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Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs.

ABSTRACT: A comprehensive understanding of the cellular heterogeneity and molecular mechanisms underlying the development, homeostasis, and disease of human intervertebral disks (IVDs) remains challenging. Here, the transcriptomic landscape of 108 108 IVD cells was mapped using single-cell RNA sequencing of three main compartments from young and adult healthy IVDs, including the nucleus pulposus (NP), annulus fibrosus, and cartilage endplate (CEP). The chondrocyte subclusters were classified based on their potential regulatory, homeostatic, and effector functions in extracellular matrix (ECM) homeostasis. Notably, in the NP, a PROCR⁺ resident progenitor population showed enriched colony-forming unit-fibroblast (CFU-F) activity and trilineage differentiation capacity. Finally, intercellular crosstalk based on signaling network analysis uncovered that the PDGF and TGF-β cascades are important cues in the NP microenvironment. In conclusion, a single-cell transcriptomic atlas that resolves spatially regulated cellular heterogeneity together with the critical signaling that underlies homeostasis will help to establish new therapeutic strategies for IVD degeneration in the clinic.

SUBMITTER: Gan Y

PROVIDER: S-EPMC8368097 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cell-based regenerative approaches have been suggested as primary or adjuvant procedures for the treatment of degenerated intervertebral disc (IVD) diseases. Our aim was to evaluate the regenerative and immunogenic properties of mildly and severely degenerated cervical nucleus pulposus (NP) cells with regard to cell isolation, proliferation and differentiation, as well as to cell surface markers and co-cultures with autologous or allogeneic peripheral blood mononuclear cells (PBMC) including changes in their immunogenic properties after 3-dimensional (3D)-culture. Tissue from the NP compartment of 10 patients with mild or severe grades of IVD degeneration was collected. Cells were isolated, expanded with and without basic fibroblast growth factor and cultured in 3D fibrin/poly (lactic-co-glycolic) acid transplants for 21 days. Real-time reverse-transcription polymerase chain reaction (RT-PCR) showed the expression of characteristic NP markers ACAN, COL1A1 and COL2A1 in 2D- and 3D-culture with degeneration- and culture-dependent differences. In a 5,6-carboxyfluorescein diacetate N-succinimidyl ester-based proliferation assay, NP cells in monolayer, regardless of their grade of degeneration, did not provoke a significant proliferation response in T cells, natural killer (NK) cells or B cells, not only with donor PBMC, but also with allogeneic PBMC. In conjunction with low inflammatory cytokine expression, analyzed by Cytometric Bead Array and fluorescence-activated cell sorting (FACS), a low immunogenicity can be assumed, facilitating possible therapeutic approaches. In 3D-culture, however, we found elevated immune cell proliferation levels, and there was a general trend to higher responses for NP cells from severely degenerated IVD tissue. This emphasizes the importance of considering the specific immunological alterations when including biomaterials in a therapeutic concept. The overall expression of Fas receptor, found on cultured NP cells, could have disadvantageous implications on their potential therapeutic applications because they could be the targets of cytotoxic T-cell activity acting by Fas ligand-induced apoptosis.

Project description:Intervertebral disc (IVD) degeneration is a pathological process, which may lead to lower back pain. The present study aimed to investigate the pathogenesis of IVD degeneration. GSE42611 was downloaded from Gene Expression Omnibus, including 4 nucleus pulposus samples isolated from degenerated IVDs and 4 nucleus pulposus samples separated from normal IVDs. The differentially expressed genes (DEGs) between the degenerated and normal samples were screened using the limma package in R. Functional and pathway enrichment analyses were conducted separately for the upregulated and downregulated genes, using Database for Annotation, Visualization and Integrated Discovery software. In addition, protein‑protein interaction (PPI) networks were constructed using the Search Tool for the Retrieval of Interacting Genes database and Cytoscape software. Finally, module analyses were conducted for the PPI networks using the MCODE plug‑in in Cytoscape. A total of 558 DEGs were identified in the degenerated nucleus pulposus cells: 253 upregulated and 305 downregulated. Pathway enrichment analysis revealed that downregulated thrombospondin 1 (THBS1) was enriched in extracellular matrix‑receptor interaction. Interleukin (IL)‑6 in the PPI network for the upregulated genes and vascular endothelial growth factor A (VEGFA) in the PPI network for the downregulated genes had higher degrees. Additionally, four modules (µM1, µM2, µM3 and µM4) were identified from the PPI network for the upregulated genes. Four modules (dM1, dM2, dM3 and dM4) were identified from the PPI network for the downregulated genes. In the dM2 module, collagen genes and integrin subunit α4 (ITGA4) may interact with each other. Additionally, functional enrichment indicated that collagen genes were enriched in extracellular matrix organization. In conclusion, IL‑6, VEGFA, THBS1, ITGA4 and collagen genes may contribute to the progression of IVD degeneration. These results suggested that the manipulation of these genes and their products may have potential as a novel therapeutic strategy for the treatment of patients with IVD.

Project description:STUDY DESIGN:Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD). PURPOSE:To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis. OVERVIEW OF LITERATURE:Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed. METHODS:IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases. RESULTS:A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins. CONCLUSIONS:This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.

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Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs.

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