Single-Cell RNA Sequencing Reveals the Difference in Human Normal and Degenerative Nucleus Pulposus Tissue Profiles and Cellular Interactions

Abstract

Background: The nucleus pulposus is a constituent structure of the human intervertebral disc, and its degeneration can cause intervertebral disc degeneration (IDD). However, the cellular and molecular mechanisms involved remain elusive.Methods: Through bioinformatics analysis, the single-cell transcriptome sequencing expression profiles of human normal nucleus pulposus (NNP) cells and human degenerative nucleus pulposus (DNP) cells were compared to clarify the transcriptome differential expression profiles of human NNP and DNP. The single-cell sequencing results of the two samples were analyzed using bioinformatics methods to compare the differences in histiocytosis between human NNP and DNP, map the histiocytes of NNP and DNP, perform cell differentiation trajectories for the cell populations of interest and predict cell function, and explore their heterogeneity by pathway analysis and Gene Ontology analysis.Results: Nine cell types were identified, which were chondrocyte 1, chondrocyte 2, chondrocyte 3, chondrocyte 4, chondrocyte 5, endothelial, macrophage, neutrophil, and T cells. Analysis of the proportion of chondrocytes in different tissues revealed that chondrocyte 1 accounted for a higher proportion of NNP cells and highly expressed COL2A1 compared with DNP cells; chondrocyte 2, chondrocyte 3, chondrocyte 4, and chondrocyte 5 accounted for a higher proportion of DNP cells compared with NNP cells. Among them, chondrocyte 2 was an inhibitory calcified chondrocyte with high expression of MGP, chondrocytes 3 were fibrochondrocytes with high expression of COL1A1, chondrocytes 4 were chondrocytes that highly express pain inflammatory genes such as PTGES, and chondrocytes 5 were calcified chondrocytes with high expression of FN1 (chondrocytes 4 and chondrocytes 5 were found for the first time in a study of single-cell transcriptome sequencing of disc tissue). Cell trajectory analysis revealed that chondrocyte 1 was at the beginning of the trajectory and chondrocyte 3 was at the end of the trajectory, while chondrocyte 5 appeared first in the trajectory relative to chondrocyte 2 and chondrocyte 4.Conclusion: After functional identification of the specifically expressed genes in five chondrocytes, it was found that chondrocyte 1 was a chondrocyte with high expression of COL2A1, COL9A2, COL11A2, and CHRDL2 in a high proportion of NNP cells, and chondrocyte 3 was a fibrochondrocyte with high expression of COL1A1, COL6A3, COL1A2, COL3A1, AQP1, and COL15A1 in an increased proportion during nucleus pulposus cell degeneration. Through cell trajectory analysis, it was found that chondrocytes 5 specifically expressing FN1, SESN2, and GDF15 may be the key cells leading to degeneration of nucleus pulposus cells. Chondrocytes 2 expressing MGP, MT1G, and GPX3 may play a role in reversing calcification and degeneration, and chondrocytes 4 expressing PTGES, TREM1, and TIMP1 may play a role in disc degeneration pain and inflammation.

Integrated proteome sequencing, bulk RNA sequencing and single-cell RNA sequencing to identify potential biomarkers in different grades of intervertebral disc degeneration

“…But in C5, the expression of SERPINA1 was inverse, and the communication stayed in a low strength. Chondrocyte 4 (C4) is an inflamed chondrocyte ( Li et al, 2022 ), and SERPINA1 expressed abundantly in C4. Although there were few NPH cells in C4, the expression of SERPINA1 was much greater in NPH compared with NPD.…”

“…GEO database entries for scRNA-seq GSE199866 ( Cherif et al, 2022 ) and GSE205535 ( Li et al, 2022 ) were downloaded. The R package Seurat ( https://satijalab.org/seurat/ ) was utilized to analyze scRNA-seq data.…”

“…Using the “tinyarray” ( https://github.com/xjsun1221/tinyarray ) R package, the expression profiles were compared. The NP cells were annotated according to previews study ( Li et al, 2022 ). The cell-cell communication analysis was conducted using “CellChat” R package.…”

Specific PFKFB3 Inhibitor Memorably Ameliorates Intervertebral Disc Degeneration via Inhibiting NF-κB and MAPK Signaling Pathway and Reprogramming of Energy Metabolism of Nucleus Pulposus Cells

“…Whether KAN regulated one or more of them to exert energy metabolism reprogramming and anti-inflammation effect during IVDD is worthy to be explored. Thirdly, Li et al reported that nine cell types were identified in human normal nucleus pulposus (NNP) cells and human degenerative nucleus pulposus (DNP) by single-cell transcriptome sequencing [ 50 ]. According to the effect of PFKFB3 inhibition on ECs and OCs that had been proved in other research [ 20 , 23 ], the inhibitory role of KAN on ECs and macrophages in NP could be partly forecasted.…”

Matrix Stiffness Activating YAP/TEAD1-Cyclin B1 in Nucleus Pulposus Cells Promotes Intervertebral Disc Degeneration

“…A chondral subcluster highly expressing fibrotic genes, including COL1A1, MMP13 and FOSL1, has been identified in human NPCs and only exists in degenerated tissues [64,65]. Fibroblast proliferation has been confirmed [29], and the proportion of fibro-chondrocytes gradually increases with the progression of degeneration [66]. Through analysis of a deposited scRNA-seq database, we identified one cluster of human NPCs highly expressing both YAP-and fibrosis-related genes, named fibro NPCs.…”