Pro-Inflammatory Priming of Umbilical Cord Mesenchymal Stromal Cells Alters the Protein Cargo of Their Extracellular Vesicles

Abstract

Umbilical cord mesenchymal stromal cells (UCMSCs) have shown an ability to modulate the immune system through the secretion of paracrine mediators, such as extracellular vesicles (EVs). However, the culture conditions that UCMSCs are grown in can alter their secretome and thereby affect their immunomodulatory potential. UCMSCs are commonly cultured at 21% O 2 in vitro, but recent research is exploring their growth at lower oxygen conditions to emulate circulating oxygen levels in vivo. Additionally, a pro-inflammatory culture environment is known to enhance UCMSC anti-inflammatory potential. Therefore, this paper examined EVs from UCMSCs grown in normal oxygen (21% O 2 ), low oxygen (5% O 2 ) and pro-inflammatory conditions to see the impact of culture conditions on the EV profile. EVs were isolated from UCMSC conditioned media and characterised based on size, morphology and surface marker expression. EV protein cargo was analysed using a proximity-based extension assay. Results showed that EVs had a similar size and morphology. Differences were found in EV protein cargo, with pro-inflammatory primed EVs showing an increase in proteins associated with chemotaxis and angiogenesis. This showed that the UCMSC culture environment could alter the EV protein profile and might have downstream implications for their functions in immunomodulation.

Therapeutic Effects of Hypoxic and Pro-Inflammatory Priming of Mesenchymal Stem Cell-Derived Extracellular Vesicles in Inflammatory Arthritis

“…EV-Pro-Inflam were the least effective treatment methodology for T cell deactivation, however they did reduce swelling and immune cell recruitment to the synovium. We hypothesise that these effects are due to alterations in the EV cargo prompted by the culture conditions, with our previous studies demonstrating modulations such as increased chemotaxis and angiogenesis prompted through proinflammatory priming of MSCs prior to EVs collection 44 . In contrast, hypoxically primed MSCs produced EVs that reduced swelling but failed to have any impact upon the histological outcomes measured in AIA joints.…”

Mesenchymal Stem Cell-Derived Extracellular Vesicles Reduce Disease Severity and Immune Responses in Inflammatory Arthritis

“…Most studies utilise pro-in ammatory IFN-γ (70,71) and/or TNF-α (72,73) to facilitate priming, although IL-1β (74) and IL-17a(75) have also been applied. Regardless of cytokine selection, the broad outcomes of exposing cells in culture to a pro-in ammatory environment is to initiate strategies that would re-establish homeostatic control in vivo, such as anti-in ammatory effects prompted through alteration of the EV cargo (76) and signalling to reduce recruitment of in ammatory mediators, expression of proin ammatory cytokines, T cell polarisation and proliferation (71,72,77), and restoration of homeostatic ratios of leukocytes and T cells (73,78). A primary mechanism involved here is IFN-γ-mediated upregulation of indoleamine 2,3-dioxygenase (IDO) (70,72,73).…”

Mesenchymal Stromal Cell Secretome for the Treatment of Immune-Mediated Inflammatory Diseases: Latest Trends in Isolation, Content Optimization and Delivery Avenues

“…According to the latest research, EV concentration and cargo could also vary when hypoxic preconditioning is employed. Hypoxic conditions have been reported to not alter the average size, morphological appearance and surface markers of MSC-derived EVs [106,107]. Not so well established is whether hypoxia could promote or demote EV production by MSCs.…”

“…It has been shown that IFN-γ is capable of inducing minor changes on EV shape and size when being used in high concentrations [142]. However, these changes do not always occur [106,107] and there is still the need to confirm if minor modifications on EV size would affect their therapeutic potential. The concentration of EVs released by licensed MSCs has also been shown to be higher.…”