Introduction: Low back pain (LBP) episodes are common and affect everyday life. A major cause of chronic LBP is intervertebral disc degeneration. Notochordal cells (NCs), the juvenile disc cells only present in young individuals and not in degenerated IVDs, possess regenerative potential that could be exploited for therapeutic approaches. Previous work indicated that porcine NCs secrete extracellular vesicles (EVs) that may mediate this effect (1). To ensure that the observed effects in biological studies are associated with EVs and not associated with soluble bioactive molecules, characterization of the EV preparation is essential (2). This study aims to perform EV characterization of multiple species using a bead-based western technology to identify NC-derived EV-associated protein markers.
Methodology: NC-conditioned medium (NCCM) was generated by culturing NC-rich tissue of porcine, canine, and human origin. EVs were isolated through differential centrifugation followed by size exclusion chromatography (SEC). EV containing fractions were identified based on protein content and pooled for analysis. The SEC fractions from porcine NCCM were pooled in sets of three and subjected to analysis. NC-derived EVs were characterized using DigiWest technology, a high-throughput bead-based multiplex platform (3).
Results: Using the DigiWest technology, a panel of 33 proteins was determined in NCCM-derived EVs. The analysis of porcine and canine NCCM-derived EVs revealed the presence of 12 EV-associated protein markers in common. In human NCCM-derived EVs, only two proteins (MFGE8 and fibronectin) were identified, most probably due to technical limitations related to low starting protein quantities. These two proteins were also present in porcine/canine NCCM-derived EVs. Possible co-isolated proteins that were detectable in control tissue samples were not detected in the EV-fractions of these tissues.
In addition, the DigiWest platform was used to detect EV-associated proteins in SEC fractions containing relatively small protein quantities (0.9-9 µg input). An enrichment in EV markers (CD9, TSG101, flotillin 1, and HSPA8) was seen in fractions 7-12 compared to later fractions, as expected based on the reported EV-marker profiles.
Conclusion: Altogether, for porcine and canine NCCM-derived EVs, several transmembrane, GPI-anchored, and cytosolic proteins were identified, which is recommended for EV characterization according to the standards of the International Society for Extracellular Vesicles (2). Additionally, the EV samples were devoid of some non-EV-associated tissue proteins that could be co-isolated. Based on these results, a panel of 19 proteins was composed to characterize NC-derived EVs from different species.
The identification of EVs in separate SEC fractions has major challenges with, amongst others, low protein quantities in the fractions and the need for parallel identification of multiple protein markers for proper EV characterization (2). However, the DigiWest technology has been shown to successfully identify multiple EV-associated proteins in a sample containing small protein quantities. It may therefore be used to identify and characterize EVs in SEC fractions.
This project received funding from the European Union’s Horizon 2020 program iPSpine (No. 825925).
1. Bach, F.C. et al., Oncotarget 8, 88845-88845 (2017)
2. Théry, C. et al. J. Extracell. Vesicles 7, (2018)
3. Treindl, F. et al., Nat. Commun. 7, 1-11 (2016)