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

## TL;DR

This study uses advanced sequencing to identify specific cell populations in rat intervertebral discs after injury, revealing how oxidative stress and cell migration contribute to disc degeneration.

## Contribution

The study introduces a novel combination of spatial and single-nucleus transcriptomics to identify puncture-induced cell subpopulations and their roles in disc degeneration.

## Key findings

- Oxidative stress in a stress zone is the primary reaction initiating intervertebral disc degeneration.
- Lcn2+ annulus fibrosus cells propagate injury signals and trigger fibrosis and immune reactions.
- Pdgfra+ cells in the nucleus pulposus have a dual origin from annulus fibrosus and nucleus pulposus cells.

## Abstract

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

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 PdgfraCreERT2;R26tdTomato mice were adopted to track platelet‐derived growth factor receptor alpha (Pdgfra) positive cells.

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 PdgfraCreERT2;R26tdTomat 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.

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.

Puncture induced oxidative stress in a Stress Zone is the primary reaction in initiating IVDD.Puncture induced several IVD cell subpopulations, including Lcn2+ AFC, Col3hi NPC and Pdgfra+ AFC.Lcn2+ AFC plays a pivotal role in connecting oxidative stress with other pathological processes.Pdgfra+ cells in the NP derived from both Pdgfra+ AFC and Col3hi NPC, highlighting dual origin of NP fibrosis.

Puncture induced oxidative stress in a Stress Zone is the primary reaction in initiating IVDD.

Puncture induced several IVD cell subpopulations, including Lcn2+ AFC, Col3hi NPC and Pdgfra+ AFC.

Lcn2+ AFC plays a pivotal role in connecting oxidative stress with other pathological processes.

Pdgfra+ cells in the NP derived from both Pdgfra+ AFC and Col3hi NPC, highlighting dual origin of NP fibrosis.

Summary of key events initiated from Stress Zone and related cell populations in puncture‐induced IVD degeneration.