Single nuclear-spatial transcriptomic sequencing reveals distinct puncture-induced cell subpopulations in the intervertebral disc of a rat model

Abstract

Objective: We aim to investigate the spatiotemporal dynamics of intervertebral disc (IVD) cell subpopulations in IVD degeneration (IVDD).

Methods: To gain combined spatial and transcriptomic insights into IVDD, we employed both spatial transcriptomic sequencing (stRNA-seq) and single nucleus RNA sequencing (snRNA-seq) in a rat puncture-induced IVDD model. The findings were verified in rat and human IVD by immunostaining and qRT-PCR. Tamoxifen-administered Pdgfra^CreERT2;R26^tdTomato mice were adopted to track platelet-derived growth factor receptor alpha (Pdgfra) positive cells.

Results: Puncture response regions were revealed on day 1 post-puncture, for which oxidative stress emerged as a prominent pathway in a Stress Zone consisting of lipocalin-2 (Lcn2)+ annulus fibrosus (AF) cells (AFC), which propagated and migrated into nucleus pulposus (NP), playing a key role in delivering injury signals and triggering pathological processes, including ferroptosis, fibrosis, and immune reactions. In the NP, Collagen 3-high (Col3hi) NP cells (NPC) were another induced population demonstrating a fibrochondrocyte-like phenotype and high epithelial–mesenchymal transition activation, an important pathway involved in tissue fibrosis. Crucially, lineage tracing experiments in Pdgfra^CreERT2;R26^tdTomat mice revealed the significant migration and proliferation of Pdgfra+ AFCs from the AF into the NP following puncture. These findings provide direct evidence that both Pdgfra+ AFCs and Col3hi NP cells may contribute to NP fibrosis.

Conclusion: Puncture-induced oxidative stress in a stress zone is the primary reaction playing an important role in initiating IVDD. Several puncture-induced cell subpopulations were identified, including Lcn2+ AFC, Col3hi NPC, and Pdgfra+ AFC. Lcn2+ AFC plays a pivotal role in connecting oxidative stress with other pathological processes. Our results clarified the dual origin of Pdgfra+ cells, highlighting the contribution of AF-derived cells to the NP during degeneration and emphasizing the complexity of cellular changes underlying NP fibrosis. Further investigation into the specific contributions of Pdgfra+ cells from different origins to fibrosis is warranted.

Keywords

intervertebral disc degeneration / LCN2 / oxidative stress / PDGFRA / single-nucleus RNA sequencing / spatial transcriptomic

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