Transcriptional profiling of human cartilage endplate cells identifies novel genes and cell clusters underlying degenerated and non-degenerated phenotypes.

BACKGROUND: Low back pain is a leading cause of disability worldwide and is frequently attributed to intervertebral disc (IVD) degeneration. Though the contributions of the adjacent cartilage endplates (CEP) to IVD degeneration are well documented, the phenotype and functions of the resident CEP cells are critically understudied. To better characterize CEP cell phenotype and possible mechanisms of CEP degeneration, bulk and single-cell RNA sequencing of non-degenerated and degenerated CEP cells were performed. METHODS: Human lumbar CEP cells from degenerated (Thompson grade ≥ 4) and non-degenerated (Thompson grade ≤ 2) discs were expanded for bulk (N=4 non-degenerated, N=4 degenerated) and single-cell (N=1 non-degenerated, N=1 degenerated) RNA sequencing. Genes identified from bulk RNA sequencing were categorized by function and their expression in non-degenerated and degenerated CEP cells were compared. A PubMed literature review was also performed to determine which genes were previously identified and studied in the CEP, IVD, and other cartilaginous tissues. For single-cell RNA sequencing, different cell clusters were resolved using unsupervised clustering and functional annotation. Differential gene expression analysis and Gene Ontology, respectively, were used to compare gene expression and functional enrichment between cell clusters, as well as between non-degenerated and degenerated CEP samples. RESULTS: Bulk RNA sequencing revealed 38 genes were significantly upregulated and 15 genes were significantly downregulated in degenerated CEP cells relative to non-degenerated cells (|fold change| ≥ 1.5). Of these, only 2 genes were previously studied in CEP cells, and 31 were previously studied in the IVD and other cartilaginous tissues. Single-cell RNA sequencing revealed 11 unique cell clusters, including multiple chondrocyte and progenitor subpopulations with distinct gene expression and functional profiles. Analysis of genes in the bulk RNA sequencing dataset showed that progenitor cell clusters from both samples were enriched in "non-degenerated" genes but not "degenerated" genes. For both bulk- and single-cell analyses, gene expression and pathway enrichment analyses highlighted several pathways that may regulate CEP degeneration, including transcriptional regulation, translational regulation, intracellular transport, and mitochondrial dysfunction. CONCLUSIONS: This thorough analysis using RNA sequencing methods highlighted numerous differences between non-degenerated and degenerated CEP cells, the phenotypic heterogeneity of CEP cells, and several pathways of interest that may be relevant in CEP degeneration.

Engineered extracellular vesicle-based gene therapy for the treatment of discogenic back pain.

Painful musculoskeletal disorders such as intervertebral disc (IVD) degeneration associated with chronic low back pain (termed "Discogenic back pain", DBP), are a significant socio-economic burden worldwide and contribute to the growing opioid crisis. Yet there are very few if any successful interventions that can restore the tissue's structure and function while also addressing the symptomatic pain. Here we have developed a novel non-viral gene therapy, using engineered extracellular vesicles (eEVs) to deliver the developmental transcription factor FOXF1 to the degenerated IVD in an in vivo model. Injured IVDs treated with eEVs loaded with FOXF1 demonstrated robust sex-specific reductions in pain behaviors compared to control groups. Furthermore, significant restoration of IVD structure and function in animals treated with FOXF1 eEVs were observed, with significant increases in disc height, tissue hydration, proteoglycan content, and mechanical properties. This is the first study to successfully restore tissue function while modulating pain behaviors in an animal model of DBP using eEV-based non-viral delivery of transcription factor genes. Such a strategy can be readily translated to other painful musculoskeletal disorders.

Lipid-correction models for δ13C values across small pelagic fishes (Clupeiformes) from the Atlantic Ocean.

The interpretation of δ13C values in trophic ecology requires standardization of the lipid content of organisms estimated through their C:N ratio. To avoid time-consuming lipid extractions, the use of mathematical corrections has been developed for many years, and the conclusions generally point in the direction of species-specific adjustment of the models. This study aimed at defining the maximum taxonomic level required to obtain the best corrected δ13C values in small pelagic fish of the order Clupeiformes. δ13C values of six species were analyzed bulk and lipid-free, and were used to fit and validate linear and mass-balance models at different taxonomic levels. Despite a species effect combined with the C:N ratio effect, the corrected δ13C values produced by a global model for the Clupeiformes were as good as or better when compared to lipid-free samples than those produced by species-specific models, paving the way for possible generalization to other species in this order. At the order level, the linear model outperformed the mass-balance model.