Isolation and characterisation of immune modulatory extracellular vesicles from CD362-selected human umbilical cord mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) exert numerous therapeutic effects, including immunomodulatory and anti-inflammatory effects in vitro and in vivo, which are being explored as a treatment for many unmet medical needs. These therapeutic effects can be attributed, to a large extent, to the paracrine actions and soluble factors released by MSCs - including extracellular vesicles (EVs), which represent a novel and promising “off-the-shelf” advanced therapy. However, the field of MSC- EV therapeutics faces some significant hurdles, including scalability, heterogeneity, and potency, that must be overcome before the clinical benefits can be fully realised. In the project described in this thesis, EVs were isolated and characterised from a novel, clinically-relevant human umbilical cord (hUC) MSC product by utilising a combined process of ultrafiltration (UF) and size exclusion chromatography (SEC). In the first results section (Chapter 2), a panel of assay techniques was used to characterise hUC-MSC-EVs according to best practices proposed by the International Society for Extracellular Vesicles (ISEV). Results of these assays showed that the isolation methodology was effective in achieving high purity of the eluted EV fractions. Selected sub-fractions displayed consistent expression of eight hUC-MSC surface markers in addition to stereotypical EV surface markers. In the second results section (Chapter 3), the scalability of the hUC-MSC-EV isolation process was investigated by generating EVs using a research laboratory-scale hollow-fibre bioreactor culture system under hypoxic culture conditions. The resulting EVs were extensively characterised and compared with those produced using a conventional, flask-based culture method. The results demonstrated consistent technical success in establishing prolonged hUC-MSC culture in the bioreactor and in isolating EVs from sequential medium harvests. The profiles of bioreactor-isolated EVs did not differ significantly from those isolated from flask-based culture. However, calculation of total hUC-MSC-EV yields compared to those of flask-based hUC-MSC cultures revealed that this culture system did not convey a benefit for scalability. In the third and final results section (Chapter 4), pooled EVs subfractions isolated by UF/SEC from various hUC-MSC xvi donors were functionally investigated using a range of in vitro assays with an emphasis on immune modulatory and anti-inflammatory functions. Results of the assays demonstrated that hUC-MSC-EVs: (a) Retained the functional enzyme, CD73, that is expressed by the parent cells and is associated with known immune modulatory properties. (b) Modulated the key pro-inflammatory intracellular signalling pathway, NFB, in a reporter cells line. (c) Were bound or internalised by a high proportion of primary human monocytes and a small proportion of primary human T cells from fresh blood samples. (d) Limited the proliferation of primary human CD4+ T-cells following polyclonal stimulation. The modulatory effects of hUC-MSC-EVs were also investigated in a lipopolysaccharide-stimulated mouse macrophage cell line assay. Although some modulations of cytokine secretion and surface marker expression were observed, consistent and robust anti-inflammatory effects did not occur with concurrent or pre-emptive EV addition. The assay did reveal evidence of donor-to-donor variability among the hUC-MSC preparations used. Overall, the project contributed novel methodologies for and insights into the isolation, scalability and phenotypic and functional properties of EVs derived from a clinical-grade human MSC therapeutic product. The diversity of findings from functional assays highlights the importance of further elucidating the complex interactions between EVs and the immune system and of defining one or more mechanisms of action prior to developing an “off-the-shelf” product intended for clinical use