"EVs in equine regenerative medicine – challenges and potential therapeutic implications.
Mesenchymal stem/stromal cell (MSC) derived EVs have shown an equivalent therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. However, non-standardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict comparability and standardisation of animal experiments.
We therefore performed a study to establish an EV isolation and characterisation protocol for equine MSC-derived EVs and define minimal criteria based on the ISEV criteria, which will facilitate reproducibility and comparison of equine derived EVs between studies in the future. Furthermore, we studied the therapeutic effect of equine bone marrow MSC (bmMSC) derived EVs on inflamed horse tenocytes in vitro and evaluated the influence of inflammatory preconditioning (pc) of the EV donor MSC on the content and biological activities of their EVs.
Equine bmMSC dericed EVs from 3 donors were isolated using two different techniques, size exclusion chromatography (SEC) and ultracentrifugation technique (UC). To establish minimal criteria for equine EVs, we validated markers (CD63 and CD9) for Western blot analysis and tested a combination of methods including techniques that do not require antibodies, such as Nanoparticle Tracking Analysis (NTA) and trans electron microscopy (TEM), with Fluorescence-triggered flow cytometry (FT-FC).
To evaluate their therapeutic effect, EVs were obtained from supernatants of bmMSCs preconditioned in serum free media with (pcMSC) or without (npcMSC) addition of 10ng/ml IL1β and 10ng/ml TNFα. Chemically inflamed (10ng/ml IL1β and 10ng/ml TNFα ) tenocytes (400.000 cells) were treated with 1ml of medium containing 1x10^9 autologous pcMSC or npcMSC derived EVs. Untreated chemically inflamed tenocytes and healthy tenocytes served as control groups. RNA-Sequencing (RNA-Seq) of EVs and tenocytes was performed. Adjusted p-value was set at 0.25.
The isolated particles stained positive for CD9 and CD63 as demonstrated by Western blot and for CD81 using FT-FC. FT-FC, NTA and TEM confirmed the EV-appropriate size range (between 30 and 150 nm) and TEM the presence of the characteristic lipid bilayers surrounding EVs.
Application of pcMSCs as well as npcMSC derived EVs significantly reduced expression of inflammation markers like CXCL6 (-1.4 and - 1.6 logFC), CSF3 (-3.3 and - 4.1 logFC), CXCL8 (-2.2 and - 2.2 logFC), CXCL6 (-1.4 and -1.6 logFC), TNFAIP6 (-1.4 and - 1.4 logFC) in treated tenocytes as compared to untreated controls. miRNA-Seq of the EVs showed significant down-regulation of miR-146a (-0.6 and -0.7 log2FC) which has been shown to play an important role in the negative regulation of inflammation in rheumatoid arthritis.
The obtained data allows to suggest minimal criteria for the standardized isolation and characterization of equine MSC derived EVs. The results further indicate that the cargo of pcMSC-EVs and npcMSC-EVs may reduce tenocyte inflammation and may have an immunomodulatory effect on the recipient cells which is independent of pre-conditioning of the EV donor cells."