Single-Cell RNA Sequencing of the Nucleus Pulposus Reveals Chondrocyte Differentiation and Regulation in Intervertebral Disc Degeneration

Abstract

The nucleus pulposus (NP), a heterogeneous tissue, is an essential functional component of the intervertebral disc. However, NP cell development route and regulation mechanism in intervertebral disc degeneration (IVDD) remain unknown. Here, we performed single-cell RNA sequencing of six NP samples with normal control, mild degeneration, and severe degeneration. Based on unbiased clustering of gene expression patterns from 30,300 single-cell RNA sequencing, we identified three cell lineage families of macrophages, endothelial, and chondrocyte cells and characterized seven chondrocyte subtypes, and defined two developmental pathways of the chondrocyte cell lineage families in the process of IVDD. Additionally, CellPhoneDB analysis revealed potential interactions between chondrocyte cells and other cells in IVDD. Chondrocytes in one of the differentiated orientations interact with macrophages and endothelial cells and have an inflammatory amplification effect, which were key factors causing IVDD. Collectively, these results revealed the dynamic cell landscape of IVDD development and offered new insights into the influence of NP cells differentiation on extracellular matrix homeostasis during degeneration, providing potential treatment targets for IVDD.

Single-Cell RNA-Seq Analysis of Cells from Degenerating and Non-Degenerating Intervertebral Discs from the Same Individual Reveals New Biomarkers for Intervertebral Disc Degeneration

“…These findings were in accordance with previous studies reporting a profound change in metabolic processes in disc cells linked to IVD degeneration [ 89 , 90 ]. Although no scRNA-seq and cell cluster annotation had previously been performed in iAF cells from degenerating and non-degenerating IVD tissue, our results are generally in agreement with previous studies of NP cells [ 48 , 49 , 54 , 55 , 56 ].…”

“…These subpopulations showed significant expression of genes involved in matrix regulation, response to stress and inflammation, cell cycle and metal binding. Similar cell subpopulations were identified by scRNA-seq analysis in human NP cells [ 48 , 49 , 54 , 81 ] and in OA [ 55 , 88 ].…”

“…The function of the clusters and gene-set activity can be predicted based on published papers applying IVD cells to single-cell sequencing [ 48 , 49 , 54 , 55 , 56 ] and using Quantitative Set Analysis for Gene Expression (QuSAGE) [ 57 ]. Pathway enrichment analysis allowed us to classify the cell clusters into four major groups.…”

“…The focus and strength of our analysis were the exploration of NP and iAF cells as a source of biomarkers with a potential to contribute to IVD degeneration. To avoid inter-donor variability observed in genome-wide association studies or other large-scale evaluations based on cell pooling and population averaging technologies [ 33 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 81 ], the present study provides the first scRNA-seq database comparison between cells of non-degenerating and degenerating human discs from the same individual. Validation of scRNA-seq expression profiling highlights potential markers that can be identified also by bulk RT-qPCR, immunohistochemistry and Western Blot.…”

“…Informative, transcriptomic profiles of cells from degenerating (D) and non-degenerating (nD) IVDs have relied either on bulk RNA sequencing, microarray-based approaches [ 30 , 31 , 32 , 33 , 34 , 35 , 36 ], or animal studies [ 18 , 28 , 37 , 38 , 39 , 40 , 41 , 42 ]. These findings are not always translatable because of interspecies differences and large variability observed when cell populations are pooled, averaging out important changes [ 33 , 43 , 44 , 45 , 46 , 47 , 48 , 49 ]. Our goal was to define transcriptional profiles of the cell populations found in D and nD adult human IVD tissue that could provide insight into disease etiology and treatment options.…”