Oral Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

INTRODUCTION: Notochordal cells (NCs) are derived from the notochord and remain in the core of the developing intervertebral disc (IVD). These cells secrete factors that have regenerative effects on diseased IVD. Among these regenerative secreted factors are extracellular vesicles (EVs) [1]. EVs are small lipid bilayer-enclosed delimited particles naturally released by cells, involved in intercellular signaling. They do so by interacting with recipient cells and releasing their biological cargo (e.g., mRNA, miRNA, DNA, protein, lipid). What biological cargo contributes to the regenerative instructions remains unknown. Therefor the aim of this study was to unravel the regenerative cargo of the NC-EVs. Within the context of the IVD we focused on sonic hedgehog (SHH) and brachyury (TBXT), since these factors have an essential role in healthy IVD development and maintenance.  

METHODS: EVs were isolated from conditioned medium from porcine NC-rich tissue (N=7) using differential centrifugation, size exclusion chromatography and sucrose density gradients. To decode the full spectrum of proteins in the EV fraction, proteomic analysis was performed using mass spectrometry (LC-MS/MS). Data was analyzed using Perseus and visualized using Cytoscape. To test the bioactivity of both NC-EVs we established nano luciferase synthetic response element reporters for SHH and TBXT pathway activation.  

RESULTS: Proteomic data on NC-EVs reveals the presence of 3,274 different proteins in all NC-EV fractions isolated from 7 different donors. To associate the function of these EV proteins, we used the database gene ontology for functional enrichment analysis. The top biological pathway that is overrepresent in the EV associated proteins is vesicle-mediated transport (454 proteins, FDR=2.27e-46). Proteins that are associated to this pathway include proteins that are often identified in EVs, such as  tetraspanins (CD63 and CD59), endomembrane network-associated proteins (FLOT1 and TSG101) and heat shock proteins (HSPA8 and HSP90AA1). Besides known EV associated proteins, NC-EVs harbor proteins involved in healthy IVD maintenance. Therefore, we first established a protein-protein network around known proteins involved in healthy IVD and integrated data from multiple databases such as UniProt, STRING, HIPPIE, reactome, human phenotype ontology and international mouse phenotyping consortium. Candidate proteins were validated by text mining the PubMed database (Figure 1A). We identified multiple proteins involved in SHH pathway activation, including SMO a key transducer of the SHH pathway activation (Figure 1B). Interestingly, also the key transcription factor required for notochordal cell development, TBXT was associated with NC-EVs (Figure 1B).  

Figure 1 – NC-EV proteomics data shows presence of proteins involved in healthy IVD.

DISCUSSION: This study shows that SHH and TBXT signaling molecules are associated with NC-EVs. SHH has previously been associated with a specific EV fraction from primary chick cells derived from the notochord [2]. Follow up work focuses on validating the SHH and TBXT pathway activating using nano luciferase reporters. Overall, these preliminary data suggest that NC-EVs partly contribute their regenerative potential via SHH and TBXT pathway activation. To translate these findings towards a more clinical approach, we will test how synthetic lipid nanoparticles can be used to deliver SHH and TBXT RNA into degenerated disc cells.