Dual role of fibroblasts in fibrous scar formation after spinal cord injury: Single-cell sequencing and experimental verification

Spinal cord injury induces fibrotic scar formation, which impedes axonal regeneration and functional recovery. Fibroblasts play a central role in scar formation by secreting extracellular matrix components such as collagen and fibronectin; their heterogeneity and functional specialization remain poorly understood. Recent studies suggest that meningeal-derived and perivascular-derived fibroblasts contribute differentially to fibrotic scars; however, controversies persist regarding the subpopulations of fibroblasts and their corresponding functions after spinal cord injury. The DAN family protein NBL1, a bone morphogenetic protein antagonist, has been implicated in scarless wound healing, but its role in spinal cord injury remains unexplored. Given the critical barrier posed by fibrotic scars to spinal cord injury recovery and the gaps in understanding fibroblast subpopulation diversity, the present study aimed to systematically characterize fibroblast heterogeneity in a spinal cord injury mouse model using integrated single-cell RNA sequencing data and clarify the differentiation trajectories and intercellular signaling networks of distinct fibroblast subpopulations. We further investigated the potential role of NBL1 in regulating fibroblast-mediated fibrosis and scar formation after spinal cord injury. We integrated single-cell RNA sequencing datasets (GSE162610, GSE205037, and GSE222082) to analyze fibroblast heterogeneity in the spinal cord injury mouse model. Fibroblast subpopulations were identified via UMAP clustering and annotated using marker genes. Pseudotime analysis and cell-cell communication (CellChat) were used to map differentiation trajectories and signaling networks. Functional validation was performed in TGF-β-stimulated fibroblast cultures and by intrathecal NBL1 administration in a T9 injured mouse model. Single-cell RNA sequencing identified four distinct fibroblast subpopulations: Group A (characterized by high Col4a2 expression), Group B (high NBL1 expression), Group C (high Pcolce2 expression), and Group D (high Apod expression). Pseudotime trajectory analysis positioned Group B upstream of Group A and suggested that bone morphogenetic protein pathway inhibition mediates the crosstalk between these two groups. In vitro, NBL1 attenuated transforming growth factor-β-induced fibronectin expression and impaired fibroblast migration. Correspondingly, in vivo administration of NBL1 led to a reduction in extracellular matrix production. In summary, our study identifies four fibroblast subpopulations in spinal cord injury, reveals the role of NBL1 in inhibiting fibroblast activation via the bone morphogenetic protein pathway, and provides a potential target for mitigating fibrotic scars after spinal cord injury.