Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially affect symptoms in an advanced murine Graft-versus-Host-Disease model

Abstract

Extracellular vesicles (EVs) harvested from cell culture supernatants of human mesenchymal stromal cells (MSCs) suppress acute inflammation in preclinical models of various diseases. Furthermore, they promote regeneration of damaged tissues. Following successful clinical treatment of a steroid-refractory Graft-versus-Host-Disease (GvHD) patient with EVs prepared from conditioned media of human bone marrow (BM)-derived MSCs, we aim to improve MSC-EV production and quality control towards clinical application. Observing functional differences of independent MSC-EV preparations in vitro, we established an optimized murine GvHD model for the analysis of independent MSC-EV preparations in vivo. To this end, T cell depleted allogeneic BM cells co-transplanted with naive allogeneic spleen-derived T cells induced GvHD symptoms with reproducible strengths in mice being preconditioned by ionizing irradiation. Administration of MSC-EV preparations with confirmed in vitro immune modulatory properties at three consecutive days significantly suppressed GvHD symptoms. In contrast, application of MSC-EV preparations lacking these in vitro immune modulating capabilities failed to suppress GvHD symptoms. Thus, our results reveal therapeutic differences among independent MSC-EV preparations that had been produced in a standardized manner. Thus, given this functional heterogeneity, any individual MSC-EV preparation considered for the clinical application should be evaluated for its potency prior to administration to patients.

Anti-Inflammatory Mesenchymal Stromal Cell-Derived Extracellular Vesicles Improve Pathology in Niemann–Pick Type C Disease

“…Indeed, despite treatment with similar particle numbers of EVs isolated from donor 16.3 MSCs compared to 41.5, the investigated features were not reduced and were comparable to NPC1 −/− mice treated with vehicle or platelet derived EVs. These results are in agreement with the in vitro immunomodulatory capacity of both MSC-EV fractions [ 32 , 33 ]. Consequently, functional testing of any MSC-EV preparation is necessary prior to clinical application.…”

“…Although both obtained MSC-EV preparations (MSC16.3-EV and MSC41.5-EV) revealed comparable EV characteristics, we previously reported that they exert different activities in an ischemic stroke as well as an optimized murine graft-versus.-host disease (GvHD) model and differed in their ability to modulate allogenic immune responses in vitro. Indeed, while the MSC41.5-EV preparation showed immunomodulatory activities in both animal models and in vitro, MSC16.3-EV failed to show therapeutic efficacy [ 32 , 33 ]. To compare their impact on typical systemic and neurologic features of the early-onset NPC1 pathology, aliquots of both MSC-EV preparations and of an EV preparation obtained from fresh, human platelet lysate supplemented MSC cultivation media (PL EV) were repetitively administered to NPC1 −/− mice.…”

“…However, important to keep in mind is that huge differences among independent MSC-EV preparations are reported, e.g., in cytokine profiles [ 11 , 26 ]. Recently, Madel et al showed therapeutic differences among independent MSC-EV preparations that had been produced in a standardized manner [ 32 ]. Also in our study, we show that the effects observed upon treatment with MSV-EV type 41.5 are in shear contrast to the results obtained upon treatment with EVs isolated from MSC of donor 16.3.…”

Imaging flow cytometry challenges the usefulness of classically used EV labelling dyes and qualifies that of a novel dye, named Exoria™ for the labelling of MSC-EV preparations

“…Following 5 days in culture, approximately a quarter of all CD4 + T cells express the interleukin-2 receptor (CD25) and the intercellular adhesion molecule-1 (CD54), indicating T cell activation (Figure 4). As previously described, MSC-EV preparations with immunomodulatory capabilities effectively reduce the content of activated CD4 + T cells (Madel et al, 2020). Consistently, in the presence of the non-labelled MSC-EV preparations only 16% of the monitored CD4 + T cells were found to display the activation cell surface markers (Figure 4B).…”

“…The immunomodulatory potential of Exoria labelled and non-labelled MSC-EV preparations were compared in a multi-donor mixed lymphocyte reaction assay (MLR) exactly as described previously (Madel et al, 2020). Briefly, Ficoll prepared peripheral blood mononuclear cells (PBMC) of 12 healthy donors were mixed in equal proportions, aliquoted and stored in the vapour phase of liquid nitrogen until usage.…”