaluation of ovine intervertebral disc cells as an in vitro model to explore degenerative processes

Intervertebral disc degeneration (IVDD) is a leading cause of chronic back pain and disability. Understanding the cellular and molecular mechanisms underlying disc degeneration is crucial for developing effective therapies. This study investigates the relevance of a cellular model using annulus fibrosus (AF) and nucleus pulposus (NP) cells isolated from lamb and aged sheep discs. Histological analysis and MRI were used to confirm moderate degeneration in 7–8-year-old sheep discs. High-throughput RNA sequencing (RNAseq) was performed on four samples per condition: AF lamb, NP lamb, AF sheep, and NP sheep. Transcriptomic analysis using Gene Set Enrichment Analysis (GSEA) and Weighted Gene Co-Expression Network Analysis (WGCNA) revealed significant differences, including a higher propensity for cellular senescence and altered metabolism in sheep samples. Further in vitro functional studies elucidated these differences, providing deeper insights into the mechanisms of IVDD. Overall design: Intervertebral discs (IVDs) were collected from 4 female 6-month-old lambs and 4 female sheep between 7-8 years old (Vendée breed, GAEC HEAS, Les Rabelais, Ligné, France) in the accredited Centre of Research and Pre-Clinical Investigations (ONIRIS, National Veterinary School of Nantes). IVDs were dissected from the vertebral bodies, the AF and NP were separated from each other and diced into pieces. The tissue fragments obtained were rinsed three times in PBS with 2% penicillin/streptomycin (P/S) for 2 minutes. The extracellular matrix was digested with 0.05% hyaluronidase H4272 in PBS at 37 °C for 15 minutes. After two rinses with PBS, 15–20 mL 0.2% trypsin T9935 in PBS were added and incubated at 37 °C for 30 minutes. After two more rinses with PBS, 15–20 mL collagenase C2674 in complete medium were added and the mixtures were incubated at 37 °C overnight. The recovered suspension was filtered through a 70-µm-pore filter and centrifuged for 5 minutes at 300 g. The cells were counted and seeded in T75 flasks. AF and NP cells were grown at 37 °C and 5% CO2 in T75 flasks in complete culture medium (DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S)). Total RNA was extracted from AF and NP cells at passage 1 (P1) of lamb and sheep was extracted using NucleoSpin RNA XS kit (Macherey-Nagel, 740902). Library preparation was performed with Illumina® Stranded mRNA Prep kit. Sequencing was then performed on an Illumina HiSeq with NovaSeq 6000 SP Reagent Kit (200 cycles) v1.5. Sequencing data was then aligned with STAR using Ovis_aries_rambouillet.ARS-UI_Ramb_v2.0.111 genome.