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Single-cell transcriptome profiling reveals nucleus pulposus heterogeneity and immunity during degeneration progression

ABSTRACT: The heterogeneity of nucleus pulposus cells (NPCs) and the molecular mechanisms underlying NP degeneration is still unclear. We performed unbiased transcriptome-wide single-cell RNA sequencing (scRNA-seq) on progressing degenerative grades of human NP. Cell subsets, their gene signatures, differentiation trajectory, and cell interactions were determined and characterized.We uncover seven populations of human NP cells and referred to these novel cell clusters with markers and their distinct functions. Monocle pseudospace, cytoTRACE, and scVelo analysis showed potential transition among cell subsets. We revealed novel markers for NP progenitors (NPPs), and found a close relationship between NPPs and fibrosis NP (FNPs). Neutrophils showed heterogeneity, which may be a resultsresult of activation and maturation. Identified granulocytic myeloid-derived suppressor cells (G- MDSCs) are correlated with disease progression. CD11b, OLR1, and CD24 were shown to be the markers in NP-derived G-MDSCs.Our study revealing NPCs type complexity and phenotypic characteristics of immune cells inside NP, providing new insights and clues for treatment of IVDD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE165722 | GEO | 2021/12/26

REPOSITORIES: GEO

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Project description:Purpose: Degeneration and aging of the nucleus pulposus (NP) of the intervertebral disc (IVD) is accompanied by alterations in NP cell phenotype marked by reduced cellularity and a shift towards a fibroblast-like state. We have recently demonstrated an ability to manipulate the phenotypic expression of adult degenerative NP cells by culture upon poly(ethylene glycol) (PEG) based hydrogels dually functionalized with integrin- and syndecan-binding laminin-mimetic peptides. In the present study, we seek to understand the transcriptome changes elicited by NP cell interactions with the presented hydrogel system. Methods: mRNA profiles of NP from 3 human tissue samples were cultured for 4 days on either tissue culture polysyrene (TCPS) or the functionalized gel before RNA was isolated and sequenced, using Illumina NovaSeq. Unaligned reads were trimmed based on quality score (minimum quality level (Phred) = 20, minimum read length = 25) and aligned to the whole human genome (STAR 2.6.1d; hg19) using Partek Flow software. This software suite was also used to calculate the counts/normalized counts of genes and to perform gene specific analysis (GSA), principle component analysis (PCA), hierarchical clustering, and gene set enrichment. Differentially regulated genes were considered to be those with a fold change value (gel/TCPS) that was greater than or equal to 2 or less than or equal to negative 2 and a p-value of less than or equal to 0.05. Results: The data corroborate and expand on the previous findings by demonstrating that degenerative adult human NP cells cultured upon these gels have upregulations in some markers of NP and notochordal cells but also downregulations of some NP-specific markers and several fibroblastic markers. Furthermore, through gene set enrichment analysis we have shown that pathways related to cell differentiation and notochord morphogenesis were upregulated in the gel condition. Additionally, 13 genes associated with G protein-coupled receptors, many of which are known drug targets, were identified as up or downregulated following periods of culture upon the gel condition. Conclusions: The data from this RNA-sequencing demonstrate that culture on laminin-mimetic-peptide presenting gels can modulate cell phenotype and promotes upregulation of NP and notochordal markers.

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Single-cell transcriptome profiling reveals nucleus pulposus heterogeneity and immunity during degeneration progression

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