Single-cell extracellular vesicle-program scoring maps immunometabolic rewiring and immune crosstalk of mesenchymal stromal cells in intervertebral disc degeneration, prioritizing AP2S1 and CSTB.

Background: Intervertebral disc degeneration (IDD) is increasingly viewed as an immune-perturbed and metabolically stressed niche rather than a purely mechanical or aging-related disorder. Mesenchymal stromal cells (MSCs) and their extracellular vesicles (EVs) may shape local immune signaling, yet MSC EV-associated transcriptional programs and their immune-network context remain poorly defined at single-cell resolution. Methods: We analyzed single-cell RNA sequencing (scRNA-seq) data from GSE230809 (8 IDD and 3 control nucleus pulposus samples) with standard preprocessing and batch correction. We derived a transcriptome-inferred MSC EV-associated program score by integrating complementary gene-set scoring strategies, stratified MSCs into EV-score-high versus -low states, and prioritized candidate regulators using a consensus LASSO/SVM-RFE workflow. CellChat was used to infer ligand-receptor communication, while pathway-level immunometabolic remodeling was assessed with GSVA/GSEA and immune-signature correlation analyses. Selected genes were examined by RT-qPCR in an Results: We resolved seven major cell populations and observed a marked shift toward higher EV-program scores in MSCs from IDD tissues. EV-score-high MSCs showed increased incoming signaling and interaction potential in CellChat networks, consistent with an "immune cue-responsive" state. Machine-learning prioritization converged on five hub genes (AP2S1, CSTB, GSTP1, RPL28, and TSG101). The hub-gene program aligned with immune-metabolic axes including IL6/JAK-STAT3 and interferon-related signaling, together with oxidative phosphorylation and ROS stress signatures, and was systematically associated with immune mediators (notably chemokines and checkpoint-related genes). AP2S1 and CSTB displayed dynamic expression along MSC pseudotime trajectories and were supported by RT-qPCR under inflammatory stimulation. A hub-gene model distinguished IDD from control samples within the discovery cohort (AUC = 0.836). Docking suggested a plausible interaction between CSTB and ripasudil (-5.98 kcal/mol). Conclusions: This study maps an MSC EV-associated program at single-cell resolution and places it within an immune-communication and immunometabolic framework in IDD. AP2S1 and CSTB are candidate nodes linking EV-program states to immune signaling and metabolic stress, providing candidate biomarkers and tractable intervention hypotheses.