Investigating the characteristics and functions of extracellular vesicles from umbilical cord mesenchymal stem cells

Abstract

Rheumatoid Arthritis (RA) is a chronic autoimmune disease which is characterised by a loss of immune tolerance, and an infiltration of immune cells into joints causing inflammation. Current pharmaceutical treatments for RA have adverse side effects and many patients do not respond favourably to them. Human umbilical cord mesenchymal stem cells (UCMSCs) present a promising alternative therapeutic due to their innate anti-inflammatory properties. Evidence from in vitro and in vivo studies of autoimmune diseases show that UCMSCs exert strong immunosuppressive effects. The therapeutic mechanism of action has been attributed to paracrine signalling, by which nanosized acellular particles called ‘extracellular vesicles’ (EVs) are one of the essential components. As UCMSC-mediated immunosuppression involves the use of EVs, and there are some safety concerns associated with MSC transplants, the antiinflammatory properties of their EVs were explored with the aim to see if they too were capable of immunosuppression.
In this thesis, UCMSCs were sourced from previous work looking at large scale cell expansion in the Quantum® bioreactor. UCMSCs were characterised and analysed based on their growth, morphology, and surface marker production before isolating EVs. Bone Marrow derived MSCs were also characterised and served as an MSC control. UCMSCs proliferated well in culture, had a spindle-like morphology, and produced cell surface markers in line with the International Society for Cell Therapy (ISCT) criteria for MSCs. The growth of UCMSCs in hypoxic and pro-inflammatory environments has been associated with a stronger anti inflammatory response. Therefore, UCMSCs were cultured in normoxia (21% O2), hypoxia (5% O2) and ‘primed’ with pro inflammatory cytokines in both normoxia and hypoxia. The aim was to see if their EVs, like the cells, had enhanced anti inflammatory properties. Analysis of the EV protein cargo identified an increase in proteins associated with chemotaxis (MCP2, MCP4 CCL3, CCL11) in the pro-inflammatory primed EVs from both normoxic and hypoxic conditions. There was also an increased expression of the anti-inflammatory proteins MMP-10 in the normoxic/primed EVs, and LIF and TRAIL in the hypoxic/primed EVs. Normoxic, hypoxic and normoxic/primed EVs were analysed through RNA sequencing. RNA sequencing of UCMSC-EVs had not been carried out before on hypoxic and normoxic/pro-inflammatory primed EVs and it identified differentially expressed miRNAs with potential anti-inflammatory properties (miR-139-5p, miR-140-5p, miR 214-5p) in the normoxic/primed EVs only. Findings also showed that the upregulated miRNAs had a low expression and were associated with cancer pathways.
These normoxic and normoxic/primed UCMSC-EVs were used in functional in vitro studies and co-cultured with individual cultures of peripheral blood mononuclear cells (PBMCs) from healthy donors, and with Jurkat lymphoblastic T-cells. The co-culturing of UCMSC-EVs with Jurkat cells is the first of its kind and results revealed that the pro-inflammatory primed EVs reduced the production of the inflammatory cytokine IL-17A in Jurkat cells as assessed via flow cytometry. Furthermore, the normoxic/primed EVs increased the production of the antiinflammatory protein FoxP3 in PBMCs. Neither normoxic nor normoxic/primed EVs exerted a pro-inflammatory effect on PBMCs and Jurkat cells. Instead, the findings point towards potential anti-inflammatory properties of normoxic/primed UCMSC-EVs, but further work is needed to establish their suitability for treating RA.