Extracellular vesicles from human mesenchymal stem cells expedite chondrogenesis in 3D human degenerative disc cell cultures (1)
Chronic low back pain (LBP) is caused by multifactorial pathologies and further affected by psychosocial factors. A certain interest has been directed towards inter-vertebral disc (IVD) degeneration, believed to be one of the major underlying pathologies. It involves loss of proteoglycan and extracellular matrix (ECM) and in- creased expressions of pro-inflammatory cytokine and matrix metalloproteinases (MMPs), which affects the viability of the disc cells (DCs) in the IVDs. Mesenchymal stem cell (MSC)-based therapies are of interest in tissue repair and regeneration due to their multilineage differentiation and immunomodulatory abilities. Extracellular vesicles (EVs) are secreted by the MSCs as mediators of intercellular communication, and they have shown a potential to drive regenerative processes in many diseases.
Human MSCs were isolated and expanded from bone marrow aspirates and further characterized using flow cytometry to confirm the mesenchymal lineage. The surface markers CD73, CD90, and CD105 of hMSCs were detected, and the hematopoietic lineage markers CD45, CD34, CD11b, CD19, and HLA-DR were absent confirming the phenotypical characteris- tic of the isolated hMSCs. NTA revealed that the size of sEVs ranges between 100 and 250nm. Flow cytometry of EVs bound to CD63 beads shows that the tetraspanins CD9, CD63, and CD81 are detected on the membrane of the EVs.
Approximately 200,000 DCs were placed in a polypropylene conical tube with 0.5 ml of chondrogenic media. The cells were centrifuged (470×g at 4 °C for 5 min) and incubated (37 °C and 5% CO2) for 3–4 h to allow spheroid formation. For the EV treatment group, the media were replaced with 500 μl of chondrogenic media containing sEVs (5 × 10^10 vesicles/ ml). The pellets were then harvested at days 7, 14, and 28.
GAG assay showed an early production of GAG after EV treatment, with significantly higher levels in DCs treated with sEVs compared to control at days 7 and 14 (p < 0.01). However, a continuous increase in GAG production was observed in the control group from day 7 to 28, while in the EV group, a significantly high production was observed from day 7 to day 14 where the pellets yielded highest production already at day 14. Alcian Blue van Gieson (ABvG) histology staining suggested that DCs treated with sEVs showed high proteoglycan accumulation as early as in day 7 and throughout the culture period, confirming that sEVs from hMSCs promoted an increased and early production of ECM in degenerated DCs.
Monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), and interleukin 8 (IL-8) were detected in the media. The level of IL-6 and IL-8 in the medium from EV-treated pellets was highest at day 7 and decreased at later time points. MMP-1, which is a collagenase responsible for degradation of collagens in the IVDs during IVD degeneration, decreased over time in both EV-treated and control.
EV treatment induced early production of crucial ECM components such as proteoglycan, aggrecan, and colla- gen type II while suppressing apoptosis and the secretion of MMP-1, a process that is essential for proper IVD regeneration. sEVs can improve cell viability and proliferation and expedite chondrogenesis in DCs from degenerated IVDs of patients suffering severe LBP.