VARIATION IN EXTRACELLULAR VESICLES AMONG CLONAL AND POLYCLONAL MESENCHYMAL STROMAL CELL POPULATIONS – AN EXPANDED ANALYSIS

• Published in: Cytotherapy (Oxford, England) England), 2024-06, Vol.26 (6), p.S83-S84
• Main Authors: Carson, E.J., Bonfield, T., Krishna, V., Midura, R., Swamydas, M., Duginski, G., Narayanan, P., Ranga, S., Furyes, A., Sandhir, S., Muschler, G.F.
• Format: Article
• Language: English
• Subjects: Exosomes; Extracellular Vesicles; Mesenchymal Stromal Cell
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2024.03.157

Tissues contain a heterogeneous population of native cells capable of in vitro proliferation and differentiation into connective tissue phenotypes. Defined as connective tissue progenitors (CTPs), these cells may qualify as mesenchymal stromal cells (MSCs) based on established criteria. The cell therapy field is focused on safety and functional relevancy of MSC extracellular vesicles (EVs). Donor variation likely contributes to inefficient clinical trial success. Our work interrogates MSC heterogeneity by quantifying MSC derived EV variability. We now demonstrate an innovative approach to identify and rigorously characterize the variation present in 4 MSC donor populations, the latest step towards establishing criteria for performance-based selection, culture, and manufacture of MSC functionally active EV products. MSCs derived from femoral metaphyseal bone of 4 human donors were cultured via limiting dilution in fetal bovine serum media for 8 days (imaged daily). A total of 15 clonal colonies were then picked with the Cell XTM cell processing system from these 4 donors and individually expanded to reach > 3M cells. A total of 11 polyclonal populations (multiple founding cells) from these 4 donors were similarly expanded to reach > 3M cells. Exogenous serum derived EVs were removed via 48-hour serum free media wash. MSC EVs were isolated from media via ultracentrifugation and banked at -80°C (consistent with MISEV guidelines). EV isolation was confirmed by electron microscopy (Fig 1). 32 biochemical analytes were measured via Luminex multiplex on lysed/whole EVs and residual non-EV secretome from each MSC clone/polyclonal population. Results showed differences in levels of the 32 analytes between clones and polyclonal populations (EVs/secretomes) from each patient (Table 1). There were 9 analytes elevated in whole EVs, 2 analytes elevated in lysed EVs, 11 analytes elevated in non-EV secretome, and 10 analytes with variable expression. There was higher elevation of IFN-α in lysed EVs and Neuropilin-1 had comparable quantities in both lysed EVs and non-EV secretome. These results indicate MSC EVs/secretomes can be harnessed to signify unique biologically active cytokine distributions that may be important for consistency in therapeutic immunomodulatory and tissue repair potency. Relevant differences between contents of EVs and non-EV secretomes derived from individual clonal populations may provide a new innovative way to optimize MSC therapeutic potency.