Single-cell RNA sequencing reveals resident progenitor and vascularization-associated cell subpopulations in rat annulus fibrosus

BACKGROUND: One of the main causes of low back pain is intervertebral disc degeneration (IDD). Annulus fibrosus (AF) is important for the integrity and functions of the intervertebral disc (IVD). However, the resident functional cell components such as progenitors and vascularization-associated cells in AF are yet to be fully identified. PURPOSE: Identification of functional AF cell subpopulations including resident progenitors and vascularization-associated cells. METHODS: In this study, the single-cell RNA sequencing data of rat IVDs from a public database were analyzed using Seurat for cell clustering, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional analysis, StemID for stem cell identification, Monocle and RNA velocity for pseudotime differentiation trajectory validation, single-cell regulatory network inference and clustering (SCENIC) for gene regulatory network (GRN) analysis, and CellChat for cell–cell interaction analysis. Immunostaining on normal and degenerated rat IVDs, as well as human AF, was used for validations. RESULTS: From the data analysis, seven AF cell clusters were identified, including two newly discovered functional clusters, the Grem1(+) subpopulation and the Lum(+) ​subpopulation. The Grem1(+) subpopulation had progenitor characteristics, while the Lum(+) ​subpopulation was associated with vascularization during IDD. The GRN analysis showed that Sox9 and Id1 were among the key regulators in the Grem1(+) subpopulation, and Nr2f2 and Creb5 could be responsible for the vascularization function in the Lum(+) ​subpopulation. Cell–cell interaction analysis revealed highly regulated cellular communications between these cells, and multiple signaling networks including PDGF and MIF signaling pathways were involved in the interactions. CONCLUSIONS: Our results revealed two new functional AF cell subpopulations, with stemness and vascularization induction potential, respectively. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: These findings complement our knowledge about IVDs, especially the AF, and in return provide potential cell source and regulation targets for IDD treatment and tissue repair. The existence of the cell subpopulations was also validated in human AF, which strengthen the clinical relevance of the findings.