Sheep cells as a suitable in vitro tool to evaluate intervertebral disc biotherapies

Jul 1, 2022, 11:40 AM
10m
Room: S3 B

Room: S3 B

Speaker

Humbert, Paul (Nantes Université, Oniris, CHU Nantes, INSERM, Regenerative Medicine and Skeleton, RMeS, UMR 1229)

Description

"Introduction:
Intervertebral disc (IVD) degeneration is among the leading cause of low back pain, disability and morbidity worldwide. As the world population ages and only symptomatic treatment exist, IVD regeneration is a major public health challenge for the upcoming years. Human IVD cells are difficult to obtain, especially healthy ones, and murine IVDs present numerous differences with human ones (size, mechanical loading, presence of notochordal cells). The sheep spine exhibits biological and biomechanical similarities with the human one and is thus recognized as an appropriate model for translational applications1. With the goal of reducing our reliance on animal models, and for economic, regulatory, and ethical reasons, we have set up an in vitro platform based on sheep annulus fibrosus (AF) and nucleus pulposus (NP) cells to evaluate cell and extracellular vesicles (EVs) therapies.

Methodology:
Cells from both the AF and NP were isolated from the IVDs of five young sheep (≈ 3 months old). Their metabolic activity and gene expression were evaluated by CCK-8 assay and RT-qPCR. To simulate a degenerative IVD microenvironment, disc cells were treated with either recombinant sheep IL-1β (10 ng/mL) or H2O2 (500 µM) or maintained in culture for over 10 passages. EVs from human adipose-derived mesenchymal stromal cells (hASCs) were produced in a turbulent flow as previously described2 and their effect on degenerative-like NP and AF cells was evaluated. In a separate experiment, hASCs cells were co-cultured with NP or AF cells, in direct contact or transwells. The use of two species allowed us to analyze RNA expression from disc cells in direct co-culture with hASCs by using sheep-specific primers.

Results:
Prolonged culture and treatments with IL-1β or H2O2 led to a significant overexpression of inflammatory cytokines (IL6, CXCL8), matrix metalloproteinases (MMP1, MMP2, MMP3, or MMP13 depending on the treatment), and downregulation of key components of the extracellular matrix (COL1A1 & COL2A1) at the transcriptional level. While EVs consistently increased basal metabolic activity of both AF and NP cells at early and late passages, they had little effect on gene expression. On the other hand, direct cocultures with human ASCs profoundly affected disc cells’ transcriptional profile. Notably, both types of cocultures led to a drastic downregulation of CXCL8 in disc cells, reduced by over 60% in indirect coculture and even undetectable in direct coculture, while we observed an upregulation of COL1A1 but also MMP1. Surprisingly, IL6 expression showed a slight increase with hASCs on transwells but a sharp decrease by over 80% when hASCs were in direct contact.

Conclusion:
We demonstrated that healthy sheep cells expressed markers of degeneration after IL-1β and H2O2 treatment, or after numerous passages in culture. They showed biological responses to hASCs and, to a lesser extent, to hASC-derived extracellular vesicles. These results confirm the suitability of sheep disc cells to model IVD degeneration in vitro and assess biotherapies.

  1. Fusellier, M. et al., Eur. Cells Mater. 39, 17–48 (2020).
  2. Berger, A. et al., Nanoscale 13, 218–232 (2021)."
    83767226284

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