Clayton Sade W, Sebastian Aimy, Wilson Stephen P, Hum Nicholas R, Walk Remy E, Easson Garrett W D, Vaidya Rachana, Broz Kaitlyn S, Loots Gabriela G, Tang Simon Y
Washington University in St. Louis, St. Louis MO.
Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore CA.
bioRxiv. 2025 Jan 4:2024.08.10.607363. doi: 10.1101/2024.08.10.607363.
Intervertebral disc (IVD) degeneration contributes to disabling back pain. Degeneration can be initiated by injury, and progressively leads to irreversible cell loss and loss of IVD function. Attempts to restore IVD function through cell replacement therapies have had limited success due to knowledge gaps in the critical cell populations and molecular crosstalk after injury. Here, we used single cell RNA sequencing to identify the transcriptional changes of endogenous cells of the IVD and infiltrating cell populations following IVD injury. Control and Injured coccygeal IVDs were extracted from 12 week old female C57BL/6J mice 7 days post injury and subjected to single-cell resolution transcriptomic sequencing. Clustering, gene ontology, and pseudotime trajectory analyses determined transcriptomic divergences in the cells of the Injured IVD, flow cytometry identified they types of infiltrating immune cells, and immunofluorescence was utilized to define mesenchymal stem cell (MSC) localization. Clustering analysis revealed 11 distinct cell populations that included IVD, immune, vascular cells, and MSCs. Differential gene expression analysis determined that Outer Annulus Fibrosus, Neutrophils, Saa2-High MSCs, Macrophages, and Krt18 Nucleus Pulposus (NP) cells were the major drivers of transcriptomic differences between Control and Injured cells. Gene ontology revealed that the most upregulated biological pathways were angiogenesis and T cell-related while wound healing and ECM regulation categories were downregulated. Pseudotime trajectory analyses revealed that IVD injury directed cells towards increased differentiation in all clusters, except for Krt18 NP cells which remained in a less mature cell state. Saa2-High and Grem1-High MSCs populations drifted towards more differentiated IVD cells profiles with injury and localized distinctly within the IVD. This study revealed novel MSC populations in a heterogeneous landscape of IVD cell populations during injury, and these cells may be leveraged for future IVD repair studies.
椎间盘（IVD）退变会导致使人丧失能力的背痛。退变可由损伤引发，并逐渐导致不可逆转的细胞丢失和IVD功能丧失。由于在关键细胞群和损伤后分子相互作用方面存在知识空白，通过细胞替代疗法恢复IVD功能的尝试取得的成功有限。在这里，我们使用单细胞RNA测序来确定IVD损伤后IVD内源性细胞和浸润细胞群的转录变化。在损伤后7天，从12周龄雌性C57BL/6J小鼠中提取对照和损伤的尾椎IVD，并进行单细胞分辨率转录组测序。聚类、基因本体和伪时间轨迹分析确定了损伤IVD细胞中的转录组差异，流式细胞术鉴定了浸润免疫细胞的类型，并利用免疫荧光来确定间充质干细胞（MSC）的定位。聚类分析揭示了11个不同的细胞群，包括IVD、免疫、血管细胞和MSC。差异基因表达分析确定，外层纤维环、中性粒细胞、Saa2高表达的MSC、巨噬细胞和Krt18髓核（NP）细胞是对照细胞和损伤细胞之间转录组差异的主要驱动因素。基因本体显示，上调最多的生物学途径是血管生成和T细胞相关途径，而伤口愈合和细胞外基质调节类别则下调。伪时间轨迹分析表明，IVD损伤使所有簇中的细胞分化增加，但Krt18 NP细胞除外，其保持在较不成熟的细胞状态。Saa2高表达和Grem1高表达的MSC群体在损伤时向更分化型的IVD细胞谱漂移，并在IVD内明显定位。这项研究揭示了损伤期间IVD细胞群异质性景观中的新型MSC群体，这些细胞可能用于未来的IVD修复研究。
