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Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone

ABSTRACT: The nucleus pulposus (NP) is composed of notochordal NP cells (NCs) and chondrocyte-like NP cells (CLCs). CLCs and chondrocytes have been reported to be similar. However, the interactions between CLCs and NCs remain unclear. In this study, to clarify how cells in NP and chondrocytes are regulated, we performed single-cell RNA sequencing (scRNA-seq) analysis of articular cartilage (AC) and NP of cynomolgus monkeys and found that part of CLCs and articular chondrocytes had similar gene expression profiles. These cells were enriched for genes related to GLI1, the nuclear mediator of the hedgehog pathway. In the NP, cell–cell interaction analysis revealed sonic hedgehog (SHH) expression in NCs, resulting in hedgehog signaling to CLCs, whereas no chondrocytes in our AC samples expressed hedgehog ligands.

ORGANISM(S): Macaca fascicularis

PROVIDER: GSE222449 | GEO | 2023/06/07

REPOSITORIES: GEO

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Project description:Objective: Aging and early degeneration of the intervertebral disc (IVD) involves the substition of notochordal cells (NCs) in the nucleus pulposus (NP) by chondrocyte-like cells (CLCs). This study investigated the gene expression profiles involved in this process using NP tissue from both non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occuring IVD degeneration. Methods: Dual channel DNA microarrays were used to compare 1) healthy NP tissue, 2) NP tissue with a mixed population of NCs and CLCs, and 3) NP tissue containing solely CLCs. Canonical Wnt-signaling was validated using qPCR of relevant Wnt target genes. Caveolin-1, a known regulator of canonical Wnt signaling, was investigated further in tissue sections using qPCR and immunohistochemistry, and in cultured NCs by qPCR and immunofluorescence. Also, the NP of 3-month-old caveolin-1 knock-out mice was histopathologically evaluated and compared with wild type mice of the same age. Results: Early IVD degeneration involved significant regulations in numerous pathways, such as extracellular matrix remodeling, Bone Morphogenetic Protein- , and Wnt/beta-catenin-signaling. With regard to Wnt/beta-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant downregulation of axin2 gene expression and caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of 3-month-old WT mice were rich in viable NCs, whereas the NPs of 3-month-old caveolin-1 knock-out mice contained chondroid-like matrix with small, rounded cells, the majority of which showed morphological signs of apoptosis. Conclusions: Aging and the onset of degeneration of the IVD involve significant downregulation of canonical Wnt signaling and caveolin-1, which appears to be essential in the physiology and preservation of NCs. DNA microarrays were used to compare nucleus palposus (NP) tissue of healthy and chondrodystrophic individuals. Furthermore, the situation of the tissue was divided into three stages: NCR: notochordal cell(NC) rich; CR: tissue containing solely chondrocyte-like-cells (CLC) and T: tissue with a mixed population of NCs and CLCs. Comparisons were analysed on a 2-color platform against a common reference sample, consisting of a multitude of canine organs, including liver, spleen, kidney, lung, hart, intestine and bone.

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.

Project description: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 molecular mechanisms involved remain elusive. Methods: Through bioinformatics analysis, the single-cell transcriptome sequencing expression profiles of human normal nucleus pulposus cells (NNP) and human degenerative nucleus pulposus cells (DNP) were compared to clarify the transcriptome differential expression profiles of human normal and human degenerative nucleus pulposus cells. The single-cell sequencing results of the two samples were analyzed using bioinformatics methods to compare the differences in histiocytosis between human normal nucleus pulposus and human degenerated nucleus pulposus, 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 Chondrocyte1, Chondrocyte2, Chondrocyte3, Chondrocyte4, Chondrocyte5, 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 normal nucleus pulposus cells and highly expressed COL2A1 compared with degenerated nucleus pulposus cells; chondrocyte 2, chondrocyte 3, chondrocyte 4, and chondrocyte 5 accounted for a higher proportion of degenerated nucleus pulposus cells compared with normal nucleus pulposus 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 are chondrocytes that highly express pain inflammatory genes such as PTGES, 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 normal nucleus pulposus 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.

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Chondrocyte-like cells in nucleus pulposus and articular chondrocytes have similar transcriptomic profiles and are paracrine-regulated by hedgehog from notochordal cells and subchondral bone

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