Background: Human endometrium harbors a rare population of SUSD2+ perivascular/highly clonogenic mesenchymal stem cells (eMSC) that can be harvested as an office-based procedure without anaesthesia.
Aim: To define the in vitro secretory and phenotype profile eMSC that can predict their mechanism of action and functional behaviour in vivo, and to determine eMSC-derive exosomes-cargo and their therapeutic potential.
Methods: SUSD2+ eMSC were expanded in serum-free media/TGF-βR inhibitor (A83-01). Exosomes were isolated from the conditioned medium using differential ultra-centrifugation. They were characterised using Western blot analysis for exosome markers, ZetaView® for size analysis, particle count and transmission electron microscopy (TEM) for morphology. Quantitative mass spectrometry was performed to identify exosome cargoes and differential protein content. eMSC/A83-01 were also primed with inflammatory cytokines TNF-α and IFN-g for 72 hours. Conditioned media and cells were assessed for secretory and surface expression phenotype.
Results and conclusions: Our comprehensive protein cargo analysis of eMSC exosomes showed that eMSC/A83-01 secreted a wide range of pro-angiogenic, anti-fibrotic molecules and anti-inflammatory cytokines compared to untreated eMSC. sEV expressing Alix, TSG101 and Syntenin-1 with ~120nm and central depression were identified. eMSC/A83-01 had low immunogenicity before and after exposure to inflammatory cytokines with no expression of HLA-DR and CD86 but high expression of immunomodulatory molecules HLA-ABC, CD200 and CD274. eMSC are at immune haemostasis and only secreted IDO and PGE2 after priming with inflammatory cytokines demonstrating dampening effect on multiple immune cells including T-cell proliferation and NK-mediated apoptosis. In addition, eMSC/A83-01 had significantly decreased CD142 expression, indicating a low procoagulant effect however, this increased after inflammatory activation indicating a pro-angiogenic effect in an inflammatory environment. In conclusion, A83-01-treated eMSCs have enhanced therapeutic properties, suggesting their broad potential for allogenic cell-therapies or cell-free exosomes in regenerative medicine.