Hypotheses and aims: Mesenchymal stem cells have been shown to reduce inflammation, enhance bacterial clearance, and improve survival in sepsis. Recent studies on the immunomodulatory function of mesenchymal stem cell-derived EVs (MSC-EVs) highlight their crucial role in attenuating the activated immune response. Interaction of MSC-EVs with activated immune cells (monocytes, macrophages, T-cells, NK-cells) induces anti-inflammatory cytokines such as interleukin-10 (IL-10) and lipid mediators such as prostaglandin E2 (PGE2), driving the differentiation towards an immunosuppressive phenotype. Adipose-derived MSCs have been shown to modulate monocyte subsets in sepsis patients via the PGE2 pathway and to reduce the level of CD16⁺ inflammatory monocytes.

We therefore postulate that MSC-EVs can prevent or revert the shift towards CD16⁺ monocyte subsets that is induced by platelet-derived EVs (pEVs) and, presumably, also by red blood cell-derived EVs (rbcEVs) (see PhD project 1). We further propose that differences in the EV composition related to the culture conditions during MSC expansion can influence their inflammatory potential. In particular, we will assess the influence of 2D vs. 3D MSC culture (hydrogels and spheroids) and the influence of medium supplements (bovine and human serum) on the ability of MSC-EVs to reduce CD16⁺ monocyte subsets. In addition, we will characterize TF expression on MSC-EVs from these different culture conditions, as TF might counteract the anti-inflammatory effect of MSC-EVs and induce a shift towards CD16⁺ monocytes, as discussed in PhD project 1. The dependance of MSC-EV-related immunomodulation on the activation of different Toll-like receptors (TLR2, TLR3, TLR4) will be studied in PhD project 3.

Methods: MSCs will be isolated from the placental amnion of healthy delivering women and further cultured in 2D and 3D culture systems. We will compare 2D culture on plastic surfaces with two different 3D culture systems, spheroids, and hydrogels. We will use spheroids generated by micro-structured spherical plates and gelatin-methacryloyl (GelMA)-based hydrogels which allow the construction of 3D scaffolds. Effects of the medium composition will be addressed using animal (fetal bovine serum) and human serum (AB-serum). MSC-EVs will be enriched by differential centrifugation and characterized using nanoparticle tracking analysis and flow cytometry (CD73, CD90, CD105 expression; negative for CD34, CD45, HLA-DR, CD14, CD16). Surface expression of TF on MSCs and MSC-EVs from different culture conditions will be determined by flow cytometry. Untouched monocytes will be isolated by depletion of non-monocytes from peripheral blood mononuclear cells (PBMCs) using magnetic beads. MSC-EVs will be added to CD16⁺ monocytes obtained by incubation of monocytes with platelets. The monocyte subset distribution will be characterized by flow cytometry as established in PhD project 1 to test whether MSC-EVs can reduce CD16 expression (reversible effect). To assess whether MSC-EVs can prevent the pro-inflammatory shift, MSC-EVs, monocytes and platelets will be co-cultured using the set-up described in PhD project 1.

Main supervisor: M.B. Fischer (University for Continuing Education Krems)

Proposed mobility: M. Dominici, Modena (characterization of MSC-EVs)