Extracellular Vesicles Mediate Mesenchymal Stromal Cell-Dependent Regulation of B Cell PI3K-AKT Signaling Pathway and Actin Cytoskeleton.

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different in silico approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated in vitro as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches.

Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T, B and NK cell functions.

Mesenchymal stromal cells (MSCs) are multipotent cells, immunomodulatory stem cells that are currently used for regenerative medicine and treatment of a number of inflammatory diseases, thanks to their ability to significantly influence tissue microenvironments through the secretion of large variety of soluble factors. Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease. Here, we used a standardized methodological approach to dissect the immunomodulatory effects exerted by MSC-derived EVs on unfractionated peripheral blood mononuclear cells and purified T, B and NK cells. We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division. Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia.

Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML.

Adipose-derived stromal cells (ASCs).

Adipose-derived stromal cells (ASCs) are now emerging as a good alternative to bone marrow derived mesenchymal stromal cells (BM-MSC) for cellular therapy. Similarly to BM-MSC, ASCs can be easily isolated as adherent fibroblastoid cell population after processing lipoaspirate samples. Lipoaspiration provides a great number of cells, without extensive manipulation. ASCs express classical mesenchymal markers and only at early passages express CD34. ASCs can differentiate in cells of mesodermal lineages, such as adipocytes, osteocytes and condrocytes. ASCs share with BM-MSC the same ability to inhibit the proliferation of allogeneic, activated immune cells, thus affecting in vivo in animal models the onset and course of rheumatoid arthritis (RA), experimental autoimmune encephalomyelitis (EAE), Crohn's disease (CD), ulcerous colitis (UC) and graft-versus-host disease (GvHD). On the other hand, the main molecular pathway involved in this effect is still unclear. On the basis of this functional property, ASCs are used in different clinical trials to treat RA, CD, UC and GvHD. However, the most promising field of clinical application is represented by bone defect repair. Despite the ability to regenerate injured tissues and to block the progression of inflammatory disorders, some authors reported that ASCs can also induce, in in vivo animal models, the growth and vascularization of solid and hematological tumors. Conversely, ASCs have been shown to hamper tumor cell proliferation, reduce cell viability and induce necrosis. Thus, more accurate studies, collaborative protocols, high standardization of methods, and safety controls are required to exclude transformation of transplanted ASCs.